Principles and methods

Ion exchange
chromatography
Contents
Introduction 4 Steps in an IEX separation 31 SOURCE: high-throughput, high-resolution 61
• Column and media preparation 31 purification, and easy scale-up
Symbols 5 • Sample preparation 32 • Media characteristics 62
Common acronyms and abbreviations 5 • Sample application and wash 32 • Purification options 62
• Sample load 33 • Purification examples 64
• Elution of target protein 34 • Performing a separation 67
01 Principles of ion exchange 7
• Linear gradient elution 34 • Cleaning 69
• Step elution 36 • Chemical stability 69
Net surface charge and pH 8
• pH elution 37 • Storage 69
Steps in an IEX separation 9
• Regeneration and re-equilibration 37 Sepharose High Performance: purification with high 70
• Equilibration 9
Detergents, denaturing agents and other additives 38 resolution
• Sample application and wash 9
Analysis of results and further steps 40 • Media characteristics 70
• Elution 9
Scaling-up 40 • Purification options 71
• Regeneration 9
Equipment selection 41 • Purification examples 72
Resolution 11
Care of IEX media 41 • Performing a separation 74
• Efficiency 12
Troubleshooting 42 • Cleaning 75
• Selectivity 14
• Chemical stability 76
Components of IEX media 16
• Storage 76
• Matrix
• Functional groups
16
18
03 Ion exchange chromatography media 47
Sepharose Fast Flow: purification with good 77
resolution and easy scale-up
Binding capacity and recovery 20 Introduction 48
• Media characteristics 78
Chromatofocusing 20 MiniBeads: purification or analysis of microgram 48
• Purification options 78
to milligram quantities with high resolution
• Purification examples 81
• Media characteristics 48
02 Ion exchange in practice 21
• Purification options 49
• Performing a separation
• Cleaning
84
86
• Purification examples 50
Introduction 22 • Chemical stability 86
• Performing a separation 51
Selecting chromatography media 22 • Storage 86
• Cleaning 52
• Capture 22 Sepharose XL: for selected proteins that require 87
• Chemical stability 53
• Intermediate purification 22 very high binding capacity to increase productivity,
• Storage 53
• Polishing 22 easy scale-up
MonoBeads: purification of milligram quantities 54
• Fast IEX media selection and method development 24 • Media characteristics 87
with high resolution
Practical considerations for IEX separation 25 • Purification options 88
• Media characteristics 55
• Buffer pH and ionic strength 25 • Purification examples 89
• Purification options 55
• Anion or cation exchanger 26 • Performing a separation 91
• Purification examples 57
• Strong or weak ion exchangers 27 • Cleaning 93
• Performing a separation 58
• Buffer selection and preparation 28 • Chemical stability 93
• Cleaning 59
• Flow rates 30 • Storage 93
• Chemical stability 60
• Flow control 30 Sepharose Big Beads: purification from crude, 94
• Storage 60 2
 viscous samples at large scale • Desalting 131 Appendix 8
• Media characteristics 95 Specific sample preparation steps 132 Analytical assays during purification 149
• Performing a separation 96 • Fractional precipitation 132 Total protein determination 149
• Cleaning 96 • Ammonium sulfate precipitation 134 Purity determination 149
• Chemical stability 97 • Resolubilization of protein precipitates 135 Functional assays 150
• Storage 97 Buffer exchange and desalting 136 Detection and assay of tagged proteins 150
Capto: high-flow media with high resolution 98 Removal of lipoproteins 137
• Media characteristics 99 Removal of phenol red 138
Appendix 9
• Purification options 99 Removal of low molecular weight contaminants 138
Storage of biological samples 151
• Purification examples 102
General recommendations 151
• Intermediate purification of insulin 103
Appendix 2 Specific recommendations for purified proteins 151
• Polishing of MAb 104
Nonvolatile and volatile buffer systems 139
Nonvolatile buffers for anion exchange chromatography 139
Appendix 10
04 Ion exchange in a Purification Strategy (Cipp) 108 Nonvolatile buffers for cation exchange chromatography 140
Volatile buffer systems 140
Column cleaning 152
Removing precipitated proteins, lipids, hydrophobically 152
Applying Cipp 110
bound proteins, or lipoproteins
Selection and combination of purification techniques 111
Appendix 3 • To remove precipitated proteins 152
Ion exchange as a capture step 114
Column packing and preparation 141 • To remove lipids, hydrophobically bound proteins, 153
Capture using Sepharose Q XL 115
Column selection 143 or lipoproteins
Ion exchange for intermediate purification 116
Column packing and efficiency 143 Extended cleaning procedures 153
Ion exchange as a polishing step 118
Alternative techniques for polishing 119
Appendix 4 Appendix 11
Selection of purification equipment 144 Media selection 154
05 Large-scale purification 120
Selection of media for automated purification 154
Selection of media for manual purification 155
BioProcess media for IEX 121 Appendix 5
Using PD-10 columns for media selection and 156
• Capture purification 123 Converting from flow velocity to volumetric 146
method development
• Polishing purification 124 flow rates
Prepacked, disposable solutions speed up the 127 From flow velocity (cm/h) to volumetric flow rate (mL/min) 146
downstream process From volumetric flow rate (mL/min) to flow velocity (cm/h) 146 Product index 157
Custom Designed Media 127 From volumetric flow rate (mL/min) to using a syringe 146

Related literature 159

06 Appendix 128 Appendix 6
Conversion data: proteins, column pressures 147
Apendix 1 Proteins 147 Ordering information 160
Sample preparation 129 Column pressures 147
Sample stability 129
Sample clarification 130 Accessories and spare parts 162
Appendix 7
• Centrifugation 130 Table of amino acids 148
• Filtration 130 3
Introduction
Biomolecules are purified using chromatography techniques that separate them according to differences in their specific
properties, as shown in Figure I.1. Ion exchange chromatography (IEX) separates biomolecules according to differences in
their net surface charge.

Property Technique
Charge Ion exchange chromatography (IEX)
Size Size exclusion chromatography (SEC), also called gel filtration (GF)
Hydrophobicity Hydrophobic interaction chromatography (HIC), Reversed phase chromatography (RPC)
Biorecognition (ligand specificity) Affinity chromatography (AC) Size exclusion Hydrophobic interaction Ion exchange

IEX for the separation of biomolecules was introduced in the 1960s and continues to play a major role in the separation
and purification of biomolecules. Today, IEX is one of the most frequently used techniques for purification of proteins,
peptides, nucleic acids, and other charged biomolecules, offering high resolution and group separations with high
loading capacity. The technique is capable of separating molecular species that have only minor differences in their
charge properties, for example two proteins differing by one charged amino acid. These features make IEX well suited for
capture, intermediate purification, or polishing steps in a purification protocol and the technique is used from microscale
purification and analysis through to purification of kilograms of product.
This handbook describes both theoretical and practical principles of the technique, the chromatography media
(resins) available and how to select them, application examples, and detailed instructions for the most commonly
performed procedures. Practical information, with many tips and hints drawn from over 50 yr of experience in
chromatography purification, guides beginners and experts towards obtaining optimal results from the latest
chromatography media.
Cytiva's business offers a wide variety of prepacked columns and ready-to-use chromatography media. A range of
handbooks ensure that purification with any chromatographic technique becomes a simple and efficient procedure at
Affinity Reversed phase
most scales and in most laboratories.

Fig I.1. Separation principles in chromatographic purification.

4
Symbols Common acronyms and abbreviations
T his symbol indicates general advice on how to improve A280  UV absorbance at specified wavelength EGTA ethylene glycol-O,
procedures or recommends measures to take in specific (in this example, 280 nm) O’-bis-[2-amino-ethyl]-N,N,N’,N’,-tetraacetic acid
situations
AC affinity chromatography ELISA enzyme-linked immunosorbent assay
This symbol indicates where special care should be taken
AIEX anion exchange chromatography F(ab’)2 fragment fragment with two antigen binding sites, obtained
H
 ighlights chemicals, buffers, and equipment by pepsin digestion
APMSF 4-aminophenyl-methylsulfonyl fluoride
Outline of experimental protocol Fab fragment antigen binding fragment obtained by
AU absorbance units
papain digestion
BSA bovine serum albumin
Fc fragment crystallizable fragment obtained by
cGMP current good manufacturing practice papain digestion
CF chromatofocusing Fv fragment unstable fragment containing the
CHO Chinese hamster ovary antigen binding domain

CIEX cation exchange chromatography GF gel filtration; also called size exclusion
chromatography
CIP cleaning-in-place
GST glutathione S-transferase
Cipp capture, intermediate purification, polishing
HCP host cell protein
CV column volume
HIC hydrophobic interaction chromatography
Dab domain antibody, the smallest functional entity of
an antibody HMW high molecular weight

DNA deoxyribonucleic acid HSA human serum albumin

DNAse deoxyribonuclease IEX ion exchange chromatography

DOC deoxycholate IgA, IgG etc. different classes of immunoglobulin

DoE design of experiments IMAC Immobilized metal ion affinity chromatography

DS desalting (group separation by size exclusion LC-MS liquid chromatography–mass spectrometry

chromatography; buffer exchange) LMW low molecular weight
EDTA ethylene diaminetetraacetic acid MAb monoclonal antibody

5
MPa megaPascal TCEP tris(2-carboxyethyl) phosphine hydrochloride
Mr relative molecular weight TFA Trifluoroacetic acid
MS mass spectrometry Tris tris-(hydroxymethyl)-aminomethane
n native, as in nProtein A UV ultraviolet
NC nitrocellulose v/v volume to volume
NHS N-hydroxysuccinimide w/v weight to volume
PAGE polyacrylamide gel electrophoresis
PBS phosphate buffered saline
PEG polyethylene glycol
pI isoelectric point, the pH at which a protein has
zero net surface charge
PMSF phenylmethylsulfonyl fluoride
psi pounds per square inch
PVDF polyvinylidene fluoride
PVP polyvinylpyrrolidine
r recombinant, as in rProtein A
RNA ribonucleic acid
RNAse ribonuclease
RPC
scFv
reversed phase chromatography
single chain Fv fragment
Principles and methodology
SDS sodium dodecyl sulfate
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel
handbooks from Cytiva
electrophoresis
Cytiva offers a wide range of handbooks that provides practical tips and in-depth information about common
SEC size exclusion chromatography methodologies used in the lab. Visit cytiva.com/handbooks to view the complete list and download your copies today.

6
01
Principles of
ion exchange

7
This chapter provides a general introduction to the theoretical principles that underlie every ion exchange separation. +
An understanding of these principles will enable the separation power of ion exchange chromatography (IEX) to be fully
appreciated. Practical aspects of performing a separation are covered in Chapter 2.
Cation
Net surface charge and pH

Surface net charge

IEX separates molecules on the basis of differences in their net surface charge. Molecules vary considerably in their
charge properties and will exhibit different degrees of interaction with charged chromatography media according to
differences in their overall charge, charge density, and surface charge distribution. The charged groups within a molecule that 0 pH
contribute to the net surface charge possess different pKa values (acid ionization constant) depending on their structure
and chemical microenvironment.
Since all molecules with ionizable groups can be titrated, their net surface charge is highly pH dependent. In the case of Anion
proteins, which are built up of many different amino acids containing weak acidic and basic groups, net surface charge
will change gradually as the pH of the environment changes, that is, proteins are amphoteric. Each protein has its own
unique net charge versus pH relationship which can be visualized as a titration curve. This curve reflects how the overall -
net charge of the protein changes according to the pH of the surroundings. Figure 1.1 illustrates several theoretical
protein titration curves (these curves can be generated using a combination of isoelectric focusing and electrophoresis, Fig 1.1. Theoretical protein titration curves, showing how net surface charge varies with pH.
but with modern solutions for rapid method development, actual titration curves are rarely used).
IEX takes advantage of the fact that the relationship between net surface charge and pH is unique for a specific protein.
In an IEX separation, reversible interactions between charged molecules and oppositely charged IEX media are controlled
in order to favor binding or elution of specific molecules and achieve separation. A protein that has no net charge at a pH
equivalent to its isoelectric point (pI) will not interact with a charged medium. However, at a pH above its pI, a protein
will bind to a positively charged medium or anion exchanger and, at a pH below its pI, a protein will bind to a negatively
charged medium or cation exchanger. In addition to the ion-exchange interaction, other types of binding can occur, but
these effects are very small and mainly due to van der Waals forces and nonpolar interactions.

8
Steps in an IEX separation Regeneration
An IEX medium comprises a matrix of spherical particles substituted with ionic groups that are negatively or positively A final wash with high ionic strength buffer regenerates the column and removes
charged. The matrix is usually porous to give a high internal surface area. The medium is packed into a column to form any molecules still bound. This ensures that the full capacity of the stationary phase
a packed bed. The bed is then equilibrated with buffer which fills the pores of the matrix and the space in between the is available for the next run. The column is then re-equilibrated in start buffer before
particles. starting the next run.

Equilibration The above describes a typical IEX separation. Alternatively, conditions can be chosen
to maximize the binding of contaminants to allow the target protein(s) to first pass
The first step is the equilibration of the stationary phase to the desired start conditions. When equilibrium is reached, through the column to be collected.
all stationary phase charged groups are bound with exchangeable counterions, such as chloride or sodium. The pH and
ionic strength of the start buffer are selected to ensure that, when sample is loaded, proteins of interest bind to the
medium and as many impurities as possible do not bind.

Sample application and wash

The second step is sample application and wash. The goal in this step is to bind the target molecule(s) and wash out all
unbound material. The sample buffer should have the same pH and ionic strength as the start buffer in order to bind all
charged target proteins. Oppositely charged proteins bind to ionic groups of the IEX medium, becoming concentrated
on the column. Uncharged proteins, or those with the same charge as the ionic group, pass through the column at the
same speed as the flow of buffer, eluting during or just after sample application, depending on the total volume of sample
loaded.

Elution
When all the sample has been loaded and the column washed with start buffer so that all nonbinding proteins have
passed through the column, conditions are altered in order to elute the bound proteins. Most frequently, proteins are
eluted by increasing the ionic strength (salt concentration) of the buffer or, occasionally, by changing the pH. As ionic
strength increases the salt ions (typically Na+ or Cl-) compete with the bound components for charges on the surface
of the medium and one or more of the bound species begin to elute and move down the column. The proteins with the
lowest net charge at the selected pH will be the first ones eluted from the column as ionic strength increases. Similarly,
the proteins with the highest charge at a certain pH will be most strongly retained and will be eluted last. The higher the
net charge of the protein, the higher the ionic strength that is needed for elution. By controlling changes in ionic strength
using different forms of gradient, proteins are eluted differently in a purified, concentrated form.

9
Low ionic strength buffer Equilibration Elution 2

Matrix Further increases in ionic

Absorption

Absorption
IEX medium equilibrated strength displace proteins that
with start buffer. are more highly charged (more
Positively tightly bound)
charged ionic
groups

Time/Volume Time/Volume

Sample application and wash Elution 3

Oppositely charged proteins bind

to ionic groups of the IEX medium,
becoming concentrated on the

Absorption
Absorption

column. Uncharged proteins, or

Negatively charged proteins those with the same charge as the
ionic groups, elute during sample
application or just after, during the
wash with start buffer.

Time/Volume Time/Volume
Neutral or positively
charged proteins

Elution 1 Regeneration

Increasing ionic strength Final high ionic strength wash

Absorption
Absorption

(using a gradient) displaces removes any ionically bound

bound proteins as ions in the proteins before re-equilibration
buffer compete for binding sites.

Time/Volume Time/Volume

Fig 1.2. Principles of an anion exchange separation.

10
Resolution
The resolution of an IEX separation is a combination of the degree of separation between the peaks eluted from the
column (the selectivity of the medium), the ability of the column to produce narrow, symmetrical peaks (efficiency)
and, of course, the amount (mass) of sample applied. These factors are influenced by practical issues such as matrix
properties, binding and elution conditions, column packing, and flow rates which are covered in detail in Chapter 2,
Ion exchange in practice.
Resolution (Rs ) is defined as the distance between peak maxima compared with the average base width of the two peaks.
Rs can be determined from a chromatogram, as shown in Figure 1.3.
Elution volumes and peak widths are measured with the same units to give a dimensionless resolution value. Rs gives
a measure of the relative separation between two peaks and can be used to determine if further optimization of the
chromatographic procedure is necessary. If Rs = 1.0 (Fig 1.4) then 98% purity has been achieved at 98% of peak recovery,
provided the peaks are symmetrical and approximately equal in size. Baseline resolution requires that Rs ≥ 1.5. At this
value, peak purity is 100%.
 single, well-resolved peak is not necessarily a pure substance, but might represent a series of components that
A
could not be separated under the chosen elution conditions.

A B
2 ( VR2 – VR1 )
Rs = wb1 + wb2 A B

R s = 1.5
R s = 1.0

Volume
VR1 VR2
96% A 96% B Volume Volume
wb1 wb2 2% B 2% A ~100% A ~100% B

Fig 1.3. Determination of the resolution (Rs) between two peaks. Fig 1.4. Separation results with different resolutions.

11
 Rapid exchange of counter-ions, Buffer flow
typically Na+ or Cl-, and solute molecules
Efficiency
Column efficiency (the ability to elute narrow, symmetrical peaks from a packed bed) relates to the zone broadening
which occurs on the column and is frequently stated in terms of the number of theoretical plates (see Appendix 3 for
determination of column efficiency). One of the main causes of zone broadening is longitudinal diffusion of the solute
molecules, that is, proteins, peptides, or oligonucleotides. Zone broadening can be minimized if the distances available
Rapid diffusion
for diffusion are minimized. In all situations, a well-packed column will contribute significantly to resolution. Columns
that are packed unevenly, too tightly, too loosely, or that contain air bubbles will lead to channeling (uneven passage
of buffer through the column), zone broadening and hence loss of resolution. Figure 1.5 illustrates the parameters that
contribute to good column efficiency. Obviously particle size is a significant factor in resolution and, in general, the
smallest particles will produce the narrowest peaks under the correct elution conditions and in a well-packed column.
Small bead size
Uniform pore size distribution

Even buffer flow distribution

Uniform packing
Narrow particle size distribution

Narrow, symmetrical peaks

Minimal zone broadening

Fig 1.5. Factors that affect column efficiency.

12
Figure 1.6 demonstrates the influence of particle size on efficiency by comparing
A 280

several different IEX media under exactly the same running conditions. Note that Mini Q™ (column 4.6 × 50 mm)
3 µm Sample: Pancreatin
different media selectivities also influence the final resolution.
Gradient elution
Although resolution in terms of efficiency can be improved by decreasing 5.0 10.0 ml 15. 0

the particle size of the matrix, using a smaller particle size often creates an A 280

increase in back pressure so that flow rates need to be decreased, lengthening Mono Q™ (column 5 × 50 mm)
the run time. Hence the need to match the medium with the requirements for 10 µm Sample: Pancreatin
Gradient elution
the purification (speed, resolution, recovery, and capacity).
0 10 min 20 30

The viscosity of highly concentrated samples might reduce resolution if large A 280

sample volumes are loaded onto columns packed with small particles. Samples

Resolution increases
RESOURCE™ Q, 1 mL

Particle size increases

may be diluted or, alternatively, a larger particle size should be used. 15 µm Sample: Pancreatin
Gradient elution

0 10 min 20 30

A 280

SOURCE™ 30Q (XK16 × 50 mm)

30 µm Sample: Pancreatin
Gradient elution

0 10 min 20 30

A 280

Q Sepharose™ High Performance

34 µm (XK16 × 50 mm)
Sample: Pancreatin
Gradient elution
0 10 min 20 30

A 280

Q Sepharose Fast Flow (XK16 × 50 mm)

90 µm Sample: Pancreatin
Gradient elution

0 10 min 20 30

Fig 1.6. Examples of the influence of particle size and selectivity on final resolution.

13
Selectivity
Good selectivity (the degree of separation between peaks) is a more important factor than high efficiency in determining Most acidic pH: all three proteins are below their Most alkaline pH: all three proteins are above their
pI, positively charged, and bind only to a cation pI, negatively charged, and bind only to the anion
resolution (Fig 1.7) and depends not only on the nature and number of the functional groups on the matrix, but also on exchanger. Proteins are eluted in the order of their exchanger. Proteins are eluted in the order of their
the experimental conditions, such as pH (influencing the protein charge), ionic strength, and elution conditions. It is the net charge. net charge.

ease and predictability with which these experimental conditions can be manipulated, when using a suitably designed
chromatography medium, that gives IEX the potential of extremely high resolution.
Abs Abs Abs Abs
Good selectivity Bad selectivity

high efficiency high efficiency

V V V V

Cation

Surface net charge

low efficiency low efficiency
0 pH

Anion

–
V V
Fig 1.7. Effect of selectivity and efficiency on resolution. Abs Abs Abs Abs

Selectivity and pH V V V V

Good selectivity is achieved by performing IEX separations at pH values carefully selected to maximize the differences in
net charge of the components of interest. Figure 1.8 emphasizes the significance of pH.
Optimum selectivity can be expected at a pH where there is maximum separation between the titration curves for the
Less acidic pH: blue protein is above its pI, negatively Less alkali pH: red protein below its pI, positively
individual proteins (i.e., the difference in net charges between the species is greatest) and when using an ion exchanger charged, other proteins are still positively charged. Blue charged. Red protein binds to cation exchanger and
with a charge opposite to the charge of the proteins at the particular pH. protein binds to an anion exchanger and can be separated can be separated from the other proteins which
from the other proteins which wash through. Alternatively, wash through. Alternatively, blue and green proteins
red and green proteins can be separated on a cation can be separated on an anion exchanger and the red
The order in which proteins are eluted cannot always be predicted with absolute certainty since a titration curve exchanger and the blue protein washes through. protein washes through.
(produced in practice by measuring electrophoretic mobility in a gel) reflects the total net charge on a protein and IEX
depends on the net charge on the surface of the protein. Fig 1.8. Effect of pH on protein binding and elution patterns.

14
Selectivity and elution equilibration
sample
injection
gradient regeneration re-equilibration
elution
volume
The figures to the right illustrate the most common forms of IEX separation in which high salt wash high salt wash
proteins are eluted by increasing the ionic strength of a buffer (typically with NaCl) 1M
5 CV tightly bound
1M 5 CV
elution
using linear gradient or step elution. The UV absorbance and conductivity traces molecules
elute in high
elution of of target
unbound unwanted molecule
show the elution of protein peaks and the changes in salt concentration, respectively, unbound molecules elute
before gradient begins
salt wash molecules material
elute
during elution. sample 5 CV
injection tightly bound

NaCl

NaCl
molecules
Buffer volumes used during sample application, elution, washing and volume
elute
re-equilibration are expressed in column volumes (CV), for example 5 CV = 5 mL 10–20 CV
5 CV

for a column with a 1 mL bed volume. Using CV to describe a separation profile equilibration re-equilibration
facilitates method development and transfer of methods to columns of different 5–10 CV
5–10 CV
5–10 CV
5–10 CV
0 0
dimensions when scaling-up. Column volumes [CV] Column volumes [CV]

Gradient elution (Fig 1.9) is often used when starting with an unknown sample Fig 1.9. Typical high-resolution IEX separation using linear gradient Fig 1.10. Typical IEX separation using step elution. .
(as many components as possible are bound to the column and eluted differentially elution.
to see a total protein profile) and for high-resolution separation or analysis.
Step elution is used in several ways. When an IEX separation has been optimized
high salt wash high salt wash elutes
using gradient elution, changing to a step elution speeds up separation times and 5–10 CV contaminants 5–10 CV
reduces buffer consumption while retaining the required purity level (Fig 1.10). 1M 1M

Step elution can also be used for group separation in order to concentrate the
unbound
proteins of interest and rapidly remove them from unwanted substances (Fig 1.11). molecules target molecules
elution of elute in wash
The target protein(s) is eluted in an enriched, concentrated form. sample elute
target
sample

NaCl
NaCl
injection injection
molecules
volume
Occasionally, step elution is used to remove contaminants by choosing conditions volume
5 CV

that maximize binding of the contaminants and allow the target protein(s) to pass
equilibration re-equilibration equilibration re-equilibration
through the column (Fig 1.12). Care must be taken to ensure that the binding 5–10 CV 5–10 CV 5–10 CV 5–10 CV
capacity of the column is sufficient to bind all contaminants. 0 0
Column volumes [CV] Column volumes [CV]

Fig 1.11. Typical IEX separation using a step elution to separate groups of Fig 1.12. Contaminant removal: target protein(s) elute in the wash,
proteins with very different charge properties. contaminants bind to the column.

15
Components of IEX media Table 1.1 Ion exchange matrices
Form Mean particle size (μm)
Chromatography media for ion exchange are made from porous or nonporous matrices, chosen for their physical stability,
MiniBeads™ Polystyrene/divinyl benzene 3
their chemical resistance to stringent cleaning conditions, and their low level of nonspecific interaction. The matrices are
substituted with functional groups that determine the charge of the medium. MonoBeads™ Polystyrene/divinyl benzene 10
SOURCE 15 Polystyrene/divinyl benzene 15
Matrix SOURCE 30 Polystyrene/divinyl benzene 30
 igh porosity offers a large surface area covered by charged groups and so ensures a high binding capacity. High porosity
H Sepharose High Performance Agarose 6% 34
is also an advantage when separating large biomolecules. Nonporous matrices are preferable for extremely high-resolution
Sepharose Fast Flow Agarose 6% 90
separations when diffusion effects must be avoided
Sepharose 4 Fast Flow Agarose 4% 90
An inert matrix minimizes nonspecific interactions with sample components
Sepharose XL Agarose 6%, dextran chains 90
 igh physical stability ensures that the volume of the packed medium remains constant despite extreme changes in
H coupled to agarose
salt concentration or pH thus improving reproducibility and avoiding the need to repack columns Sepharose Big Beads Agarose 6% 200
 igh physical stability and uniformity of particle size facilitate high flow rates, particularly during cleaning or
H Capto™ ImpRes High-flow agarose 40
re-equilibration steps, to improve throughput and productivity
Capto ImpAct High-flow agarose 50
High chemical stability ensures that the matrix can be cleaned using stringent cleaning solutions if required Capto High-flow agarose 90
 odern IEX media use either polymer or agarose-based matrices to fulfill not only the requirements for high binding
M
capacity, chemical and physical stability, but to generate media with suitable particle sizes for a range of applications
(Table 1.1)
MiniBeads is a matrix made from polystyrene, with divinyl benzene as cross-linker, to produce highly spherical
(monodispersed), very small (3 µm), nonporous particles that facilitate micropreparative or analytical separations when
extremely high resolution is more important than high binding capacity or high flow rates.
MonoBeads and SOURCE are matrices made from polystyrene with divinyl benzene to produce highly spherical
(monodispersed), small (10, 15, or 30 µm), porous particles (Fig 1.13) that facilitate high resolution separations at
high flow rates.

16
Sepharose media are based on chains of agarose, arranged in bundles and with different degrees of cross-linking
(Fig 1.14), to give a range of rigid, macroporous matrices with good capacity and low non-specific adsorption. The most
suitable matrix can be selected according to the degree of resolution, binding capacity and flow rates desired for the
separation. For example, gradient elution on Sepharose High Performance (34 µm) will give a high resolution separation
whereas the larger particles of Sepharose Fast Flow (90 µm) or Sepharose Big Beads (200 µm) would be most suited for
high capacity, step elution at high flow rate.
Capto media are based on a chemically modified, high-flow agarose matrix. This matrix provides particle rigidity without
compromising pore size, outstanding pressure/flow properties, and high chemical stability to support CIP procedures.
Capto media are suitable for scaling up and for use in large-scale bioprocess purifications. The basic characteristics of
Capto (90 µm), Capto ImpAct (50 µm), and Capto ImpRes (40 µm) IEX media are summarized in Chapter 3.

Fig 1.13. Electron micrograph of MonoBeads showing spherical, monodispersed particles.

Fig 1.14. Structure of cross-linked agarose media (Sepharose).

17
Functional groups Table 1.2. Functional groups used on ion exchangers

The functional groups substituted onto a chromatographic matrix (Table 1.2) determine the charge of an IEX medium, Anion exchangers Functional group
that is, a positively charged anion exchanger or a negatively charged cation exchanger. Quaternary ammonium (Q) strong -CH2-N+-(CH3)3
The terms strong and weak refer to the extent that the ionization state of the functional groups varies with pH. Diethylaminoethyl (DEAE)* weak -CH2-CH2-N+-(CH2-CH3)2
The terms strong and weak do not refer to the strength with which the functional groups bind to proteins. Strong Diethylaminopropyl (ANX)* weak -CH2-CHOH-CH2-N+-(CH2-CH3)2
ion exchangers show no variation in ion exchange capacity with change in pH (Fig 1.15). These exchangers do not
take up or lose protons with changing pH and so have no buffering capacity, remaining fully charged over a broad pH Cation exchangers Functional group
range. Strong ion exchangers include Q (anionic), S, and SP (cationic). Sulfopropyl (SP) strong -CH2-CH2-CH2-SO3-
There are several advantages to working with strong ion exchangers: Methyl sulfonate (S) strong -CH2-SO3-
D
 evelopment and optimization of separations is fast and easy since the charge characteristics of the medium do not Carboxymethyl (CM) weak -CH2-COO -
change with pH * The active end of the charged group is the same for DEAE and ANX. The difference between them is in the
length of the carbon chain of the charged group. DEAE has a diethylaminoethyl group bound to the agarose.
The mechanism of interaction is simple since there are no intermediate forms of charge interaction ANX has a diethylaminopropyl group attached which prevents the formation of quaternary groups, giving a
Sample loading (binding) capacity is maintained at high or low pH since there is no loss of charge from the ion exchanger different selectivity compared to DEAE.

12 12 12 12

10 10 10 10
Q Sepharose
Q Sepharose
Fast Fast
FlowFlow SP Sepharose
SP Sepharose
Fast Fast
FlowFlow
8 8 8 8

6 6 6 6

4 4 4 4

2 2 2 2
1 12 23 34 45 56 67 7 1 12 23 34 45 56 67 7
100 mM
100NaOH
mM NaOH
(mL) (mL) 100 mM
100NaOH
mM NaOH
(mL) (mL)

Fig 1.15. Titration curves show the ion exchange capacity of strong ion exchangers Q and SP. Approximately
5 mL of Q or SP Sepharose Fast Flow were equilibrated in 1 M KCl and titrated with 100 mM NaOH.

18
The majority of proteins have pI within the range 5.5 to 7.5 and can be separated on either strong or weak ion exchangers. DEAE Sepharose Fast Flow CM Sepharose Fast Flow
12 12
An advantage of a weak ion exchanger, such as DEAE (anionic), ANX (anionic), and CM (cationic) is that they can offer a
different selectivity compared to strong ion exchangers. A disadvantage is that because weak ion exchangers can take up 10 10
or lose protons with changing pH, their ion exchange capacity varies with pH (Fig 1.16).
8 8
T ry a weak ion exchanger such as DEAE, CM, or ANX Sepharose Fast Flow, if a strong ion exchanger (substituted
with Q, S, or SP) does not give the required selectivity. 6 6

4 4

2 2
0 200 400 600 800 1000 0 200 400 600 800 1000
100 mM NaOH (mL) 100 mM NaOH (mL)

ANX Sepharose 4 Fast Flow (high sub)

12

10

0
0 1 2 3 4 5 6 7 8 9 10 11 12
100 mM NaOH (mL)

Fig 1.16. Titration curves show how the ion exchange capacity of weak ion exchangers varies with pH.

19
Binding capacity and recovery
The capacity of an IEX medium is a quantitative measure of its ability to take up counterions (proteins or other charged
molecules). The total ionic capacity is the number of charged functional groups/mL medium, a fixed parameter of each
medium. Of more practical relevance is the actual amount of protein that can bind to an IEX medium, under defined
experimental conditions. The capacity of a chromatography medium can be measured in two ways: static binding
capacity (SBC) and dynamic binding capacity (DBC). SBC is the maximum amount of protein that can bind at given
conditions. SBC is often obtained during excess load of sample. DBC is measured during given conditions, including flow
rate, and is the amount of protein that binds before a significant breakthrough of the target protein appears. Figures for
binding capacity in this handbook refer to the dynamic binding capacity.
The static and dynamic binding capacities depend upon the properties of the protein, the IEX medium, and the
experimental conditions. The capacity of an IEX medium will vary according to the molecular size of the specific protein
(which affects its ability to enter all the pores of the matrix) and its charge/pH relationship (the protein must carry the
correct net charge at a sufficient surface density at the chosen pH). With earlier IEX media, larger biomolecules had
limited access to the functional groups, significantly reducing the binding capacity. Nowadays, ion exchange matrices
such as MonoBeads, Capto, SOURCE, and Sepharose media all have exclusion limits for globular proteins in excess of
1 × 106 and are therefore suitable for the majority of biomolecule separations. Binding capacities will still vary according
to the molecular size of the biomolecules. For example, a matrix with a high degree of small pores will exhibit a higher
binding capacity for smaller molecules. Experimental conditions such as pH, ionic strength, counterion, flow rate, and
temperature should all be considered when comparing binding capacities of different IEX media.
Modern IEX media show very low levels of nonspecific adsorption so that sample recovery under suitable separation
conditions is very high, typically between 90% and 100%.

Chromatofocusing
Chromatofocusing (CF) is a purification method separating proteins on the basis of differences in their isoelectric
points (pI). The matrix is usually a weak anion exchanger in which the functional groups are amines. The eluent is a
buffer containing a large number of buffering substances which together give a uniform buffering capacity over a broad
pH range. A pH gradient is generated on the column as buffer and medium interacts. Proteins with different pI values
migrate at different rates as the pH gradient develops, continually binding and dissociating while being focused into
narrow bands and finally eluted.
CF is a powerful method and can resolve very small differences in pI (down to 0.02 pH units) and thus separates very
similar proteins. However, the capacity of the method is low and should preferably only be used for partially pure samples.
CF can be considered if IEX or other methods do not give satisfactory purification.
20
02
Ion exchange
in practice

21
Introduction Polishing
This chapter includes practical advice on how to control experimental conditions to achieve a successful separation and When IEX is used for polishing, most impurities have been removed except for
guidelines for selection of the most appropriate medium or prepacked column for each application. The final resolution of trace amounts or closely related substances such as structural variants of the
an ion exchange (IEX) separation is determined by selectivity and column efficiency. These parameters are influenced in target protein, nucleic acids, viruses, or endotoxins. The purpose of the separation
turn by factors such as particle size, porosity and column packing. The separation is influenced by a number of factors, for is to reduce these variants and trace contaminants to acceptable levels for the
example, the way in which the net surface charge of each protein in the sample varies with pH, the pH and ionic strength application. In contrast to capture steps where fast, high capacity, step elution is
of buffers, and the elution conditions. Understanding the role and importance of each parameter ensures that every most commonly used, a polishing step will therefore focus on achieving the highest
separation can be performed with the required resolution, throughput and speed. Additional application examples and possible resolution (Fig 2.1).
product-related information are found in Chapter 3.

Selecting chromatography media

The origin and differences between modern IEX matrices are explained in Chapter 1. Choice of a suitable matrix depends
on factors such as the scale of the final purification, the purpose of the separation (for example to concentrate sample in
a capture step or to achieve high resolution in a final polishing step) and the throughput required. Refer to Chapter 4 for
more details on the use of capture, intermediate purification, and polishing steps in a purification strategy.

Capture
When IEX is used as a capture step, the objective is to quickly absorb the protein(s) of interest from the crude sample and
isolate them from critical contaminants such as proteases. The target protein(s) are concentrated and transferred to an
environment that will conserve potency/activity. Removal of other critical contaminants can also be achieved by careful
optimization of pH and elution conditions.
The focus is on capacity and speed in a capture step. It is advisable to compromise on the potential resolution that can
be achieved by an IEX separation to maximize the capacity and/or speed of the separation in this first step (Fig 2.1).

Intermediate purification
When IEX is used for intermediate purification, the objective is to remove most of the significant impurities such as
proteins, nucleic acids, endotoxins, and viruses. In a typical intermediate purification step, speed is less critical since
sample volume has been reduced and damaging contaminants have been removed during capture. Focus is on capacity
and resolution in order to maintain productivity and to achieve as high selectivity (purity) as possible (Fig 2.1).

22
 A280

Mini Q (column 4.6 × 50 mm) Sample:

Use for intermediate purification
Pancreatin
Highest resolution μg/run MiniBeads (Q or S) if column capacity is sufficient
Gradient elution
and no scale-up is required.
5.0 10.0 15.0 ml

Use for intermediate purification A280

Polishing Mono Q (column 5 × 50 mm) Sample:

if column capacity is sufficient
Pancreatin
Highest resolution mg/run MonoBeads (Q or S) and no scale-up is required. Can
Remove trace impurities or Gradient elution
be used for capture steps if sample
closely-related substances
is free from particulate matter.
Sample condition: 0 10 20 30 min

almost pure A 280

RESOURCE Q, 1 mL Sample:
Use SOURCE 15 when resolution Pancreatin
SOURCE 15 (Q or S)
is top priority. Gradient elution

High resolution and throughput 0 10 20 30 min

Wider pH stability window A 280

SOURCE 30Q (HR 16 × 50 mm) Sample:

Use SOURCE 30 when speed is Pancreatin
SOURCE 30 (Q or S)
top priority. Gradient elution

0 10 20 30 min

Intermediate

Resolution
purification Use HiTrap columns prepacked A280

Sample:
Q Sepharose High Performance
with Sepharose High Performance, (HR 16 × 50 mm) Pancreatin
High resolution Sepharose High Performance
Remove bulk impurities Sepharose XL and Sepharose Gradient elution
Easy scale-up (Q or SP)
Sample condition: Fast Flow for media selection and
partially purified pH scouting. 0 10 20 30 min

Use Capto ImpRes and

High resolution and throughput Capto ImpRes (Q or SP) Capto S ImpAct for increased
Flexibility of process design Capto S ImpAct productivity in large-scale
production

A 280

Try weak ion exchangers Q Sepharose Fast Flow Sample:

Easy scale-up, Broad choice of such as DEAE, CM, or ANX (HR 16 × 50 mm) Pancreatin
Sepharose Fast Flow
selectivity, including alternatives if the selectivity of Q or S is Gradient elution
(Q, SP, DEAE, CM, ANX)
to Q or S IEX media unsatisfactory.
Capture
0 10 20 30 min

Isolate, concentrate, and

stabilize target protein(s)
Use high bed heights for
Sample conditions: High volume throughput and
Capto (Q, S, DEAE, adhere, MMC) increased productivity. Use
clarified or nonclarified high capacity, Easy scale-up Capto MMC for high salt feed.

A 280

Q Sepharose XL Sample:
Use for capture with high binding Recombinant α-amylase
High binding capacity for
Sepharose XL (Q or SP) capacity/rapid separation of Pilot scale:
selected proteins, Easy scale-up
proteins from clarified samples Gradient elution begins
0 5.0 10.0 15.0 20.0 Volume (l) after 20 L

Large scale, viscous samples Sepharose Big Beads (Q or SP) Use with step elution.

Fig 2.1. A typical purification strategy has three phases: Capture, Intermediate Purification, and Polishing (Cipp). Each phase has a specific objective, dependent largely on the properties of the starting
material. The appropriate IEX medium is selected according to the objective of the purification step and the condition of the starting material.
23
Fast IEX media selection and method development
Time and sample can be saved in the early stages of development by using small, prepacked columns such as those in
the HiTrap IEX Selection Kit (Fig 2.2). The kit allows quick and efficient screening for the most suitable charge group and
enables development of the basic separation method (see Appendix 11, Media selection). This approach is particularly
helpful if the properties of the target protein(s) are unknown.
HiTrap columns can be run with a syringe, a peristaltic pump, or any ÄKTA™ chromatography system. HiTrap columns can
be used for small-scale purification as well as fast method development and are supplied with detailed protocols for use.
The HiScreen™ column format has been specially designed for screening and optimizing before scaling up the
purification. The higher bed height of HiScreen (10 cm), in comparison with HiTrap (2.5 cm), is suitable for scaling up while
keeping the bed height constant.

Fig 2.2. HiTrap™ IEX Selection Kit contains seven HiTrap columns prepacked with different
Sepharose Fast Flow media. The kit is an excellent choice for screening of the most appropriate media
and conditions to use in application and development work.

24
Practical considerations for IEX separation
This section covers detailed aspects of each step in an IEX separation, together with practical hints and tips to improve
resolution and overall performance.

Buffer pH and ionic strength

Buffer pH and ionic strength must be compatible with protein stability and activity. The most suitable pH should allow the
proteins of interest to bind, but should be as close to the point of release (elution) as possible. If the pH is too low or too
high, elution becomes more difficult and high salt concentrations might be needed. This should be avoided since some
proteins begin to precipitate at high ionic strength and high salt concentrations can interfere with assays or subsequent
chromatographic steps.
Avoid extreme changes in pH or other conditions that can cause inactivation or even precipitation.
The pH and ionic strength of the sample are extremely important in order to achieve the most effective high resolution
or group separations and to make the most of the high loading capacity. Samples should preferably be buffered in the
start buffer (see Appendix 1, Sample preparation for details). When working with small volumes during screening and
scouting, it might be sufficient to dilute the sample in start buffer in order to lower the ionic strength and adjust the pH
to a value similar to that of the start buffer.
Proteins often begin to dissociate from IEX media about 0.5 pH units from their pI at an ionic strength around 100 mM.
The pH of the start buffer should be at least 0.5 to 1.0 pH unit above the pI of the target substance when using an anion
exchanger (Q, DEAE, or ANX) or 0.5 to 1.0 pH unit below the pI of the target substance when using a cation exchanger
(SP or CM).
For samples with unknown charge properties, try the following strong ion exchangers first:
anion exchange (Q)
start buffer: pH 8.0
elution buffer: start buffer including 1 M NaCl, pH 8.0
cation exchange (S, SP)
start buffer: pH 6.0
elution buffer: start buffer including 1 M NaCl, pH 6.0
See Appendix 2 for recommendations on volatile and nonvolatile buffer systems for anion and cation exchangers.
Whenever possible, check for stability at the pH and ionic strength values selected, especially if recovery of
biological activity is a priority.

25
 +
Anion or cation exchanger
For molecules such as nucleic acids, which carry only negatively charged groups, an anion exchanger is the obvious Range of
choice. However, since the net charge of molecules such as proteins (carrying positively and negatively charged groups) stability

Net charge of protein

depends on pH, the choice is based on which type of exchanger and pH give the desired resolution within the constraints

pl
of sample stability. For example, Figure 2.3 shows a theoretical protein which has a net positive charge below its pI, and Attached to
can bind to a cation exchanger. Above its pI, the protein has a net negative charge and can bind to an anion exchanger. anion exchangers
However, the protein is only stable in the pH range between 5.0 and 8.0 and so an anion exchanger has to be used.
If sample components are most stable below their pI, use a cation exchanger. Attached 4 6 8 10
pH
If sample components are most stable above their pI, use an anion exchanger. to cation
exchangers
If stability is high over a wide pH range on both sides of the pI, use either type of ion exchanger.

Fig 2.3. Considerations when selecting a suitable IEX medium.

26
Strong or weak ion exchangers Table 2.1. Functional groups used on ion exchangers

Table 2.1 shows the functional groups used on IEX media. The terms strong and weak refer to the extent that the Anion exchangers Functional group
ionization state of the functional groups varies with pH. The terms strong and weak do not refer to the strength with Quaternary ammonium (Q) strong -CH2-N+-(CH3)3
which the functional groups bind to proteins. Diethylaminoethyl (DEAE)1 weak -CH2-CH2-N-(CH2-CH3)2
Begin with a strong ion exchanger to enable development work to be performed over a broad pH range. Use a Diethylaminopropyl (ANX)1 weak -CH2-CHOH-CH2-N-(CH2-CH3)2
strong anion exchanger (Q) to bind the protein(s) of interest if their pI is below pH 7.0 or unknown.
Cation exchangers Functional group
Use a strong ion exchanger in cases where maximum resolution occurs at an extreme pH and the proteins of
Sulfopropyl (SP) strong -CH2-CH2-CH2-SO3-
interest are stable at that pH.
Methyl sulfonate (S) strong -CH2-SO3-
Consider using a weak exchanger if the selectivity of the strong ion exchanger is unsatisfactory, but remember that the Carboxymethyl (CM) weak -CH2-COO-
ion exchange capacity of a weak ion exchanger varies with pH. As a result:
1
T he active end of the charged group is the same for DEAE and ANX. The difference between them is in the
Sample loading (binding) capacity can vary with increasing pH due to loss of charge from the exchanger length of the carbon chain of the charged group. DEAE has a diethylaminopropyl group bound to the agarose.
ANX has a diethylaminopropyl group attached, which prevents the formation of quaternary groups giving a
R
 esolution is more readily affected by changes in flow rate or sample load due to the intermediate forms of charge
different selectivity compared to DEAE.
interaction that can occur
P
 redicted results (based on known information about the sample components such as their pI and how their net
surface charge changes with pH) might not correlate with actual results since the number of charged groups on weak
ion exchangers can vary with pH
Longer equilibration times might be required in order to titrate the weak ion exchange functional groups
When using a weak exchanger, work within the pH values given below to minimize variations in performance:
DEAE: pH 2.0 to 9.0
ANX: pH 2.0 to 9.0
CM: pH 6.0 to 10.0

27
Buffer selection and preparation
Buffer ions
Buffering ions should have the same charge as the functional groups on the IEX medium (buffering ions that carry a
charge opposite to that of the functional groups will take part in the ion exchange process and can cause significant
pH fluctuations during elution) and, preferably, a pKa value within 0.6 pH units of the working pH. An exception to this
rule is seen in the frequent use of phosphate buffers with anion exchange separations. However, phosphate buffers must
be very carefully prepared to ensure reproducibility between batches.
Use a buffer concentration that is sufficient to maintain buffering capacity and constant pH, typically 20 to 50 mM.
Use volatile buffers if the purified product is to be lyophilized.
See Appendix 2 for recommendations on volatile and nonvolatile buffer systems for anion and cation exchangers.
F ilter buffers after all salts and additives have been included. Use high quality water and chemicals. Filter solutions
through 1 µm filters for particle sizes above 90 µm, 0.45 µm filters for 34 µm particles, or 0.22 µm filters for particle
sizes below 15 µm or when sterile or extra clean samples are required. To avoid formation of air bubbles in a packed
column, ensure that column and buffers are at the same temperature when preparing for a run.

Effect of temperature on buffer pH

Select buffers that have appropriate pKa values for the working temperature. The pKa of a buffering substance varies with
temperature. For example, Tris has a pKa of 8.85 at 0°C, 8.06 at 25°C and 7.72 at 27°C. Using Tris at 4°C at a pH 7.9 would
give a very low buffering capacity and the working pH would be outside the useful pH range (pKa ± 0.5) of the buffer.
Prepare buffers at the same temperature at which they will be used.
T emperatures < 10°C can minimize aggregation caused by hydrophobic interactions between sample components.
Working at these lower temperatures can be an alternative solution to using a detergent to improve solubility.

28
 Counterions  se the following procedure if the medium is to be used with counterions other
U
than Na+ or Cl-: 1 1
Counterions (salt ions) used in IEX are almost always Na+ for cation exchange and Cl– for anion exchange.
1 1 3 3
2 2 3
Salts such as NaCl have a chaotropic character (i.e., an ability to make water less polar) and therefore a lower ‘salting-out’ 3 4 4

Elution buffer concentration

Elution buffer concentration

2

Elution buffer concentration

Elution buffer concentration

1. Wash 2
effect on hydrophobic molecules. This ensures maximum solubility during elution and improves recovery. Chaotropic 4 4 0.35 M the
0.35
packed column with 10 column volumes 0.5 to 1 M salt solution
NaCl
M NaCl
salts can also be used in the presence of organic solvents if required. Salts such as (NH4)2SO4 or K3PO4 should be avoided containing the new counterion. Flow rate: see relevant 0.28
IEX media
M
0.28 NaBr section
M NaBr
in Chapter 3.

A 280 nm

A 280 nm
A 280 nm

A 280 nm
as they are most likely to cause precipitation at high concentrations. 6 6
In certain applications alternative counterions such as Li+, Br–, I–, SO42–, CH3COO –, or HCOO – can improve and even 2.6 14.8
6
Wash with 10 column volumes of start buffer at the same
mLmL
14.8
flow
5 5 14.6 rate as in step 1.
mLmL
14.6
alter, selectivity since they exhibit different elution strengths, but it should be noted that using these ions might affect 5 5
3. Repeat steps 1 and 2 several times.
the binding capacity of the medium. Figure 2.4 shows how selectivity and resolution can vary when using different
counterions.
Perform a blank run to check conductivity and pH.

Column: Mono Q HR 5/5

Volume (mL)
Volume (mL) Volume (mL)
Volume (mL)
Samples: carbonic anhydrase, transferrin, ovalbumin, a-lactalbumin, b-lactoglobulin A and B

(A) (B) (C) (D)

2 2 3 3
1 1 1 1 4 4
1 1 3 3 1 1
2 2 3 3 0.70.7
M NaOAc
M NaOAc
4 4 2 2
3–43–4
Elution buffer concentration
Elution buffer concentration

Elution buffer concentration

Elution buffer concentration
2 2

Elution buffer concentration

Elution buffer concentration

Elution buffer concentration

Elution buffer concentration

4 4 0.35
0.35
M NaCl
M NaCl
0.28
0.28
M NaBr
M NaBr
0.25 M Nal
0.25 M Nal
6 6
A 280 nm
A 280 nm

A 280 nm
A 280 nm

6 6

A 280 nm

A 280 nm
A 280 nm

A 280 nm
14.6 mLmL
14.6 6 6
6 6
14.8
14.8
mLmL 5 514.6
14.6
mLmL 15.0 mLmL
15.0
5 5 5 5 5 5

Volume
Volume
(mL)
(mL) Volume
Volume
(mL)
(mL) Volume (mL)
Volume (mL) Volume (mL)
Volume (mL)

Fig 2.4. Effect of salt ions (counterions) (A) sodium chloride, (B) sodium bromide, (C) sodium iodide, and (D) sodium acetate on selectivity and resolution (Mono Q HR 5/5 now available as Mono Q 5/50 GL). Note the variation in elution order of peaks 3 and 4.
2 2 3 3
1 1 4 4
1 1 29
0.70.7
M NaOAc
M NaOAc
2 2
3–43–4
Elution
Elution

Elution
Elution
Flow rates Column:
Sample:
SOURCE 30Q, 10 mm i.d. × 50 mm (4 mL)
Mixture of lactoglobulin B and amyloglucosidase
The maximum flow rate applied during a separation can vary according to the stage of the separation. For example, Sample load: 1 mg/mL of bed volume
during sample application and elution, lower flow rates allow time for sample components to diffuse in and out of the Start buffer: 20 mM BIS-Tris PROPANE, pH 7.0
Elution buffer: 500 mM sodium chloride, 20 mM BIS-TRIS PROPANE, pH 7.0
pores as they bind to or dissociate from the functional groups. Figure 2.5 shows an example of the influence of flow rate
Flow rates (flow velocities): (A) 4 mL/min (300 cm/h)
on resolution. Higher flow rates can be used for equilibration, washing and re-equilibration, limited primarily by the rigidity (B) 13 mL/min (1000 cm/h)
of the media and by pressure specifications of the equipment. Gradient: 0% to 100% elution buffer, 20 CV
Recommended flow rates for each chromatography medium are given in Chapter 3. Working from these recommendations, (A) (B)
select the highest flow rate that maintains resolution and minimizes separation time. For example, if peaks are well
separated at a low flow rate, increase the flow rate or alternatively, increase the sample volume to benefit from a higher
capacity without significant loss of resolution.
Flow rate can be measured as volumetric flow rate, that is, volume per unit time (mL/min). When comparing results

A 280 nm

A 280 nm
between columns of different sizes or when scaling-up, it is useful to use flow velocity, which measures the flow rate
(mL/min) divided by the cross-sectional area of the column and is expressed as flow velocity time (for example, cm/h,
see Appendix 5). Results obtained at the same flow velocity on different size columns will be comparable.
Save time by using higher flow rates during the high salt wash and re-equilibration steps. Do not exceed the
maximum recommended flow for the medium.
Higher flow rates and viscous buffers increase operating pressures (remember that buffer viscosity increases when 0 10 20 0 2 4 6 8
running at 4°C). Check the maximum operating pressure of the packed column and set the upper pressure limit on Time (min) Time (min)

the chromatography system accordingly. Fig 2.5. Influence of increasing flow rate on resolution.

Flow control
Accurate, reproducible flow control is essential for good resolution and reproducibility.
Use a pump within a chromatography system (rather than a peristaltic pump) to fully utilize the high rigidity and
excellent flow properties of the medium.
If you have packed the column yourself, always use a flow rate for separation that is less than the flow rate used for
column packing in order to avoid shrinking of the column bed by pressure increases that can occur when running
a sample.

30
Steps in an IEX separation Column and media preparation
The six steps listed are described in more detail throughout this section. Using prepacked columns is highly recommended to ensure the desired high
performance and reproducible results. An evenly packed column ensures that
1. Equilibrate column with 5 to 10 CV of start buffer or until the baseline, eluent pH, and conductivity are stable. component peaks are not unnecessarily broadened as sample passes down the
2. Adjust the sample to the chosen starting pH and ionic strength and apply to the column. column so that optimal resolution can be achieved.
3. Wash with 5 to 10 CV of start buffer or until the baseline, eluent pH, and conductivity are stable, that is, when all  Allow buffers, media or prepacked columns to reach the same temperature
unbound material has washed through the column. before use. Rapid changes in temperature, for example removing packed
columns from a cold room and then applying buffer at room temperature, can
4. B
 egin elution using a gradient volume of 10 to 20 CV with an increasing ionic strength up to 500 mM NaCl (50%B).
cause air bubbles in the packing and affect the separation.
Alternatively, elute bound proteins with 5 CV of start buffer + NaCl at chosen ionic strength. Repeat at higher ionic
strengths until the target protein(s) has been eluted.  Wash away storage solutions and preservatives before using any IEX medium.
5. Regeneration: Wash with 5 CV of 1 M NaCl (100%B) to elute any remaining ionically bound material.  Increase the volumes used for column equilibration before the first run if using
buffers containing detergents or a different counterion to the one in which the
6. Re-equilibrate with 5 to 10 CV of start buffer or until eluent pH and conductivity reach the required values.
medium has been stored.
Buffer volumes referred to are expressed in column volumes, for example 3 CV = 3 mL for a column with a 1 mL

Appendix 3 gives details on column packing. The volume required for the packed bed
bed volume. Using CV to describe a separation profile facilitates method development and transfer of methods to
is determined by the amount of sample to be purified and the binding capacity of the
columns of different dimensions.
medium. Pack a column that will have approximately five-fold excess of the binding
The number of CV used at each stage of the separation can often be reduced by optimization. For example, capacity required with a bed height up to 20 cm.
less buffer is required to equilibrate a strong ion exchanger, the gradient volume can be reduced if resolution can
Check column performance regularly by determining column efficiency and
be maintained and less buffer might be required for washing when separating less complex and reasonably clean 

peak symmetry. See Appendix 3. Note that this does not apply to HiTrap or
samples.
HiPrep™ columns.

31
Sample preparation Sample application and wash
Correct sample and buffer preparation is essential in order to achieve optimal separation and avoid any deterioration Starting conditions should maximize binding of the target proteins near the top of
in column performance. Simple steps to clarify a sample before application to a column will avoid the risk of blockage the column and, when possible, minimize binding of contaminants so that they pass
and reduce the need for stringent washing procedures. Appendix 1 contains a detailed overview of sample preparation through the column.
techniques.
For efficient binding the sample should be at the same pH and ionic strength
 Desalt samples and transfer into the chosen start buffer (see Buffer exchange and desalting in Appendix 1). The pH as the start buffer. The sample volume can be relatively large without affecting
and ionic strength of the sample are extremely important in order to achieve the most effective high resolution or the separation since the sample will bind at the top of the column as long as
group separations and to make the most of the high loading capacity. equilibration and sample conditions are correct.
 For small sample volumes in a high salt concentration and with no major contaminants such as lipids or ionic Apply samples directly to the column via a chromatography system, a
detergents, it might be sufficient to dilute the sample with start buffer in order to lower the salt concentration to a peristaltic pump, or a syringe. The choice of equipment depends largely on the
level that does not interfere with binding to the medium. However, buffer exchange and desalting is the only way to sample volume, the size of column, the type of IEX medium, and the requirements
guarantee the correct pH and ionic strength conditions of a sample. for accuracy in gradient elution. Ensure that the top of the column bed is not
disturbed during sample application.
 Samples must be clear and free from particulate matter, particularly when working with particle sizes of 34 µm
or less. For small sample volumes, a syringe-tip filter of cellulose acetate or PVDF can be sufficient for sample Do not change buffer conditions until all unbound material has been washed
filtration. through the column (monitored by UV absorbance) and until UV and
conductivity values have returned to starting conditions.
Sample concentration and viscosity
The solubility or viscosity of the sample can limit the quantity that can be applied to a column. High sample viscosity can
cause instability of the separation and an irregular flow pattern resulting in broad, distorted peaks, and problems with
back pressure. The critical parameter is the viscosity of the sample relative to the viscosity of the eluent.
 Dilute viscous samples with start buffer. If high viscosity is caused by the presence of nucleic acid contaminants,
see Appendix 1 for advice on their removal. Remember that viscosity varies with temperature. If dilution is not an
option, using a medium with a larger particle size can help to overcome viscosity problems.
 Samples should generally not exceed 50 to 70 mg/mL protein, but can vary according to the type of sample and the
type of chromatographic medium.

32
Sample load Column: SOURCE 30S, 5 mm i.d. × 50 mm (1 mL)
Sample: Mixture of chymotypsinogen, cytochrome C, and lysozyme
Sample load (mass) is of greater importance than sample volume. The amount of sample which can be applied to a Sample load: (A) 1 mg
column depends on the dynamic binding capacity of the IEX medium and the degree of resolution required. Sample load (B) 10 mg
Start buffer: 20 mM sodium phosphate, pH 6.8
has a major influence on resolution since the width of the peaks is directly related to the amount of substance present,
Elution buffer: 500 mM sodium chloride, 20 mM sodium phosphate, pH 6.8
as shown in Figure 2.6. Consequently, in order to achieve satisfactory resolution, the total amount of protein applied and Flow rate (flow velocity): 1 mL/min (300 cm/h)
bound to the medium should not exceed the total binding capacity of the packed column. Gradient: 0% to 100% elution buffer, 20 CV

Apply up to 30% of the total binding capacity of the column for optimal resolution with gradient elution. Sample
(A) (B)
loads can be increased if resolution is satisfactory or when using a step elution.
If sample volumes are large compared to the total CV, the sample buffer composition, in particular the ionic
strength, should be the same as that of the start buffer to ensure adequate binding conditions.
Chapter 3 gives typical binding capacities for each medium as a guideline for total binding capacity. The actual (dynamic)

A 280 nm

A 280 nm
binding capacity is also affected by factors such as size and shape of the molecules, the pore size of the matrix, flow rate,
sample concentration, pH/protein charge, and ionic strength. Capacity will decrease for molecules of very large diameter
or length such as protein complexes > Mr 400 000 asymmetric proteins, and DNA. These molecules are unable to penetrate
the matrix pores, limiting their binding primarily to the charged groups on the surface of the matrix. Since the exact
distribution of pore sizes in some matrices can vary and the apparent size of a molecule can vary according to the buffer
conditions, there is no distinct molecular weight cut-off point when molecules can or cannot penetrate the matrix pores.
The binding step and the dynamic binding capacity can be increased by applying sample at a pH where the target

0 10 20 0 10 20
protein has a higher charge than if the optimal pH for separation was used. Time (min) Time (min)

Fig 2.6. The influence of increasing sample load on resolution.

33
Sample volume
As a binding technique, IEX is independent of sample volume as long as the ionic strength of the sample is the same  Select the steepest gradient to give acceptable resolution at the selected pH.
or as low as the start buffer and the target proteins are sufficiently charged at the selected pH. Large volumes of dilute
solutions such as fractions from a desalting step or a cell culture supernatant can be applied directly to an IEX medium
sample
without prior concentration. equilibration injection
gradient regeneration re-equilibration
elution
volume
Elution of target protein high salt wash
Bound proteins are eluted by controlled changes in ionic strength or pH. The way in which these changes take place, 1M
5 CV tightly bound
by using a linear or step elution, is selected according to the aim of the separation. molecules
Linear gradient elution allows high-resolution separation/analysis. Step elution ensures faster separation time with elute in high
unbound molecules elute salt wash
reduced buffer consumption as well as group separation.

NaCl concentration
before gradient begins

Linear gradient elution

For high-resolution separation/analysis, elution is performed using a linear gradient volume of 10 to 20 CV with an
increasing ionic strength up to 500 mM NaCl (50%B). 10 to 20 CV
Linear ionic strength gradients, as shown in Figure 2.7, are the most frequently used type of elution and should always be
used when starting with an unknown sample (when as many components as possible are bound to the column and eluted
5 to 10 CV
differentially to see a total protein profile). At low ionic strengths, competition for charged groups on the IEX medium is 5 to 10 CV
at a minimum. Increasing the ionic strength increases competition and reduces the interaction between the medium and 0
Column volumes (CV)
the bound substances, which begin to elute. The elution buffer is usually the same buffer salt and pH as the start buffer,
but contains additional salt, most often sodium chloride. Fig 2.7. Typical IEX separation using linear gradient elution. The UV (protein) and conductivity (salt) traces
show the elution of protein peaks and the changes in salt concentration during elution.
Use of linear gradient elution during method development is strongly recommended. Linear ionic strength
gradients are easy to prepare and very reproducible when generated by a suitable chromatography system.
The results obtained can then serve as a base from which to optimize the separation.
The retention of charged proteins on the IEX medium is related to the volume of the column and the concentration
difference across it:
L ong, shallow gradients give maximum separation between peaks, but separation times will be longer and there will
be greater peak broadening
S
 hort, steep gradients give faster separations and sharper peaks, but peaks will be eluted closer together
P
 eaks eluted later in the gradient tend to be slightly broader than those eluted early on

34
The effects of gradient slope are shown in Figure 2.8. 1M
high salt wash

5 CV
If gradient elution volumes are decreased, it might be necessary to decrease the sample load proportionally


in order to maintain the same resolution. Similarly, if sample load is increased (within the total capacity of the
shallow step
column), gradient volumes might need increasing to maintain resolution. gradient gradient

NaCl concentration
Gradients are most efficiently formed using ÄKTA chromatography systems with preprogrammed method
templates, which automatically control the mixing of solutions being supplied to a column.
sample
 Accurate buffer preparation, efficient mixing, and the shortest possible flow path between a mixer and the top of injection
a column will help to ensure accurate gradient formation. volume

For certain separations, when conditions for a high-resolution separation using a linear gradient have been established, 10 to 20 CV
it might be possible to reduce the total separation time by using a more complex elution profile while maintaining equilibration re-equilibration

resolution (Fig 2.9). Shallow gradients can be used where maximum resolution is required while steeper gradients can 5 to 10 CV 5 to 10 CV
Gradient 0% to 100% elution buffer in 20 CV Gradient 0% to 40% elution
Gradient 0% tobuffer
100%inelution
20 CV buffer
0 in 20 CV Gradient 0% to 40% elution buffer in 20 CV
be used in areas where resolution is satisfactory. Column volumes (CV)

0.10 Fig 2.9. Complex gradient profiles can reduce0.10

total separation time for certain separations.
A 280 nm 0.15 0.15

A 280 nm

A 280 nm

A 280 nm
0.10 0.10
Column: Mono Q HR 5/5 0.05 0.05
Sample: Partially purified dynorphin-converting enzyme
0.05 0.05
Start buffer: 20 mM Tris, pH 7.0
Elution buffer: 20 mM Tris, 1 M NaCl, pH 7.0
0.00 0.00 0.00 0.00
Flow rate: 1 mL/min
0.0 10.0 20.0 30.0 0.0 10.0
0.0 20.0
10.0 30.0
20.0 30.0 0.0 10.0 20.0 30.0
(A) Time (min) (B) Time (min) Time (min) Time (min)
Gradient 0% to 100% elution buffer in 20 CV Gradient
Gradient
0% to0%
100%
to 40%
elution
elution
buffer
buffer
in 20inCV
20 CV Gradient 0% to 40% elution buffer in 20 CV

0.10 0.10
0.15 0.15
Ionic strength

Ionic strength

Ionic strength

Ionic strength
A 280 nm

A 280 nm

A 280 nm

A 280 nm
0.10 0.10
0.05 0.05

0.05 0.05

0.00 0.00 0.00 0.00

0.0 10.0 20.0 30.0 0.0 0.0 10.0 10.0 20.0 Elution
20.0 volume
30.0 30.0 0.0 10.0 20.0 Elution volumevolume
30.0Elution Elution volume
Time (min) Time (min)
Time (min) Time (min)

Fig 2.8. Effect of gradient slope on resolution, in theory and in practice.

35
Ionic strength

Ionic strength

Ionic strength

Ionic strength
Step elution
As shown in Figure 2.10, step elutions are performed by sequential addition of the same buffer at increasing ionic of contaminants during sample application. The target protein(s) is then eluted by
strengths. Step elution is technically simple, but care must be taken in the design of the steps and the interpretation a single buffer change in an enriched, concentrated form. Figure 2.11 shows an
of results since substances eluted by a sharp change in ionic strength elute close together, giving a false peak that can example of such a separation in which a HiTrap Q HP column is used to separate
contain several components. Peaks tend to have sharp fronts and pronounced tailing since they frequently contain more human serum proteins from the unwanted IgG fraction, which passes directly through
than one component. Tailing can lead to the appearance of false peaks if a change in ionic strength is introduced too early. the column.
For these reasons, use a linear ionic strength gradient when developing a new method.
If starting conditions have been chosen to maximize the binding of contaminants,
When an IEX separation has been optimized using gradient elution, changing to step elution reduces the total number then no change in elution conditions is required since the target protein(s)
of CV used for a separation. This speeds up separation times and reduces buffer consumption while retaining the required will pass through the column. For many applications, it is preferable to discard
purity level. Step elutions of this type are often used for routine, large-scale separation. An added advantage of a step the column rather than spend time and effort removing unwanted bound
elution when used at larger scale is that it is often possible to apply a greater amount of sample, since the molecules substances.
which would elute early in a gradient separation no longer take up binding capacity on the column.
In a group separation the molecules of interest are concentrated and rapidly removed from unwanted substances. When
binding and elution conditions for a target protein(s) and contaminants have been determined, usually during preliminary
gradient elution separations, conditions are chosen to maximize binding of the target protein(s) and minimize binding

high salt wash 100% Buffer B

1.0 Column: HiTrap Q, 1 mL
1M 5 CV Sample: Human serum, filtered (0.45 µm filter) and buffer exchanged to start buffer
elution on a PD-10 column
elution of of target 0.8 Sample volume: 1.0 mL
unbound unwanted molecule pool 1
pool 2 Start buffer: 75 mM Tris-HCl, pH 8.0
molecules material
NaCl concentration

Elution buffer: 75 mM Tris-HCl, 1.0 M NaCl, pH 8.0

elute 0.6
sample 5 CV Flow rate (flow velocity): 0.5 mL/min, (75 cm/h)

A 280 nm
injection tightly bound
volume molecules 0.4
elute
5 CV
0.2
equilibration re-equilibration
5 to 10 CV 0
5 to 10 CV 5 10 15 20 25 30 35 40
0 Volume (mL)
Column volumes (CV)

Fig 2.10. Typical IEX separation using step elution. The UV (protein) and conductivity (salt) traces show the Fig 2.11. Group separation of serum proteins on HiTrap Q HP.
elution of protein peaks and the changes in salt concentration during elution.
36
pH elution Increase flow rates during wash and re-equilibration steps to save time


between runs.
Since the net charge on a protein is pH-dependent, samples can also be eluted from an IEX medium by altering the pH
of the elution buffer. As there is no salt gradient, samples are simply retained on the column at one pH and eluted by If ionic detergents have been used, wash the column with 5 CV of distilled


increasing or decreasing the pH. The various charged groups in the sample or on the column are titrated until they are water, followed by 2 CV of 2 M NaCl. Re-equilibrate with at least 10 CV of start
neutral or of opposite charge to the medium and the sample elutes. buffer until the UV baseline, eluent pH, and/or conductivity are stable. Organic
solvents such as ethanol can be used to remove nonionic detergents. When
Proteins bound to an anion exchanger (Q, DEAE, ANX) will elute as pH is decreased
selecting an organic solvent, check the chemical stability of the medium to
Proteins bound to a cation exchanger (SP, S, CM) will elute as pH is increased determine a suitable concentration.
Since pH elution will involve working at pH values close to the pI of a protein and since many proteins show minimum
solubility close to their pI, precautions must be taken to avoid precipitation on the column (see Detergents, denaturing
agents, and other additives later in this chapter for information on the use of additives to avoid precipitation).
Always test in advance the solubility of sample components at the pH and salt concentrations to be used during
separation.
For any type of pH elution, care must be taken in the selection and mixing of buffer systems in order to achieve
reproducibility. Stepwise pH elution is easier to produce and more reproducible than using a linear pH gradient. Note
that for weak ion exchangers the buffer might have to titrate the charged groups on the medium and there will be a short
period of re-equilibration before the new pH is reached.
Linear pH gradients are very difficult to produce at constant ionic strength, since simultaneous changes in ionic strength,
although small, also occur. These gradients cannot be obtained simply by mixing buffers of different pH in linear volume
ratios since the buffering capacities of the systems produced are pH-dependent. A relatively linear gradient can be
produced over a narrow pH interval (maximum 2.0 pH units) by mixing two solutions of the same buffer salt adjusted to
1.0 pH unit above and 1.0 pH unit below the pKa for the buffer.
In general, separation of proteins according to their pI, using chromatofocusing, is likely to provide a more reliable
and higher resolution result than attempting to elute proteins from an IEX column using a pH gradient.

Regeneration and re-equilibration

Include a wash step (regeneration) at the end of every run in order to remove any molecules that are still bound to the
medium. Monitor UV absorbance so that the wash step can be shortened or prolonged, as necessary.
A re-equilibration step after washing returns the column to start conditions before applying further samples. Whenever
possible, monitor pH and conductivity to check when start conditions have been reached. The re-equilibration step can
then be shortened or prolonged as necessary.

37
Detergents, denaturing agents and other Table 2.2. Commonly used detergents and denaturing agents

additives Detergent Type Typical conditions for use Compatibility

Urea 2 to 8 M anion or cation exchangers
Any additives used for dissociation, solubilization, metal chelation, and enzyme
Guanidine hydrochloride 3 to 6 M anion or cation exchangers
inhibition should always be checked for their charge characteristics at the
working pH. Run blank gradients with additives included in order to check their Triton™ X-100 nonionic 2% anion or cation exchangers
effect on the chromatographic profile. N-Octylglucoside nonionic 2% anion or cation exchangers
Additives used during sample preparation will be separated from the sample Sodium dodecyl sulfate ionic 0.1% to 0.5% exchange for nonionic detergent during first
components during IEX. If proteins are seen to precipitate, elute later than chromatography step, avoid anion exchangers
expected, or are poorly resolved, add a suitable concentration of the additives Sarcosyl1 anionic 1.5% cation exchangers
used for initial solubilization to the start and elution buffers. Nonidet P40 nonionic anion or cation exchangers
Zwitterionic additives such as betaine can prevent precipitation and can be used at Polyoxyethylene ethers nonionic anion or cation exchangers
high concentrations without interfering with the gradient elution. (e.g., Brij 35)
Detergents are useful as solubilizing agents for proteins with low aqueous solubility Polyoxyethylene sorbitans nonionic anion or cation exchangers
such as membrane components. Anionic, cationic, zwitterionic, and nonionic (e.g., Tween™ 80)
(neutral) detergents can be used during IEX. CHAPS zwitterionic, derivative anion or cation exchangers
of cholic acid (pH-dependent)
Denaturing agents such as guanidine hydrochloride or urea can be used for initial
solubilization of a sample and during separation. However, they should be avoided CHAPSO zwitterionic, derivative anion or cation exchangers
unless denaturation is a requirement. Note that, at the pH values used for separation, of cholic acid (pH-dependent)
guanidine is a charged molecule with a counterion and will therefore participate in Deoxycholate cation anion exchangers
the ion exchange process in the same way as NaCl. 1
Sarcosyl is strongly protein-denaturing.
Examples of commonly used detergents and denaturing agents are given in Table 2.2.
 Temperatures < 10°C can minimize aggregation caused by hydrophobic
interactions between sample components. Working at these lower
temperatures is an alternative to using a detergent to improve solubility.

38
Developing or optimizing a separation using buffers that contain detergents
A single peak obtained from a ‘detergent run’ often contains more than one


component and should be analyzed carefully. Selecting a different detergent

1. Select detergents that are compatible with the sample. A detergent must be neutral, zwitterionic, or have might improve the separation.
the same charge as the IEX medium. Detergents that bind to the medium can be difficult to remove and can
affect protein loading capacity, pH, conductivity, and resolution. Detergent concentrations that are too high will increase buffer viscosity so


that flow rates must be reduced to avoid overpressure of the column. The
2. Determine the minimum concentration that is likely to keep the sample in solution during the separation. concentration of detergent required for solubilization can often be reduced
Note that different detergents will have different solubilization properties resulting in different peak profiles. during the separation.
3. E
 quilibrate the column thoroughly with the detergent solution, using a concentration that is below the critical Use detergents of the highest quality that are free from salts. Filter buffers that

micelle concentration for the specific detergent. contain detergents under weak suction and ultrasonication for degassing to
4. R
 un blank salt gradients to determine the UV absorbance profile of the detergent and to detect any effect pH. avoid foaming.
Micelle formation causes light scattering and the appearance of a peak during UV monitoring. If micelle Wash previously used columns thoroughly using recommended procedures

formation is a problem try the following: before working with buffers that contain detergents.
- decrease detergent concentration as far as possible without impairing sample solubility
- increase detergent concentration to run the gradient above the critical micelle concentration (this creates a
gradual rather than abrupt UV increase)
- change the salt gradient so that the sudden change in UV absorption does not occur during the run
- change to highly chaotropic salts such as lithium perchlorate or sodium trichloroacetate that can be used at
higher concentrations without causing micelle formation
5. Perform test runs with sample to find the detergent that gives optimal solubilization and resolution.

39
Reagents to reduce polarity Table 2.3. Guidelines for scaling-up

Monoethylene glycol, glycerol and similar mild reagents that reduce polarity can be included in buffers. Avoid high Maintain Increase
concentrations (> 40% w/w) as buffer viscosity will increase and can overpressure the column. Column bed height Column volume, i.e., column diameter
Flow velocity (cm/h) Flow rate (mL/min)
Metal chelators: EDTA, EGTA
Sample concentration Sample load
EDTA (ethylenediaminetetracetic acid) and EGTA (ethylene glycol-bis-[2-aminoethyl]-N,N,N’,N’-tetraacetic acid) are often
Gradient elution volume , i.e., number of column
used in buffers as metal chelators and can be used with IEX. EDTA and EGTA contain several carboxylic acid groups that
volumes used for the gradient
interact with anion exchangers. During anion exchange separations, EDTA and EGTA can concentrate as a band on the
column and elute during a salt gradient. Both molecules absorb UV and will appear as a peak or as background noise in
the chromatogram.

Analysis of results and further steps

The analysis of results from the first separation will indicate if conditions can be improved to increase the yield, achieve
higher purity, speed up the separation, or increase the amount of sample that can be processed in a single run.
Samples eluted using a salt gradient will contain a range of salt concentrations. Dilute or desalt fractions before analysis,
if the assay is sensitive to changes in salt concentration.
Commonly used analytical assays are outlined in Appendix 8.

Scaling-up
For fast separations, it might be easier to repeat a separation several times on a small column and pool the fractions
of interest, rather than scale-up to a larger column. However, a larger column can be preferred for routine processing
of large sample volumes. General guidelines for scaling-up are shown in Table 2.3.

40
When scaling-up an IEX separation, follow the points below to ensure the same cycle time for small scale and larger scale Equipment selection
separations.
Appendix 4 provides a guide to the selection of systems for IEX.

1. Optimize the separation at small scale. Care of IEX media

2. Maintain bed height, sample concentration, and the ratio of sample volume: volume of medium. When IEX media have been in use for some time, it might become necessary to
3. Increase the column volume by increasing the cross-sectional area (diameter) of the column. remove precipitated proteins or other contaminants that have built up in the
column. The need for cleaning can be seen by the appearance of a colored band
4. R
 un the separation at the same flow velocity (see Appendix 5) as used on the smaller column with the same ratio at the top of the column, a space between the upper adapter and the bed surface,
of gradient volume: column volume. a loss in resolution, or a significant increase in back pressure. A general cleaning
procedure for each IEX medium is given in Chapter 3 and Appendix 10 also contains
recommended procedures for severe contamination by precipitated proteins, lipids,
 The HiScreen column format has been especially designed for screening and optimizing before scaling up. The hydrophobically bound proteins, or lipoproteins. In all cases, prevention is better than
higher bed height of HiScreen (10 cm), in comparison with HiTrap (2.5 cm), is suitable for scaling up while keeping cure and routine cleaning is recommended.
the bed height constant.
Always use filtered buffers and samples to reduce the need for additional
During method development, a small particle size may be used to improve resolution. However, smaller particles

column maintenance. See Appendix 1 for further details on sample preparation.
can also result in increased back pressure and this factor can become restrictive when scaling-up. Consider using
larger particles, preferably of the same medium, to utilize lower back pressures and higher flow rates. Always degas buffers and keep buffers, columns and samples at the same
temperature to avoid the formation of air bubbles in the column.
 When scaling-up, the salt concentrations at which peaks elute can decrease with increased sample loads. As sample
is applied to the column, components with a low net charge are displaced by components with a higher net charge. Filter cleaning solutions before use and always re-equilibrate the column with
Molecules will elute in the same order, but at a different point in the elution profile. start buffer before the next separation.

 When method scouting, develop the method, whenever possible, on the medium that will be used at the larger scale. If an increase in back pressure is observed, either on the pressure monitor or
by seeing the surface of the medium move downwards, check that the problem
 For production-scale separations which must satisfy throughput and cleaning-in-place (CIP) requirements of the is actually caused by the column before starting the cleaning procedure.
bioprocess industry, transfer an optimized method as early as possible to a matrix designed for bioprocessing such Disconnect one piece of equipment at a time (starting at the fraction collector),
as SOURCE, Sepharose High Performance, Sepharose Fast Flow, Capto, or Sepharose Big Beads. start the pump, and check the pressure after each piece is disconnected.
 See Appendix 3 for column selection and packing. A dirty on-line filter is a common cause of increased back pressure. Check back
pressure at the same stage during each run, since the value can vary within a
run during sample injection or when changing to a different buffer.

41
Troubleshooting
The desired IEX separation: target proteins Sample elutes before salt gradient begins Sample still eluting when gradient begins
well-resolved by gradient elution Ensure that buffers are in the correct containers. Reduce ionic After sample application the UV trace must return to baseline
If only certain peaks are of interest in this well-resolved, gradient-elution strength of sample by desalting (see Buffer exchange and desalting before elution begins, otherwise proteins that do not bind to the
separation, it can be advantageous to transfer to a step elution in in Appendix 1), or dilution with start buffer. For an anion exchanger, column interfere with the separation. Increase the volume of start
order to save time and buffer. The rest of this section focuses on increase buffer pH; for a cation exchanger, decrease buffer pH. buffer (equilibration step) before starting the gradient elution.
practical problems that might lead to a suboptimal IEX separation. If proteins still do not bind at any pH, the column might be
sample
contaminated by detergent.
equilibration gradient regeneration re-equilibration
injection
elution
volume
high salt wash high salt wash high salt wash
5 to 10 CV 5 to 10 CV
1M 1M 1M
5 CV tightly bound
molecules
elute in high sample sample
unbound molecules elute salt wash injection injection
NaCl concentration

NaCl concentration
NaCl concentration
before gradient begins volume volume

equilibration equilibration
5 to 10 CV 5 to 10 CV
10 to 20 CV
re-equilibration re-equilibration
5 to 10 CV 5 to 10 CV
5 to 10 CV
5 to 10 CV
0 0 0
Column volumes (CV) Column volumes (CV) Column volumes (CV)

Sample elutes during high salt wash Protein(s) of interest eluting late in gradient
Proteins are binding too strongly. Ensure that buffers are in the Proteins are binding too strongly. Increase ionic strength of gradient. It is preferable to alter pH if a very high salt concentration is required
correct containers. If using an anion exchanger, decrease buffer pH; for elution. For an anion exchanger, decrease buffer pH and for a cation exchanger, increase buffer pH. Refer also to Table 2.4.
if using a cation exchanger, increase buffer pH.
high salt wash Protein(s) of interest eluting too early in gradient
5–10 CV
1M
Proteins are not binding strongly. Check ionic strength of gradient. Alter pH, for an anion exchanger, increase buffer pH and for a cation
sample exchanger, decrease buffer pH. Refer also to Table 2.4.
injection
volume
Protein(s) of interest not sufficiently resolved
NaCl

equilibration Refer to the contents of this chapter to review key parameters for improving resolution. Refer also to Table 2.4.
5–10 CV

re-equilibration
5–10 CV

0
Column volumes [CV]
42
Table 2.4. Troubleshooting

Situation Cause Remedy

Reduced or no flow through the column Outlet closed or pumps not working. Open outlet. Check pumps for signs of leakage (if using a peristaltic pump, check tubing also).
Blocked filter, end-piece, adapter, or tubing. Remove and clean or replace if possible. Always filter samples before use.
Lipoproteins or protein aggregates have precipitated. Remove lipoproteins and aggregates during sample preparation, see Appendix 1. Follow cleaning procedures, see Appendix 10.
Protein precipitation in the column. Modify buffer, pH and/or salt conditions during the run to maintain stability. Follow cleaning procedures, see Appendix 10.
Protein precipitation in the column caused by removal of Modify start buffer and elution buffer to maintain stability.
stabilizing agents during separation.
Microbial growth has occurred in the column. Store in the presence of 20% ethanol to prevent microbial growth when not in use. Always filter buffers. Follow cleaning
procedures, see Appendix 10.
Peak of interest is poorly resolved from Sample applied incorrectly. Check bed surface and top filter for possible contamination.
other major peaks. Large mixing spaces at top of or after column. Adjust top adapter to surface of medium if necessary. Reduce all post-column volumes.
Incorrect buffer pH and/or ionic strength. Check pH and ionic strength to ensure that column was re-equilibrated after previous run. Check conditions required.
Prepare new solutions.
Suboptimal elution conditions, e.g., incorrect pH, Alter elution conditions: alter pH, use shallower gradient, reduce flow rate (listed in priority order).
gradient too steep, flow rate too high.
Sample is too viscous. Dilute with buffer. Maintain protein concentration below 50 mg/mL.
Column is poorly packed. Check column efficiency, see Appendix 3. Repack if needed. Use prepacked columns.
Column overloaded. Decrease sample load.
Lipoproteins or protein aggregates have precipitated. Remove lipoproteins and aggregates during sample preparation (see Appendix 1).
Precipitation of proteins in the column. Modify buffer, pH and/or salt conditions during the run to maintain stability.
Microbial growth has occurred in the column. Store in the presence of 20% ethanol to prevent microbial growth. Always filter buffers. Follow cleaning procedures,
see Appendix 10.
Proteins do not bind or elute as expected. Proteins or lipids have precipitated on the column or column filter. Clean the column and exchange or clean the filter. Check pH and salt stability of sample.
Sample not filtered properly. Clean the column, filter the sample and repeat.
Sample has changed during storage. Prepare fresh samples.

continues on following page

43
Situation Cause Remedy
Protein might be unstable or inactive in the elution buffer. Determine the pH and salt stability of the protein.
Column equilibration incomplete. Repeat or prolong the equilibration step until conductivity and pH are constant.
Incorrect buffer pH and/or ionic strength. Check conditions required. Prepare new solutions.
Proteins are forming aggregates and binding strongly to the medium. Use urea or zwitterions, betaine up to 10%, taurine up to 4%.
Sample or buffer conditions are different from previous runs. Check sample and buffer conditions.
Microbial growth has occurred in the column. Store in the presence of 20% ethanol to prevent microbial growth when not in use. Always filter buffers. Follow
cleaning procedures, see Appendix 10.
Protein elutes later than expected or not at all. Incorrect buffer pH. Check pH meter calibration. Use a buffer pH closer to the pI of the protein.
Ionic strength too low. Increase salt concentration in elution buffer.
Ionic interactions between protein and matrix. Maintain ionic strength of buffers above 50 mM.
Hydrophobic interactions between protein and matrix. Reduce salt concentration to minimize hydrophobic interaction. Increase pH. Add suitable detergent or organic
solvent, e.g., 5% isopropanol.
Protein elutes earlier than expected Ionic strength of sample or buffer is too high. Decrease ionic strength of sample or buffer.
(during the wash phase) Incorrect pH conditions. Increase pH (anion exchanger). Decrease pH (cation exchanger).
Column equilibration incomplete. Repeat or prolong the equilibration step until conductivity and pH are constant.
Leading or very rounded peaks in chromatogram. Channeling in the column. Repack column using a thinner slurry of medium.
Check column packing (see Appendix 3).
Column overloaded. Decrease sample load and repeat.
Column contaminated. Clean using recommended procedures.
Peaks are tailing. Incorrect start buffer conditions, sample is not binding to column. Adjust pH. Check salt concentration in start buffer.
Sample too viscous. Dilute in application buffer.
Column packing too loose. Check column efficiency (see Appendix 3). Repack using a higher flow rate. Use prepacked columns.

continues on following page

44
Situation Cause Remedy
Peaks have a leading edge. Column packing compressed. Check column efficiency (see Appendix 3). Repack using a lower flow rate. Use prepacked columns.
Medium/beads appears in eluent. Column packing compressed. Check column efficiency (see Appendix 3). Repack using a slower flow rate. Use prepacked columns.
Bed support end piece is loose or broken. Replace or tighten.
Column operated at too high pressure. Do not exceed recommended operating pressure for medium or column.
Medium has been damaged during column packing. Do not use magnetic stirrers when equilibrating loose IEX medium
Low recovery of activity, but normal recovery Protein might be unstable or inactive in the buffer. Determine the pH and salt stability of the protein.
of protein. Enzyme separated from cofactor or similar. Test by pooling aliquots from the fractions and repeating the assay.
Protein yield lower than expected. Protein might have been degraded by proteases. Add protease inhibitors to the sample and buffers to prevent proteolytic digestion. Run sample through a medium such as
Benzamidine Sepharose 4 Fast Flow (high sub) to remove trypsin-like serine proteases.
Adsorption to filter during sample preparation. Use another type of filter.
Sample precipitates. Check pH and salt conditions, adjust to improve sample solubility.
Hydrophobic proteins. Add denaturing agents, polarity reducing agents or detergents. Add 10% ethylene glycol to running buffer to prevent
hydrophobic interactions.
Nonspecific adsorption. Reduce salt concentration to minimize hydrophobic interaction. Add suitable detergent or organic solvent, e.g., 5%
isopropanol. If necessary, add 10% ethylene glycol to running buffer to prevent hydrophobic interactions.
Peaks too small. Sample absorbs poorly at chosen wavelength. If appropriate, check absorbance range on monitor.
If satisfactory, use a different wavelength, e.g., 214 nm instead of 280 nm.
Different assay conditions have been used before and after the Use same assay conditions for all assays.
chromatographic step.
Excessive band broadening. Check column packing. Repack if necessary.
More sample is recovered than expected. Protein co-eluting with other substances. Optimize conditions to improve resolution.
Check buffer conditions used for assay before and after the run. Check selection of medium.
More activity is recovered than was applied Different assay conditions have been used before and after the Use same assay conditions for all assays.
to the column. chromatography step.
Removal of inhibitors during separation.

continues on following page

45
Situation Cause Remedy
Back pressure increases during a run or Bed compressed. If possible, repack the column or use a new column. Check sample preparation.
during successive runs. Microbial growth. Store in the presence of 20% ethanol to prevent microbial growth. Always filter buffers. Follow cleaning procedures,
see Appendix 10.
Turbid sample. Improve sample preparation (see Appendix 1). Improve sample solubility: add betaine (max. 10% w/v at 25°C), taurine
(max. 4% w/v at 25°C, below pH 8.5) or glycerol (1% to 2 %). For hydrophobic samples, add ethylene glycol, urea, detergents,
or organic solvents.
Precipitation of protein in the column filter and/or at the top of Clean using recommended methods. If possible, exchange or clean filter or use a new column. Include any additives that were
the bed. used for initial sample solubilization in the running buffer.
Incorrect pH is causing precipitation. Calibrate pH meter, prepare new solutions and try again. Change pH.
Precipitation of lipoproteins at increased ionic strength. Lipoproteins are removed prior to chromatography by the addition of 10% dextran sulfate (final 0.2%), and 1 M calcium
chloride (final 0.5 M).
Air bubbles in the bed. Buffers not properly degassed. Degas buffers thoroughly.
Column packed or stored at cool temperature and then Remove small bubbles by passing degassed buffer through the column; take special care if buffers are used after storage in
warmed up. a fridge or cold room.
Do not allow column to warm in sunlight or heating system.
Repack column if possible (see Appendix 3).
Cracks in the bed. Large air leak in column. Check all connections for leaks.
Repack the column if possible (see Appendix 3).
Negative peaks at solvent front. Refractive index effects. Exchange the sample into start buffer.
Unexpected peaks in chromatogram. Buffer impurities. Clean the buffer by running it through a precolumn. Use high quality reagents.
Peaks appear on gradients. Incomplete elution of previous sample. Wash the column according to recommended blank methods.
Spikes in chromatogram. Air bubble trapped in UV monitor flow cell. Always use degassed buffers.
UV baseline rises with gradient. Micelle formation as salt concentration changes. Work below or above the critical micelle concentration of any detergents being used or change the gradient so that the
increase in UV absorption does not occur while the samples are eluting.
Buffer impurities. Use high quality reagents.

46
03
Ion exchange
chromatography
media

47
Introduction MiniBeads are based on a nonporous, monodispersed matrix of rigid, hydrophilic
polymer particles, substituted with quaternary amino (Q) or methyl sulfonate (S)
Historically, several different types of material have been used as a base matrix to which positively or negatively charged groups. The very small size (3 µm), uniformity and physical rigidity of the particles
groups are covalently attached to form an IEX medium. Chapter 1 describes how the matrix characteristics determine create excellent conditions for extremely high-resolution ion exchange separations at
chromatographic properties such as efficiency, capacity, and recovery as well as chemical and physical stability and flow relatively high flow rates and low back pressures (nonuniform, porous particles would
properties. create higher back pressures, reduce flow rate and impair achievable resolution).
IEX media such as Sepharose High Performance, Sepharose 6 Fast Flow, SOURCE, and Capto have much improved flow Such high resolution is essential for successful separation of complex samples in the
properties compared to earlier media and show no change in bed volume under conditions of changing ionic strength picogram (pg) to microgram (μg) scale. The strong ion exchange groups (Q and S)
or pH. Stringent conditions can be used for cleaning the media when required and there is no need for frequent column maintain their charge over a broad pH range, allowing selection of the most suitable
repacking. Most of these media are also designed to meet the throughput and cleaning-in-place requirements for pH for each application.
large-scale industrial chromatography. The following descriptions of different IEX media will start from high-resolution
media for purification and analysis in small scale, continue with media for many standard applications, and end up with
Media characteristics
media for larger scales. Composition: rigid, nonporous matrix of monodisperse, hydrophilic polymer particles
(3 µm) substituted with quaternary amino (Q) or methyl sulfonate (S) groups (Table 3.1).
MiniBeads: purification or analysis of microgram to milligram
quantities with high resolution Table 3.1. Characteristics of MiniBeads media

Use MiniBeads for polishing steps at microscale when high resolution is essential and the capacity of the

Product Functional group pH stability1 Mean particle size
prepacked column is sufficient. Strong anion
 Use MiniBeads for intermediate purification if only microgram to milligram quantities are required, if there is no exchanger
requirement for scale-up, and if the capacity of the prepacked column is sufficient. To avoid column blockage, it is Mini Q -CH2N+-(CH3)3 Long term: 3–11 3 µm (monosized)
especially important to remove particulate matter before using MiniBeads. Short term: 1–14

 Use MiniBeads for faster, higher resolution separations compared to MonoBeads, if the capacity of the prepacked Strong cation
column is sufficient. exchanger

 Run MiniBeads on ÄKTA chromatography systems such as ÄKTA pure 25 and HPLC systems. HPLC systems are Mini S -CH2-SO3– Long term: 3–11 3 µm (monosized)
recommended for optimal performance of the smallest columns (PC 3.2/3). Appendix 4 provides guidance on Short term: 1–14
selecting the right ÄKTA system. 1
L ong-term pH stability refers to the pH interval where the medium is stable over a long period of time
without adverse side effects on the chromatography performance. Short-term pH stability refers to the pH
interval for regeneration, cleaning-in-place, and sanitization procedures. All ranges are estimates based on
experience and knowledge gained at Cytiva.

48
Purification options Table 3.2. Purification options for Mini Q and Mini S prepacked columns
Maximum
Mini Q and Mini S™ media are available prepacked in Precision (PC 3.2/3) and Recommended operating back
Tricorn™ (4.6/50 PE) columns for high-resolution purification of biomolecules (Fig 3.1). working flow pressure2
The purification options for the prepacked columns are shown in Table 3.2. Maximum flow rate range Working (MPa/psi)
Product, column volume Binding capacity per column rate (mL/min) (mL/min) pH range1 1 MPa = 10 bar
Strong anion exchangers
Mini Q PC 3.2/3, 0.24 mL3 1.44 mg (a-amylase, Mr 49 000) 1.0 0.1 to 1.0 3 to 11 10/1450
1.44 mg (trypsin inhibitor, Mr 20 100)
Mini Q 4.6/50 PE, 0.8 mL 4.8 mg (a-amylase, Mr 49 000) 2.0 0.5 to 2.0 3 to 11 18/2600
4.8 mg (trypsin inhibitor, Mr 20 100)
Strong cation exchangers
Mini S PC 3.2/3, 0.24 mL3 1.2 mg (ribonuclease, Mr 13 700) 1.0 0.1 to 1.0 3 to 11 10/1450
1.2 mg (lysozyme, Mr 14 300)
Mini S 4.6/50 PE, 0.8 mL 4 mg (ribonuclease, Mr 13 700) 2.0 0.5 to 2.0 3 to 11 18/2600
Fig 3.1. Mini Q and Mini S media are available prepacked in Precision (PC 3.2/3) and Tricorn (4.6/50 PE) 4 mg (lysozyme, Mr 14 300)
columns. 1
Working pH range refers to the pH interval where the medium can be operated as intended without adverse long-term effects.
2
Maximum operating back pressure refers to the pressure above which the medium begins to compress.
3
Requires a Precision Column Holder for attachment to HPLC systems, see Appendix 4.

49
Purification examples
Fast separations at high resolution Purity check
Column: Mini S 4.6/50 PE Column: Mini S PC 3.2/3 Column: Mini Q 4.6/50 PE
Sample: a-chymotrypsinogen A (25 µg/mL), Sample:  a-chymotrypsinogen A, ribonuclease A, Start buffer: 10 mM NaOH
ribonuclease A (75 µg/mL), lysozyme (25 µg/mL) cytochrome C, lysozyme (6:10:6:5), 25 µg/mL Elution buffer: 10 mM NaOH, 2 M NaCl
Sample volume: 200 µL Sample load: 6 µg Flow rate: 1.0 mL/min
Start buffer: 20 mM sodium acetate, pH 5.0 Start buffer: 20 mM acetic acid, pH 5.0
Elution buffer: 20 mM sodium acetate, 400 mM NaCl, pH 5.0 Elution buffer:  20 mM acetic acid, 500 mM lithium chloride,
Flow rate: 0.83 mL/min pH 5.0
Gradient: 0% to 100% elution buffer in 12 CV Flow rate (velocity): 0.80 mL/min (10 cm/min)
Gradient: 0% to 100% elution buffer in 6 min (20 CV)
Biotinylated 20-mer
100
0.25
100
40.0 40.0
0.0030 50.0
50.0
0.20

Elution buffer (%)

Conductivity (mS/cm)

Conductivity (mS/cm)
A260 nm

A260 nm
Elution buffer (%)

0.15
A 280 nm

0.0020 Crude
A 280 nm
20.0 20.0
synthesis
After
0.10 mixture
purification

0.0010
0.05 0.0 0.0

0.0 0.0
0.0000 0.0 0.00 0.0
20.0 30.0 40.0 20.0 30.0 40.0
0.0 5.0 10.0 15.0 0.0 5.0
Time (min) Volume (mL) Volume (mL)
Time (min)

Fig 3.2. Separation of a protein mixture on Mini S 4.6/50. Fig 3.3. Mini S PC 3.2/3 gives fast, high-resolution separation. Fig 3.4. Purity check of 5’-biotinylated synthetic oligonucleotide 20-mer on Mini Q 4.6/50 PE before and after
purification on a RESOURCE RPC column.

50
Long term reproducibility Performing a separation
Column: Mini S PC 3.2/3 Guidelines for selection of media, buffer, pH, and ionic strength conditions and method optimization are given in Chapter 2.
Sample: Chymotrypsinogen A, ribonuclease A, lysozyme, 6 mg in ratio 1:3:1 Use the instructions given here as a basis from which to optimize a separation.
Start buffer: 20 mM acetic acid, pH 5.0
Elution buffer: 20 mM acetic acid, 400 mM NaCl, pH 5.0 Correct sample and buffer preparation is essential in order to achieve optimal separation and avoid any
Flow rate: 0.4 mL/min deterioration in column performance, especially when using small particles such as MiniBeads. Samples must be
Gradient: 0% to 100% elution buffer in 12 min (20 CV) fully dissolved and free from particles or other material likely to interfere with the separation. Refer to Chapter 2
and Appendix 1 for recommendations and advice on sample preparation.
Filter buffers after all salts and additives have been included. Use high quality water and chemicals. Filter solutions
through 0.22 µm filters. To avoid formation of air bubbles in a packed column, ensure that column and buffers are
at the same temperature when preparing for a run.
Injection The pH of the start buffer should be at least 0.5 to 1.0 pH unit above the pI of the target substance when using an
anion exchanger (Q) and 0.5 to 1.0 pH unit below the pI of the target substance when using a cation exchanger (S).
1 See Appendix 2 for recommendations on volatile and nonvolatile buffer systems for anion and cation exchangers.
For samples with unknown charge properties, try the following:
anion exchange (Q)
A 280 nm

start buffer: 20 mM Tris-HCl, pH 8.0

elution buffer: start buffer including 1 M NaCl, pH 8.0
cation exchange (S)
51 start buffer: 20 mM 2-(N-Morpholino)ethanesulfonic acid (MES), pH 6.0
elution buffer: start buffer including 1 M NaCl, pH 6.0
Users of ÄKTA systems with automatic buffer preparation functionality can select one of the buffer recipes
recommended for anion exchange chromatography at pH 8.0 or cation exchange chromatography at pH 6.0,
see ÄKTA Laboratory-scale Chromatography Systems: Instrument Management Handbook, 29010831.

201

5.0 10.0 15.0

Time (min)

Fig 3.5. Chromatograms from the first, 51st, and 201st separation of a series run on the same Mini S PC 3.2/3
column. The same consistent reproducibility has been confirmed on Mini Q PC 3.2/3 (data not shown).

51
First-time use or after long-term storage Separation by step elution
Although separations by step elution (see Chapter 2) can be performed using
1. To remove ethanol, wash with 4 CV of distilled water at 0.1 mL/min (Mini Q and S PC 3.2/3, 0.24 mL columns) MiniBeads, gradient elution is recommended to maximize resolution.
or 0.5 mL/min (Mini Q and S 4.6/50 PE, 0.8 mL columns). This step ensures removal of ethanol and avoids If ionic detergents have been used, wash the column with 5 CV of distilled
the risk of precipitation if buffer salts were to come into contact with the ethanol. The step can be omitted if water, followed by 2 CV of 2 M NaCl. Re-equilibrate with at least 10 CV of start
precipitation is not likely to be a problem. buffer until the UV baseline, eluent pH, and/or conductivity are stable. Organic
2. Wash with 4 CV of start buffer at 0.4 mL/min (Mini Q and S PC 3.2/3, 0.24 mL columns) or 0.8 mL/min (Mini Q and solvents such as ethanol can be used to remove nonionic detergents. When
S 4.6/50 PE, 0.8 mL columns). selecting an organic solvent, check the chemical stability of the medium to
determine a suitable concentration.
3. Wash with 4 CV of elution buffer, same flow as step 2.
Refer to Chapter 2 for advice on optimizing the separation. Check column
4. Wash with 4 CV of start buffer, same flow as step 2. performance regularly by determining column efficiency and peak symmetry.
See Appendix 3.
Separation by gradient elution Cleaning
Correct preparation of samples and buffers and application of a high salt wash
Flow rates: 0.4 mL/min (PC 3.2/3, 0.24 mL columns) or 0.8 mL/min (4.6/50 PE, 0.8 mL columns). Collect fractions (1 M NaCl) at the end of each separation should keep most columns in good condition.
throughout the separation. However, reduced performance, a slow flow rate, increasing back pressure or
complete blockage are all indications that the medium needs to be cleaned using
1. Equilibrate column with 5 to 10 CV of start buffer or until the baseline, eluent pH, and conductivity are stable.
more stringent procedures in order to remove contaminants.
2. Adjust the sample to the chosen starting pH and ionic strength and apply to the column.
Reverse the direction of flow during column cleaning so that contaminants do
3. W
 ash with 5 to 10 CV of start buffer or until the baseline, eluent pH, and conductivity are stable, that is, when all not need to pass through the entire length of the column. The number of
unbound material has washed through the column. column volumes and time required for each cleaning step can vary according
4. Begin elution using a gradient volume of 10 to 20 CV and an increasing ionic strength up to 0.5 M NaCl (50%B). to the degree of contamination.

5. Wash with 5 CV of 1 M NaCl (100%B) to elute any remaining ionically bound material.
6. Re-equilibrate with 5 to 10 CV of start buffer or until eluent pH and conductivity reach the required values.

52
Removing common contaminants

1. Wash with 2 CV of 2 M NaCl at 0.2 mL/min.

2. Wash with 4 CV of 1 M NaOH at 0.2 mL/min.
3. Wash with 2 CV of 2 M NaCl at 0.2 mL/min.
4. Rinse with at least 2 CV of distilled water at 0.2 mL/min until the UV-baseline and eluent pH are stable.
5. W
 ash with at least 4 CV of start buffer or storage buffer at 0.2 mL/min until pH and conductivity values have
reached the required values.

To remove precipitated proteins, lipids, hydrophobically bound proteins, or lipoproteins, refer to Appendix 1.

Chemical stability
For daily use, MiniBeads are stable in all common aqueous buffers in the pH range of 3 to 11 and in the presence of
additives such as denaturing agents (8 M urea or 6 M guanidine hydrochloride), nonionic or ionic detergents, and up to
30% acetonitrile in aqueous buffers. Note that aqueous solutions of urea, ethylene glycol, and similar compounds will
increase the back pressure due to increased viscosity.
MiniBeads can be used with organic solutions such as dimethylsulfoxide, dimethylformamide, or formic acid, but the
separation properties of the media will change.
Avoid anionic detergents with Mini Q. Avoid cationic detergents with Mini S. Avoid oxidizing agents.

Storage
For column storage, wash with 4 CV of distilled water followed by 4 CV of 20% ethanol. Degas the ethanol/water mixture
thoroughly and apply at a low flow rate to avoid overpressuring the column. Store at room temperature or, for long
periods, store at 4°C to 8°C. Whenever possible, use the storage and shipping device if supplied by the manufacturer.
Ensure that the column is sealed well to avoid drying out. Do not freeze.

53
MonoBeads: purification of milligram quantities with
high resolution
Use MonoBeads for polishing steps at laboratory scale when high resolution is essential and a higher capacity than
MiniBeads is required.
Use MonoBeads for capture or intermediate purification when milligram quantities are required, when there is no
requirement for scale-up, and/or when prepacked MiniBead columns do not offer sufficient capacity. Note that, to
avoid column blockage, it is especially important to remove particulate matter before using MonoBeads.
Run MonoBeads on ÄKTA chromatography systems and HPLC systems. Appendix 4 provides guidance on selecting
the right ÄKTA system. HPLC systems are recommended for optimal performance of the smallest columns (PC 1.6/5).
Mono Q and Mono S™ IEX media are based on a hydrophilic matrix made from monodispersed, rigid, polystyrene/divinyl
benzene particles, substituted with quaternary ammonium (Q) or methyl sulfonate (S) groups (Fig 3.6). This combination
confers extreme chemical and physical stability to the media. The small particle sizes (10 µm) allow fast binding and
dissociation to facilitate high resolution while the uniformity of the particles ensures high flow rates at low back
pressures. The strong ion exchange groups (Q and S) maintain their charge over a broad pH range (Fig 3.7), allowing Fig 3.6. Electron micrograph of MonoBeads showing their distinct monodispersity.
selection of the most suitable pH for each application.
Mono Q Mono S
1.0 mL ion exchanger in 1 M KCl 1.0 mL ion exchanger in 1 M KCl
12 12

10 10

8 8

pH

pH
6 6

4 4

2 2
0 0.1 0.2 0.3 0.4 0 0.1 0.2 0.3 0.4
HCl (mmol) HCl (mmol)

Fig 3.7. Titration curves for Mono Q and Mono S. Binding capacity remains constant over a broad pH
working range.

54
Media characteristics
Composition: rigid, monodisperse, polystyrene/divinyl benzene particles (10 μm) with an optimized pore size distribution.
The base matrix is substituted with quaternary amino (Q) or methyl sulfonate groups (S), see Table 3.3.

Table 3.3. Characteristics of MonoBeads media

Product Functional group pH stability1 Mean particle size (μm)

Strong anion exchanger
Mono Q -CH2-N+-(CH3)3 Long term: 2 to 12 10 (monosized)
Short term: 2 to 12
Strong cation exchanger
Mono S -CH2-SO3- Long term: 2 to 12 10 (monosized)
Short term: 2 to 14
1
L ong-term pH stability refers to the pH interval where the medium is stable over a long period of time without adverse side effects on chromatographic
performance. Short-term pH stability refers to the pH interval for regeneration, cleaning-in-place, and sanitization procedures. All ranges are estimates
based on experience and knowledge gained at Cytiva.

Purification options
MonoBeads (Q and S) are available in convenient prepacked Tricorn PE (PEEK) and Tricorn GL (glass) columns (Fig 3.8).
Purification options for the prepacked columns are described in Table 3.4.

Fig 3.8. MonoBeads (Q and S) are available prepacked in Tricorn PC (Precision Column), PE (PEEK), and
Tricorn GL (glass) columns.

55
Table 3.4. Purification options for Mono Q and Mono S prepacked columns

Recommended working Maximum flow rate Maximum operating back

Product, column volume Binding capacity per column flow rate range (mL/min) (mL/min) Working pH range1 pressure2 (MPa/psi) 1 MPa = 10 bar
Strong anion exchangers
Mono Q PC 1.6/5, 0.1 mL3 2.5 mg (thyroglobulin, Mr 669 000) 0.01 to 0.4 0.4 2 to 12 5/725
Mono Q 5/50 GL, 1 mL 25 mg (thyroglobulin, Mr 669 000) 0.5 to 3.0 3.0 2 to 12 4/580
65 mg (HSA, Mr 68 000)
80 mg (a-lactalbumin, Mr 14 300)
Mono Q 4.6/100 PE, 1.7 mL 40 mg (thyroglobulin, Mr 669 000) 0.5 to 3.0 3.0 2 to 12 4/580
110 mg (HSA, Mr 68 000)
140 mg (a-lactalbumin, Mr 14 300)
Mono Q 10/100 GL, 8 mL 200 mg (thyroglobulin, Mr 669 000) 2.0 to 6.0 10.0 2 to 12 4/580
520 mg (HSA, Mr 68 000)
640 mg (a-lactalbumin, Mr 14 300)
Mono Q HR 16/10, 20 mL 500 mg (thyroglobulin, Mr 669 000) up to 10.0 10.0 2 to 12 3/435
1300 mg (HSA, Mr 68 000)
1600 mg (a-lactalbumin, Mr 14 300)
Strong cation exchangers
Mono S PC 1.6/5, 0.1 mL3 7.5 mg (human IgG, Mr 160 000) 0.01 to 0.4 0.4 2 to 12 5/725
Mono S 5/50 GL, 1 mL 75 mg (human IgG, Mr 160 000) 0.5 to 3.0 3.0 2 to 12 4/580
75 mg (ribonuclease, Mr 13 700)
Mono S 4.6/100 PE, 1.7 mL 130 mg (human IgG, Mr 160 000) 0.5 to 3.0 3.0 2 to 12 4/580
130 mg (ribonuclease, Mr 13 700)
Mono S 10/100 GL, 8 mL 600 mg (human IgG, Mr 160 000) 2.0 to 6.0 10.0 2 to 12 4/580
600 mg (ribonuclease, Mr 13 700)
Mono S HR 16/10, 20 mL 1500 mg (human IgG, Mr 160 000) up to 10.0 10.0 2 to 12 3/435
1500 mg (ribonuclease, Mr 13 700)
1
Working pH range refers to the pH interval where the medium can be operated as intended without adverse long-term effects.
2
Maximum operating back pressure refers to the pressure above which the medium begins to compress.
3
Requires a Precision Column Holder for attachment to HPLC systems.

56
Purification examples
Two-step purification using complementary selectivities
Column: Mono Q HR 5/5
0.5 0.5 Column: Mono S HR 5/5
Sample: 500 mL of T. reesei crude cellulases in start buffer, 2.5 mg
Sample: Peak 3 from Mono Q HR 5/5
Start buffer: 20 mM Tris-HCl, pH 7.6
Start buffer: 20 mM acetate, pH 3.6
Elution buffer: 20 mM Tris-HCl, 500 mM NaCl, pH 7.6
Elution buffer: 20 mM acetate, 200 mM NaCl, pH 3.6
Flow rate: 1.0 mL/min
Flow rate: 1.0 mL/min
Gradient: 0% elution buffer (4 CV), 0% to 40% elution buffer (21 CV),
Gradient: 0% to 100% elution buffer (26 CV)
40% to 100% elution buffer (15 CV)

A 280 nm
A 280 nm

0 10 20 30 0 10 20
Time (min) Time (min)

Fig 3.9. Purification of cellulose on Mono Q and Mono S HR 5/5 columns (now available as Mono Q 5/50 GL and Mono S 5/50 GL).

High-resolution, polishing step

(A) (B)
100
Column: Mono S 5/50 GL
1 2 3 4 5
1200 Sample: Recombinant transposase TniA partially
purified on SOURCE 15Q 4.6/100 PE
1000 Mr Lane 1. Sample, clarified extract diluted five-fold
Elution buffer (%)

and HiTrap Heparin HP, 5 mL

97 000 Lane 2. Pooled from SOURCE 15Q 4.6/100 PE
A 280 nm

800 Sample load: 14.5 mL

Lane 3. Pooled from HiTrap Heparin HP
Start buffer: 20 mM MES, 1 mM EDTA, 2 mM MgCl2, 66 000
600 Lane 4. Pooled from Mono S 5/50 GL
1 mM DTT, pH 6.5 TniA
45 000 Lane 5. LMW-SDS Marker Kit
400 Elution buffer: 20 mM MES, 1 mM EDTA, 2 mM MgCl2,
1 mM DTT, 1 M NaCl, pH 6.5
200 30 000
Flow rate: 1 mL/min
0 Pool 0 Gradient: 0% to 100% elution buffer in 20 CV
0.0 10 20 30 40
Volume (mL)

Fig 3.10. Final polishing step in purification of a DNA-binding protein, transposase TniA. Two well-resolved peaks after separation on Mono S 5/50 GL. (A) SDS-PAGE analysis shows fractions from each of the three steps used
in this protocol. (B) PhastSystem™ electrophoresis unit using SDS-PAGE PhastGel™ Homogeneous – 12.5 and Coomassie™ staining.
57
Long-term reproducibility Performing a separation
Guidelines for selection of media, buffer, pH, and ionic strength conditions and
Column: Mono Q 5/50 GL (Tricorn) method optimization are given in Chapter 2. Use these instructions as a basis from
Injection 2000 Sample: Conalbumin (3.0 mg/mL), α-lactalbumin (4 mg/mL), STI (6 mg/mL)
Injection 1000 Sample volume: 200 µL
which to optimize a separation.
Injection 1 Buffer A: 200 mM Tris, pH 7.0
Correct sample and buffer preparation is essential to achieve optimal
Buffer B: Buffer A + 500 mM NaCl
separation and to avoid any deterioration in column performance, especially
A280 (mAU)

Gradient: Linear, 0% to 100% B in 20 CV

Column equilibration: 5 CV when using small particles such as MonoBeads. Samples must be fully
Flow rate: 1.0 mL/min dissolved and free from particles or other material likely to interfere with the
System: ÄKTA system separation. Refer to Chapter 2 and Appendix 1 for recommendations and
advice on sample preparation.
Filter buffers after all salts and additives have been included. Use high
0 5 10 15 20 25 quality water and chemicals. Filter solutions through 0.22 µm filters. To avoid
Volume (mL) formation of air bubbles in a packed column, ensure that column and buffers
are at the same temperature when preparing for a run.
Fig 3.11. Chromatograms illustrating run to run reproducibility for Mono Q 5/50 GL (Tricorn column). Runs 1, 1000, and 2000 are shown.
The pH of the start buffer should be at least 0.5 to 1.0 pH unit above the pI of
the target substance when using an anion exchanger (Q) and 0.5 to 1.0 pH unit
Separation in organic solvents below the pI of the target substance when using a cation exchanger (S). See
Appendix 2 for recommendations on volatile and nonvolatile buffer systems for
Column: Mono S HR 5/5 anion and cation exchangers.
Sample: Bacitracin 4 mg/mL in start buffer
Sample load: 200 µL For samples with unknown charge properties, try the following:
Start buffer: 90% methanol, 50 mM formic acid/lithium hydroxide (LiOH), pH 3.8
Elution buffer: 90% methanol, 50 mM formic acid/LiOH, 350 mM lithium perchlorate (LiClO4), anion exchange (Q)
A 280 nm

pH 3.8 start buffer: 20 mM Tris-HCl, pH 8.0

Flow rate: 1 mL/min elution buffer: start buffer including 1 M NaCl, pH 8.0
Gradient: 0% to 100% elution buffer in 20 CV
cation exchange (S)
start buffer: 20 mM 2-(N-Morpholino)ethanesulfonic acid (MES), pH 6.0
elution buffer: start buffer including 1 M NaCl, pH 6.0
Users of ÄKTA systems with automatic buffer preparation functionality
can select one of the buffer recipes recommended for anion exchange
Time chromatography at pH 8.0 or cation exchange chromatography at pH 6.0, see
Fig 3.12. Separation of the peptide bacitracin on Mono S HR 5/5 (now available as Mono S 5/50 GL).
ÄKTA Laboratory-scale Chromatography Systems: Instrument Management
Handbook, 29010831.

58
First-time use or after long-term storage Separation by step elution
Although separations by step elution (see Chapter 1) can be performed using
1. To remove ethanol, wash with 5 CV of distilled water at 0.1 mL/min (PC 1.6/5, 0.1 mL columns), 1 mL/min (5/50 GL, MonoBeads, gradient elution is recommended in order to achieve the highest
1 mL and 4.6/100 PE, 1.7 mL columns), 2 mL/min (10/100 GL, 8 mL columns) or 4 mL/min (HR 16/10, 20 mL possible resolution.
columns). This step ensures removal of ethanol and avoids the risk of precipitation if buffer salts were to come Save time by using higher flow rates during the high salt wash and re-equilibration
into contact with the ethanol. The step can be omitted if precipitation is not likely to be a problem. steps. Do not exceed the maximum recommended flow for the column.
2. W
 ash with 5 CV of start buffer at 0.1 mL/min (PC 1.6/5, 0.1 mL columns), 2 mL/min (5/50 GL, 1 mL and 4.6/100 If ionic detergents have been used, wash the column with 5 CV of distilled
PE, 1.7 mL columns), 4 mL/min (10/100 GL, 8 mL columns), or 8 mL/min (HR 16/10, 20 mL columns). water, followed by 2 CV of 2 M NaCl. Re-equilibrate with at least 10 CV of start
3. Wash with 5 CV of elution buffer, same flow as step 2. buffer until the UV baseline, eluent pH and/or conductivity are stable. Organic
solvents such as ethanol can be used to remove nonionic detergents. When
4. Wash with 5 CV of start buffer, same flow as step 2. selecting an organic solvent, check the chemical stability of the medium to
determine a suitable concentration.

Separation by gradient elution Check column performance regularly by determining column efficiency and
peak symmetry. See Appendix 3. Refer to Chapter 2 for advice on optimizing
the separation.
Flow rates: 0.1 mL/min (PC 1.6/5, 0.1 mL columns), 2 mL/min (5/50 GL, 1 mL and 4.6/100 PE, 1.7 mL columns),
4 mL/min (10/100 GL, 8 mL columns) or 8 mL/min (HR 16/10, 20 mL columns). Collect fractions throughout the Cleaning
separation. Correct preparation of samples and buffers and application of a high salt wash
1. Equilibrate column with 5 to 10 CV of start buffer or until the baseline, eluent pH, and conductivity are stable. (1 M NaCl) at the end of each separation should keep most columns in good condition.
However, reduced performance, a slow flow rate, increasing back pressure, or
2. Adjust the sample to the chosen starting pH and ionic strength and apply to the column.
complete blockage are all indications that the medium needs to be cleaned using
3. W
 ash with 5 to 10 CV of start buffer or until the baseline, eluent pH, and conductivity are stable, that is, when all more stringent procedures to remove contaminants.
unbound material has washed through the column.
Reverse the direction of flow during column cleaning so that contaminants
4. Begin elution using a gradient volume of 10 to 20 CV and an increasing ionic strength up to 0.5 M NaCl (50%B). do not need to pass through the entire length of the column. The number of
5. Wash with 5 CV of 1 M NaCl (100%B) to elute any remaining ionically bound material. column volumes and time required for each cleaning step may vary according
to the degree of contamination. If the cleaning procedure to remove common
6. Re-equilibrate with 5 to 10 CV of start buffer or until eluent pH and conductivity reach the required values. contaminants does not restore column performance, change the top filter
before trying alternative cleaning methods. Care should be taken when
changing a filter as this can affect the column packing and interfere with
performance.

59
Removing common contaminants

Flow rates: 0.05 mL/min (PC 1.6/5, 0.1 mL columns), 0.5 mL/min (5/50 GL, 1 mL columns), 0.2 mL/min (4.6/100 PE,
1.7 mL columns), 2 mL/min (10/100 GL, 8 mL columns), or 5 mL/min (HR 16/10, 20 mL columns).
1. Wash with at least 2 CV of 2 M NaCl.
2. Wash with at least 4 CV of 1 M NaOH.
3. Wash with at least 2 CV of 2 M NaCl.
4. Rinse with at least 2 CV of distilled water until the UV-baseline and the eluent pH are stable.
5. W
 ash with at least 4 CV of start buffer or storage buffer until pH and conductivity values have reached the
required values.

To remove precipitated proteins, lipids, hydrophobically bound proteins, or lipoproteins, refer to Appendix 1.

Chemical stability
For daily use, MonoBeads are stable in all common, aqueous buffers in the pH range 2 to 12, and in the presence of
additives such as denaturing agents (8 M urea or 6 M guanidine hydrochloride), nonionic or ionic detergents, and up to
20% acetonitrile in aqueous buffers. Note that aqueous solutions of urea, ethylene glycol and similar compounds will
increase the back pressure due to increased viscosity.
MonoBeads can be used with organic solutions such as dimethylsulfoxide, dimethylformamide, or formic acid, but the
separation properties of the media will change.
Avoid anionic detergents with Mono Q. Avoid cationic detergents with Mono S. Avoid oxidizing agents.

Storage
For column storage, wash with 5 CV of distilled water followed by 5 CV of 20% ethanol. Degas the ethanol/water mixture
thoroughly and apply at a low flow rate to avoid overpressuring the column. Store at room temperature or, for long
periods, store at 4°C to 8°C. Ensure that the column is sealed well to avoid drying out. Whenever possible, use the storage
and shipping device if supplied by the manufacturer. Do not freeze.

60
SOURCE: high-throughput, high-resolution purification, and easy
scale-up
Use SOURCE 15 for intermediate purification or polishing steps in laboratory- or large-scale applications that
require high resolution and high throughput (flow velocities up to 1800 cm/h).
Use SOURCE 30 as an alternative to SOURCE 15 for intermediate purification or polishing steps in large-scale
applications where speed rather than resolution is a priority (flow velocities up to 2000 cm/h).
Use SOURCE 30 as an alternative to SOURCE 15 for large sample volumes where speed rather than resolution is a
priority. The larger particle size slightly reduces resolution, but separations can be performed at higher flow rates.
Run SOURCE columns on ÄKTA chromatography systems, HPLC systems, or systems using peristaltic pumps.
Appendix 4 provides guidance on selecting the right ÄKTA system.
SOURCE media are based on a hydrophilic matrix made from monodispersed, rigid, polystyrene/divinyl benzene and
substituted with quaternary ammonium (Q) or methyl sulfonate (S) groups (Figure 3.13). This combination confers
extreme chemical and physical stability to the media. The small particle sizes allow fast binding and dissociation to
facilitate high resolution while the uniformity and stability of the particles ensures high flow rates at low back pressure.
The strong ion exchange groups (Q and S) maintain their charge over a broad pH range, allowing selection of the most
suitable pH for each application. The high flow rates that can be used with SOURCE media are more likely to be limited by
the equipment available rather than the physical properties of the media.
Fig 3.13. Uniform size distribution of SOURCE monodispersed particles.
Separation methods can be easily scaled up from columns such as RESOURCE Q or S, 1 mL prepacked with SOURCE 15,
to large-scale columns such as FineLINE™.

61
Media characteristics
Composition: rigid, monodisperse, polystyrene/divinyl benzene particles with an optimized pore-size distribution.
The base matrix is substituted with quaternary amino groups (Q) or methyl sulfonate groups (S), see Table 3.5.

Table 3.5. Characteristics of SOURCE 15 and 30 media

Product Functional group pH stability1 Mean particle size (μm, monosized)

SOURCE 15Q -CH2-N+-(CH3)3 Long term: 2 to 12 15
Short term: 1 to 14
SOURCE 30Q -CH2-N+-(CH3)3 Long term: 2 to 12 30
Short term: 1 to 14
SOURCE 15S -CH2-SO3- Long term: 2 to 13 15
Short term: 1 to 14
SOURCE 30S -CH2-SO3- Long term: 2 to 13 30
Short term: 1 to 14
1
L ong-term pH stability refers to the pH interval where the medium is stable over a long period of time without adverse side effects on the chromatography
performance. Short-term pH stability refers to the pH interval for regeneration, cleaning-in-place, and sanitization procedures. All ranges are estimates
based on experience and knowledge gained at Cytiva. Fig 3.14. SOURCE is available in media packs and is prepacked in Tricorn and RESOURCE columns.

Purification options
SOURCE Q and S media are available in media packs and in convenient prepacked Tricorn (PE) and RESOURCE columns
(Fig 3.14). Purification options for the media and prepacked columns are described in Table 3.6.

62
Table 3.6. Purification options for SOURCE media and prepacked columns Use prepacked RESOURCE columns (1 mL or 6 mL) for fast media selection,


Maximum method scouting, group separations, sample concentration or clean-up.

operating back
Use SOURCE 15Q PE 4.6/100 PE to improve resolution by increasing column
Recommended pressure3 

Binding capacity per working flow Maximum Working pH (MPa/psi)

length with further optimization and as the first step towards scaling up.
Product, column volume column or per mL medium rate range1 flow1 range2 1 MPa = 10 bar For column packing in XK columns, see Table 3.7. Select a production column such as
Strong anion exchangers FineLINE for larger volumes.
SOURCE 15Q 45 mg/mL 150 to 900 cm/h 1800 cm/h 2 to 12 0.5/72
(BSA, Mr 67 000) Table 3.7. Packing of SOURCE 15 and SOURCE 30 chromatography media in XK columns
SOURCE 30Q 40 mg/mL 300 to 1000 cm/h 2000 cm/h 2 to 12 0.5/72
Volume (mL) Bed height (cm)
(BSA, Mr 67 000)
SOURCE 15
RESOURCE Q, 1 mL 45 mg 1.0 to 10 mL/min 10 mL/min 2 to 12 1.5/220
(BSA, Mr 67 000) Tricorn 10/100 up to 8 up to 10
RESOURCE Q, 6 mL 270 mg 1.0 to 60 mL/min 60 mL/min 2 to 12 0.6/87 Tricorn 10/150 up to 12 up to 15
(BSA, Mr 67 000) Tricorn 10/200 up to 16 up to 20
SOURCE 15Q 75 mg 0.5 to 2.5 mL/min 5 mL/min 2 to 12 4/580 SOURCE 30
4.6/100 PE, 1.7 mL (BSA, Mr 67 000)
XK 16/20 up to 30 up to 15
Strong cation exchangers
XK 26/20 up to 80 up to 15
SOURCE 15S 80 mg/mL 150 to 900 cm/h 1800 cm/h 2 to 13 0.5/72
XK 26/40 up to 196 > 15
(lysozyme, Mr 14 500)
SOURCE 30S 80 mg/mL 300 to 1000 cm/h 2000 cm/h 2 to 13 0.5/72
(lysozyme, Mr 14 500)
RESOURCE S, 1 mL 80 mg 1.0 to 10 mL/min 10 mL/min 2 to 13 1.5/220
(lysozyme, Mr 14 500)
RESOURCE S, 6 mL 480 mg 1.0 to 60 mL/min 60 mL/min 2 to 13 0.6/87
(lysozyme, Mr 14 500)
SOURCE 15S 140 mg 0.5 to 2.5 mL/min 5 mL/min 2 to 13 4/580
4.6/100 PE, 1.7 mL (lysozyme, Mr 14 500)
1
See Appendix 5 to convert flow velocity (cm/h) to volumetric flow rate (mL/min) and vice versa.
2
Working pH range refers to the pH interval where the medium can be operated as intended without adverse long-term effects.
3
Maximum operating back pressure refers to the pressure above which the medium begins to compress.

63
 Purification examples
Fast, high resolution separations Scaling up: resolution maintained

Column: RESOURCE Q 1 mL Column: RESOURCE S 1 mL Columns: (A) SOURCE 15S, 2.2 mL

Sample: Pancreatin 5 mg/mL Sample: Snake venom, 4 mg/mL (B) SOURCE 15S, FineLINE 100, 390 mL
Sample volume: 200 µL Sample volume: 100 µL Samples: Ribonuclease, cytochrome C and lysozyme
Start buffer: 20 mM bis-Tris-propane, pH 7.5 Start buffer: 20 mM sodium phosphate, pH 6.8 Sample load: (A) 0.46 mg in 200 µL
Elution buffer: 20 mM bis-Tris-propane, 500 mM NaCl, pH 7.5 Elution buffer: 20 mM sodium phosphate, 400 mM NaCl, pH 6.8 (B) 80.5 mg in 350 mL
Flow rate, flow velocity: 9.6 mL/min, 1800 cm/h Flow rate, flow velocity: 1 mL/min, 180 cm/h Start buffer: 20 mM sodium phosphate, pH 6.8
Gradient: 0% to 80% elution buffer in 20 CV Gradient: 0% to 100% elution buffer in 20 CV Elution buffer: 20 mM sodium phosphate, 400 mM NaCl, pH 6.8
Flow rates (flow velocities): (A) 2.2 mL/min (300 cm/h)
(B) 385 mL/min (300 cm/h)
Gradient: 0% elution buffer (2 CV)
0% to 100% elution buffer (21 CV)
(A) (B)
100 100
0.05

0.10
100 0.05 100 0.04
A 280 nm

A 280 nm

A 280 nm
A 280 nm

Elution buffer (%)

Elution buffer (%)

Elution buffer (%)
0.03
Elution buffer (%)

50 50

0.05 0.02

0.01

0 0.00 0
0 0 0
0 1 2 3 0 10 20 30 0 10 20 0 20

Time (min) T ime (min) Time (min) Time (min)

Fig 3.15. Separation of pancreatin on RESOURCE Q, 1 mL in 3 min. Fig 3.16. Separation of snake venom on RESOURCE S, 1 mL at 1 mL/min (180 cm/h). Fig 3.17. Separation of proteins scaled up from a 2.2 mL column to a 390 mL column.

64
Intermediate purification Separations under extreme pH conditions
Figure 3.18 shows an example of SOURCE 30Q used for an intermediate purification step in a large-scale process. The high pH stability of SOURCE media makes them well-suited for applications
Recombinant P. aeruginosa exotoxin A, produced as a periplasmic protein in E. coli, was initially purified with requiring conditions of extreme pH such as purification of certain peptides and
STREAMLINE DEAE expanded bed adsorption, followed by hydrophobic interaction chromatography (HIC) on synthetic oligo-nucleotides, as shown in Figures 3.19 and 3.20.
Phenyl Sepharose 6 Fast Flow (high sub). The fraction of interest was then further purified on SOURCE 30Q before
a final HIC polishing step on SOURCE 15PHE to remove the final contaminants. Column: RESOURCE S, 1 mL
Sample: Partially purified bacitracin, 5 mg/mL
Sample volume: 100 µL
Columns: SOURCE 30Q, FineLINE 100 (375 mL) Start buffer: 5 mM potassium phosphate, pH 2.8, 30% acetonitrile
Sample: Partially purified recombinant P. aeruginosa exotoxin A, diluted 1:3 with water Elution buffer: 5 mM potassium phosphate, 400 mM KCl, pH 2.8, 30% acetonitrile
Sample: 1.8 g total protein (0.29 g exotoxin A) in 1.5 L Flow rate (flow velocity): 1 mL/min (180 cm/h)
Start buffer: 20 mM sodium phosphate, pH 7.4 Gradient: 0% to 100% elution buffer in 10 CV
Elution buffer: 20 mM sodium phosphate, 1 M NaCl, pH 7.4
Flow rate (flow velocity): 785 mL/min (600 cm/h)
Gradient: 0% to 50% elution buffer in 20 CV

0.50

0.40
A 280 nm

0.30

0.20

0.10 1 2 3

Pool Native PAGE results, Coomassie staining

0.00 Lane 1. Pool from step 2 on Phenyl Sepharose Fast Flow
(high sub) 0.0 10.0 T ime (min)
0 2 4 6 8 10 12
Lane 2. Pool from step 3 on SOURCE 30Q
Volume (l) Lane 3. Pool from step 4 on SOURCE 15PHE

Fig 3.18. Intermediate purification of recombinant Pseudomonas aeruginosa exotoxin A on SOURCE 30Q. Fig 3.19. Intermediate purification of the peptide bacitracin from Bacillus subtilis on RESOURCE Q, 1 mL.
65
Method optimization Batch-to-batch reproducibility
Sample: Crude oligonucleotide 20-mer (10 µg) Batch-to-batch reproducibility is particularly important for media used for scaling-up
Start buffer: 10 mM NaOH, pH 12 and large scale industrial applications which are under strict regulatory control. Figures
Elution buffer: 10 mM NaOH, 2 M NaCl, pH 12 3.21 and 3.22 demonstrate the high batch-to-batch reproducibility of SOURCE 15 and
Flow rate: 1 mL/min
SOURCE 30 media.
(A) (B) (C) A 280 nm
Columns: SOURCE 30S, 2.2 mL, four separate
Column: RESOURCE Q, 1 mL Column: RESOURCE Q, 1 mL Column: SOURCE 15Q 4.6/100 PE, 1.7 mL
batches
Gradient: 0% to 50% elution buffer in 30 CV Gradient: 20% to 35% elution buffer in 50 CV Gradient: 20% to 35% elution buffer in 50 CV
Sample: Chymotrypsinogen, cytochrome C,
and lysozyme
Sample load: 0.32 mg/mL bed volume
Conductivity
Start buffer: 20 mM sodium phosphate, pH 6.8
800 70 Elution buffer: 20 mM sodium phosphate, 500 mM
150 Conductivity
NaCl, pH 6.8
Flow rate (flow velocity): 2.2 mL/min (300 cm/h)
Conductivity 60 Gradient: 0% to 100% elution buffer in 20 CV

Conductivity
600
A 260 nm

A 260 nm

100

400 50

50 40
200
0.0 10.0 20.0 Time (min)

30 Fig 3.21. Selectivity tests on four production batches of SOURCE 30S.

0 0

0 10 20 30 0 20 40 0 20 40 60
Time (min) Time (min) Time (min) 100
Columns: SOURCE 15Q, 2.2 mL, four separate
0.060 batches
Fig 3.20. Manipulation of gradient slope and shape to maximize resolution. Initial purification of a 20 mer oligonucleotide was optimized on RESOURCE Q, Sample: Ovalbumin (3 mg/mL),
1 mL and transferred to SOURCE Q PE 4.6/100 to further increase resolution by increasing bed height to 10 cm. β-lactoglobulin (3 mg/mL)

Elution buffer (%)

A 280 nm
0.040
50 Sample volume: 200 µL
Start buffer: 20 mM bis-Tris-propane, pH 7.0
Elution buffer: 20 mM bis-Tris-propane, 350 mM
0.020
NaCl, pH 7.0
Flow rate (flow velocity): 2.2 mL/min (300 cm/h)
0
0 10 20
Gradient: 0% elution buffer (2 CV)
Ti me (min) 0% to 100% elution buffer (21 CV)

Fig 3.22. Selectivity tests on four production batches of SOURCE 15Q.

66
Performing a separation
Guidelines for selection of media, buffer, pH and ionic strength conditions, and method optimization are given in Chapter 2.
Use the instructions given here as a basis from which to optimize a separation.
Correct sample and buffer preparation is essential in order to achieve optimal separation and avoid any deterioration
in column performance. Samples must be fully dissolved and free from particles or other material likely to interfere
with the separation. Refer to Chapter 2 and Appendix 1 for recommendations and advice on sample preparation.
Filter buffers after all salts and additives have been included. Use high quality water and chemicals. Filter solutions
using filters of 0.45 µm or 0.22 µm for 30 µm particles and 0.22 µm filters for 15 µm particles. To avoid formation of air
bubbles in a packed column, ensure that column and buffers are at the same temperature when preparing for a run.
The pH of the start buffer should be at least 0.5 to 1.0 pH unit above the pI of the target substance when using an
anion exchanger (Q) and 0.5 to 1.0 pH unit below the pI of the target substance when using a cation exchanger (S).
See Appendix 2 for recommendations on volatile and nonvolatile buffer systems for anion and cation exchangers.
For samples with unknown charge properties, try the following:
anion exchange (Q)
start buffer: 20 mM Tris-HCl, pH 8.0
elution buffer: start buffer including 1 M NaCl, pH 8.0
cation exchange (S)
start buffer: 20 mM 2-(N-Morpholino)ethanesulfonic acid (MES), pH 6.0
elution buffer: start buffer including 1 M NaCl, pH 6.0
Users of ÄKTA systems with automatic buffer preparation functionality can select one of the buffer recipes
recommended for anion exchange chromatography at pH 8.0 or cation exchange chromatography at pH 6.0,
see ÄKTA Laboratory-scale Chromatography Systems: Instrument Management Handbook, 29010831.

67
First-time use or after long-term storage Separation by step elution

1. To remove ethanol, wash with 5 CV of distilled water at 2 mL/min (SOURCE 15 4.6/100 PE), 4 mL/min Flow rates: 2 mL/min (SOURCE 15 4.6/100 PE), 4 mL/min (RESOURCE 1 mL),
(RESOURCE 1 mL), 6 mL/min (RESOURCE 6 mL), or 200 cm/h for SOURCE packed in larger columns. This step 6 mL/min (RESOURCE 6 mL), or 200 cm/h for SOURCE packed in larger
ensures removal of ethanol and avoids the risk of precipitation if buffer salts were to come into contact with the columns.
ethanol. The step can be omitted if precipitation is not likely to be a problem. Collect fractions throughout the separation.
2. W
 ash with 5 CV of start buffer, at 2 mL/min (SOURCE 15 4.6/100 PE), 4 mL/min (RESOURCE 1 mL), 6 mL/min 1. Equilibrate column with 5 to 10 CV of start buffer or until the baseline,
(RESOURCE 6 mL), or 200 cm/h for SOURCE packed in larger columns. eluent pH, and conductivity are stable.
3. Wash with 5 CV of elution buffer, same flow as step 2. 2. A
 djust the sample to the chosen starting pH, and ionic strength and apply
4. Wash with 5 CV of start buffer, same flow as step 2. to the column.
3. W
 ash with 5 to 10 CV of start buffer or until the baseline, eluent pH, and
conductivity are stable, that is, when all unbound material has washed
Separation by gradient elution
through the column.
4. Elute the target protein with 5 CV of start buffer containing NaCl at chosen
Flow rates: 2 mL/min (SOURCE 15 4.6/100 PE), 4 mL/min (RESOURCE 1 mL), 6 mL/min (RESOURCE 6 mL), or ionic strength.
200 cm/h for SOURCE packed in larger columns.
5. I f necessary: repeat step 4 at higher ionic strengths until the target
Collect fractions throughout the separation. protein(s) has been eluted.
1. Equilibrate column with 5 to 10 CV of start buffer or until the baseline, eluent pH, and conductivity are stable. 6. W
 ash with 5 CV of a high salt solution (1 M NaCl in start buffer) to elute any
2. Adjust the sample to the chosen starting pH and ionic strength and apply to the column. remaining ionically bound material.
3. W
 ash with 5 to 10 CV of start buffer or until the baseline, eluent pH, and conductivity are stable, that is, when all 7. R
 e-equilibrate with 5 to 10 CV of start buffer or until eluent pH and
unbound material has washed through the column. conductivity reach the required values.
4. Begin elution using a gradient volume of 10 to 20 CV and an increasing ionic strength up to 0.5 M NaCl (50%B).
5. Wash with 5 CV of 1 M NaCl (100%B) to elute any remaining ionically bound material.
6. Re-equilibrate with 5 to 10 CV of start buffer or until eluent pH and conductivity reach the required values.

68
 Save time by using higher flow rates during the high salt wash and re-equilibration steps. Do not exceed the maximum To remove precipitated proteins, lipids, hydrophobically bound proteins, or lipoproteins,
recommended flow for the medium. refer to Appendix 1.

If ionic detergents have been used, wash the column with 5 CV of distilled water, followed by 2 CV of 2 M NaCl. Chemical stability
Re-equilibrate with at least 10 CV of start buffer until the UV baseline, eluent pH, and/or conductivity are stable.
Organic solvents such as ethanol can be used to remove nonionic detergents. When selecting an organic solvent, For daily use, SOURCE media are stable in all common, aqueous buffers pH 2 to 12,
check the chemical stability of the medium to determine a suitable concentration. denaturing agents (8 M urea, 6 M guanidine hydrochloride), 75% acetic acid, 1 M NaOH,
1 M HCl, 70% ethanol, 30% acetonitrile, and with additives such as nonionic
Check column performance regularly by determining column efficiency and peak symmetry. See Appendix 3. detergents.
Cleaning Avoid cationic detergents with SOURCE S. Avoid anionic detergents with
SOURCE Q. Avoid oxidizing agents.
Correct preparation of samples and buffers and application of a high salt wash (1 M NaCl) at the end of each separation
should keep most columns in good condition. However, reduced performance, a slow flow rate, increasing back pressure Storage
or complete blockage are all indications that the medium needs to be cleaned using more stringent procedures in order
to remove contaminants. For column storage, wash with 5 CV of distilled water followed by 5 CV of 20%
ethanol. Include 200 mM sodium acetate in the 20% ethanol solution for SOURCE S.
Reverse the direction of flow during column cleaning so that contaminants do not need to pass through the entire Degas the ethanol/water mixture thoroughly and apply at a low flow rate to avoid
length of the column. The number of column volumes and time required for each cleaning step varies according to overpressuring the column. Store at room temperature or, for long periods, store at
the degree of contamination. If the cleaning procedure to remove common contaminants does not restore column 4°C to 8°C. Ensure that the column is sealed well to avoid drying out. Whenever possible,
performance, change the top filter (when possible) before trying alternative cleaning methods. Care should be use the storage and shipping device if supplied by the manufacturer. Store unused
taken when changing a filter as this can affect the column packing and interfere with performance. media at 4°C to 30°C in 20% ethanol. Do not freeze.
Removing common contaminants

Flow rates: 0.2 mL/min (SOURCE 15 4.6/100 PE), 1 mL/min (RESOURCE 1 mL), 6 mL/min (RESOURCE 6 mL), or 40
cm/h with a contact time of 1 to 2 h for SOURCE packed in larger columns
1. Wash with at least 2 CV of 2 M NaCl.
2. Wash with at least 4 CV of 1 M NaOH.
3. Wash with at least 2 CV of 2 M NaCl.
4. Rinse with at least 2 CV of distilled water until the UV-baseline and the eluent pH are stable.
5. W
 ash with at least 4 CV of start buffer or storage buffer until eluent pH and conductivity have reached the
required values.

69
Sepharose High Performance: purification with high resolution Table 3.8. Characteristics of Sepharose High Performance media

Use Sepharose High Performance for intermediate purification steps that require high capacity and high resolution Product Functional group pH stability1 Mean particle size (µm)


(flow velocities up to 150 cm/h). Q Sepharose -CH2-N+-(CH3)3 Long term: 2 to 12 34

High Performance Short term: 1 to 14
Run Sepharose High Performance columns on ÄKTA chromatography systems. Appendix 4 provides guidance on
selecting the right ÄKTA system. SP Sepharose -CH2-SO3- Long term: 4 to 13 34
High Performance Short term: 3 to 14
Sepharose High Performance media are based on a matrix of 34 µm particles made from 6% agarose and highly 1
L ong-term pH stability refers to the pH interval where the medium is stable over a long period of time
cross-linked for chemical and physical stability. The small particle size ensures fast binding and dissociation even at high without adverse side effects on chromatography performance. Short-term pH stability refers to the pH
sample loads and flow rates which, in combination with high selectivity, give high-resolution separations. Particle size interval for regeneration, cleaning-in-place, and sanitization procedures. All ranges are estimates based on
and bed volumes remain stable, despite changes in ionic strength or pH, to ensure fast separations at high flow rates. experience and knowledge gained at Cytiva.
The strong ion exchange groups (Q and SP) maintain their charge over a broad pH range, allowing selection of the most
suitable pH for each application.

Media characteristics
Composition: sulfopropyl (SP) or quaternary amino (Q) groups coupled to highly cross-linked 6% agarose via chemically
stable ether bonds, see Table 3.8.

70
Purification options
Sepharose Q and SP High Performance are available in chromatography media packs, in convenient prepacked HiTrap
columns for small-scale work, HiPrep columns for purification scale-up, and HiScreen columns (Fig 3.23). The purification
options for these prepacked formats are found in Table 3.9.

Table 3.9. Purification options for Sepharose High Performance media and prepacked columns
Maximum
operating back
pressure3
Binding capacity per column Recommended Working (MPa/psi)
Product or per mL medium working flow1 Maximum flow1 pH range2 1 MPa = 10 bar
Strong anion exchangers
Q Sepharose High Performance 70 mg/mL (HSA, Mr 68 000) 30 to 150 cm/h 150 cm/h 2 to 12 0.5/72
HiTrap Q HP, 1 mL 50 mg (HSA, Mr 68 000) up to 1 mL/min 4 mL/min 2 to 12 0.3/43
HiTrap Q HP, 5 mL 250 mg (HSA, Mr 68 000) up to 5 mL/min 20 mL/min 2 to 12 0.3/43
HiPrep Q HP, 20 mL 1000 mg (BSA, Mr 68 000) up to 5 mL/min 20 mL/min 2 to 12
HiScreen Q HP, 4.7 mL 70 mg/mL (BSA, Mr 68 000) 0.6 mL/min 1.2 mL/min 2 to 12 0.3/43
Strong cation exchangers
SP Sepharose High Performance 55 mg/mL (ribonuclease, 30 to 150 cm/h 150 cm/h 4 to 13 0.5/72
Mr 13 700)
HiTrap SP HP, 1 mL 55 mg (ribonuclease, up to 1 mL/min 4 mL/min 4 to 13 0.3/43
Mr 13 700)
HiTrap SP HP, 5 mL 275 mg (ribonuclease, up to 5 mL/min 20 mL/min 4 to 13 0.3/43
Mr 13 700)
Fig 3.23. Q and SP Sepharose High Performance media are available prepacked in HiTrap, HiPrep, and
HiPrep SP HP 16/10, 20 mL 1100 mg (ribonuclease, up to 5 mL/min 20 mL/min 4 to 13 HiScreen columns or in media packs.
Mr 13 700)
HiScreen S HP, 4.7 mL 55 mg/mL (ribonuclease, 0.6 mL/min 1.2 mL/min 4 to 12 0.3/43
Mr 13 500)
1
See Appendix 5 to convert flow velocity (cm/h) to volumetric flow rate (mL/min) and vice versa.
2
Working pH range refers to the pH interval where the medium can be operated as intended without adverse long-term effects.
3
Maximum operating back pressure refers to the pressure above which the medium begins to compress.

71
  Use prepacked HiTrap columns (1 mL or 5 mL) for media selection, method Purification examples
scouting, group separations, small-scale purification, sample concentration, Figure 3.24 shows scaling up from HiTrap SP HP to HiPrep SP HP 16/10. A number of factors were kept constant, such as
or clean-up. Connect up to three HiTrap columns in series to scale-up. sample load/mL medium, flow rates and number of CV in the gradient. The separation was maintained through a 20-fold
 Use prepacked HiPrep columns (20 mL) for method development, group scale-up. The scale-up resulted in similar separation of the sample, which comprised four standard proteins.
separations, larger scale purification, or sample concentration.
 Use prepacked HiScreen columns (4.7 mL, bed height 10 cm) for method HiTrap SP HP, 1 mL HiPrep SP HP 16/10, 20 mL
development and screening before scaling up with bed height maintained. Sample: Concanavalin A, ribonuclease A, Sample: Concanavalin A, ribonuclease A,
α‑chymotrypsinogen A, lysozyme, α‑chymotrypsinogen A, lysozyme,
For column packing in Tricorn and XK columns, see Table 3.10. 4 mg protein/mL (3:3:1:1) in start buffer 4 mg protein/mL (3:3:1:1) in start buffer
Sample load: 1 mg protein/mL medium Sample load: 1 mg protein/mL medium
Sample volume: 0.25 mL, 25% of CV Sample volume: 5.0 mL, 25% of CV
Table 3.10. Packing volumes and bed heights for Sepharose Q and SP High Performance media packed in
Flow rate (flow velocity): 0.5 mL/min (75 cm/h) Flow rate (flow velocity): 2.5 mL/min (75 cm/h)
Tricorn and XK columns
Start buffer: 50 mM MES, pH 6.0 Start buffer: 50 mM MES, pH 6.0
Column Volume (mL) Bed height (cm) Elution buffer: 50 mM MES, 1 M NaCl, pH 6.0 Elution buffer: 50 mM MES, 1 M NaCl, pH 6.0
Gradient: 0% to 43% elution buffer over 10 mL (10 CV) Gradient: 0% to 43% elution buffer over 200 mL (10 CV)
Tricorn 10/100 up to 8 up to 10
Tricorn 10/150 up to 12 up to 15
Tricorn 10/200 up to 16 up to 20
XK 16/20 up to 30 up to 15 0.16 0.16
XK 26/20 up to 80 up to 15
0.14 0.14 Conductivity Conductivity
0.14 0.14 Conductivity Conductivity
XK 26/40 up to 196 mL > 15 cm
0.12 0.12 0.12
A280 0.12 A280 A280 A280
0.10 0.10 0.10
0.10

A280nm

A280nm
0.08

A280nm

A280nm
0.08 0.08 0.08

0.06 0.06 0.06 0.06

0.04 0.04 0.04 0.04

0.02 0.02 0.02 0.02

0 0 0 0
2 4 6 8 10 2 12 4 14 6 16 8 10 12 14 1640 80 120 160 20040 24080 280120 320
160 200 240 280 320
Volume (mL) Volume (mL) Volume (mL) Volume (mL)

Fig. 3.24. Scaling up the separation of four standard proteins from HiTrap SP to HiPrep SP HP 16/10.

72
Group separations Sample concentration
Figure 3.25 shows a group separation of human serum proteins on HiTrap Q HP using Concentrating a sample prior to SEC minimizes sample volume and facilitates a rapid, high-resolution size separation.
a one-step elution that had been optimized to ensure that IgG flowed through the HiTrap columns offer a convenient, ready-to-use solution for sample concentration. Table 3.11 gives examples of the
column leaving other serum components to be eluted separately. high concentration factors achieved when concentrating proteins from very dilute starting material using HiTrap columns
prepacked with Sepharose HP medium. Similar results can be achieved with HiTrap columns prepacked with Sepharose
Column: HiTrap Q HP 1 mL
Mr
Fast Flow or Sepharose XL media.
Sample: Human serum, filtered (0.45 µm filter)
and buffer exchanged to start buffer 97 000
on a PD-10 Desalting column 66 000
Table 3.11. Sample concentration using 1 mL HiTrap ion exchange columns
Sample volume: 1.0 mL 45 000
Flow rate (flow velocity): 0.5 mL/min (75 cm/h)
30 000 Sample Eluted
Start buffer: 75 mM Tris-HCl, pH 8.0
Elution buffer: 75 mM Tris-HCl, 1.0 M NaCl, pH 8.0 20 100 concentration Sample concentration Volume Concentration
14 400 Column Sample (µg/mL) volume (mL) (µg/mL) eluted (mL) factor (volume) Yield (%)
100% elution buffer HiTrap Q HP, 1 mL Human IgG 23 450 3180 3.0 150 92
1.0 1 2 3 4
10 100 4700 2.0 50 93
Lane 1. Low Molecular Weight (LMW)
Calibration Kit, Cytiva 1010 10 3370 3.0 3 100
0.8
Lane 2. Start material, buffer exchanged
pool 1
pool 2 HiTrap SP HP, 5 mL Lysozyme 333 150 3170 16.0 9 100
human serum, diluted 1:75
Lane 3. Flowthrough, pool 1, diluted 1:10 33 1500 3720 13.2 114 98
A 280 nm

0.6
Lane 4. Desorbed material, pool 2, diluted 1:25

0.4

0.2

0
0 5 10 15 20 25 30 35 40

Volume (mL)

Fig 3.25. Separation of IgG from human serum proteins on HiTrap Q HP 1 mL, using one-step elution. Analysis
by SDS-PAGE (silver staining).

73
Performing a separation First-time use or after long-term storage
Guidelines for selection of media, buffer, pH, and ionic strength conditions and method optimization are given in Chapter 2.
Use the instructions given here as a basis from which to optimize a separation. 1. To remove ethanol, wash with 1 CV of distilled water at 1 mL/min (HiTrap 1 mL),
Correct sample and buffer preparation is essential in order to achieve optimal separation and avoid any deterioration 5 mL/min (HiTrap 5 mL), 0.6 mL/min (HiScreen 4.7 mL), 0.8 mL/min (HiPrep
in column performance. Samples must be fully dissolved and free from particles or other material likely to interfere 20 mL), or at 25 cm/h for Sepharose High Performance packed in larger
with the separation. Refer to Chapter 2 and Appendix 1 for recommendations and advice on sample preparation. columns. This step ensures removal of ethanol and avoids the risk of
precipitation if buffer salts were to come into contact with the ethanol.
Filter buffers after all salts and additives have been included. Use high-quality water and chemicals. Filter solutions

The step can be omitted if precipitation is not likely to be a problem.
through 0.45 µm or 0.22 µm filters. To avoid formation of air bubbles in a packed column, ensure that column and
buffers are at the same temperature when preparing for a run. 2. Wash with 5 CV of start buffer at 1 mL/min (HiTrap 1 mL), 5 mL/min (HiTrap
5 mL) 0.6 mL/min (HiScreen 4.7 mL), 3 mL/min (HiPrep 20 mL) or at 50 cm/h
The pH of the start buffer should be at least 0.5 to 1.0 pH unit above the pI of the target substance when using an

for Sepharose High Performance packed in larger columns.
anion exchanger (Q) and 0.5 to 1.0 pH unit below the pI of the target substance when using a cation exchanger (S).
See Appendix 2 for recommendations on volatile and nonvolatile buffer systems for anion and cation exchangers. 3. Wash with 5 CV of elution buffer, same flow as step 2.
For samples with unknown charge properties, try the following: 4. Wash with 5 CV of start buffer, same flow as step 2.
anion exchange (Q) 5. Run a blank elution before applying sample.
start buffer: 20 mM Tris-HCl, pH 8.0
elution buffer: start buffer including 1 M NaCl, pH 8.0
cation exchange (S)
start buffer: 20 mM 2-(N-Morpholino)ethanesulfonic acid (MES), pH 6.0
elution buffer: start buffer including 1 M NaCl, pH 6.0
Users of ÄKTA systems with automatic buffer preparation functionality can select one of the buffer recipes
recommended for anion exchange chromatography at pH 8.0 or cation exchange chromatography at pH 6.0,
see ÄKTA Laboratory-scale Chromatography Systems: Instrument Management Handbook, 29010831.

74
Separation by gradient elution
Save time by using higher flow rates during the high salt wash and re-equilibration
Flow rates: 1 mL/min (HiTrap 1 mL), 5 mL/min (HiTrap 5 mL), 0.6 mL/min (HiScreen 4.7 mL), 3 mL/min (HiPrep 20 mL), steps. Do not exceed the maximum recommended flow for the medium.
or at 50 to 100 cm/h for Sepharose High Performance packed in larger columns. Collect fractions throughout the If ionic detergents have been used, wash the column with 5 CV of distilled
separation. water, followed by 2 CV of 2 M NaCl. Re-equilibrate with at least 10 CV of start
1. Equilibrate column with 5 to 10 CV of start buffer or until the baseline, eluent pH, and conductivity are stable. buffer until the UV baseline, eluent pH and/or conductivity are stable. Organic
solvents such as ethanol can be used to remove nonionic detergents. When
2. Adjust the sample to the chosen starting pH and ionic strength and apply to the column. selecting an organic solvent, check the chemical stability of the medium to
3. W
 ash with 5 to 10 CV of start buffer or until the baseline, eluent pH, and conductivity are stable, that is, when determine a suitable concentration.
all unbound material has washed through the column. Check column performance regularly by determining column efficiency and


4. Begin elution using a gradient volume of 10 to 20 CV and an increasing ionic strength up to 0.5 M NaCl (50%B). peak symmetry. See Appendix 3. Note that this does not apply to HiTrap
columns.
5. Wash with 5 CV of 1 M NaCl (100%B) to elute any remaining ionically bound material.
6. Re-equilibrate with 5 to 10 CV of start buffer or until eluent pH and conductivity reach the required values. Cleaning
Correct preparation of samples and buffers and application of a high salt wash (1 M NaCl)
at the end of each separation should keep most columns in good condition. However,
Separation by step elution reduced performance, a slow flow rate, increasing back pressure or complete blockage
are all indicators that the medium needs to be cleaned using more stringent
Flow rates: 1 mL/min (HiTrap 1 mL), 5 mL/min (HiPrep 20 mL), 0.6 mL/min (HiScreen 4.7 mL), or at 50 to 100 cm/h for procedures to remove contaminants.
Sepharose High Performance packed in larger columns. Reverse the direction of flow during column cleaning so that contaminants
Collect fractions throughout the separation. do not need to pass through the entire length of the column. The number of
column volumes and time required for each cleaning step varies according to
1. Equilibrate column with 5 to 10 CV of start buffer or until the baseline, eluent pH, and conductivity are stable. the degree of contamination. If the cleaning procedure to remove common
2. Adjust the sample to the chosen starting pH and ionic strength and apply to the column. contaminants does not restore column performance, change the top filter
(when possible) before trying alternative cleaning methods. Care should be
3. W
 ash with 5 to 10 CV of start buffer or until the baseline, eluent pH and conductivity are stable, that is, when
taken when changing a filter as this can affect column packing and interfere
all unbound material has washed through the column.
with performance.
4. Elute the target protein with 5 CV of start buffer containing NaCl at chosen ionic strength.
5. If necessary: repeat step 4 at higher ionic strengths until the target protein(s) has been eluted.
6. Wash with 5 CV of a high salt solution (1 M NaCl in start buffer) to elute any remaining ionically bound material.
7. Re-equilibrate with 5 to 10 CV of start buffer or until eluent pH and conductivity reach the required values.

75
Removing common contaminants Storage
For column storage, wash with 2 CV of distilled water followed by 2 CV of 20%
Flow rates: 1 mL/min (HiTrap 1 mL), 5 mL/min (HiTrap 5 mL), 0.6 mL/min (HiScreen 4.7 mL), 3 mL/min (HiPrep 20 mL), ethanol. Include 0.2 M sodium acetate in the 20% ethanol solution for columns
or at 40 cm/h with a contact time of 1 to 2 h for Sepharose High Performance packed in larger columns. packed with SP Sepharose High Performance. Degas the ethanol/water mixture
thoroughly and apply at a low flow rate to avoid overpressuring the column. Store at
1. Wash with at least 2 CV of 2 M NaCl at 0.6 mL/min (HiScreen 4.7 mL) 1 mL/min (HiTrap 1 mL), 5 mL/min (HiTrap room temperature or, for long periods, store at 4°C to 8°C. Ensure that the column
5 mL), 3 mL/min (HiPrep 20 mL), or at 40 cm/h with a contact time of 1 to 2 h for Sepharose High Performance is sealed well to avoid drying out. Whenever possible, use the storage and shipping
packed in larger columns. device if supplied by the manufacturer. Store unused media at 4°C to 30°C in 20%
2. Wash with at least 4 CV of 1 M NaOH. ethanol. Do not freeze.
3. Wash with at least 2 CV of 2 M NaCl. To avoid formation of air bubbles in a packed column, ensure that column and
buffers are at the same temperature when preparing for a run.
4. Rinse with at least 2 CV of distilled water until the UV-baseline and the eluent pH are stable.
5. W
 ash with at least 4 CV of start buffer or storage buffer until eluent pH and conductivity have reached the
required values.

To remove precipitated proteins, lipids, hydrophobically bound proteins, or lipoproteins, refer to Appendix 1.

Chemical stability
For daily use, Sepharose High Performance media are stable in all common, aqueous buffers, 1 M NaOH, denaturing
agents (8 M urea, 6 M guanidine hydrochloride), 70% ethanol, 1 M acetic acid, 30% acetonitrile, and with additives such
as nonionic detergents.
Sepharose High Performance can be used with organic solvents such as dimethylsulfoxide, dimethylformamide,
tetrahydrofuran, acetone, chloroform, dichloromethane, dichloroethane, and dichloroethane/pyridine (50:50) as well as
polar solvents and aqueous/organic solutions. The water in the medium can be exchanged by the alternative solvent with
very little effect on the pore size of the matrix.
Avoid cationic detergents with SP Sepharose High Performance. Avoid anionic detergents with Q Sepharose High
Performance. Avoid oxidizing agents.

76
Sepharose Fast Flow: purification with good resolution and easy (A)
HiTrap AN X FF
II

III
50
Columns: (A) HiTrap ANX FF (high sub), 1 mL
(B) HiTrap Q XL, 1 mL
scale-up
300 (high sub), 1 mL
I (C) HiTrap Q FF, 1 mL
(D) HiTrap DEAE FF, 1 mL
Use Sepharose Fast Flow for capture or intermediate purification steps that require good resolution (flow velocity Sample: 0.4 mg conalbumin (pI = 6.3),
 40
up to 300 cm/h). 250 0.8 mg a-lactoglobulin (pI = 5.8),
II
1.2 mg soya bean trypsin inhibitor
 Use a weak ion exchanger such as DEAE, CM, or ANX Sepharose Fast Flow, if a strong ion exchanger (substituted (B) III (pI = 4.5) dissolved in 2 mL start buffer
with Q or SP) does not give the required selectivity. 200
HiTrap Q XL, 1 mL
I
Sample volume: 2 mL

Conductivity (mS/cm)
30 Start buffer: 20 mM Tris-HCl pH 7.4
 Run Sepharose Fast Flow columns on ÄKTA chromatography systems, HPLC systems, or systems using peristaltic Elution buffer: 20 mM Tris-HCl, 500 mM NaCl pH 7.4

A 280 nm
II
pumps. Appendix 4 provides guidance on selecting the right ÄKTA system. Flow rate: 1 mL/min (150 cm/h)
150
III Gradient: 0% elution buffer (25 CV),
(C)
Sepharose Fast Flow media are based on a matrix of 90 µm particles made from 6% agarose and highly cross-linked for HiTrap Q FF, 1 mL I 20
0% to 80% elution buffer (40 CV)
chemical and physical stability. ANX Sepharose 4 Fast Flow (high sub) is based on 4% agarose to form a medium that Wash: 5 mL start buffer
maintains a high binding capacity when separating large molecules such as thyroglobulin (Mr = 650 000), particularly 100 Elution:  40 mL, linear gradient,
0% to 80% elution buffer
suitable for large-scale production when total binding capacity becomes economically significant. II
III
(D)
Sepharose Fast Flow matrices are substituted with a range of ion exchange groups (Q, DEAE, ANX, SP, and CM) giving the 50
HiTrap DEAE FF, 1 mL

I
10

opportunity to test and use different selectivities (see Chapter 1 for an explanation of strong and weak ion exchangers).
Ion exchangers containing strong ion exchange groups (Q and SP) maintain their charge over a broad pH range, allowing
selection of the most suitable pH for each application. 0
0

Ion exchangers containing weak ion exchange groups (DEAE, CM, and ANX) offer alternative selectivities, but over a 0 10 20 30 40 50
Volume (mL)
narrower pH working range. Figure 3.26 illustrates how the selectivity of Sepharose Fast Flow media changes according
to the anion exchange group. Fig 3.26. Separation of conalbumin (I), a-lactalbumin (II) and soya bean trypsin inhibitor (III) on a range of anion
Particle size and bed volumes remain stable, despite changes in ionic strength or pH, to ensure fast separations at high exchange HiTrap columns demonstrates the difference in selectivity according to the anion exchange group.
flow rates with good resolution. Methods can be easily scaled up from columns such as HiTrap Q FF (1 mL, prepacked
with Q Sepharose Fast Flow) through to large-scale columns such as FineLINE. The performance of Sepharose Fast Flow
is well documented and there are many examples of the smooth transfer from the laboratory to pilot scale and on to
production.

77
Media characteristics
Composition:
S
 ulfopropyl (SP), carboxymethyl (CM), quaternary amino (Q) or diethylaminoethyl (DEAE) groups coupled to highly
cross-linked 6% agarose via chemically stable ether bonds
Diethylaminopropyl (ANX) group coupled to highly cross-linked 4% agarose via chemically stable ether bonds
The characteristics of Sepharose Fast Flow media are shown in Table 3.12.

Table 3.12. Characteristics of Sepharose Fast Flow media

Mean particle
Product Functional group pH stability1 size (µm)
Q Sepharose Fast Flow -CH2-N+-(CH3)3 Long term: 2 to 12 90
Short term: 2 to 14
SP Sepharose Fast Flow -CH2-CH2-CH2-SO3- Long term: 4 to 13 90
Short term: 3 to 14
DEAE Sepharose Fast Flow -CH2-CH2-N+-(CH2-CH3)2 Long term: 2 to 12 90
Short term: 2 to 14
ANX Sepharose 4 Fast Flow -CH2-CHOH-CH2-N+-(CH2-CH3)2 Long term: 3 to 13 90
Short term: 2 to 14
CM Sepharose Fast Flow -CH2-COO- Long term: 4 to 13 90
Short term: 2 to 14
1
L ong-term pH stability refers to the pH interval where the medium is stable over a long period of time without adverse side effects on the chromatography
performance. Short-term, pH stability refers to the pH interval for regeneration, cleaning-in-place, and sanitization procedures. All ranges are estimates
based on experience and knowledge gained at Cytiva. Fig 3.27. The range of Sepharose Fast Flow is available in media packs and prepacked HiTrap, HiPrep,
and HiScreen columns. The media are also available in high-throughput process development (HTPD) format,
see Chapter 5, Large-scale purification.
Purification options
Sepharose Fast Flow media, with a range of selectivities, are available prepacked in HiTrap, HiScreen, and HiPrep columns
and in media packs (Fig 3.27). Purification options for the media and prepacked columns are shown in Table 3.13.

78
Table 3.13. Purification options for Sepharose Fast Flow media and prepacked columns

Maximum operating back pressure3

Product Binding capacity per column or per mL medium Recommended working flow1 Maximum flow1 Working pH range2 (MPa/psi) 1 MPa = 10 bar

Strong anion exchangers

Q Sepharose Fast Flow 3 mg/mL (thyroglobulin, Mr 669 000) 50 to 400 cm/h 750 cm/h 2 to 12 0.3/43
120 mg/mL (HSA, Mr 68 000)
110 mg/mL, (a–lactalbumin, Mr 14 300)
HiTrap Q FF, 1 mL 3 mg (thyroglobulin, Mr 669 000) up to 1 mL/min 4 mL/min 2 to 12 0.3/43
120 mg (HSA, Mr 68 000)
110 mg (a–lactalbumin, Mr 14 300)
HiTrap Q FF, 5 mL 15 mg (thyroglobulin, Mr 669 000) up to 5 mL/min 20 mL/min 2 to 12 0.3/43
600 mg (HSA, Mr 68 000)
550 mg (a–lactalbumin, Mr 14 300)
HiScreen Q FF, 4.7 mL 120 mg/mL (HSA, Mr 68 000) 2.3 mL/min 3.5 mL/min 2 to 12 0.15/22
HiPrep Q FF 16/10, 20 mL 60 mg (thyroglobulin, Mr 669 000) 2 to 10 mL/min 10 mL/min 2 to 12 0.15/22
2400 mg (HSA, Mr 68 000)
2200 mg (a–lactalbumin, Mr 14 300)

Weak anion exchangers

DEAE Sepharose 100 mg/mL (a–lactalbumin, Mr 14 300) 50 to 400 cm/h 750 cm/h 2 to 9 0.3/43
Fast Flow 110 mg/mL (HSA, Mr 68 000)
ANX Sepharose 4 Fast Flow 43 mg/mL (BSA, Mr 67 000) 50 to 300 cm/h 400 cm/h 3 to 10 0.1/14
(high sub) 5 mg/mL (thyroglobulin, Mr 669 000)
HiTrap DEAE FF, 1 mL 100 mg (a–lactalbumin, Mr14 300) up to 1 mL/min 4 mL/min 2 to 9 0.3/43
110 mg (HSA, Mr 68 000)
HiTrap DEAE FF, 5 mL 500 mg (a–lactalbumin, Mr 14 300) up to 5 mL/min 20 mL/min 2 to 9 0.3/43
550 mg (HSA, Mr 68 000)
HiScreen DEAE FF 110 mg (HSA, Mr 68 000) 2.3 mL/min 3.5 mL/min 2 to 9 0.15/22
HiPrep DEAE FF 16/10, 20 mL 2000 mg (a–lactalbumin, Mr 14 300) 2 to 10 mL/min 10 mL/min 2 to 9 0.15/22
2200 mg (HSA, Mr 68 000)
HiTrap ANX FF 43 mg (BSA, Mr 67 000) up to 1 mL/min 4 mL/min 3 to 10 0.3/43
(high sub), 1 mL 5 mg (thyroglobulin, Mr 669 000)
HiTrap ANX FF 215 mg (BSA, Mr 67 000) up to 5 mL/min 20 mL/min 3 to 10 0.3/43
(high sub), 5 mL 25 mg (thyroglobulin, Mr 669 000)
79
Continues on following page
Table 3.13 cont.

Maximum operating back

Product Binding capacity per column or per mL medium Recommended working flow1 Maximum flow1 Working pH range2 pressure3 (MPa/psi) 1 MPa = 10 bar

Strong cation exchangers

SP Sepharose Fast Flow 50 mg/mL (bovine COHb, Mr 69 000) 50 to 400 cm/h 750 cm/h 4 to 13 0.3/43
50 mg/mL (human IgG, Mr 160 000)
70 mg/mL (ribonuclease A, Mr 13 700)
HiTrap SP FF, 1 mL 50 mg (bovine COHb, Mr 69 000) up to 1 mL/min 4 mL/min 4 to 13 0.3/43
50 mg (human IgG, Mr 160 000)
70 mg (ribonuclease A, Mr 13 700)
HiTrap SP FF, 5 mL 250 mg (bovine COHb, Mr 69 000) up to 5 mL/min 20 mL/min 4 to 13 0.3/43
250 mg (human IgG, Mr 160 000)
350 mg (ribonuclease A, Mr 13 700)
HiScreen SP FF, 4.7 mL 110 mg/mL (HSA, Mr 68 000) 2.3 mL/min 3.5 mL/min 4 to 13 0.15/22
HiPrep SP FF 16/10, 20 mL 1000 mg (bovine COHb, Mr 69 000) 2 to 10 mL/min 10 mL/min 4 to 13 0.15/22
1000 mg (human IgG, Mr 160 000)
1400 mg (ribonuclease A, Mr 13 700)

Weak cation exchangers

CM Sepharose Fast Flow 50 mg/mL medium (ribonuclease A, Mr 13 700) 50 to 400 cm/h 750 cm/h 6 to 10 0.3/43
HiTrap CM FF, 1 mL 50 mg (ribonuclease A, Mr 13 700) up to 1 mL/min 4 mL/min 6 to 10 0.3/43
HiTrap CM FF, 5 mL 250 mg (ribonuclease A, Mr 13 700) up to 5 mL/min 20 mL/min 6 to 10 0.3/43
HiPrep CM FF 16/10, 20 mL 1000 mg (ribonuclease A, Mr 13 700) 2 to 10 mL/min 10 mL/min 6 to 10 0.15/22
1
See Appendix 5 to convert flow velocity (cm/h) to volumetric flow rate (mL/min) and vice versa.
2
Working pH range refers to the pH interval where the medium can be operated as intended without adverse long-term effects.
3
Maximum operating back pressure refers to the pressure above which the medium begins to compress.

80
  Use prepacked HiTrap columns (1 mL or 5 mL) for media selection, method
scouting, group separations, small-scale purification, sample concentration,
Purification examples
or clean-up. Connect up to three HiTrap columns in series to scale-up. Media scouting
 Use prepacked HiPrep columns (20 mL) for method development, group Using 1 mL HiTrap columns the most suitable matrix and charged group for a separation can be quickly and easily selected
separations, larger scale purification, sample concentration or clean-up. Connect before optimization and scale-up. In Figure 3.28, a comparison of elution profiles for the same sample separated under
several HiPrep columns in series to increase binding capacity. identical conditions on three different Sepharose media illustrates the differences in selectivity and resolution that can
result from changing the charge group and the particle size. The most suitable medium can be selected and conditions
Use prepacked HiScreen columns (4.7 mL, bed height 10 cm) for method

optimized according to the requirements for the separation, for example to isolate a single, well-resolved peak or to
development and screening before scaling up with bed height maintained.
maximize resolution between several peaks of interest.
For column packing in Tricorn and XK columns, see Table 3.14. Select a production
Begin by scouting on the strong ion exchangers (Q, S, or SP) in order to find the greatest differences in charge
column such as BPG or Chromaflow for larger volumes. 

between the molecules of interest.

Table 3.14. Packing volumes and bed heights for Sepharose Fast Flow media packed in Tricorn and Columns: (A) HiTrap SP XL, 1 mL
I
XK columns (B) HiTrap SP FF, 1 mL
(C) HiTrap CM FF, 1 mL
Column Volume (mL) Bed height (cm) (A)
III
Sample: 3 mg ribonuclease A (pI = 9.3),
HiT rap SP XL, 1 mL
Tricorn 10/100 up to 8 up to 10 0.8 mg cytochrome C (pI = 10.3),
0.8 mg lysozyme (pI > 11.0)
Tricorn 10/150 up to 12 up to 15 II
40.0
Sample volume: 2 mL in start buffer
150
Tricorn 10/200 up to 16 up to 20 Start buffer: 20 mM sodium phosphate, pH 6.8
Elution buffer: 20 mM sodium phosphate, 500 mM NaCl pH 6.8
XK 16/20 up to 30 up to 15 Flow rate (flow velocity): 1 mL/min (150 cm/h)

Conductivity (mS/cm)
XK 26/20 up to 80 up to 15 30.0
Gradient: 0% elution buffer (25 CV),
(B) I
0% to 100% elution buffer (40 CV)
XK 26/40 up to 196 > 15 100
HiTrap SP FF, 1 mL

A 280 nm
II+III
XK 50/20 up to 274 up to 14
XK 50/30 up to 559 up to 28.5
20.0

(C) I
50 III
HiTrap CM FF, 1 mL
II
,

10.0

0 20 40 60 Fig 3.28. Media scouting: separation of ribonuclease A (I), cytochrome C (II), and lysozyme (III)
Volume (mL) on HiTrap CM FF 1 mL, HiTrap SP FF 1 mL, and HiTrap SP XL 1 mL.
81
Capture
An example of capture using Sepharose DEAE Fast Flow medium prepacked in HiPrep DEAE FF 16/10 is shown in Figure 3.29.

Column: HiPrep DEAE FF 16/10, 20 mL

Sample:  200 mL clarified E. coli supernatant, diluted 1:2 with water, pH 6.6,
conductivity 2.6 mS/cm
Start buffer: 25 mM Tris-HCl, 10% glycerol, 1 mM EDTA, 2 mM DTT, pH 7.4
Elution buffer: 1 M NaCl, 25 mM Tris-HCl, 10% glycerol, 1 mM EDTA, 2 mM DTT, pH 7.4
Flow rate (flow velocity): 5 mL/min (150 cm/h)
Gradient: 0% elution buffer (6 CV)
0% to 50% elution buffer (20 CV)
50% elution buffer (1 CV)
100% elution buffer (2 CV)

2.0 4.0
The phosphatase activity is
represented by the green bars.
3.0
A 280 nm

A 405 nm
2.0

1.0

0
300 500 700 900
Volume (mL)

Fig 3.29. A HiPrep DEAE FF 16/10 column is used as the capture step to concentrate rPhosphatase and remove most of the contaminants.

82
 3 3

A 280 nm
A 280 nm
10 10
1 1

5 5

Scaling-up 0 0
Figure 3.30 shows the ease with which separations can be scaled up on columns prepacked with Sepharose Fast Flow.
Beginning with a 1 mL HiTrap column the reproducibility of the separation has been maintained through a0 20-fold
0 5 scale-up.
5 10 10 15 15 20 20 25 25 30 30
Volume
Volume (mL)(mL)
(A) (B) (C)
Sample: 1. Conalbumin, 2 mg/mL 2 2 2 2 2
2. a-lactalbumin, 4 mg/mL HiTrap Q FF, 1 mL HiTrap Q FF, 5 mL HiPrep
HiPrep Q FFQ16/10,
FF 16/10, 20 mL
20 mL
25 25HiTrap Q FF, 5 mL 25 25
3. Soy trypsin inhibitor,
15
6 mg/mL
Sample volume: 1 CV 20 20 20 20
3
(A) 1 mL, (B) 5 mL, (C) 20 mL

A 280 nm

A 280 nm
3 3

A 280 nm

A 280 nm
A 280 nm
Start buffer: 50 mM Tris-HCl, pH 7.3 10 15 15 15 15 3 3
Elution buffer: 50 mM Tris-HCl, 500 mM 1
NaCl, pH 7.3 1 1 1 1
10 10 10 10
Flow velocity (flow rates): 150 cm/h (1 mL/min using 5
HiTrap 1 mL, 5 mL/min using
HiTrap 5 mL and HiPrep 50 50 5 5
16/10 columns) 0
Gradient: 0% to 100% elution buffer in 0 0 0 0
20 CV (A) 20 mL,
0 5 10 15 20 25 30 0 0 20 20 40 40 60 60 80 80 100100 120120 140140 0 0 100100 200200 300300 400400 500500
(B) 100 mL, (C) 400 mL
Volume (mL) Volume
Volume (mL)(mL) Volume
Volume (mL)(mL)

Fig 3.30. Five-fold and 20-fold scale-up using prepacked Q Sepharose Fast Flow prepacked columns. 2
2
HiTrap Q FF, 5 mL HiPrep Q FF 16/10, 20 mL
25 25

Sample concentration 20 20
It can be an advantage to concentrate a sample prior to SEC in order to minimize sample3 volume and facilitate a rapid,
A 280 nm

A 280 nm
15
15 a convenient, ready-to-use solution for sample concentration. 3
high-resolution size separation. HiTrap columns offer
Table 3.11 earlier in the chapter gives examples of the high concentration
1 factors achieved when concentrating proteins
10 10 Similar 1
from very dilute starting material using HiTrap columns prepacked with Sepharose High Performance medium.
results can be achieved with HiTrap columns prepacked with Sepharose Fast Flow or Sepharose XL media.
50 5

0 0

0 20 40 60 80 100 120 140 0 100 200 300 400 500

Volume (mL) Volume (mL)

83
Performing a separation
Guidelines for selection of media, buffer, pH, and ionic strength conditions and method optimization are given in Chapter 2.
Use the instructions given here as a basis from which to optimize a separation.
Correct sample and buffer preparation is essential in order to achieve optimal separation and avoid any
deterioration in column performance. Samples must be fully dissolved and free from particles or other material
likely to interfere with the separation. Refer to Chapter 2 and Appendix 1 for recommendations and advice on
sample preparation.
Filter buffers after all salts and additives have been included. Use high quality water and chemicals. Filter solutions
using filters of 1 µm or less. To avoid formation of air bubbles in a packed column, maintain buffers and columns at
a constant temperature before and during a run.
The pH of the start buffer should be at least 0.5 to 1.0 pH unit above the pI of the target substance when using an
anion exchanger (Q, DEAE, or ANX) and 0.5 to 1.0 pH unit below the pI of the target substance when using a cation
exchanger (SP, CM). See Appendix 2 for recommendations on volatile and nonvolatile buffer systems for anion and
cation exchangers.
For samples with unknown charge properties, try the following:
anion exchange (Q)
start buffer: 20 mM Tris-HCl, pH 8.0
elution buffer: start buffer including 1 M NaCl, pH 8.0
cation exchange (SP)
start buffer: 20 mM 2-(N-Morpholino)ethanesulfonic acid (MES), pH 6.0
elution buffer: start buffer including 1 M NaCl, pH 6.0
If selectivity is not satisfactory when using a strong ion exchanger (Q or SP),
try a weak ion exchanger (DEAE, ANX, or CM) instead.
Users of ÄKTA systems with automatic buffer preparation functionality can select one of the buffer recipes
recommended for anion exchange chromatography at pH 8.0 or cation exchange chromatography at pH 6.0,
see ÄKTA Laboratory-scale Chromatography Systems: Instrument Management Handbook, 29010831.

84
First-time use or after long-term storage Separation by step elution

1. To remove ethanol, wash with 1 CV of distilled water at 1 mL/min (HiTrap 1 mL, HiScreen 4.7 mL), 5 mL/min Flow rates: 1 mL/min (HiTrap 1 mL), 5 mL/min (HiTrap 5 mL), 2.3 mL/min
(HiTrap 5 mL), 2 mL/min (HiPrep 20 mL), or at 50 cm/h for Sepharose Fast Flow packed in larger columns. This (HiScreen 4.7 mL), 5 mL/min (HiPrep 20 mL), or at 150 cm/h for Sepharose Fast
step ensures removal of ethanol and avoids the risk of precipitation if buffer salts were to come into contact Flow packed in larger columns.
with the ethanol. The step can be omitted if precipitation is not likely to be a problem. Collect fractions throughout the separation.
2. W
 ash with 5 CV of start buffer at 1 mL/min (HiTrap 1 mL), 2.3 mL/min (HiScreen 4.7 mL), 5 mL/min (HiTrap 5 mL), 1. Equilibrate column with 5 to 10 CV of start buffer or until the baseline,
or 5 mL/min (HiPrep 20 mL). eluent pH, and conductivity are stable.
3. Wash with 5 CV of elution buffer, same flow as step 2. 2. A
 djust the sample to the chosen starting pH and ionic strength and apply to
4. Wash with 5 CV of start buffer, same flow as step 2. the column.
3. W
 ash with 5 to 10 CV of start buffer or until the baseline, eluent pH, and
conductivity are stable, that is, when all unbound material has washed
Separation by gradient elution through the column.
4. E
 lute the target protein with 5 CV of start buffer containing NaCl at chosen
Flow rates: 1 mL/min (HiTrap 1 mL), 5 mL/min (HiTrap 5 mL), 2.3 mL/min (HiScreen 4.7 mL), 5 mL/min (HiPrep 20 mL), ionic strength.
or at 150 cm/h for Sepharose Fast Flow packed in larger columns. Collect fractions throughout the separation.
5. I f necessary: repeat step 4 at higher ionic strengths until the target
1. Equilibrate column with 5 to 10 CV of start buffer or until the baseline, eluent pH, and conductivity are stable. protein(s) has been eluted.
2. Adjust the sample to the chosen starting pH and ionic strength and apply to the column. 6. W
 ash with 5 CV of a high salt solution (1 M NaCl in start buffer) to elute any
3. W
 ash with 5 to 10 CV of start buffer or until the baseline, eluent pH, and conductivity are stable, i.e., when all remaining ionically bound material.
unbound material has washed through the column. 7. R
 e-equilibrate 5 to 10 CV of start buffer or until eluent pH and conductivity
4. Begin elution using a gradient volume of 10 to 20 CV and an increasing ionic strength up to 0.5 M NaCl (50%B). reach the required values.

5. Wash with 5 CV of 1 M NaCl (100%B) to elute any remaining ionically bound material.
6. Re-equilibrate with 5 to 10 CV of start buffer or until eluent pH and conductivity reach the required values.

85
 Save time by using higher flow rates during the high salt wash and re-equilibration steps. Do not exceed the To remove precipitated proteins, lipids, hydrophobically bound proteins, or lipoproteins,
maximum recommended flow for the medium. refer to Appendix 1.

If ionic detergents have been used, wash the column with 5 CV of distilled water, followed by 2 CV of 2 M NaCl. Chemical stability
Re-equilibrate with at least 10 CV of start buffer until the UV baseline, eluent pH and/or conductivity are stable.
Organic solvents such as ethanol can be used to remove nonionic detergents. When selecting an organic solvent, For daily use, Sepharose Fast Flow media are stable in all common, aqueous buffers,
check the chemical stability of the medium to determine a suitable concentration. 1 M NaOH, denaturing agents (8 M urea, 6 M guanidine hydrochloride), with additives
such as nonionic detergents, 70% ethanol, 1 M acetic acid, and 30% isopropanol.
Refer to Chapter 2 for advice on optimizing the separation.
Sepharose Fast Flow can be used with organic solvents such as dimethylsulfoxide,
Check column performance regularly by determining column efficiency and peak symmetry, see Appendix 3. dimethylformamide, tetrahydrofuran, acetone, chloroform, dichloromethane,
Note that Appendix 3 does not apply to HiTrap or HiPrep columns. dichloroethane, and dichloroethane/pyridine (50:50) as well as polar solvents and
aqueous/organic solutions. The water in the medium can be exchanged by the
Cleaning alternative solvent with very little effect on the pore size of the matrix.
Correct preparation of samples and buffers and application of a high salt wash (1 M NaCl) at the end of each separation
Avoid cationic detergents with SP or CM Sepharose Fast Flow. Avoid anionic
should keep most columns in good condition. However, reduced performance, a slow flow rate, increasing back pressure
detergents with Q, DEAE, or ANX Sepharose Fast Flow. Avoid oxidizing agents.
or complete blockage are all indications that the medium needs to be cleaned using more stringent procedures in order
to remove contaminants. Storage
Reverse the direction of flow during column cleaning so that contaminants do not need to pass through the entire For column storage, wash with 2 CV of distilled water followed by 2 CV of 20%
length of the column. The number of column volumes and time required for each cleaning step varies according to ethanol. Include 0.2 M sodium acetate in the 20% ethanol solution for
the degree of contamination. If the cleaning procedure to remove common contaminants does not restore column SP Sepharose Fast Flow. Degas the ethanol/water mixture thoroughly and apply at
performance, change the top filter (when possible) before trying alternative cleaning methods. Care should be a low flow rate to avoid over-pressuring the column. Store at room temperature or,
taken when changing a filter as this can affect the column packing and interfere with performance. for long periods, store at 4°C to 8°C. Ensure that the column is sealed well to avoid
drying out. Whenever possible, use the storage and shipping device if supplied by the
Removing common contaminants manufacturer. Store unused media at 4°C to 30°C in 20% ethanol. Do not freeze.
To avoid formation of air bubbles in a packed column, ensure that column
Flow rates: 1 mL/min (HiTrap 1 mL, HiScreen 4.7 mL), 5 mL/min (HiTrap 5 mL), 5 mL/min (HiPrep 20 mL), or at 40 cm/h and buffers are at the same temperature when preparing for a run.
with a contact time of 1 to 2 h for Sepharose Fast Flow packed in larger columns.
1. Wash with at least 2 CV of 2 M NaCl.
2. Wash with at least 4 CV of 1 M NaOH.
3. Wash with at least 2 CV of 2 M NaCl.
4. Rinse with at least 2 CV of distilled water (same flow as step 1) until the UV-baseline and the eluent pH are stable.
5. W
 ash with at least 4 CV of start buffer or storage buffer until eluent pH and conductivity have reached the
required values.
86
Sepharose XL: for selected proteins that require very high binding Table 3.15. Characteristics of Sepharose XL media

capacity to increase productivity, easy scale-up Product Functional group pH stability1 Mean particle size (µm)
Q Sepharose XL -CH2-N+-(CH3)3 Long term: 2 to 12 90
Use Sepharose XL media for purification of proteins when improved binding capacity compared to other Sepharose

Short term: 2 to 14
media has been confirmed for the selected protein.
SP Sepharose XL -CH2-CH2-CH2-SO3- Long term: 4 to 13 90
 Use Sepharose XL at the beginning of a purification scheme for initial capture when a high binding capacity and Short term: 3 to 14
rapid separation is required for a selected protein from clarified samples. 1
L ong-term pH stability refers to the pH interval where the medium is stable over a long period of time
 Run columns packed with Sepharose XL on systems such as ÄKTA, HPLC, or systems using peristaltic pumps. without adverse side effects on the chromatography performance. Short-term pH stability refers to the pH
Appendix 4 provides guidance on selecting the right ÄKTA system. interval for regeneration, cleaning-in-place, and sanitization procedures. All ranges are estimates based on
experience and knowledge gained at Cytiva.
Sepharose XL media are based on a matrix of 90 µm particles, made from 6% agarose and highly cross-linked for
chemical and physical stability, substituted with quaternary ammonium (Q) or sulfopropyl (SP) groups. The ionic groups
are bound to long, flexible dextran chains which have been coupled to the agarose. This increases the exposure of the
Q or SP groups thereby raising the binding capacity to a very high level without restricting the passage of charged
molecules. The strong ion exchange groups maintain their charge over a broad pH range, allowing selection of the most
suitable pH for each application. Particle size and bed volumes remain stable, despite changes in ionic strength or pH,
to ensure fast separations at high flow rates with good resolution.

Media characteristics
Composition: sulfopropyl (SP) or quaternary amino (Q) groups attached via chemically stable ether bonds to long, flexible
dextran chains that are covalently coupled to highly cross-linked 6% agarose (Table 3.15).

87
Purification options
Q and SP Sepharose XL are available in chromatography media packs as well as convenient prepacked HiTrap columns
for small-scale purification and HiPrep columns for scale-up (Fig 3.31). Purification options for the media and prepacked
columns are shown in Table 3.16.

Table 3.16. Purification options for Sepharose XL media and prepacked columns
Maximum
operating
back pressure3
Binding capacity Recommended Working (MPa/psi) Fig 3.31. Q and SP Sepharose XL are available in prepacked HiTrap and HiPrep columns, in chromatography
Product per column or per mL medium working flow1 Maximum flow 1
pH range2 1 MPa = 10 bar media packs, and in the HiTrap IEX Selection Kit, 17600233.
Strong anion exchangers
Q Sepharose XL > 130 mg/mL (BSA, Mr 67 000) 300 to 500 cm/h 700 cm/h 2 to 12 0.3/43 For column packing in Tricorn and XK columns, see Table 3.17. Select a production
HiTrap Q XL, 1 mL > 130 mg (BSA, Mr 67 000) up to 1 mL/min 4 mL/min 2 to 12 0.3/43 column such as BPG or Chromaflow for larger volumes.
HiTrap Q XL, 5 mL > 650 mg (BSA, Mr 67 000) up to 5 mL/min 20 mL/min 2 to 12 0.3/43
HiPrep Q XL 16/10, 20 mL > 2600 mg (BSA, Mr 67 000) 2 to 10 mL/min 10 mL/min 2 to 12 0.15/22 Table 3.17. Packing volumes and bed heights for Sepharose XL media packed in Tricorn and XK columns

Strong cation exchangers Column Volume (mL) Bed height (cm)

SP Sepharose XL > 160 mg/mL (lysozyme, Mr 14 500) 300 to 500 cm/h 700 cm/h 4 to 13 0.3/43 Tricorn 10/100 up to 8 up to 10
HiTrap SP XL, 1 mL > 160 mg (lysozyme, Mr 14 500) up to 1 mL/min 4 mL/min 4 to 13 0.3/43 Tricorn 10/150 up to 12 up to 15
HiTrap SP XL, 5 mL > 800 mg (lysozyme, Mr 14 500) up to 5 mL/min 20 mL/min 4 to 13 0.3/43 Tricorn 10/200 up to 16 up to 20
HiPrep SP XL 16/10, 20 mL > 3200 mg (lysozyme, Mr 14 500) 2 to 10 mL/min 10 mL/min 4 to 13 0.15/22 XK 16/20 up to 30 up to 15
1
See Appendix 5 to convert linear flow (cm/h) to volumetric flow rate (mL/min) and vice versa. XK 26/20 up to 80 up to 15
2
Working pH range refers to the pH interval where the medium can be operated as intended without adverse long-term effects.
XK 26/40 up to 196 > 15
3
Maximum operating back pressure refers to the pressure above which the medium begins to compress.
XK 50/20 up to 274 up to 14
XK 50/30 up to 559 up to 28.5
 Use prepacked HiTrap columns (1 mL or 5 mL) for media selection, method scouting, group separations, small-scale
purification, sample concentration, or clean-up. Connect up to three HiTrap columns in series to scale-up.
 Use prepacked HiPrep columns (20 mL) for method development, group separations, larger scale purification,
sample concentration or clean-up. Connect several HiPrep columns in series to increase binding capacity.

88
 Purification examples
Media selection Capture
Capture of alkaline phosphatase from a clarified lysate of E. coli using a HiTrap Q XL
The most suitable matrix and charged group for a separation can be selected quickly and easily by using 1 mL HiTrap
1 mL column is shown in Figure 3.33. Separation was monitored at A280 nm and
columns. In Figure 3.32, a comparison of elution profiles for the same sample separated under identical conditions on three
phosphatase activity assayed by a spectrophotometric method at A405 nm.
different media illustrates the differences in selectivity and resolution that can result from changing the charge group
and matrix. The most suitable medium can be selected and conditions optimized according to the requirements for the
purification. In this example, Sepharose XL resolves the three components and optimization of elution conditions could Column: HiTrap Q XL, 1 mL
further improve the resolution. However, any of these media would be suitable if the aim was to isolate the first major peak Sample: 2 mL of E.coli lysate clarified by centrifugation
Sample volume: 2 mL
(ribonuclease A). Start buffer: 20 mM Tris-HCl, pH 7.4
I Elution buffer: 20 mM Tris-HCl, 500 mM NaCl, pH 7.4
Columns: (A) HiTrap SP XL, 1 mL Flow rate: 1 mL/min (150 cm/h)
(A) (B) HiTrap SP FF, 1 mL Gradient: 0% elution buffer (30 CV), 0% to 100% elution buffer (40 CV)
HiTrap SP XL, 1 mL
III
(C) HiTrap CM FF, 1 mL
Sample: 3 mg ribonuclease A (pI = 9.3), 50
40 0.8 mg cytochrome C (pI = 10.3), A 405 nm
150 II 0.8 mg lysozyme (pI > 11) 600
Sample volume: 2 mL in start buffer 40

Conductivity (mS/cm)
Start buffer: 20 mM sodium phosphate, pH 6.8 500
Conductivity (mS/cm)
Elution buffer: 20 mM sodium phosphate,
30

A 280 nm
A 280 nm

30 500 mM NaCl pH 6.8 400

(B) I
HiTrap SP FF, 1 mL Flow rate (flow velocity): 1 mL/min (150 cm/h)
Gradient: 0% elution buffer (25 CV), 0% to 300
100 II+III 100% elution buffer (40 CV) 20
20 200
10
100
50 (C) I III
HiTrap CM FF, 1 mL II 0
0
10 0 10 20 30 40 50 60
Volume (mL)

0 Fig 3.33. Clarified E. coli lysate on HiTrap Q XL, absorbance values at 450 nm relate to phosphatase activity in
eluted fractions.
0 20 40 60
Volume (mL)
Fig 3.32. Media scouting: separation of ribonuclease A (I), cytochrome C (II), and lysozyme (III) on a range of anion exchange HiTrap columns.

89
Capture and scale-up Sample concentration
Figure 3.34 shows a pilot scale purification performed on a Sepharose XL ion exchanger. The separation was developed It can be an advantage to concentrate a sample prior to SEC in order to minimize
on Q Sepharose XL packed in an XK 16/20 column in order to select optimal pH and to determine maximum binding sample volume and facilitate a rapid, high resolution size separation. HiTrap columns
capacity available. Adding CaCl2 to the sample precipitated DNA and so increased the binding capacity for the target offer a convenient, ready-to-use solution for sample concentration. Table 3.9 earlier
protein. Final loading was reduced to 75% of the maximum capacity and the result verified before scaling-up to a larger, in the chapter gives examples of the high concentration factors achieved when
INdex column. concentrating proteins from very dilute starting material using HiTrap columns
prepacked with Sepharose High Performance medium. Similar results can be achieved
with HiTrap columns prepacked with Sepharose Fast Flow or Sepharose XL media.

Column: Q Sepharose XL in INdEX 70 column, 385 mL bed volume

Sample: Recombinant a-amylase produced in E. coli, homogenized, 2.2 L diluted in distilled water to
15.4 L, 7.2 mS/cm, 10 mM CaCl2, centrifuged
Start buffer: 20 mM Tris-HCl, pH 8.0, 10 mM CaCl2
Elution buffer: 20 mM Tris-HCl, pH 8.0, 1 M NaCl, 10 mM CaCl2
Flow flow: 300 cm/h, 12 L/h
Gradient: 20 CV 0 to 1 M NaCl
Eluent: 1.48 L, 3.8 CV Spec.
Spec. act. a-amylase 6420 U/L

Conductivity (mS/cm)
A 280 nm
0 500 1000
Volume (mL)
Fig 3.34. Capture of recombinant a-amylase from E. coli on Q Sepharose XL pilot scale column. The first peak
during gradient elution contained α-amylase.
90
Performing a separation
Guidelines for selection of media, buffer, pH and ionic strength conditions, and method optimization are given in Chapter 2.
Use the instructions given here as a basis from which to optimize a separation.
Correct sample and buffer preparation is essential in order to achieve optimal separation and avoid any deterioration
in column performance. Samples must be fully dissolved and free from particles or other material likely to interfere
with the separation. Refer to Chapter 2 and Appendix 1 for recommendations and advice on sample preparation.
Filter buffers after all salts and additives have been included. Use high quality water and chemicals. Filter solutions
using filters of 1 µm or smaller. To avoid formation of air bubbles in a packed column, maintain buffers and columns
at a constant temperature before and during a run.
The pH of the start buffer should be at least 0.5 to 1.0 pH unit above the pI of the target substance when using an
anion exchanger (Q) and 0.5 to 1.0 pH unit below the pI of the target substance when using a cation exchanger (SP).
See Appendix 2 for recommendations on volatile and nonvolatile buffer systems for anion and cation exchangers.
For samples with unknown charge properties, try the following:
anion exchange (Q)
start buffer: 20 mM Tris-HCl, pH 8.0
elution buffer: start buffer including 1 M NaCl, pH 8.0
cation exchange (SP)
start buffer: 20 mM 2-(N-Morpholino)ethanesulfonic acid (MES), pH 6.0
elution buffer: start buffer including 1 M NaCl, pH 6.0
Users of ÄKTA systems with automatic buffer preparation functionality can select one of the buffer recipes
recommended for anion exchange chromatography at pH 8.0 or cation exchange chromatography at pH 6.0,
see ÄKTA Laboratory-scale Chromatography Systems: Instrument Management Handbook, 29010831.

91
First-time use or after long-term storage Separation by step elution

1. T o remove ethanol, wash with 1 CV of distilled water at 1 mL/min (HiTrap 1 mL), 5 mL/min (HiTrap 5 mL), 5 mL/min Flow rates: 1 mL/min (HiTrap 1 mL), 5 mL/min (HiTrap 5 mL), 5 mL/min (HiPrep 20 mL),
(HiPrep 20 mL), or at 50 cm/h for Sepharose XL packed in larger columns. This step ensures removal of ethanol or at 150 cm/h for Sepharose XL packed in larger columns.
and avoids the risk of precipitation if buffer salts were to come into contact with the ethanol. The step can be Collect fractions throughout the separation.
omitted if precipitation is not likely to be a problem.
1. Equilibrate column with 5 to 10 CV of start buffer or until the baseline,
2. W
 ash with 5 CV of start buffer at 1 mL/min (HiTrap 1 mL), 5 mL/min (HiTrap 5 mL), 5 mL/min (HiPrep 20 mL), or at eluent pH, and conductivity are stable.
150 cm/h for Sepharose XL packed in larger columns.
2. A
 djust the sample to the chosen starting pH and ionic strength and apply to
3. Wash with 5 CV of elution buffer, same flow as step 2. the column.
4. Wash with 5 CV of start buffer, same flow as step 2. 3. W
 ash with 5 to 10 CV of start buffer or until the baseline, eluent pH, and
5. Run a blank elution before applying sample. conductivity are stable, that is, when all unbound material has washed
through the column.
4. E
 lute the target protein with 5 CV of start buffer containing NaCl at chosen
Separation by gradient elution ionic strength.
5. I f necessary: repeat step 4 at higher ionic strengths until the target
Flow rates: 1 mL/min (HiTrap 1 mL), 5 mL/min (HiTrap 5 mL), 5 mL/min (HiPrep 20 mL), or at 150 cm/h for Sepharose XL protein(s) has been eluted.
packed in larger columns. Collect fractions throughout the separation.
6. W
 ash with 5 CV of a high salt solution (1 M NaCl in start buffer) to elute any
1. Equilibrate column with 5 to 10 CV of start buffer or until the baseline, eluent pH and conductivity are stable. remaining ionically bound material.
2. Adjust the sample to the chosen starting pH and ionic strength and apply to the column. 7. R
 e-equilibrate with 5 to 10 CV of start buffer or until eluent pH and
3. W
 ash with 5 to 10 CV of start buffer or until the baseline, eluent pH, and conductivity are stable, that is, when all conductivity reach the required values.
unbound material has washed through the column.
4. Begin elution using a gradient volume of 10 to 20 CV and an increasing ionic strength up to 0.5 M NaCl (50%B).  Save time by using higher flow rates during the high salt wash and re-equilibration
5. Wash with 5 CV of 1 M NaCl (100%B) to elute any remaining ionically bound material. steps. Do not exceed the maximum recommended flow for the medium.
6. Re-equilibrate 5 to 10 CV of start buffer or until eluent pH and conductivity reach the required values.  If ionic detergents have been used, wash the column with 5 CV of distilled
water, followed by 2 CV of 2 M NaCl. Re-equilibrate with at least 10 CV of start
buffer until the UV baseline, eluent pH and/or conductivity are stable.

92
Organic solvents such as ethanol can be used to remove nonionic detergents. When selecting an organic solvent, Chemical stability
check the chemical stability of the medium to determine a suitable concentration.
For daily use, Sepharose XL media are stable in all common, aqueous buffers,
 Check column performance regularly by determining column efficiency and peak symmetry. See Appendix 3. 1 M NaOH, denaturing agents (8 M urea, 6 M guanidine hydrochloride), with additives
such as nonionic detergents, 70% ethanol, 1 M acetic acid, and 30% isopropanol.
Cleaning
Sepharose XL can be used with organic solvents such as dimethylsulfoxide,
Correct preparation of samples and buffers and application of a high salt wash (1 M NaCl) at the end of each separation dimethyl-formamide, tetrahydrofuran, acetone, chloroform, dichloromethane,
should keep most columns in good condition. However, reduced performance, a slow flow rate, increasing back pressure, dichloroethane, and dichloroethane/pyridine (50:50) as well as polar solvents and
or complete blockage are all indications that the medium needs to be cleaned using more stringent procedures in order aqueous/organic solutions. The water in the medium can be exchanged by the
to remove contaminants. alternative solvent with very little effect on the pore size of the matrix.
 Reverse the direction of flow during column cleaning so that contaminants do not need to pass through the entire Avoid cationic detergents with SP Sepharose XL. Avoid anionic detergents with
length of the column. The number of column volumes and time required for each cleaning step varies according to Q Sepharose XL. Avoid oxidizing agents.
the degree of contamination. If the cleaning procedure to remove common contaminants does not restore column
performance, change the top filter (when possible) before trying alternative cleaning methods. Care should be Storage
taken when changing a filter as this can affect the column packing and interfere with performance.
For column storage, wash with 2 CV of distilled water followed by 2 CV of 20%
Removing common contaminants ethanol. Include 0.2 M sodium acetate in the storage solution for SP Sepharose XL.
Degas the ethanol/water mixture thoroughly and apply at a low flow rate to avoid
overpressuring the column. Store at room temperature or, for long periods, store
Flow rates: 1 mL/min (HiTrap 1 mL), 5 mL/min (HiTrap 5 mL), 5 mL/min (HiPrep 20 mL), or at 40 cm/h with a contact at 4°C to 8°C. Ensure that the column is sealed well to avoid drying out. Whenever
time of 1 to 2 h for Sepharose XL packed in larger columns. possible, use the storage and shipping device if supplied by the manufacturer. Store
unused media at 4°C to 30°C in 20% ethanol. Do not freeze.
1. Wash with at least 2 CV of 2 M NaCl.
To avoid formation of air bubbles in a packed column, ensure that column and
2. Wash with at least 4 CV of 1 M NaOH (same flow as step 1). 

buffers are at the same temperature when preparing for a run.

3. Wash with at least 2 CV of 2 M NaCl (same flow as step 1).
4. Rinse with at least 2 CV of distilled water (same flow as step 1) until the UV-baseline and the eluent pH are stable.
5. W
 ash with at least 4 CV of start buffer or storage buffer (same flow as step 1) until eluent pH and conductivity
have reached the required values.

To remove precipitated proteins, lipids, hydrophobically bound proteins, or lipoproteins, refer to Appendix 1.

93
Sepharose Big Beads: purification from crude, viscous samples 3000 BPG 300/20 cm/dist. water/25°C

at large scale PP113/30 cm/65% ethanol/20°C,

viscosity 2.5 times water

 Use Sepharose Big Beads for purification of proteins from crude, viscous samples.

Flow velocity (cm/h)

2000
 Use Sepharose Big Beads when handling large volumes of crude or viscous samples that must be bound rapidly and
when resolution is less important.
 Use Sepharose Big Beads for capture steps, when viscosity and back pressure can limit the throughput attainable
with ion exchangers of smaller particle size. 1000

 Run columns packed with Sepharose Big Beads on systems such as ÄKTA, HPLC, or systems using peristaltic pumps.
Appendix 4 gives guidance on how to select the most suitable ÄKTA system.
Sepharose Big Beads are ion exchangers designed for large-scale industrial applications. Sepharose Big Beads are based 0
0 1 2 3 4
on 100 to 300 µm, cross-linked 6% agarose particles, substituted with quaternary ammonium (Q) or sulfopropyl (SP) C/Co, % (concentration in eluate, C Pressure (bar)
as fraction of concentration in sample, Co)
groups. The large particle size, together with a high degree of cross-linking for extreme physical and chemical stability, concentration in eluate (C) as fraction of
Fig 3.35. Sepharose Big Beads allow
concentration high flow
in sample (Co) rates with high-viscosity samples.
ensures that high flow rates can be maintained when processing very viscous samples. For example, a flow of 500 cm/h
can be maintained in an industrial process at viscosities up to 2.5 times the viscosity of water. More dilute samples can
be run at 1000 cm/h. Particle size and bed volumes remain stable, despite changes in ionic strength or pH. The strong ion BSA (300 cm/h)
exchange groups (Q and SP) maintain their charge over a broad pH range, allowing selection of the most suitable pH for 80
β-lactogl. (300 cm/h)
BSA (12 cm/h)
each application.
β -lactogl. (12 cm/h)
Figures 3.35 and 3.36 show the excellent flow characteristics and typical binding capacities for Sepharose Big Beads media.
60

C/Co, (%)1
40

20

0
0 100 200
Binding capacity (mg protein/mL medium)
1
Concentration in eluate (C) as fraction of concentration in sample (Co).

Fig 3.36. Typical binding capacities of SP Sepharose Big Beads. Binding capacity measured in acetate pH 5.0
for bovine serum albumin (BSA) and formate pH 4.1 for b-lactoglobulin at flow velocities of 12 and 300 cm/h.

94
Media characteristics
Composition: sulfopropyl (SP) or quaternary amino (Q) groups coupled to highly cross-linked 6% agarose via chemically For column packing in XK columns during method development, particularly when
stable ether bonds, see Table 3.18. handling crude, viscous samples, see Table 3.20.

Table 3.18. Characteristics of Sepharose Big Beads Table 3.20. Packing volumes and bed heights for Sepharose Q and SP High Performance packed in XK columns

Product Functional group pH stability1 Mean particle size (µm) Column Volume (mL) Bed height (cm)
Strong anion exchanger XK 16/20 up to 30 up to 15
Q Sepharose Big Beads -CH2-N+-(CH3)3 Long term: 2 to 12 200 XK 26/20 up to 80 up to 15
Short term: 2 to 14
XK 26/40 up to 196 > 15
Strong cation exchanger
SP Sepharose Big Beads -CH2-CH2-CH2-SO3- Long term: 4 to 13 200 XK 50/20 up to 274 up to 14
Short term: 3 to 14 XK 50/30 up to 559 up to 28.5
1
L ong-term pH stability refers to the pH interval where the medium is stable over a long period of time without adverse side effects on the chromatography
performance. Short-term pH stability refers to the pH interval for regeneration, cleaning-in-place, and sanitization procedures. All ranges are estimates
based on experience and knowledge gained at Cytiva. Select a production scale column such as BPG or Chromaflow for larger volumes.
Sepharose Big Beads can be packed in large scale columns by applying constant
pressure between 0.1 to 0.3 MPa (1.0 to 3.0 bar, 14.5 to 43.5 psi) by slurry
Purification options sedimentation followed by adapter compression, or by suction packing. Follow
Purification options for Q and SP Sepharose Big Beads are described in Table 3.19. the instructions supplied with the medium.
Table 3.19. Purification options for Sepharose Big Beads
Maximum operating
Binding capacity per Recommended Maximum Working back pressure3
Product column or per mL medium working flow1 flow1 pH range2 (MPa/psi) 1 MPa = 10 bar
Strong anion exchanger
Q Sepharose Big Beads Tested for each up to 300 cm/h 1800 cm/h 2 to 12 0.3/43
specific application
Strong cation exchanger
SP Sepharose Big Beads Tested for each up to 300 cm/h 1800 cm/h 4 to 13 0.3/43
specific application
1
 ee Appendix 5 to convert flow velocity (cm/h) to volumetric flow rate (mL/min) and vice versa.
S
2
Working pH range refers to the pH interval where the medium can be operated as intended without adverse long-term effects.
3
Maximum operating back pressure refers to the pressure above which the medium begins to compress.
95
Performing a separation Cleaning
Guidelines for selection of media, buffer, pH, and ionic strength conditions, and method optimization are given in Chapter 2. Correct preparation of samples and buffers and regeneration with a high salt wash
See Appendix 2 for recommendations on volatile and nonvolatile buffer systems. (1 M NaCl) at the end of each separation should keep most columns in good
condition. However, reduced performance, a slow flow rate, increasing back pressure,
 Correct sample and buffer preparation is essential in order to achieve optimal separation and avoid any deterioration
or complete blockage are all indications that the medium needs to be cleaned using
in column performance. Refer to Chapter 2 and Appendix 1 for recommendations and advice.
more stringent procedures in order to remove contaminants.
 Filter buffers after all salts and additives have been included. Use high quality water and chemicals. Filter solutions
Reverse the direction of flow during column cleaning so that contaminants
through 1 µm filters. To avoid formation of air bubbles in a packed column, maintain buffers and columns at a 

do not need to pass through the entire length of the column. The number of
constant temperature before and during a run.
column volumes and time required for each cleaning step varies according to
First-time use or after long-term storage the degree of contamination. If the cleaning procedure to remove common
contaminants does not restore column performance, change the top filter
(when possible) before trying alternative cleaning methods. Care should
1. Wash with 5 CV of distilled water at 300 cm/h. be taken when changing a filter as this can affect the column packing and
2. Wash with 5 CV of start buffer, same flow as step 1. interfere with performance.

3. Wash with 5 CV of elution buffer, same flow as step 1. Removing common contaminants
4. Wash with 5 CV of start buffer, same flow as step 1.
1. Wash with at least 2 CV of 2 M NaCl at 40 cm/h for a contact time of 1 to 2 h.
Gradient or step elution 2. Wash with at least 4 CV of 1 M NaOH (same flow as step 1).
3. Wash with at least 2 CV of 2 M NaCl (same flow as step 1).
Conditions for a large scale-purification using Sepharose Big Beads will be determined during method development 4. R
 inse with at least 2 CV of distilled water (same flow as step 1) until the
and relate to the specific application. Refer to Chapter 2 for advice on optimizing a separation. Typical separation UV-baseline and the eluent pH are stable.
flow rates should be 200 to 500 cm/h. 5. W
 ash with at least 4 CV of start buffer or storage buffer (same flow as step 1)
until eluent pH and conductivity have reached the required values.
 If ionic detergents have been used, wash the column with 5 CV of distilled water, followed by 2 CV of 2 M NaCl.
Re-equilibrate with at least 10 CV of start buffer until the UV baseline, eluent pH and/or conductivity are stable. To remove precipitated proteins, lipids, hydrophobically bound proteins, or lipoproteins,
Organic solvents such as ethanol can be used to remove nonionic detergents. When selecting an organic solvent, refer to Appendix 1.
check the chemical stability of the medium to determine a suitable concentration.
Check column performance regularly by determining column efficiency and peak symmetry. See Appendix 3.


96
Chemical stability
For daily use, Sepharose Big Beads are stable in all common, aqueous buffers, 1 M NaOH, denaturing agents (8 M urea,
6 M guanidine hydrochloride), with additives such as nonionic detergents, 70% ethanol, 1 M acetic acid, 30% acetonitrile,
and 30% isopropanol.
Sepharose Big Beads can be used with organic solvents such as dimethylsulfoxide, dimethyl-formamide, tetrahydrofuran,
acetone, chloroform, dichloromethane, dichloroethane and dichloroethane/pyridine (50:50), as well as polar solvents and
aqueous/organic solutions. The water in the medium can be exchanged by the alternative solvent with very little effect
on the pore size of the matrix.
Avoid cationic detergents with SP Sepharose Big Beads. Avoid anionic detergents with Q Sepharose Big Beads.
Avoid oxidizing agents.

Storage
For column storage, wash with 2 CV of distilled water followed by 2 CV of 20% ethanol. Include 200 mM sodium acetate in
the storage solution for SP Sepharose Big Beads. For small-scale columns, degas the ethanol/water mixture thoroughly,
for large-scale columns ensure that an air trap is included before the column. Add storage solution at a low flow rate,
checking the back pressure as the column equilibrates. Alternatively, store at neutral pH in buffer containing 20% ethanol
or in 100 mM NaOH.
Store at room temperature or, for long periods, store at 4°C to 8°C. Ensure that the column is sealed well to avoid drying
out. Store unused media at 4°C to 30°C in 20% ethanol. Do not freeze.

97
Capto: high-flow media with high resolution 500
Capto SP ImpRes
SP Sepharose High Performance
 Use Capto media, including Capto, Capto ImpRes and Capto ImpAct, for screening of selectivity and method 400
conditions before scaling up, as well as for small-scale purification.

Flow velocity (cm/h)

 Use Capto media with high flow-properties for capture, intermediate purification, and polishing of a wide range 300
of biomolecules.
 Run Capto media in an optimal way with liquid chromatography systems such as ÄKTA. Appendix 4 provides 200
guidance on selecting the right ÄKTA system.
 Capto media are based on the high-flow agarose base matrix, which gives excellent pressure-flow properties, 100
making the media suitable for process-scale applications (Fig 3.37).
0
0 1 2 3 4 5
Pressure (bar)

Fig 3.37. The pressure-flow properties of Capto ImpRes are improved compared with
Sepharose High Performance due to the increased mechanical stability of the base matrix.
Running conditions: AxiChrom™ 300 column, 20 cm bed height with water at 20°C.

Capto media are high capacity ion exchangers where the ligands are coupled to a
chemically modified, high-flow agarose matrix. The high-flow agarose matrix provides
particle rigidity without compromising the pore size. This allows for fast mass transfer
resulting in high dynamic binding capacities at high flow rates, making the media
suitable for process-scale applications.
Capto Q, Capto S, and Capto DEAE are used for capture and intermediate purification
of proteins. Capto Q ImpRes and Capto SP ImpRes are high-resolution media
designed for intermediate purification and polishing. Capto S ImpAct is especially
suitable for intermediate purification and polishing of MAbs, where a common main
challenge is to selectively remove impurities similar to the target product.

98
Media characteristics
Composition: Quaternary amine (Q), sulfopropyl (SP), and sulfonate (S) coupled to highly cross-linked high-flow agarose.
The characteristics of Capto media are shown in Table 3.21.

Table 3.21. Characteristics Capto media

Product Functional group pH stability1 Mean particle size (µm)

Capto Q -CH2-N+-(CH3)3 Long term: 2 to 12 90
Short term: 2 to 14
Capto DEAE -CH2-CH2-N+-(CH2CH3)2 Long term: 2 to 12 90
Short term: 2 to 14
Capto S -CH2-SO3- Long term: 4 to 12 90
Short term: 3 to 14
Capto Q ImpRes -CH2-N+-(CH3)3 Long term: 2 to 12 40
Short term: 2 to 14
Capto SP ImpRes -CH2-CH2-CH2-SO3- Long term: 4 to 12 40
Short term: 3 to 14
Fig. 3.38. Capto media are available in bulk packs, and prepacked HiTrap and HiScreen columns. The media
-
Capto S ImpAct -CH2-SO 3
Long term: 4 to 12 50 are also available in high-throughput process development (HTPD) format.
Short term: 3 to 14
1
L ong-term pH stability refers to the pH interval where the medium is stable over a long period of time without adverse side effects on the chromatography
performance. Short-term pH stability refers to the pH interval for regeneration, cleaning-in-place, and sanitization procedures. All ranges are estimates
based on experience and knowledge gained at Cytiva.

Purification options
Anion and cation exchanger Capto media are available prepacked in HiTrap and HiScreen columns and in chromatography
media packs (Fig 3.38). Table 3.22 is a presentation of different Capto media for ion exchange chromatography. Some of
the media are also available in PreDictor™ filter plates, PreDictor RoboColumn™, and ReadyToProcess™ columns (see Chapter 5,
Large-scale purification).

99
Table 3.22. Purification options for Capto media

Binding capacity Recommended working flow rate Maximum flow rate Working pH range
Strong anion exchangers
Capto Q > 100 mg (BSA)/mL medium up to 700 cm/h1 700 cm/h1 2 to 12
Capto Q ImpRes > 55 mg (BSA)/mL medium up to 220 cm/h1 220 cm/h1 2 to 12
HiTrap Capto Q, 1 mL > 100 mg (BSA) 1.0 mL/min 4.0 mL/min 2 to 12
HiTrap Capto Q, 5 mL > 500 mg (BSA) 5.0 mL/min 20.0 mL/min 2 to 12
HiTrap Capto Q ImpRes, 1 mL > 55 mg (BSA) 1.0 mL/min 4.0 mL/min 2 to 12
HiTrap Capto Q ImpRes, 5 mL > 275 mg (BSA) 5.0 mL/min 20.0 mL/min 2 to 12
HiScreen Capto Q, 4.7 mL > 470 mg (BSA) 1.2 mL/min 2.3 mL/min 2 to 12
HiScreen Capto Q ImpRes, 4.7 mL > 260 mg (BSA) 1.2 mL/min 2.3 mL/min 2 to 12
Weak anion exchangers
Capto DEAE > 90 mg (ovalbumin)/mL medium up to 700 cm/h1 700 cm/h1 2 to 12
HiTrap Capto DEAE, 1 mL > 90 mg (ovalbumin) 1.0 mL/min 4.0 mL/min 2 to 12
HiTrap Capto DEAE, 5 mL > 450 mg (ovalbumin) 5.0 mL/min 20.0 mL/min 2 to 12
HiScreen Capto DEAE, 4.7 mL > 420 mg (ovalbumin) 1.2 mL/min 2.3 mL/min 2 to 12
Strong cation exchangers
Capto S > 120 mg (lysozyme)/mL medium up to 700 cm/h1 700 cm/h1 4 to 12
Capto SP ImpRes > 70 mg (lysozyme)/mL medium up to 220 cm/h1 220 cm/h1 4 to 12
Capto S ImpAct > 90 mg (lysozyme)/mL medium up to 220 cm/h1 220 cm/h1 4 to 12
HiTrap Capto S, 1 mL > 120 mg (lysozyme) 1.0 mL/min 4.0 mL/min 4 to 12
HiTrap Capto S, 5 mL > 600 mg (lysozyme) 5.0 mL/min 20.0 mL/min 4 to 12
HiTrap Capto SP ImpRes, 1 mL > 70 mg (lysozyme) 1.0 mL/min 4.0 mL/min 4 to 12
HiTrap Capto SP ImpRes, 5 mL > 350 mg (lysozyme) 5.0 mL/min 20.0 mL/min 4 to 12
HiTrap Capto S ImpAct, 1 mL > 90 mg (lysozyme) 1.0 mL/min 4.0 mL/min 4 to 12
HiTrap Capto S ImpAct, 5 mL > 450 mg (lysozyme) 5.0 mL/min 20.0 mL/min 4 to 12
HiScreen Capto S, 4.7 mL > 560 mg (lysozyme) 1.2 mL/min 2.3 mL/min 4 to 12
HiScreen Capto SP ImpRes, 4.7 mL > 330 mg (lysozyme) 1.2 mL/min 2.3 mL/min 4 to 12
HiScreen Capto S ImpAct, 4.7 mL > 420 mg (lysozyme) 1.2 mL/min 2.3 mL/min 4 to 12
1
F low velocity in a 1 m column with 20 cm bed height at 20°C using process buffers with the same viscosity as water at < 0.3 MPa (3 bar, 43.5 psi). See Appendix 5 to convert flow velocities (cm/h) to volumetric flow rate (mL/min) and vice versa.
2
Working pH range refers to the pH interval where the medium can be operated as intended without adverse long-term effects.
100
Molecular weights (Mr) of proteins in the table: BSA, 67 000; ovalbumin, 45 000; lysozyme, 14 500.
 Use prepacked HiTrap columns (1 mL or 5 mL) for media selection, group separations, and small-scale purification.


Use prepacked HiScreen columns (4.7 mL, 10 cm bed height) for method development and optimization before


scaling up.
For column packing: Capto media can be used with most modern chromatography equipment from laboratory


to production scale. Due to the higher rigidity of Capto media, packing procedures differ slightly compared with
Sepharose (for details of packing laboratory-scale columns, see the appropriate Instructions).
Table 3.23 lists suitable empty columns for packing of Capto media from Cytiva.

Table 3.23. Example of columns suitable for packing of Capto media

Column family Inner diameter (mm)

Laboratory scale
Tricorn 5, 10
HiScale™ 16, 26, 50
Pilot and production scale
AxiChrom 50 to 10001
BPG 100 to 3002
1
Maximum bed height for AxiChrom 1000 is 20 cm.
2
Note that the pressure rating of BPG 450 column is too low for Capto ImpRes media.

101
Purification examples
Capture and scale-up
Capto media belong to the BioProcess™ range of media that are developed and supported for production-scale chromatography. The small prepacked
column formats, HiTrap 1 mL and 5 mL and HiScreen (4.7 mL), are convenient to use together with a chromatography system when developing efficient and
robust separation methods. Further development and optimization using Tricorn or HiScale columns then permits straightforward scale-up. Figure 3.39
shows an example of scaling up an optimized purification of α-chymotrypsin on Capto S, starting from a Tricorn column.

Column: Tricorn 5/100 (bed height 9.7 cm, CV = 1.9 mL) Column: XK 16/40 (bed height 20.7 cm, CV = 41.5 mL) Column: AxiChrom 50 (bed height 22 cm, CV = 431 mL)
Medium: Capto S Medium: Capto S Medium: Capto S
Sample: a-chymotrypsin in E. coli homogenate, Sample: a-chymotrypsin in E. coli homogenate, Sample: a-chymotrypsin in E. coli homogenate,
4 mg/mL to 50 mL 4 mg/mL to 1040 mL 4 mg/mL to 10.8 L
Start buffer: 50 mM sodium acetate, pH 4.8 Start buffer: 50 mM sodium acetate, pH 4.8 Start buffer: 50 mM sodium acetate, pH 4.8
Elution buffer: 50 mM sodium acetate, 1 M NaCl, pH 4.8 Elution buffer: 50 mM sodium acetate, 1 M NaCl, pH 4.8 Elution buffer: 50 mM sodium acetate, 1 M NaCl, pH 4.8
Flow velocity: 285 cm/h Flow velocity: 624 cm/h Flow velocity: 645 cm/h
Gradient: 0% to 100% 0 CV, 100% 5 CV Gradient: 0% to 100% 0 CV, 100% 5 CV Gradient: 0% to 100% 0 CV, 100% 5 CV
Residence time: 2 min Residence time: 2 min Residence time: 2 min

(A) (B) (C)

3500
3500
3500
200200
200
4000
4000
4000
3000
3000
3000 150150
150
4000
4000
4000 150150
150
2500
2500
2500 150150
3000 150
3000
Conductivity (mS/cm)

Conductivity (mS/cm)

Conductivity (mS/cm)
Conductivity

Conductivity

Conductivity
3000
Conductivity (mS/cm)

Conductivity (mS/cm)

Conductivity (mS/cm)
2000
2000
2000 3000
3000
3000
100100
100 100100
A280

A280

A280
280

280

280
100
AA280

A280

A280
100100
A

A
1500
1500
1500 2000
2000
2000 100
2000
2000
2000
(mS/cm)

(mS/cm)

(mS/cm)
Re-equilibration

RRee--eeqquuiilliibbrraattiioonn
Re-equilibration
1000

RRee--eeqquuiilliibbrraattiioonn
1000
Re-equilibration
RRee--eeqquuiilliibbrraattiioonn

1000
50 50
50 50 50
50
1000
1000
1000 50 50
50 1000
1000
1000

Eluti n

EElluuttii nn
500500
Eluti n
EElluuttii nn
Elution
EElluuttiioonn

500

W ash
Waasshh
W ash
Waasshh
W ash
Waasshh

Inl t
IInnll tt
Inlet

P
IInnlleett

PP
P
PP
CIP
CCIIPP

W
W
W

0 00
0 00 0 00 0 00 0 00 0 00
0 00 20 20
20 40 40
40 60 60
60 80 80
80 100100
100 0 00 20 20
20 40 40
40 60 60
60 80 80
80 100100
100 0 00 20 20
20 40 40
40 60 60
60 80 80
80 100100
100
Time (min)
Time (min)
Time (min) Time (min)
Time (min)
Time (min) Time (min)
Time
Time (min)
(min)

Fig 3.39. A 200-fold scale up of α-chymotrypsin on Capto S packed in (A) Tricorn 5/100, (B) XK 16/40 and, (C) AxiChrom columns.

102
Intermediate purification of insulin
Capto SP ImpRes was used for intermediate purification where the purpose was to separate insulin from components
remaining from the initial capture step. The resulting chromatogram is shown in Figure 3.40. Insulin (first peak) was
well-resolved from truncated insulin impurities (middle peak) and C-peptides (third peak). Pooled fractions were analyzed
by RPC, which showed an increase of insulin purity from 64% to 91%. The truncated insulin content was reduced from
11.5% to 2.8%.

Column: Tricorn 5/50 CV 1 mL

Medium: Capto SP ImpRes
Sample: 18 mg cleaved insulin*
Start buffer: 50 mM acetate, 47.5% ethanol, pH 4
Elution buffer: 50 mM acetate, 47.5% ethanol, 1 M NaCl, pH 4
Elution: First step: 47.5% ethanol, 130 mM NaCl, pH 4 (10 CV)
Second step: 47.5% ethanol, 1 M NaCl, pH 4 (5 CV)
Flow rate (flow velocity): 0.4 mL/min (120 cm/h)
Residence time: 2.5 min
* Kindly provided by Biomm S.A. (Brazil).

Desthreonine/desamido-insulin

900 100
Pooled fractions C-peptide
800 90

700 80
Conductivity (mS/cm)

600 70
A280 (mAU)

60
500
50
400
40
300
30
200 20
100 10

0 0
0 10 20 30 40 50
Volume (mL)

Fig 3.40. The intermediate step purification of insulin on Capto SP ImpRes removes contaminants such as desthreonine- and desamido-insulin.

103
Polishing of MAb Medium:
Column:
Capto S ImpAct
Tricorn 5/100, bed height 10 cm
To evaluate Capto S ImpAct for removal of aggregates, four different MAb purified on MabSelect SuRe™ medium were Sample: MAb E, purified on MabSelect SuRe medium
run using linear gradient elution in Tricorn columns. Fractions from the elution peaks were collected and analyzed by Sample load: 85 mg/mL medium
Start buffer: 50 mM sodium acetate, pH 5.3
analytical SEC for aggregate content. HCP and protein A content were analyzed using a Gyrolab™ workstation and a
Elution buffer: 50 mM sodium acetate + 500 mM NaCl, pH 5.3
commercial ELISA assay, respectively. As can be seen in Table 3.24, Capto S ImpAct demonstrates effective aggregate Flow rate: 0.35 mL/min, residence time 5.4 min
and HCP removal at a high monomer recovery. Figure 3.41 shows the chromatogram for MAb E, illustrating that Gradient: Linear, 0 to 350 mM NaCl in 20 CV
aggregates (green) elute at the tail of the elution peak. For this MAb, the aggregate level was reduced to 0.6% at 90%
monomer recovery. The initial aggregate content was 2%.
3500 70 200
Table 3.24. Results from the purification of four MAb using Capto S ImpAct 3000 60 175

Conductivity (mS/cm)
MAb A MAb C MAb D MAb E 150
2500 50

Aggregates (%)
DBC (mg/mL medium) 108 92 118 122 125
2000 40

mAU
Load (mg/mL medium) 80 64 80 85 100
Start aggregate content (%) 7 2 4 2 1500 30
75
Aggregates at 90% monomer recovery (%) 0.9 0.6 1.2 0.6 1000 20
50
Start HCP content (ppm) 34 1800 300 454
500 10 25
HCP at 90% monomer recovery (ppm) 8 42 25 43
0 0 0
Start protein A content (ppm) 4 1 1 <1
0 20 40 60 80 100
Protein A at 90% monomer recovery (ppm) <1 <1 <1 <1 Volume (mL)
Elution pool volume (CV) 5.1 5.4 4.0 6.3 Fig 3.41. Chromatogram from purification of MAb E using Capto S ImpAct in a Tricorn 5/100 column.
Elution pool concentration at 90% 13.6 10.6 17.8 12.3 Histogram in green represents aggregates in fractions. SEC was used for aggregate content analysis, using a
monomer recovery (mg/mL) prepacked Superdex™ 200 Increase 10/300 GL column.

104
Performing a separation First-time use or after long-term storage
Guidelines for selection of media, buffer, pH and ionic strength conditions and method optimization are given in Chapter 2.
Use the instructions given here as a basis from which to optimize a separation. Flow rates: 1 mL/min (HiTrap 1 mL), 5 mL/min (HiTrap 5 mL), 1.2 mL/min
Correct sample and buffer preparation is essential in order to achieve optimal separation and avoid any deterioration

(HiScreen 4.7 mL).
in column performance. Samples must be fully dissolved and free from particles or other material likely to interfere 1. Remove the stopper and connect the column to the system (or syringe)
with the separation. Refer to Chapter 2 and Appendix 1 for recommendations and advice on sample preparation. with a drop-to-drop connection to avoid introducing air into the column.
Filter buffers after all salts and additives have been included. Use high quality water and chemicals. Filter solutions
 2. R
 emove the snap-off end at the column outlet and wash with 1 column
using filters of 1 μm or less. To avoid formation of air bubbles in a packed column, maintain buffers and columns at volume (CV) of distilled water. This step ensures removal of ethanol and
constant temperature before and during a run. avoids the precipitation of buffer salts upon exposure to ethanol. The step
The pH of the start buffer should be at least 0.5 to 1.0 pH unit above the pI of the target substance when using

can be omitted if precipitation is not likely to be a problem.
an anion exchanger (Q, DEAE) and 0.5 to 1.0 pH unit below the pI of the target substance when using a cation 3. Wash with 5 CV of start buffer.
exchanger (S, SP).
4. Wash with 5 CV of elution buffer.
For samples with unknown charge properties, try the following:
5. Wash with 5 CV of start buffer.
Anion exchange (Q)
Start buffer: 20 mM Tris-HCl, pH 8.0
Elution buffer: 20 mM Tris-HCl, 1 M NaCl, pH 8.0
Cation exchange (SP)
Start buffer: 50 mM sodium acetate, pH 5.0
Elution buffer: 50 mM sodium acetate, 1 M NaCl, pH 5.0
or
Start buffer: 50 mM 2-(N-Morpholino)ethanesulfonic acid (MES), pH 6.0
Elution buffer: 50 mM MES, 1 M NaCl, pH 6.0
Users of ÄKTA systems with automatic buffer preparation functionality can select one of the buffer recipes


recommended for anion exchange chromatography at pH 8.0 or cation exchange chromatography at pH 6.0,
see ÄKTA Laboratory-scale Chromatography Systems: Instrument Management Handbook, 29010831.

105
Separation by gradient elution Separation by step elution
Linear ionic strength gradients should always be used for method development or when starting with an unknown Reduce separation time and buffer consumption by transferring to a step elution.
sample. Linear ionic strength gradients are easy to prepare and very reproducible when generated by a suitable
chromatography system. The results obtained can then serve as a base from which to optimize the separation. Flow rates: 1 mL/min (HiTrap 1 mL), 5 mL/min (HiTrap 5 mL), 1.2 mL/min
(HiScreen 4.7 mL). Collect fractions throughout the separation.
Flow rates: 1 mL/min (HiTrap 1 mL), 5 mL/min (HiTrap 5 mL), 1.2 mL/min (HiScreen 4.7 mL). Collect fractions 1. Equilibrate the column with 5 to 10 CV of start buffer until the UV baseline,
throughout the separation. eluent pH, and conductivity are stable.
1. Equilibrate the column with 5 to 10 CV of start buffer or until the UV baseline, eluent pH, and conductivity are 2. A
 djust the sample to the chosen starting pH and conductivity and apply to the
stable. column.
2. Adjust the sample to the chosen starting pH and conductivity and apply to the column. 3. W
 ash with 5 to 10 CV of start buffer or until no material appears in the
3. Wash with 5 to 10 CV of start buffer or until no material appears in the effluent. effluent.
4. B
 egin elution using a gradient volume of 10 to 20 CV and an increasing salt concentration up to 500 mM NaCl 4. E
 lute the target protein with 5 CV of start buffer including NaCl at chosen
(50% elution buffer). concentration.
5. Wash with 5 CV of 1 M NaCl (100% elution buffer) to elute any remaining ionically bound material. 5. I f necessary: repeat step 4 at higher NaCl concentrations until the target
protein has been eluted.
6. R
 e-equilibrate with 5 to 10 CV of start buffer or until the UV baseline, eluent pH, and conductivity reach the
required values. 6. W
 ash with 5 CV of a high salt solution (1 M NaCl in start buffer) to elute any
remaining ionically bound material.
7. R
 e-equilibrate with 5 to 10 CV of start buffer or until the UV baseline, eluent
pH, and conductivity reach the required values.

Save time by using higher flow rates during the high salt wash and
re-equilibration steps.
Do not exceed the maximum recommended flow and back pressure for
the column.

106
Cleaning Storage
Correct preparation of samples and buffers, including a high salt wash (1 to 2 M NaCl) after each purification, should Wash with 2 CV of distilled water followed by 2 CV of 20% ethanol (Capto Q, Capto DEAE,
maintain columns in good condition. However, reduced performance, increased back pressure or blockage indicates that Capto Q ImpRes) or 20% ethanol containing 200 mM sodium acetate (Capto S,
the medium needs cleaning. Capto SP ImpRes, Capto S ImpAct). Store at 4°C to 30°C. Do not freeze. Ensure that
the column is sealed well to avoid drying out.
Removing common contaminants
Capto media with multimodal functionality
Flow rates: 1 mL/min (HiTrap 1 mL), 5 mL/min (HiTrap 5 mL), 1.2 mL/min (HiScreen 4.7 mL). Some Capto media have ligands with multimodal functionality, where the ligand
interacts with the target protein through two or more modes of action. This
1. Wash with at least 2 CV of 2 M NaCl.
multimodal functionality gives a different selectivity compared to standard ion
2. Wash with at least 4 CV of 1 M NaOH. exchangers, which allows multimodal ion exchangers a wider window of operation
3. Wash with at least 2 CV of 2 M NaCl. in circumstances where traditional media are not as effective as desired. Such
circumstances can be encountered, for example, when the loading conductivity
4. Rinse with at least 2 CV of distilled water. of the sample is too high for traditional IEX media, when there is a need to reduce
5. Wash with at least 5 CV of start buffer until eluent pH and conductivity have reached the required values. the number of purification steps, or when the selectivity of traditional media is
insufficient to provide the required purity of the target protein. See the Multimodal
Chromatography Handbook, 29054808 for more information about our multimodal
To remove precipitated proteins, lipids, hydrophobically bound proteins, or lipoproteins, refer to Appendix 1. Capto media.

Chemical stability
For daily use, Capto media are stable in all common, aqueous buffers, 1 M NaOH, denaturing agents (8 M urea,
6 M guanidine hydrochloride), 70% ethanol, 30% acetonitrile, and with additives such as nonionic detergents.
Avoid cationic detergents with Capto S, Capto SP ImpRes, and Capto S ImpAct.
Avoid anionic detergents with Capto Q and Capto Q ImpRes.
Avoid oxidizing agents.

107
04
Ion exchange
in a Purification
Strategy (Cipp)

108
To ensure efficient, reproducible purification giving the required degree of purity, it is beneficial to develop
a multistep process using the purification strategy of Capture, Intermediate Purification, and Polishing (Cipp),
shown in Figure 4.1.
Cipp is used in both the pharmaceutical industry and in the research laboratory to ensure faster method
development, a shorter time to pure product and good economy. This chapter gives a brief overview of this
approach which is recommended for any multi-step protein purification. The Strategies for Purification Handbook
from Cytiva is a definitive guide for planning efficient and effective protein purification strategies. An important
first step for any purification is correct sample preparation and this is covered in more detail in Appendix 1 and
Chapter 2.
IEX plays a significant and highly flexible role in most multistep purification schemes. If a specific affinity
medium is not available or if little is known about the target molecule, IEX is recommended as the first step Polishing
Achieve final high-level purity
to consider for any purification. The technique can be used for capture, intermediate purification, or polishing,
according to the demands of the specific application. Since IEX offers different selectivities (using anion or
cation exchangers) and since the pH of the purification can be modified to alter the charge characteristics of Intermediate purification

Purity
the sample components, it is possible to use the technique more than once in the same purification scheme. Remove bulk impurities
In addition, IEX can be used with step elution for a rapid capture step or with gradient elution to achieve the
highest resolution in a polishing step.
Capture
Isolate, concentrate, and stabilize

Preparation, extraction, clarification

Step

Fig 4.1. Preparation and Cipp.

109
Applying Cipp
Imagine the purification has three phases: Capture, Intermediate Purification, and Polishing.
Assign a specific objective to each step within the purification process.
The issues associated with a particular purification step will depend greatly upon the properties of the starting material.
Thus, the objective of a purification step will vary according to its position in the process.
In the capture phase, the objectives are to isolate, concentrate, and stabilize the target product. The product should be
concentrated and transferred to an environment that will conserve potency/activity.
During the intermediate purification phase, the objectives are to remove most of the bulk impurities, such as other
proteins and nucleic acids, endotoxins, and viruses.
In the polishing phase, most impurities have already been removed. The objective is to achieve final purity by removing
any remaining trace impurities or closely related substances.
The optimal selection and combination of purification techniques for Capture, Intermediate Purification, and
Polishing is crucial for an efficient purification.
C does not mean that there must always be three purification steps. For example, capture and intermediate
ipp
purification might be achievable in a single step, as might intermediate purification and polishing. Similarly, purity
demands can be so low that a rapid capture step is sufficient to achieve the desired result. For purification of
therapeutic proteins, a fourth or fifth purification step might be required to fulfill the highest purity and safety
demands. The number of steps used will always depend upon the purity requirements and intended use of the protein.

110
Selection and combination of purification techniques Resolution
Proteins are purified using purification techniques that separate according to differences in specific properties, as shown
in Table 4.1.

Table 4.1. Protein properties used during purification

Protein property Technique

Size Size exclusion chromatography (SEC)
Charge Ion exchange chromatography (IEX)
Hydrophobicity Hydrophobic interaction chromatography (HIC), Reversed phase chromatography (RPC)
Biorecognition (ligand specificity) Affinity chromatography (AC)

Speed Recovery
There are four important performance parameters to consider when planning each purification step: resolution, capacity,
speed, and recovery. Optimization of any one of these four parameters can be achieved only at the expense of the others,
and each purification step will be a compromise (Fig. 4.2). The importance of each parameter will vary depending on
whether a purification step is used for capture, intermediate purification, or polishing. Purification methods should be Capacity
selected and optimized to meet the objectives for each purification step.
Fig 4.2. Key performance parameters for protein purification. Each purification step should be optimized for
one or two of the parameters.

111
Capacity, in the simple model shown, refers to the amount of target protein loaded Table 4.2. Suitability of purification techniques for Cipp
during purification. In some cases the amount of sample that can be loaded will be
Typical characteristics Purification phase
limited by volume (as in SEC) or by large amounts of contaminants rather than the
amount of the target protein. Sample start Sample end
Method Resolution Capacity Capture Intermediate Polishing conditions conditions
Speed is most important at the beginning of purification where contaminants such
AC +++ +++ +++ ++ + Various binding Specific elution
as proteases must be removed as quickly as possible.
or or conditions conditions
Recovery becomes increasingly important as the purification proceeds because of ++ ++
the increased value of the purified product. Recovery is influenced by destructive IMAC +++ ++ +++ ++ + Low concentration of High concentration of
processes in the sample and by unfavorable conditions on the column. imidazole, pH > 7.0 imidazole, 500 mM NaCl,
Resolution is achieved by the selectivity of the technique and the efficiency and pH > 7.0
selectivity of the chromatography matrix in producing narrow peaks. In general, SEC ++ + + +++ Most conditions Buffer exchange
resolution is most difficult to achieve in the final stages of purification when acceptable, limited possible,
impurities and target protein are likely to have very similar properties. sample volume diluted sample

Select a technique to meet the objectives for the purification step. IEX +++ +++ +++ +++ +++ Low ionic strength. High ionic strength

pH depends on protein or pH changed
Choose logical combinations of purification techniques based on the main and IEX type
benefits of the technique and the condition of the sample at the beginning or HIC +++ ++ ++ +++ +++ High ionic strength, Low ionic strength
end of each step. addition of salt required
A guide to the suitability of each purification technique for the stages in Cipp is shown RPC +++ ++ + ++ Ion-pair reagents and Organic solvents (risk for
in Table 4.2. organic modifiers might loss of biological activity)
be required

Minimize sample handling between purification steps by combining techniques to avoid the need for sample
conditioning. The product should be eluted from the first column in conditions suitable for the start conditions of
the next column (see Table 4.2).
Ammonium sulfate, often used for sample clarification and concentration (see Appendix 1), leaves the sample in
a high salt environment. Consequently HIC, which requires high salt to enhance binding to the media, becomes
the excellent choice as the capture step. The salt concentration and the total sample volume will be significantly
reduced after elution from the HIC column. Dilution of the fractionated sample or rapid buffer exchange using a
desalting column will prepare it for the next IEX or AC step.
SEC is a nonbinding technique unaffected by buffer conditions, but with limited volume capacity. SEC is well-suited
for use after any of the concentrating techniques (IEX, HIC, AC) since the target protein will be eluted in a reduced
volume and the components from the buffer will not affect the size exclusion process.
112
Selection of the final strategy will always depend upon specific sample properties and (NH4)2SO4 precipitation
the required level of purification. Logical combinations of techniques are shown in
Figure 4.3.
Capture
For any capture step, select the technique showing the most effective binding


to the target protein while binding as few of the contaminants as possible,

AC AC AC IEX HIC
HIC
that is, the technique with the highest selectivity and/or capacity for the target
protein.
Intermediate
A sample is purified using a combination of techniques and alternative
IEX HIC IEX
IEX
selectivities. For example, in an IEX-HIC-SEC strategy, the capture step selects HIC
according to differences in charge (IEX), the intermediate purification step according
to differences in hydrophobicity (HIC), and the final polishing step according to
differences in size (SEC). Polishing
If nothing is known about the target protein, use IEX-HIC-SEC. This combination SEC or GF GF


of techniques can be regarded as a standard protocol. IEX SEC SEC SEC

 Consider the use of both anion and cation exchange chromatography to give
different selectivities within the same purification strategy.
Fig 4.3. Examples of logical combinations of chromatography steps.

113
Ion exchange as a capture step
When IEX is used as a capture step, the objective is to quickly adsorb the protein(s) of interest from the crude sample and
isolate them from critical contaminants such as proteases and glycosidases. The target protein(s) are concentrated and
transferred to an environment which will conserve potency/activity. Removal of other critical contaminants may also be
achieved by careful optimization of pH and elution conditions.
The focus is on capacity and speed in a capture step. It is advisable to compromise on the potential resolution that can
be achieved by an IEX separation in order to maximize the capacity and/or speed of the separation in this first step.
IEX media for capture steps should offer high speed and high capacity. The choice of a suitable IEX medium depends on
the sample properties and the scale of the purification (see also Chapter 5, Large-scale purification). Examples of IEX
capture media include:
1. Sepharose Fast Flow (90 µm particle size) – good resolution for crude mixtures at any scale using flow velocities up
to 300 cm/h and offering a wide range of selectivities.
2. C
 apto (90 µm particle size) – excellent pressure-flow properties for large-scale applications using flow velocities up
to 700 cm/h.
3. S
 epharose XL (90 µm particle size) – high capacity, good resolution for capture of selected proteins at laboratory and
process scale using flow velocities up to 300 cm/h.
4. S
 epharose Big Beads (200 µm particle size) – for viscous samples that preclude the use of IEX media with smaller
particle size, using flows up to 300 cm/h, or for fast separations of very large sample volumes when resolution is of less
importance, using flows up to 1000 cm/h.
If only milligram quantities of product are needed and the capture step will not be scaled up, use high-performance
media such as SOURCE or Sepharose High Performance according to the capacity required.
Select start conditions that avoid adsorption of contaminants and so help to maximize the binding capacity for the
target protein(s). This will facilitate a fast, simple step elution of the concentrated target protein(s).

114
Capture using Sepharose Q XL
Figure 4.4 shows optimization of a capture step used for purification of a recombinant enzyme, deacetooxycephalosporin C synthase (DAOCS). Since this
enzyme is oxygen-sensitive, it was important to rapidly remove the most harmful contaminants from the relatively unstable target protein. The isoelectric
point of DAOCS (pI = 4.8) made an anion exchanger the most suitable choice. Columns from the HiTrap IEX Selection Kit were screened to select the most
suitable medium (results not shown) before optimizing the separation on a larger HiPrep Q XL 16/10 column. After a linear gradient elution (A), a multi-stepwise
elution was tested (B). Since this caused a broader elution peak, it was decided to use a simple stepwise elution400 (C). In comparison with the gradient elution,
the speed of the purification increased, buffer consumption decreased, and the target protein was eluted in a smaller volume (note that the x-axes differ 80 3000 80
between (A) and (C)).

Conductivity (mS/cm)

Conductivity (mS/cm)
300

A 280 nm

A 280 nm
60 60
2000
Column: HiPrep Q XL 16/10 200
Sample: Clarified E. coli extract
40 40
Sample volume: 40 mL
Start buffer: 50 mM Tris-HCl, 1 mM EDTA, pH 7.5; 2 mM DTT, 200 mM benzamidine-HCl, 0.2 mM PMSF 100 1000
Elution buffer: Start buffer + 1 M NaCl 20 20
Flow rate (flow velocity): 10 mL/min (300 cm/h)
0
System: ÄKTA system 0
0 0
0 100 200 300 0 100 200
(A) (B) (C) Volume (mL) Volume (mL)

400
400
8080 3000
3000 8080
Conductivity (mS/cm)

Conductivity (mS/cm)
Conductivity (mS/cm)

Conductivity (mS/cm)
300
300 80
3000
A 280 nm

A 280 nm

Conductivity (mS/cm)
A 280 nm

A 280 nm

6060 6060
2000
2000

A 280 nm
60
200
200 2000
4040 4040
40
100
100 1000
1000 1000
2020 2020 20
00 0
00
00 00 0
00 100
100 200
200 300
300 00 100
100 200
200 0 50 100 150 200
Volume
Volume (mL)
(mL) Volume
Volume (mL)
(mL) Volume (mL)

Fig 4.4. Capture step using IEX and optimization of conditions. The elution position of DAOCS is shaded.

8080
3000
3000
Conductivity (mS/
Conductivity (mS/

115
A 280 nm
A 280 nm

6060
2000
2000

4040
1000
1000
Ion exchange for intermediate purification
When IEX is used for intermediate purification, the objective is to remove most of the significant impurities such as
proteins, nucleic acids, endotoxins, and viruses.
In a typical intermediate purification step, speed is less critical since sample volume has been reduced and damaging
contaminants have been removed during capture. Focus is on capacity and resolution in order to maintain productivity
(amount of target protein processed per column in unit time) and to achieve as high selectivity (purity) as possible.
Consequently, a gradient elution will usually be required.
Use a technique with a selectivity that is complementary to that used in the capture step.
IEX media for intermediate purification should offer high capacity and high resolution with a range of complementary
selectivities. The choice of a suitable IEX medium depends on the sample properties and the scale of the purification
(see also Chapter 5, Large-scale purification). Examples of IEX media for intermediate purification:
1. Sepharose High Performance (34 µm particle size) — high resolution in laboratory scale using flow velocities
up to 150 cm/h.
2. C
 apto ImpAct and Capto ImpRes (50 µm and 40 µm particle size) — excellent pressure-flow properties for large-scale
applications using flow velocities up to 200 cm/h.
3. S
 OURCE 15 (15 µm particle size) — high throughput, high resolution for laboratory or large-scale applications using
flow velocities up to 1800 cm/h.
4. S
 OURCE 30 (30 µm particle size) — an alternative to SOURCE 15 for large-scale applications when flow velocities
up to 2000 cm/h can be used.
5. S
 epharose Fast Flow (90 µm particle size) — fast separations using flow velocities up to 300 cm/h, broad range
of selectivities.
If only milligram quantities are required and the intermediate purification step will not be scaled-up, use MonoBeads
or MiniBeads according to the capacity required.
 Optimize the selectivity of the medium to ensure high binding capacity and to maximize resolution. Use continuous
gradient or multistep elution conditions.

116
Intermediate purification using Capto S ImpAct Column:
Medium:
Tricorn 5/100
Capto S ImpAct (B/E mode)
Capto S ImpAct was used for intermediate purification in a three-step MAb-purification process (Fig 4.5). Sample: MAb A in 50 mM sodium acetate, pH 5.3 (6.8 mS/cm)
Load: 64 g MAb/l medium (70% of QB10)
Residence time: 5.4 min
Binding buffer: 50 mM sodium acetate, pH 5.3 (6.8 mS/cm)
MabSelect SuRe LX Wash: 5 CV of binding buffer
Elution buffer: 50 mM sodium acetate, pH 5.3, 0 to 350 mM NaCl in 20 CV
System: ÄKTA system

100
Capto S ImpAct (bind/elute [B/E] mode) 5000 8000

80 7000
4000
Capto adhere ImpRes (flowthrough [FT] mode) 6000

Aggregate (% )

HCP (ng/mL)
3000 60

A280 (mAU)
5000

4000
2000 40
Fig 4.5. Three-step purification deploying MabSelect SuRe LX for capture, Capto S ImpAct for intermediate purification, and Capto Q for polishing. 3000

1000 2000
20
The intermediate purification of MAb A resulted in reduction of aggregate concentration from 2% to 0.6% and host cell
1000
protein (HCP) concentration from 1800 ppm to 42 ppm. The yield of MAb A monomer in this step was 90%.
0 0 0
The high selectivity of Capto S ImpAct between MAb monomer, aggregates, and HCP can be seen from the chromatograms
in Figure 4.6. 0 10 20 30 40 50 60 70 80
Volume (mL)

Fig 4.6. Intermediate purification of MAb A using Capto S ImpAct. Aggregates eluted in the tail of the elution
peak (red histogram), whereas most of the HCP (green histogram) eluted after the elution peak (blue UV trace).

117
Ion exchange as a polishing step Column:
Sample:
Mono S 5/50 GL
14.5 mL from HiPrep 26/10 Desalting
When IEX is used for polishing, most impurities have been removed except for trace amounts or closely related substances Start buffer: 20 mM MES pH 6.5, 1 mM EDTA, 2 mM MgCl2, 1 mM DTT
Elution buffer: 20 mM MES pH 6.5, 1 mM EDTA, 2 mM MgCl2, 1 mM DTT, 1 M NaCl
such as structural variants of the target protein, nucleic acids, viruses, or endotoxins. The purpose of the separation is to Flow rate: 1 mL/min
reduce these variants and trace contaminants to acceptable levels for the application. In contrast to capture steps where Gradient: 0% to 100% elution buffer, 20 CV
a fast, high capacity, step elution is most commonly used, a polishing step will therefore focus on achieving the highest
possible resolution. An example of this approach is shown in Figure 4.7 in which Mono S 5/50 GL was used to separate
a recombinant DNA binding protein, transposase TniA, from minor contaminants remaining after partial purification by 1200
anion exchange and heparin affinity chromatography.
1000
IEX media for polishing steps should offer high resolution. The choice of a suitable IEX medium depends on the sample
properties and the scale of the purification (see also Chapter 5, Large-scale purification). Examples of IEX polishing 800

A 280 nm
media:
1. MiniBeads (3 µm particle size) — polishing at microscale when highest resolution is essential. 600

2. M
 onoBeads (10 µm particle size) — polishing at laboratory scale when highest resolution is essential and 400
a higher capacity than MiniBeads is required.
3. S
 OURCE 15 (15 µm particle size) — rapid, high resolution polishing for laboratory or large-scale applications using flow 200
velocities up to 1800 cm/h.
0
4. S
 OURCE 30 (30 µm particle size) — an alternative to SOURCE 15 for large-scale applications when flow velocities up to 0. 0 10 .0 20 .0 30 .0 40 .0
2000 cm/h can be used. Volume (mL)
5. S
 epharose High Performance (34 μm particle size) — high resolution at laboratory scale using flow velocities Fig 4.7. High-resolution cation exchange chromatography on Mono S 5/50 GL. The desalted sample was
up to 150 cm/h. applied to Mono S 5/50 GL, and TniA was eluted as a sharp peak at approximately 500 mM NaCl.

6. C
 apto ImpAct and Capto ImpRes (50 µm and 40 µm particle size) — excellent pressure-flow properties for
large-scale applications using flow velocities up to 200 cm/h.
Optimize the gradient elution to maximize selectivity. Use high efficiency media with small bead sizes to improve
resolution.

118
Alternative techniques for polishing Column:
Sample:
XK 16/60 packed with Superdex 75 prep grade
Partly purified ZZ-brain IGF
Most commonly, separations by charge, hydrophobicity, or affinity will have been used in earlier stages of a purification Sample load: 1.0 mL
strategy so that high-resolution SEC is ideal for the final polishing step. The product can be purified and transferred into Buffer: 300 mM ammonium acetate, pH 6.0
Flow rate (flow velocity): 0.5 mL/min (15 cm/h)
the required buffer in one step and dimers and aggregates can be removed, as shown in Figure 4.6.
SEC is also the slowest of the chromatography techniques and the size of the column determines the volume of sample monomeric ZZ-Brain IGF
0.01
that can be applied. It is therefore most logical to use SEC after techniques that reduce sample volume so that smaller
columns can be used. Media for polishing steps should offer the highest possible resolution. Superdex Increase is the first
choice at laboratory scale and Superdex prep grade for large-scale applications.

A280 nm
RPC can also be considered for a polishing step, provided that the target protein can withstand the run conditions.
RPC separates proteins and peptides on the basis of hydrophobicity and is a high selectivity (high resolution) technique, 0.005
usually requiring the use of organic solvents. The technique is widely used for purity check analyses when recovery
of activity and tertiary structure are not essential. Since many proteins are denatured by organic solvents, RPC is not
generally recommended for protein purification because recovery of activity and return to a correct tertiary structure can Vo Vt
be compromised. However, in the polishing phase, when the majority of protein impurities have been removed, RPC can
be an excellent technique, particularly for small target proteins that are not often denatured by organic solvents. 0
Fraction 1 2 3 4 5 6
0 1 2 3 4
Time (h)

Fig 4.6. Final polishing step: separation of dimers and multimers on Superdex 75 prep grade.

119
05
Large-scale
purification

120
The BioProcess chromatography media family includes media widely used by biopharmaceutical manufacturers. Support
for these products comprises of validated manufacturing methods, secure long-term chromatography media supply,
safe and easy handling, and regulatory support files (RSF) to assist process validation and submissions to regulatory
authorities. In addition, the Fast Trak Training and Education team provides high-level hands-on training for all key
aspects of bioprocess development and manufacturing.
All BioProcess media have high chemical stability to allow efficient cleaning/sanitization procedures and validated
packing methods established for a wide range of large-scale columns.

BioProcess media for IEX

BioProcess media for ion exchange chromatography are designed for large scale purification and use in industrial
processes. Examples of BioProcess media for IEX purification are shown in Table 5.1 (the properties and handling of each
medium are described in detail in Chapter 3).
Most of the media, except Sepharose Big Beads, are available in HiTrap and HiScreen formats for development of efficient
and robust purification parameters before scaling up. Some of the media are also available in high-throughput process
development (HTPD) formats, such as PreDictor 96-well filter plates for fast and easy parallel screening of running
conditions and PreDictor RoboColumn (miniature columns for use with a robotic station) for testing of, for example, DBC.
By using these small-scale formats in the early stages of process development, valuable time is saved and buffer and
sample consumption reduced.
The range of BioProcess media includes capture media such as Capto, Sepharose Fast Flow, and Sepharose XL. These
media can be used at high flow rates and have high dynamic binding capacities. Media with smaller beads, such as
Capto ImpRes, Capto ImpAct, Sepharose High Performance, and SOURCE give high final resolution and are suitable for
polishing purification (see also Chapter 4 for the principle of capture and polishing purification).

121
Table 5.1. Examples of BioProcess chromatography media for IEX

Medium Chromatography method Average particle size, d50V (µm) Recommended flow rate1 Protein binding capacity1 Main usage
Q Sepharose Big Beads Strong anion 200 High low Capture
SP Sepharose Big Beads Strong cation 200 High low Capture
Capto Q Strong anion 90 High High Capture
Capto DEAE Weak anion 90 High Medium Capture
Capto S Strong cation 90 High High Capture
Q Sepharose Fast Flow Strong anion 90 Medium Medium Capture
DEAE Sepharose Fast Flow Weak anion 90 Medium Medium Capture
ANX Sepharose 4 Fast Flow (high sub) Weak anion 90 Medium Medium Capture
SP Sepharose Fast Flow Strong cation 90 Medium Medium Capture
CM Sepharose Fast Flow Weak cation 90 Medium Medium Capture
Q Sepharose XL Strong anion 90 Medium High Capture
SP Sepharose XL Strong cation 90 Medium High Capture
Capto Q ImpRes Strong anion 40 Medium Medium Polishing
Capto SP ImpRes Strong cation 40 Medium Medium Polishing
Capto S ImpAct Strong cation 50 Medium High Polishing
SOURCE 30Q Strong anion 30 High Medium Polishing
SOURCE 30S Strong cation 30 High Medium Polishing
SOURCE 15Q Strong anion 15 High Medium Polishing
SOURCE 15S Strong cation 15 High Medium Polishing
1
Recommended flow rate and protein binding capacity are expressed as high, medium, or low for easy comparison. The values for each medium can be found in Chapter 3.

122
Capture purification (A) (B)
160 200
General capture purification is performed using Capto and Sepharose Fast Flow.

Dynamic binding capacity (mg/mL)

Dynamic binding capacity (mg/mL)

140 180
When IEX is used as a capture step, the objective is to quickly adsorb the protein of Capto Q
interest from the crude sample and remove critical contaminants such as proteases. 160 Capto S
120
For modern industrial applications, Capto media are usually the preferred option 140
having benefits such as high rigidity and ability to run at high flow rates. Figure 5.1 100 120
shows a comparison of media properties. Capto media give increased DBC over a 80 100
wide range of residence times due to excellent mass transfer properties. Further, SP Sepharose Fast Flow
Q Sepharose Fast Flow 80
60
a high DBC contributes to shortening the overall processing time as the total number 60
of cycles can be reduced. 40
40
Sepharose Fast Flow media are available with a wide range of ion exchange groups 20 20
and are still widely used in biopharmaceutical processes. 0
0
The capture media Q Sepharose XL and SP Sepharose XL have greater binding 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9
capacity compared with other Sepharose media. The high binding capacity has been Residence time (min) Residence time (min)
obtained by binding the ionic group to long, flexible dextran chains coupled to the (C)
agarose matrix. 200

Dynamic binding capacity (mg/mL)

180
Sepharose Big Beads are designed for capture of large volumes of crude and/or Capto DEAE
viscous sample. Due to the large bead size of the Sepharose Big Beads matrix (200 µm 160
average particle size), a lower resolution compared with other capture media can be 140
expected. 120
100
80
DEAE Sepharose Fast Flow
60
40
20
0
0 1 2 3 4 5 6 7 8 9
Residence time (min)

Fig 5.1. Dynamic binding capacity (DBC) as a function of residence time for Capto and corresponding Sepharose Fast Flow media. Proteins: (A) BSA;
(B) α-chymotrypsin; (C) amyloglucosidase. Residence times below 2 min are not possible for Sepharose Fast Flow in large-scale columns due to lower
pressure/flow properties than for Capto media.

123
Polishing purification Columns: HiScreen Capto Q ImpRes, HiScreen Q HP, 4.7 mL
Sample:  5 mL apo-transferrin (0.3 mg/mL), α-lactoalbumin (0.4 mg/mL), soybean trypsin inhibitor (0.6 mg/mL) in start buffer
The polishing purification is performed using chromatography media with smaller Start buffer: 50 mM Tris, pH 7.4
average bead size, such as Capto ImpRes, Capto ImpAct, and SOURCE media. Elution buffer: 50 mM Tris, 500 mM NaCl, pH 7.4
Flow rate (flow velocity): HiScreen Capto Q ImpRes, 2.3 mL/min (300 cm/h); HiScreen Q HP, 1.2 mL/min (150 cm/h)
In contrast to capture purification where a fast, high-capacity step elution is most
Gradient: 0% to 100% elution buffer in 20 CV
commonly used, a polishing purification will focus on achieving the high resolution Residence time: HiScreen Capto Q ImpRes, 2 min; HiScreen Q HP, 4 min
(resulting in high purity).
(A) HiScreen Capto Q ImpRes (B) HiScreen Q HP
Although the charged groups of the S, SP, Q, and DEAE ligands are identical between
different IEX media, minor differences in selectivity can occur due to differences in 300 50 300 50
base matrix, ligand density, ligand composition, and surface extenders. Capto ImpRes
media are designed for high resolution and high throughput during polishing steps.
A comparison with Q Sepharose High Performance in HiScreen columns showed 250 250
40 40

Conductivity (mS/cm)

Conductivity (mS/cm)
similar resolution results, although HiScreen Capto Q ImpRes was run at
300 cm/h — twice the flow velocity of HiScreen Q HP (Fig 5.2). Capto ImpRes media
200 200
provide improved performance over Sepharose when scaling up due to their good

A280 (mAU)
30 30

A280 (mAU)
pressure/flow properties, allowing high flow rates.
150 150
20 20
100 100

10 50 10
50

0 0 0 0
0 20 40 60 80 100 120 0 20 40 60 80 100 120
Volume (mL) Volume (mL)
Fig 5.2. Chromatograms from resolution comparisons on two IEX media with the same Q charged group ligand. Peaks (left to right) are apo-transferrin,
α-lactoalbumin, and soybean trypsin inhibitor. The small bead size of (A) Capto Q ImpRes and (B) Q Sepharose High Performance gives high resolution in
both cases. However, the higher flow velocity possible with Capto Q ImpRes makes it a good choice for large-scale intermediate purification or polishing.

124
The difference between Capto ImpRes and Sepharose High Performance media is also illustrated in Figure 5.3. Although Column: HiScreen Capto S ImpAct, CV 4.7 mL
the bead sizes of the media are similar, the pressure/flow properties of Capto ImpRes are significantly improved as a Sample: ~ 10 mg/mL MAb A purified on MabSelect SuRe, buffer exchanged into start buffer
Sample load: 80 mg/mL medium
result of the greater mechanical stability of its high-flow base matrix. Start buffer: 50 mM sodium acetate, pH 5.0 + 50 mM NaCl
Elution buffer: 50 mM sodium acetate, pH 5.0 + 500 mM NaCl
Flow rate loading: 1.1 mL/min, residence time 4 min
500 Flow rate elution: 0.6 mL/min, residence time 8 min
Capto SP ImpRes
Gradient: Linear, 20 CV
SP Sepharose High Performance
400 200
3000
70 175
Velocity (cm/h)

Conductivity (mS/cm)
300 2500 60 150

Aggregates (%)
2000 50 125

mAU
200 40
1500 100
30 75
100 1000
20 50
500
10 25
0
0 0 0
0 1 2 3 4 5
Pressure (bar) 0 20 40 60 80 100 120 140 160 180 200 220
Volume (mL)
Fig 5.3. The pressure-flow properties of Capto ImpRes are enhanced compared with Sepharose High Performance due to the improved mechanical stability
of the base matrix. Running conditions: AxiChrom 300 column, 20 cm bed height with water at 20°C. Fig 5.4. Purification of MAb A at a high sample load (80 mg/mL medium). Histogram in green represents
aggregates in fractions.

Capto S ImpAct chromatography medium is designed for the polishing steps of MAb
and a wide range of other biomolecules. In this study, the binding capacity for MAb A
and the efficiency in the clearance of impurities was evaluated. The content of
aggregates in the sample was approximately 7% after the initial protein A capture
step. A high load, 80 mg/mL medium, was applied to the Capto S ImpAct column
and elution was performed using a linear gradient. The chromatogram illustrates
that aggregates (green) elute at the tail of the elution peak (Fig 5.4). The purification
resulted in 90% monomer recovery and reduction of aggregate content to 0.9%.

125
SOURCE 30Q and 30S are easy to pack at both laboratory and large scale and are suitable for industrial processes when SOURCE 15Q and SOURCE 15S have a uniform, 15 µm diameter, spherical shape and
large volumes of partially purified material need to be processed. The excellent scalability of SOURCE 30S was shown in a are designed for high-resolution purification at lab scale and for scaling up. The beads
scale up from a 105-mL small scale-column to a 50-l custom-designed production column (Fig 5.5). The results show that give stable packed beds with low back pressure. Figure 5.6 illustrates the maintained
performance was maintained despite high flow rates and an almost 500-fold scale-up factor. performance of high-resolution separations also at very high flow rates.
(A) Chymotrypsinogen, cytochrome C
Columns: SOURCE 30S: (A) FineLINE Pilot 35, 35 mm i.d. × 109 mm (105 mL) 0.040
0.040 Cytochrome C, lysozyme
SOURCE 30S: (B) FineLINE 100, 100 mm i.d. × 100 mm (0.78 L)
SOURCE 30S: (C) FineLINE 800, 800 mm i.d. × 100 mm (50 L)

Resolution (Rs)
0.080
0.080
Sample: Ribonuclease A, cytochrome C, and lysozyme (3.75:1:1) 0.030
0.030 2
Sample load: 0.32 mg total protein/mL media
0.060
Start buffer: 20 mM sodium phosphate, pH 6.8 0.060

nmnm
0.020

A 280
Elution buffer: 20 mM sodium phosphate + 400 mM NaCl, pH 6.8

nmnm
0.020

A 280
1

A280
Flow rate: (A) 48 mL/min (B) 0.39 L/min, (C) 25 L/min 0.040

A280
0.040
Gradient: 0% to 100% elution buffer, 20 CV
0.010
0.010
0.020
0.020

0.000
200 600 1000 1400 1800
0.000
0.000 0.000 Flow velocity (cm/h)
1000 1500 2000 2500 10 15 20
1000 1500 2000 2500 10 15 20
Volume (mL) Volume (L)
Volume (mL) Volume (L)
40 14 4 2
Separation time (min)
(B) (C)
Fig 5.6. Resolution vs flow velocity for model proteins. Column: RESOURCE S, 1 mL (6.4 mm diameter × 30 mm
bed height). Sample: chymotrypsinogen, cytochrome C, lysozyme. Total load 16 mg.
0.080 50
50

0.060 40
40
AUFS
A280 nm

AUFS

0.040 30
30

0.020 20
20

0.000 10
10
10 15 20 600 800 1000 1200 1400
600 800 1000 1200 1400
Volume (L)
Volume (L)
Volume (L)

Fig 5.5. Scale-up from a FineLINE Pilot 35 column via FineLINE 100 column (7-fold) to FineLine 800 custom-designed column (64-fold). Total scale-up factor: 476-fold.

126
Prepacked, disposable solutions speed up the downstream
process
In addition to a wide range of industrial-scale columns such as AxiChrom columns and bulk media for purification of
proteins, Cytiva offers large-scale, disposable ReadyToProcess columns. These columns are prepacked, prequalified,
and presanitized process chromatography columns available with a range of BioProcess media — including Capto,
Capto ImpAct, Capto ImpRes, Sepharose Fast Flow, and Sepharose High Performance product families. ReadyToProcess
columns are available in several different sizes (Fig 5.7) and are designed for purification of biopharmaceuticals
(e.g., proteins and antibodies, vaccines, plasmids, and viruses) for clinical phase I and II studies. Depending on the scale
of operations, the columns can also be used for manufacturing, as well as preclinical studies. ReadyToProcess columns
make column packing, qualification, and sanitization redundant in the purification process allowing significant time savings.

Custom Designed Media

Custom Designed Media (CDM) can be produced for specific industrial process separations when suitable media are not
available from the standard range. The Custom Designed Media group (CDM group) works in close collaboration with the
user to design, manufacture, test, and deliver media for specialized purification requirements.
See also Handbook of Process Chromatography: Optimization, Scale-up, and Validation, 18112156.
Fig 5.7. ReadyToProcess columns are easily connected to the system and can be disposed after completed
production.

127
06
Appendix

128
Apendix 1
Sample preparation
Samples for chromatographic purification should be clear and free from particulate matter. Simple steps to clarify a Test pH stability in steps of one pH unit between pH 2.0 and pH 9.0
sample before beginning purification will avoid clogging the column, reduce the need for stringent washing procedures,
Test salt stability with 0 to 2 M NaCl and 0 to 2 M (NH4)2SO4 in steps of 0.5 M
and extend the life of the chromatographic medium.
T est the stability towards acetonitrile and methanol in 10% steps between
Sample extraction procedures and the selection of buffers, additives, and detergents are determined largely by the
0% and 50%
source of the material, the stability of the target molecule, the chromatographic techniques that will be employed and
the intended use of the product. These subjects are dealt with in general terms in the Strategies for Protein Purification Test the temperature stability in 10°C steps from 4°C to 40°C
Handbook and more specifically according to target molecule in the Recombinant Protein Purification Handbook, and T est the stability and occurrence of proteolytic activity by leaving an aliquot of the
Antibody Purification Handbook, available from Cytiva. sample at room temperature overnight. Centrifuge each sample and measure
activity and UV absorbance at 280 nm in the supernatant
Sample stability
In the majority of cases, biological activity needs to be retained after purification. Retaining the activity of the target
molecule is also an advantage when following the progress of the purification, since detection of the target molecule
often relies on its biological activity. Denaturation of sample components often leads to precipitation or enhanced
nonspecific adsorption, both of which will impair column function. Hence there are many advantages to checking the
stability limits of the sample and working within these limits during purification.
Proteins generally contain a high degree of tertiary structure, kept together by van der Waals’ forces, ionic and
hydrophobic interactions, and hydrogen bonding. Any conditions capable of destabilizing these forces may cause
denaturation and/or precipitation. By contrast, peptides contain a low degree of tertiary structure. Their native state is
dominated by secondary structures, stabilized mainly by hydrogen bonding. For this reason, peptides tolerate a much
wider range of conditions than proteins. This basic difference in native structures is also reflected in that proteins are
not easily renatured, while peptides often renature spontaneously.
It is advisable to perform some stability tests before beginning to develop a purification protocol. The list below


shows examples of such testing:

129
Sample clarification Table A1.1. Whatman syringe filters for filtration of samples

Filter pore size (µm) Up to sample volume (mL) Whatman syringe filter1 Membrane
Centrifugation and filtration are standard laboratory techniques for sample clarification and are used routinely when
handling small samples. 0.8 100 Puradisc FP 30 CA

It is highly recommended to centrifuge and filter samples immediately before chromatographic purification. 0.45 1 Puradisc 4 PVDF
0.45 10 Puradisc 13 PVDF
Centrifugation 0.45 100 Puradisc 25 PVDF
Centrifugation removes lipids and particulate matter, such as cell debris. If the sample is still not clear after 0.45 10 SPARTAN™ 13 RC
centrifugation, use filter paper or a 5 µm filter as a first step and one of the filters below as a second-step filter. 0.45 100 SPARTAN 30 RC
For small sample volumes or proteins that adsorb to filters, centrifuge at 10 000 × g for 15 min 0.45 100 Puradisc FP 30 CA
For cell lysates, centrifuge at 40 000 to 50 000 × g for 30 min 0.2 1 Puradisc 4 PVDF
Serum samples can be filtered through glass wool after centrifugation to remove remaining lipids 0.2 10 Puradisc 13 PVDF
0.2 100 Puradisc 25 PVDF
Filtration
0.2 10 SPARTAN 13 RC
Filtration removes particulate matter. Whatman™ syringe filters, which give the least amount of nonspecific binding 0.2 100 SPARTAN 30 RC
of proteins, are composed of cellulose acetate (CA), regenerated cellulose (RA), or polyvinylidene fluoride (PVDF)
(Table A1.1). 0.2 100 Puradisc FP 30 CA
1
The number indicates the diameter (mm) of the syringe filter.

130
For sample preparation before chromatography, select a filter pore size in relation to the bead size of the Table A1.2. Selecting a sample filter based on the bead size of the chomatographic medium used
chromatographic medium (Table A1.2).
Nominal pore size of filter (µm) Particle size of chromatographic medium (µm)
Check the recovery of the target protein in a test run. Some proteins adsorb nonspecifically to filter surfaces. 1.0 90 and upwards

Desalting 0.45 30 or 34
0.22 3, 10, 15 or when extra clean samples or sterile filtration
Desalting columns are suitable for any sample volume and will rapidly remove low molecular weight contaminants in a
is required
single step at the same time as transferring the sample into the correct buffer conditions. Centrifugation and/or filtration
of the sample before desalting is still recommended. Detailed procedures for buffer exchange and desalting are given in
the section Buffer exchange and desalting later in this appendix.
At laboratory scale, when samples are reasonably clean after filtration or centrifugation, the buffer exchange and
desalting step can be avoided. For affinity chromatography or hydrophobic interaction chromatography, it might be
sufficient to adjust the pH of the sample. For IEX, it might be sufficient to dilute the sample to reduce the ionic strength.

131
Specific sample preparation steps Clarification
Bulk proteins and particulate Supernatant
Specific sample preparation steps might be required if the crude sample is known to matter precipitated
contain contaminants such as lipids, lipoproteins, or phenol red that might build up
on a column. Gross impurities, such as bulk protein, should be removed before any Chromatography
chromatographic step. Extraction, Clarification,
Note: if precipitating agent is
incompatible with next purification step,
Concentration Redissolve pellet1
Fractional precipitation Target protein precipitated
use Sephadex™ G-25 for desalting and
buffer exchange, for example,
Fractional precipitation is occasionally used at laboratory scale to remove gross with proteins of similar solubility
HiTrap Desalting or PD-10 columns
impurities from the sample. Precipitation techniques separate fractions by the principle
of differential solubility. Because proteins differ in their degree of hydrophobicity, Extraction, Clarification Concentration
increased salt concentrations can enhance hydrophobic interactions between the Bulk proteins and particulate Target protein
proteins and cause precipitation. Fractional precipitation can be applied to remove matter precipitated precipitated Redissolve pellet1
gross impurities in three different ways, as shown in Figure A1.1. with proteins of
similar solubility

1
Remember: not all proteins are easy to redissolve, yield can be reduced

Fig A1.1. Three ways to use precipitation.

 Precipitation techniques can be affected by temperature, pH, and sample concentration. These parameters should
be controlled to ensure reproducible results.
Examples of precipitation agents are reviewed in Table A1.3. The most common precipitation method using ammonium
sulfate is described in more detail.

132
Table A1.3. Examples of precipitation techniques

Precipitation agent Typical conditions for use Sample type Comment

Ammonium sulfate As described below. > 1 mg/mL proteins especially immunoglobulins. Stabilizes proteins, no denaturation, supernatant can go directly
to HIC. Helps to reduce lipid content.
Dextran sulfate Add 0.04 mL 10% dextran sulfate and 1 mL of 1 M CaCl2 Samples with high levels of lipoprotein, Precipitates lipoprotein.
per mL sample, mix 15 min, centrifuge 10 000 × g, e.g., ascites.
discard pellet.
Polyvinylpyrrolidine Add 3% (w/v), stir 4 h, centrifuge 17 000 × g, discard pellet. Samples with high levels of lipoprotein, Alternative to dextran sulfate.
e.g., ascites.
Polyethylene glycol (PEG, Mr > 4000) Up to 20% w/v. Plasma proteins. No denaturation, supernatant goes directly to IEX or AC,
complete removal might be difficult. Stabilizes proteins.
Acetone (cold) Up to 80% v/v at ± 0°C. Collect pellet after centrifugation Can denature protein irreversibly. Useful for peptide
at full speed in a microcentrifuge. precipitation or concentration of sample for electrophoresis.
Polyethyleneimine 0.1% w/v. Precipitates aggregated nucleoproteins.
Protamine sulfate 1% w/v. Precipitates aggregated nucleoproteins.
Streptomycin sulfate 1% w/v. Precipitation of nucleic acids.
Caprylic acid (X/15) g where X = volume of sample. Antibody concentration should be > 1 mg/mL. Precipitates bulk of proteins from sera or ascites, leaving
immunoglobulins in solution.
Details taken from: Scopes R. K., Protein Purification, Principles and Practice, Springer, (1994), J. C. Janson and L. Rydén, Protein Purification, Principles, High Resolution Methods and Applications, second ed. Wiley Inc, (1998). Personal communications.

133
Ammonium sulfate precipitation
Some proteins can be damaged by ammonium sulfate. Take care when adding crystalline ammonium sulfate;
high local concentrations can cause contamination of the precipitate with unwanted proteins.
For routine, reproducible purification, precipitation with ammonium sulfate should be avoided in favor of


chromatography.
In general, precipitation is rarely effective for protein concentrations below 1 mg/mL.

Solutions needed for precipitation:

Saturated ammonium sulfate solution (add 100 g ammonium sulfate to 100 mL distilled water, stir to dissolve).
1 M Tris-HCl, pH 8.0.
Buffer for first purification step.

1. Filter (0.45 µm) or centrifuge the sample (10 000 × g at 4°C).

2. Add 1 part 1 M Tris-HCl, pH 8.0 to 10 parts sample volume to maintain pH.
3. Stir gently. Add ammonium sulfate solution, drop by drop. Add up to 50% saturation1. Stir for 1 h.
4. Centrifuge 20 min at 10 000 × g.
5. R
 emove supernatant. Wash the pellet twice by resuspension in an equal volume of ammonium sulfate solution
of the same concentration (i.e., a solution that will not redissolve the precipitated protein or cause further
precipitation). Centrifuge again.
6. Dissolve pellet in a small volume of the buffer to be used for the next step.
7. A
 mmonium sulfate is removed during clarification/buffer exchange steps with Sephadex G-25, using desalting
columns (see Buffer exchange and desalting later in this appendix).
1
The percent saturation can be adjusted either to precipitate a target molecule or to precipitate contaminants.

134
The quantity of ammonium sulfate required to reach a given degree of saturation varies according to temperature. Resolubilization of protein precipitates
Table A1.4 shows the quantities required at 20°C.
Many proteins are easily resolubilized in a small amount of the buffer to be used in
the next chromatographic step. However, a denaturing agent may be required for less
Table A1.4. Quantities of ammonium sulfate required to reach given degrees of saturation at 20°C soluble proteins. Specific conditions will depend upon the specific protein. These
Final percent saturation to be obtained agents must always be removed to allow complete refolding of the protein and to
maximize recovery of mass and activity. A chromatographic step often removes a
20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
denaturant during purification. Table A1.5 gives examples of common denaturing agents.
Starting percent
Amount of ammonium sulphate to add (grams) per liter of solution at 20°C
saturation
Table A1.5. Denaturing agents used for resolubilization of relatively insoluble proteins
0 113 144 176 208 242 277 314 351 390 430 472 516 561 608 657 708 761
5 85 115 146 179 212 246 282 319 358 397 439 481 526 572 621 671 723 Denaturing agent Typical conditions for use (molar, M) Removal/comment

10 57 86 117 149 182 216 251 287 325 364 405 447 491 537 584 634 685 Urea 2 to 8 Remove using Sephadex G-25

15 28 58 88 119 151 185 219 255 293 331 371 413 456 501 548 596 647 Guanidine 3 to 6 Remove using Sephadex G-25
hydrochloride
20 0 29 59 89 121 154 188 223 260 298 337 378 421 465 511 559 609
25 0 29 60 91 123 157 191 228 265 304 344 386 429 475 522 571 Details taken from: Scopes R. K., Protein Purification, Principles and Practice, Springer, (1994), J. C. Janson and
L. Rydén, Protein Purification, Principles, High Resolution Methods and Applications, second ed. Wiley Inc, (1998)
30 0 30 61 92 125 160 195 232 270 309 351 393 438 485 533 and other sources.
35 0 30 62 94 128 163 199 236 275 316 358 402 447 495
40 0 31 63 96 130 166 202 241 281 322 365 410 457
45 0 31 64 98 132 169 206 245 286 329 373 419
50 0 32 65 99 135 172 210 250 292 335 381
55 0 33 66 101 138 175 215 256 298 343
60 0 33 67 103 140 179 219 261 305
65 0 34 69 105 143 183 224 267
70 0 34 70 107 146 186 228
75 0 35 72 110 149 190
80 0 36 73 112 152
85 0 37 75 114
90 0 37 76
95 0 38

135
Buffer exchange and desalting 0.30 15
Dialysis is frequently mentioned in the literature as a technique to remove salt or other small molecules and to exchange
the buffer composition of a sample. However, dialysis is generally a very slow technique, requiring large volumes of buffer. 0.25
There is also a risk of losing material during handling or as a result of proteolytic breakdown or nonspecific binding to the

Conductivity (mS/cm)
dialysis membranes. A simpler and much faster technique is to use a desalting column, packed with Sephadex G-25, to
perform a group separation between high and low molecular weight substances. Proteins are separated from salts and 0.20
10
other small molecules.

A280
In a fast, single step, the sample is desalted, transferred into a new buffer and low molecular weight materials are removed. 0.15 protein salt
Desalting columns are used not only to remove low molecular weight contaminants, such as salt, but also for buffer
exchange before or after different chromatographic steps and for the rapid removal of reagents to terminate a reaction. 0.10
Sample volumes up to 30% of the total volume of the desalting column can be processed. Sample concentration does not 5
influence the separation as long as the concentration of proteins does not exceed 70 mg/mL when using normal aqueous 0.05
buffers. The sample should be fully dissolved. Centrifuge or filter to remove particulate material.
For small sample volumes, it is possible to dilute the sample with the start buffer that is to be used for chromatographic 0.00
purification, but cell debris and particulate matter must still be removed. 0 1 2 3
Time (min)
Volatile buffers such as 100 mM ammonium acetate or 100 mM ammonium hydrogen carbonate can be used if it is


necessary to avoid the presence of NaCl. Fig A1.2. Buffer exchange of mouse plasma (10 mL) on HiPrep 26/10 Desalting.

Figure A1.2 shows a typical buffer exchange and desalting separation. The process can be monitored by following changes
in UV absorption and conductivity.

For laboratory-scale operations, Table A1.6 shows examples of prepacked, ready-to-use desalting and buffer exchange
columns (see Size Exclusion Chromatography Handbook, 18102218, for additional formats).

Table A1.6. Examples of desalting and buffer exchange columns

Column Sample volume (mL) Sample elution volume (mL)

PD MiniTrap™ G-25 0.2 to 0.5 0.1 to 0.5
PD-10 (gravity feed column) 1.5 to 2.5 2.5 to 3.5
HiTrap Desalting, 5 mL 0.25 to 1.5 1.0 to 2.0
HiPrep 26/10 Desalting 2.5 to 15 7.5 to 20

136
To desalt larger sample volumes:  The maximum recommended sample volume is 1.5 mL. See Table A1.7 for the
- Connect up to five HiTrap Desalting 5 mL columns in series to increase the sample volume capacity, for example, two effect of reducing the sample volume applied to the column.
columns: sample volume 3 mL, five columns: sample volume 7.5 mL.
Table A1.7. Recommended sample and elution volumes using a syringe
- Connect up to four HiPrep 26/10 Desalting columns in series to increase the sample volume capacity, for example two
columns: sample volume 30 mL, four columns: sample volume 60 mL. Even with four columns in series, the sample can Sample load Add buffer Elute and collect Yield Remaining salt Dilution
be processed in 20 to 30 min, at room temperature, in aqueous buffers. (mL) (mL) (mL) (%) (%) factor
Instructions are supplied with each column. Desalting and buffer exchange can take less than 5 min per sample with 0.25 1.25 1.0 > 95 0.0 4.0
greater than 95% recovery for most proteins. 0.50 1.0 1.5 > 95 < 0.1 3.0
1.00 0.5 2.0 > 95 < 0.2 2.0
Manual desalting with HiTrap Desalting 5 mL using a syringe 1.50 0 2.0 > 95 < 0.2 1.3

1. Fill the syringe with buffer. Remove the stop plug. To avoid introducing air into the column, connect the column Removal of lipoproteins
“drop to drop” to the syringe (via the adapter provided).
Lipoproteins and other lipid material can rapidly clog chromatography columns
2. Remove the snap-off end. and it is advisable to remove them before beginning purification. Precipitation
3. W
 ash the column with 25 mL buffer at 5 mL/min to remove completely the 20% ethanol (supplied as storage agents such as dextran sulfate and polyvinylpyrrolidine, described under Fractional
buffer). If air is trapped in the column, wash with degassed buffer until the air disappears. Air bubbles introduced precipitation earlier in this appendix, are recommended to remove high levels of
onto the column by accident during sample application do not influence the separation. lipoproteins from samples such as ascitic fluid.
4. A
 pply the sample (0.25 to 1.5 mL) using a 2 to 5 mL syringe at a flow rate between 1 to 10 mL/min. Discard the Centrifuge samples when performing precipitation to avoid the risk of nonspecific
liquid eluted from the column. binding of the target molecule to a filter.
5. I f the sample volume is less than 1.5 mL, change to buffer and proceed with the injection until a total of 1.5 mL  Samples such as serum can be filtered through glass wool to remove remaining
has been eluted. Discard the eluted liquid. lipids.
6. Elute the protein with the appropriate volume selected from Table A1.7 and collect the desalted protein.
Note: 5 mL/min corresponds to approximately 120 drops/min when using a HiTrap 5 mL column. A simple
peristaltic pump or a chromatography system can also be used for the desalting procedure.

137
Removal of phenol red
Phenol red is frequently used at laboratory scale as a pH indicator in cell culture. Although not directly interfering with
purification, phenol red binds to certain purification media and should be removed as early as possible to avoid the risk
of contamination. It is known to bind to anion exchange media at pH > 7.0.
Use a desalting column to simultaneously remove phenol red (a low molecular weight molecule) and transfer
sample to the correct buffer conditions for further purification, as described under Buffer exchange and desalting
earlier in this appendix.

Removal of low molecular weight contaminants

If samples contain a high level of low molecular weight contaminants, use a desalting column before the first
chromatographic purification step, as described under Buffer exchange and desalting earlier in this appendix.

138
Appendix 2
Nonvolatile and volatile buffer systems

Nonvolatile buffers for anion exchange chromatography

pKa1
pH 4 5 6 7 8 9 10 11 (25°C) pH interval Substance Conc. (mM) Counterion pKa (25°C)1 d(pKa)/dT (°C)
4.3 to 5.3 N-Methylpiperazine 20 Cl- 4.75 -0.015

4.8 to 5.8 Piperazine 20 Cl- or HCOO - 5.33 -0.015

N-methylpiperazine 4.75
5.5 to 6.5 l-Histidine 20 Cl- 6.04
Piperazine 5.33
6.0 to 7.0 bis-Tris 20 Cl- 6.48 -0.017
bis-Tris 6.48
6.2 to 7.2; 8.6 to 9.6 bis-Tris propane 20 Cl- 6.65; 9.10
bis-Trispropane 6.65; 9.10
7.3 to 8.3 Triethanolamine 20 Cl- or CH3COO - 7.76 -0.020
Triethhanolamine 7.76
7.6 to 8.6 Tris 20 Cl- 8.07 -0.028
Tris 8.07
8.0 to 9.0 N-Methyldiethanolamine 20 SO42- 8.52 -0.028
N-methyldiethanolamine 8.52 8.0 to 9.0 N-Methyldiethanolamine 50 Cl- or CH3COO - 8.52 -0.028
Propane-1,3-Diamino 8.88 8.4 to 9.4 Diethanolamine 20 at pH 8.4 Cl- 8.88 -0.025
Ethanolamine 9.50 50 at pH 8.8
8.4 to 9.4 Propane 1,3-Diamino 20 Cl- 8.88 -0.031
Piperazine 9.73
9.0 to 10.0 Ethanolamine 20 Cl- 9.50 -0.029
Propane-1,3-Diamino 10.55
9.2 to 10.2 Piperazine 20 Cl- 9.73 -0.026
Piperidine 11.12
10.0 to 11.0 Propane 1,3-Diamino 20 Cl- 10.55 -0.026

10.6 to 11.6 Piperidine 20 Cl- 11.12 -0.031

1
Ref: Handbook of chemistry and physics, 83rd edition, CRC, 2002 to 2003.

139
Nonvolatile buffers for cation exchange chromatography Volatile buffer systems
pKa1
ph 2.5 3 4 5 6 7 8 9 (25°C) pH range Buffer system Counterion pKa-values for buffering ions1
3.3 to 4.3 Formic acid H+ 3.75
Citric acid 3.13 3.3 to 4.3; 4.8 to 5.8 Pyridine/formic acid HCOO - 3.75; 5.25

Lactic acid 3.86 3.3 to 4.3; 9.3 to 10.3 Trimethylamine/formic acid HCOO - 4.75; 9.81

4.21 4.3 to 5.8 Pyridine/acetic acid CH3COO - 4.75; 5.25

Butanedioic acid (succinic acid)
4.3 to 5.3; 9.3 to 10.3 Trimethylamine/acetic acid CH3COO - 4.75; 9.81
Acetic acid 4.75
3.3 to 4.3; 8.8 to 9.8 Ammonia/formic acid HCOO - 3.75; 9.25
Methylmalonic acid 5.76
4.3 to 5.3; 8.8 to 9.8 Ammonia/acetic acid CH3COO - 4.75; 9.25
MES 6.27
5.9 to 6.9; 9.3 to 10.3 Trimethylamine/carbonate CO32- 6.35; 9.81
Phosphate 7.20 5.9 to 6.9; 8.8 to 9.8 Ammonium bicarbonate HCO3- 6.35; 9.25
HEPES 7.56 5.9 to 6.9; 8.8 to 9.8 Ammonium carbonate/ammonia CO32- 6.35; 9.25
BICINE 8.33 5.9 to 6.9; 8.8 to 9.8 Ammonium carbonate CO32- 6.35; 9.25

4.3 to 5.3: 7.2 to 8.2 N-Ethylmorpholine/acetate HCOO - 4.75; 7.72

1
Ref: Handbook of chemistry and physics, 83rd edition, CRC, 2002 to 2003.
pH interval Substance Conc. (mM) Counterion pKa (25°C)1 d(pKa)/dT (°C)
1.4 to 2.4 Maleic acid 20 Na+ 1.92

2.6 to 3.6 Methylmalonic acid 20 Na+ or Li+ 3.07

2.6 to 3.6 Citric acid 20 Na+ 3.13 -0.0024

3.3 to 4.3 Lactic acid 50 Na+ 3.86

3.3 to 4.3 Formic acid 50 Na+ or Li+ 3.75 +0.0002

3.7 to 4.7; 5.1 to 6.1 Succinic acid 50 Na+ 4.21; 5.64 -0.0018

4.3 to 5.3 Acetic acid 50 Na+ or Li+ 4.75 +0.0002

5.2 to 6.2 Methylmalonic acid 50 Na+ or Li+ 5.76

5.6 to 6.6 MES 50 Na+ or Li+ 6.27 -0.0110

6.7 to 7.7 Phosphate 50 Na+ 7.20 -0.0028

7.0 to 8.0 HEPES 50 Na+ or Li+ 7.56 -0.0140

7.8 to 8.8 BICINE 50 Na+ 8.33 -0.0180

1
Ref: Handbook of chemistry and physics, 83rd edition, CRC, 2002 to 2003.

140
Appendix 3
Column packing and preparation
Prepacked columns from Cytiva will ensure reproducible results and the highest performance.
 Use small prepacked columns for media scouting and method optimization, to increase efficiency in method
development, for example, HiTrap IEX Selection Kit.
Efficient column packing is essential for IEX separation, especially when using gradient elution. A poorly packed column
gives rise to poor and uneven flow, band broadening, and loss of resolution. If column packing is required, the following
guidelines will apply at all scales of operation:
 ith a high binding capacity medium, use short, wide columns (typically 5 to 15 cm bed height) for rapid purification,
W
even at low flow velocity
T he amount of IEX medium required will depend on the binding capacity of the medium and the amount of sample.
Binding capacities for each medium are given in this handbook and supplied with the product instructions. Estimate
the amount of medium required to bind the sample of interest and use five times this amount to pack the column.
The amount of medium required can be reduced if resolution is satisfactory
O
 nce separation parameters have been determined, scale up a purification by increasing the diameter of the column
to increase column volume. Avoid increasing the length of the column, if possible, as this will alter separation
conditions
IEX media can be packed in either Tricorn, XK, or HiScale columns available from Cytiva (Fig A3.1).
Fig A3.1. Column packing in progress.

141
 1. Equilibrate all materials to the temperature at which the separation will be performed. 10.  top the pump and close the column outlet. Remove the top piece
S
and carefully fill the rest of the column with buffer to form an upward
2. E
 liminate air by flushing column end pieces with the recommended buffer. Ensure no air is trapped under the
meniscus at the top.
column net. Close column outlet leaving 1 to 2 cm of buffer in the column.
11. I nsert the adapter into the column at an angle, ensuring that no air is
3. Gently resuspend the medium.
trapped under the net.
12.  lide the adapter slowly down the column (the outlet of the adaptor
S
Note that IEX media from Cytiva are supplied ready to use. Decanting of fines that could clog the column is unnecessary. should be open) until the mark is reached. Lock the adapter in position.
Avoid using magnetic stirrers since they can damage the matrix. 13.  onnect the column to the pump and begin equilibration. Reposition the
C
adapter if necessary.
4. Estimate the amount of slurry (resuspended medium) required on the basis of the recommendations supplied.
5. P
 our the required volume of slurry into the column. Pouring down a glass rod held against the wall of the column  The medium must be thoroughly washed to remove the storage solution, usually
will minimize the introduction of air bubbles. 20% ethanol. Residual ethanol can interfere with subsequent procedures.
6. Immediately fill the column with buffer. Many media equilibrated with sterile phosphate-buffered saline containing an
7. Mount the column top piece and connect to a pump. antimicrobial agent may be stored at 4°C for up to 1 mo, but always follow the
specific storage instructions supplied with the product.
8. Open the column outlet and set the pump to the desired flow rate.

When slurry volume is greater than the total volume of the column, connect a second glass column to act as
a reservoir (see Ordering information for details). This ensures that the slurry has a constant diameter during
packing, minimizing turbulence and improving column packing conditions.
If the recommended flow rate cannot be obtained, use the maximum flow rate the pump can deliver.

9. M
 aintain the packing flow rate for at least 3 CV after a constant bed height is obtained. Mark the bed height on
the column.

Do not exceed 75% of the packing flow rate during any purification.

142
Column selection
Tricorn, XK, and HiScale columns are fully compatible with the high flow rates achievable with modern media and a broad Absorbance
Ve
range of column dimensions are available. Columns most suitable for packing IEX media are listed under the column
packing section for each IEX medium (Chapter 3). In most cases the capacity of the IEX medium and the amount of
sample to be purified will determine the column size required.

Column packing and efficiency

Column efficiency is expressed as the number of theoretical plates per meter chromatography bed (N) or as H (height
wh
equivalent to a theoretical plate, HETP), which is the bed length (L) divided by the plate number. Column efficiency is
related to the band broadening that can occur on a column and can be calculated from the expression:

( )
2
VR
N = 5.54 × 50%
wh
VR = volume eluted from the start of sample application to the peak maximum
wh = peak width measured as the width of the recorded peak at half of the peak height a b
10%

H is calculated from the expression: Volume

L Fig A3.2. Measurements taken to calculate column efficiency.
H=
N
L = height of packed bed.
The asymmetry factor (As) is expressed as:
Measurements of VR and wh can be made in distance (mm) or volume (mL) but both parameters must be expressed b

As =
in the same unit. a
where
Column performance should be checked at regular intervals by injecting acetone to determine column efficiency (N) and
peak symmetry (asymmetry factor, As). Since the observed value for N depends on experimental factors such as flow rate a = 1st half peak width at 10% of peak height
and sample loading, comparisons must be made under identical conditions. In IEX, efficiency is measured under isocratic b = 2nd half peak width at 10% of peak height
conditions by injecting acetone (which does not interact with the medium) and measuring the eluted peak as shown in
Figure A3.2. As should be as close as possible to 1.0. A reasonable As value for a short column as
used in IEX is 0.80 to 1.80.
As a general rule, a good H value is about two to three times the average particle diameter of the medium being packed.
For a 90 µm particle, this means an H value of 0.018 to 0.027 cm.  An extensive leading edge is usually a sign that the medium is packed too
tightly and extensive tailing is usually a sign that the medium is packed too
loosely.
 Run at least two column volumes of buffer through a newly packed column to
ensure that the medium is equilibrated with start buffer. Use pH monitoring to
check the pH of the eluent. 143
Appendix 4
Selection of purification equipment
Simple IEX, such as elution by a step-gradient, can be performed using a syringe Table A4.1. Ways of working with standard ÄKTA chromatography systems
or peristaltic pump with prepacked HiTrap columns. A chromatography system is
required when reproducible results are important and when manual purification
becomes too time-consuming and inefficient. This can be the case when large
sample volumes are handled, or when there are many different samples to be purified.
The progress of the purification can be monitored automatically and high-resolution
Way of working ÄKTA start ÄKTAprime plus ÄKTAxpress ÄKTA pure ÄKTA avant
separations with accurately controlled linear-gradient elution can be performed.
Simple, one-step desalting, buffer exchange • • • • •

Research
Table A4.1 lists the standard ÄKTA system configurations for currently available

Process development
Automated and reproducible protein purification
systems, see also ÄKTA Laboratory-scale Systems: Instrument Management using all common techniques including support for • • • • •
Handbook, 29010831. Table 4.2 on the next page provides a summary of prepacked gradient elution

IEX columns for use with ÄKTA systems. Software compatible with regulatory requirements,
• • •
e.g., good laboratory practice (GLP)

Method development and optimization using design o • •

of experiments (DoE)

Automatic buffer preparation including pH scouting • o •

Automatic chromatography medium or column o • o •

scouting

Automatic, multistep purification o • o o

Scale-up, process development o o o •

Flow rate (mL/min) 0.5 to 5.0 0.1 to 50.0 0.1 to 65.0 0.001 to 25.0 (ÄKTA 0.001 to 25/0.01 to 150
pure 25)/ 0.01 to 150
(ÄKTA pure 150)

Max. operating pressure (MPa) 0.5 1 3 20/5 20/5

Software1 for system control and data handling UNICORN™ start PrimeView™2 UNICORN 5 UNICORN 6 or later UNICORN 6 or later
1
A specific software version might be needed for the chosen system. See the web page for each respective system at www.cytiva.com/AKTA.
2
With PrimeView, you can monitor results and evaluate data but not create methods nor control the system.
• = included
o = optional

144
Table A4.2. Summary of prepacked columns for IEX using ÄKTA systems

Chromatography technique Base matrix HiTrap HiScreen HiPrep RESOURCE Tricorn (GL/PE) Precision Columns (PC)7
Easy to use with a syringe, Optimized for method Convenient scale-up Fast with good High quality and Micro-purification
peristaltic pump, or and process development Preparative size exclusion resolution high resolution and analysis
chromatography system chromatography
Ion exchange Sepharose HP,
ü ü ü
Sepharose FF
Capto, Capto ImpRes ü ü
Capto ImpAct ü ü
MonoBeads ü ü
MiniBeads ü ü
SOURCE ü ü
System compatibility ÄKTA pure ÄKTA avant ÄKTA pure ÄKTA pure ÄKTA pure 25 ÄKTAmicro6
ÄKTA start ÄKTA pure 150 ÄKTA start5 ÄKTAmicro6 ÄKTAxpress
ÄKTAxpress ÄKTApurifier 1002 ÄKTA avant ÄKTApurifier 10² ÄKTAmicro6
ÄKTAprime plus ÄKTAexplorer 1003 ÄKTAxpress ÄKTAexplorer 103 ÄKTApurifier 102
ÄKTApurifier2 ÄKTAprime plus ÄKTAfplc4 ÄKTAexplorer 103
ÄKTAexplorer3 ÄKTApurifier2 ÄKTAfplc4
ÄKTAfplc4 ÄKTAexplorer3
ÄKTAfplc4
1
Sample volume.
2
ÄKTApurifier has been discontinued and replaced by ÄKTA pure.
3
ÄKTAexplorer has been discontinued and replaced by ÄKTA avant.
4
ÄKTAfplc has been discontinued and replaced by ÄKTA pure 25.
5
HiPrep 26/60 can be used but is not optimal.
6
ÄKTAmicro has been discontinued and replaced by ÄKTA pure 25 with microgram-scale purification flow path.
7
Precision Columns (PC) provide excellent results when used in combination with HPLC systems.

145
Appendix 5
Converting from flow velocity to volumetric flow rates
It is convenient when comparing results for columns of different sizes to express flow as flow velocity (cm/h). However,
flow is usually measured in volumetric flow rate (mL/min). To convert between flow velocity and volumetric flow rate use
one of the formulae below.

From flow velocity (cm/h) to volumetric flow rate (mL/min) From volumetric flow rate (mL/min) to flow velocity (cm/h)
Flow velocity (cm/h) Volumetric flow rate (mL/min) × 60
Volumetric flow rate (mL/min) = × c olumn cross sectional Flow velocity (cm/h) =
60 column cross sectional area (cm2)
area (cm2)
4
Y p × d2 = Z × 60 ×
= × p × d2
60 4
where
where
Z = volumetric flow rate in mL/min
Y = flow velocity in cm/h
d = column inner diameter in cm
d = column inner diameter in cm
Example:
Example:
What is the linear flow in a Tricorn 5/50 column (i.d. 0.5 cm) when the volumetric
What is the volumetric flow rate in an XK 16/70 column (i.d. 1.6 cm) when the
flow rate is 1 mL/min?
flow velocity is 150 cm/h?
Z = volumetric flow rate = 1 mL/min
Y = flow velocity = 150 cm/h
d = column inner diameter = 0.5 cm
d = inner diameter of the column = 1.6 cm
4
Flow velocity = 1 × 60 × cm/h
150 × p × 1.6 × 1.6 p × 0.5 × 0.5
Volumetric flow rate = mL/min
60 × 4
= 5.03 mL/min From volumetric flow rate (mL/min) to using a syringe
1 mL/min = approximately 30 drops/min on a HiTrap 1 mL column
5 mL/min = approximately 120 drops/min on a HiTrap 5 mL column

146
Appendix 6
Conversion data: proteins, column pressures
Proteins Column pressures
Mass (g/mol) 1 µg 1 nmol Protein A280 for 1 mg/mL The maximum pressure drop over the packed bed refers to the pressure above which the
10 000 100 pmol; 6 × 1013 molecules 10 µg IgG 1.35 column contents might begin to compress.
50 000 20 pmol; 1.2 × 1013 molecules 50 µg IgM 1.20 Pressure units may be expressed in megaPascal (MPa), bar, or pounds per square
100 000 10 pmol; 6.0 × 1012 molecules 100 µg IgA 1.30 inch (psi) and can be converted as follows: 1 MPa = 10 bar = 145 psi
150 000 6.7 pmol; 4.0 × 1012 molecules 150 µg Protein A 0.17
Avidin 1.50
Streptavidin 3.40
Bovine Serum Albumin 0.70
1 kb of DNA = 333 amino acids of coding capacity
= 37 000 g/mol
270 bp DNA = 10 000 g/mol
1.35 kb DNA = 50 000 g/mol
2.70 kb DNA = 100 000 g/mol
Average molecular weight of an amino acid = 120 g/mol.

147
Appendix 7
Table of amino acids
Middle unit
residue (-H20)

Three-letter Single-letter Three-letter Single-letter Charge at Hydrophobic Uncharged Hydrophilic

Amino acid code code Structure Amino acid code code Structure Formula Mr Formula Mr pH 6.0 to 7.0 (nonpolar) (polar) (polar)

HOOC
C3H7NO2 89.1 C3H5NO 71.1 Neutral •
HOOC
Alanine Ala A CH3 Methionine Met M CH2CH2SCH3
H 2N H 2N C6H14N4O2 174.2 C6H12N4O 156.2 Basic (+ve) •
HOOC NH2 HOOC
Arginine Arg R Phenylalanine Phe F
CH2CH2CH2NHC CH2 C 4H 8N 2O 3 132.1 C 4H 6N 2O 2 114.1 Neutral •
H 2N NH H 2N
HOOC HOOC
Proline Pro P C4H7NO4 133.1 C4H5NO3 115.1 Acidic(-ve) •
Asparagine Asn N CH2CONH2
NH
H 2N H 2N
HOOC HOOC C3H7NO2S 121.2 C3H5NOS 103.2 Neutral •
Aspartic acid Asp D CH2COOH Serine Ser S CH2OH
H 2N H 2N C5H9NO4 147.1 C5H7NO3 129.1 Acidic (-ve) •
HOOC HOOC
Cysteine Cys C CH2SH Threonine Thr T CHCH3
C5H10N2O3 146.1 C 5H 8N 2O 2 128.1 Neutral •
H 2N H 2N OH
HOOC HOOC
Tryptophan Trp W C2H5NO2 75.1 C2H3NO 57.1 Neutral •
Glutamic acid Glu E CH2CH2COOH CH2
H 2N H 2N
NH
HOOC HOOC C 6H 9N 3O 2 155.2 C 6H 7N 3O 137.2 Basic (+ve) •
Glutamine Gln Q CH2CH2CONH2 Tyrosine Tyr Y CH2 OH
H 2N H 2N C6H13NO2 131.2 C6H11NO 113.2 Neutral •
HOOC HOOC
Glycine Gly G H Valine Val V CH(CH3)2
H 2N H 2N
C6H13NO2 131.2 C6H11NO 113.2 Neutral •
HOOC N

Histidine His H CH2 C6H14N2O2 146.2 C6H12N2O 128.2 Basic (+ve) •

NH
H 2N
HOOC C5H11NO2S 149.2 C5H9NOS 131.2 Neutral •
CH(CH3)CH2CH3
Isoleucine Ile I
H 2N
C9H11NO2 165.2 C9H9NO 147.2 Neutral •
HOOC CH3
CH2CH
Leucine Leu L
H 2N CH3 C5H9NO2 115.1 C5H7NO 97.1 Neutral •
HOOC

Lysine Lys K CH2CH2CH2CH2NH2 C3H7NO3 105.1 C3H5NO2 87.1 Neutral •

H 2N

C4H9NO3 119.1 C4H7NO2 101.1 Neutral •

C11H12N2O2 204.2 C11H10N2O 186.2 Neutral •

C9H11NO3 181.2 C9H9NO2 163.2 Neutral •

C5H11NO2 117.1 C5H9NO 99.1 Neutral •

148
Appendix 8
Analytical assays during purification
 The percentage of acrylamide in the SDS gel should be selected according to
Analytical assays are essential to follow the progress of purification. They are used to assess the effectiveness of each the expected molecular weight of the protein of interest (see Table A8.1).
step in terms of yield, biological activity, and recovery as well as to help during optimization of experimental conditions.
The importance of a reliable assay for the target molecule cannot be overemphasized.
Table A8.1. Percentage of acrylamide used in SDS gels for proteins of different molecular weights
 When testing chromatographic fractions, ensure that the buffers used for purification do not interfere with
the assay. Acrylamide in resolving gel (%) Mol. weight range
Homogeneous: 5 36 000 to 200 000
Total protein determination 7.5 24 000 to 200 000
Lowry or Bradford assays are used most frequently to determine the total protein content. The Bradford assay is 10 14 000 to 200 000
particularly suited to samples where there is a high lipid content that can interfere with the Lowry assay.
12.5 14 000 to 100 000
Purity determination 15 14 000 to 60 0001
Purity is most often estimated by SDS-PAGE. Alternatively, isoelectric focusing, capillary electrophoresis, reversed phase Gradient: 5 to 15 14 000 to 200 000
chromatography, or mass spectrometry may be used. 5 to 20 10 000 to 200 000
SDS-PAGE analysis 10 to 20 10 000 to 150 000
The general steps involved in SDS-PAGE analysis are summarized below. 1
The larger proteins fail to move significantly into the gel.

The gel is usually stained after electrophoresis in order to make the protein bands
1. Prepare samples by mixing with equal volume of 2 × SDS loading buffer visible by, for example, Coomassie Blue or silver staining. A more recent way of
2. Vortex briefly and heat for 5 min at 90°C to 100°C. making protein visible is by prelabeling the proteins by fluorescent dye (Amersham™
WB Cy™5 dye reagent) before loading the sample in the gel. By doing in this way the
3. Load the samples and, optionally, a MW marker onto a SDS-polyacrylamide gel.
gel image can be acquired directly after finished electrophoresis by laser scanner or
4. Run the gel. CCD camera and the result is obtained much faster. This workflow is outlined below.
5. S
 tain the gel with Coomassie Blue (Coomassie Blue Tablets, PhastGel Blue R-350) or silver (PlusOne Silver
Staining Kit, Protein).

149
Protein prelabeling with CyDye™
 Electrophoresis, protein transfer, and probing may be accomplished using a
1. Prepare samples by prelabeling with Amersham WB Cy5 dye reagent. variety of equipment and reagents. The Amersham WB system is an automated
2. Vortex briefly and heat for 5 min at 90°C to 100°C. system that can be used for all these steps including software evaluation.
For more information, visit www.cytiva.com/westernblotting. For further
3. Load the samples and, optionally, a MW marker onto a SDS-polyacrylamide gel. information on the basic principles and methods used in Western blotting,
4. Run the gel and proceed directly to image capture. refer to the Western Blotting Handbook, 28999897 and the instruction manuals
supplied with the detection kits.
ELISAs are most commonly used as activity assays
 For information and advice on electrophoresis techniques, refer to the handbook 2-D Electrophoresis, Principles
and Methods, 80642960. For information on the Amersham WB system and accessories including Amersham WB Cy5 F unctional assays using the phenomenon of surface plasmon resonance (SPR)
prelabeling reagents, visit www.cytiva.com/westernblotting. to detect immunospecific interactions (e.g., using Biacore™ systems) enable the
determination of active concentration, epitope mapping, and studies of interaction
Functional assays kinetics

Immunospecific interactions have enabled the development of many alternative assay systems for the assessment of  The Biacore Assay Handbook, 29019400 gives a general overview of the
active concentration of target molecules. different types of SPR-based applications. The handbook also provides advice
on sample preparation, design, and optimization of different assays.
Western blot analysis is used to confirm protein identity and quantitate the level of target molecule
Detection and assay of tagged proteins
1. Separate the protein samples by SDS-PAGE. SDS-PAGE, Western blotting, and ELISA can also be applied to the detection and
2. T ransfer the separated proteins from the gel to an appropriate membrane, depending on the choice assay of genetically engineered molecules to which a specific tag has been attached.
of detection reagents. Amersham Protran™ (NC) or Amersham Hybond™ P (PVDF) membranes are recommended In some cases, an assay based on the properties associated with the tag itself can
for chemiluminescent detection using Amersham ECL™ start, Amersham ECL, Amersham ECL Prime, be developed, for example, the GST Detection Module for enzymatic detection and
or Amersham ECL Select™ Western blotting detection reagents. Amersham Protran Premium (NC) quantitation of GST-tagged proteins. Further details on the detection and quantitation
or Amersham Hybond LFP (PVDF) membranes are recommended for fluorescent detection with of GST and (his)6-tagged proteins are available in the Recombinant Protein Purification
Amersham ECL Plex™ Western blotting detection system. Handbook, 18114275 and the GST Gene Fusion System Handbook, 18115758 from Cytiva.

3. Develop the membrane with the appropriate specified reagents.

150
Appendix 9
Storage of biological samples
The advice given here is of a general nature and cannot be applied to every biological sample. Always consider
the properties of the specific sample and its intended use before following any of these recommendations.

General recommendations Specific recommendations for purified proteins

Add stabilizing agents, when necessary. Stabilizing agents are often required for storage of purified proteins Store as a precipitate in high concentration of ammonium sulfate, for example 4.0 M
Serum, culture supernatants, and ascitic fluid should be kept frozen at -20°C or -70°C, in small aliquots Freeze in 50% glycerol, especially suitable for enzymes
Avoid repeated freeze/thawing or freeze drying/redissolving that can reduce biological activity A
 void the use of preserving agents if the product is to be used for a biological
assay. Preserving agents should not be added if in vivo experiments are to be
Avoid conditions close to stability limits for example pH or salt concentrations, reducing or chelating agents
performed. Store samples in small aliquots and keep frozen
K
 eep refrigerated at 4°C in a closed vessel to minimize bacterial growth and protease activity. Above 24 h at 4°C,
Sterile filter to prolong storage time
add a preserving agent if possible (e.g., merthiolate 0.01%)
A
 dd stabilizing agents such as glycerol (5% to 20%) or serum albumin (10 mg/mL)
Sodium azide can interfere with many coupling methods and some biological assays and can be a health hazard.
to help maintain biological activity. Remember that any additive will reduce the
It can be removed by using a desalting column (see Appendix 1, Sample preparation).
purity of the protein and might need to be removed at a later stage
A
 void repeated freeze/thawing or freeze drying/redissolving that can reduce
biological activity
Certain proteins, including some mouse antibodies of the IgG3 subclass, should
not be stored at 4°C as they precipitate at this temperature. Keep at room
temperature in the presence of a preserving agent.

151
Appendix 10
Column cleaning
Correct preparation of samples and buffers and application of a high salt wash (1 M NaCl) at the end of each separation
should keep most columns in good condition. However, reduced performance, a slow flow rate, increasing back pressure,
or complete blockage are all indications that the medium needs to be cleaned using more stringent procedures in order
to remove contaminants.
Reverse the direction of flow during column cleaning so that contaminants do not need to pass through the entire 1. Wash with 2 CV of 6 M guanidine hydrochloride.


length of the column. The number of column volumes and time required for each cleaning step varies according to 2. Wash immediately with at least 5 CV of buffer at pH 7.0 to 8.0.
the degree of contamination. If the cleaning procedure to remove common contaminants does not restore column
3. R
 inse with at least 2 CV of distilled water until the UV-baseline and eluent
performance, change the top filter (when possible) before trying alternative cleaning methods. Care should be
pH are stable.
taken when changing a filter as this can affect the column packing and interfere with performance. The cleaning
procedure to remove common contaminants is included with each of the media described in Chapter 3. 4. W
 ash with at least 4 CV of start buffer or storage buffer until pH and
conductivity values have reached the required values.
Removing precipitated proteins, lipids, hydrophobically bound proteins, or lipoproteins Alternatively,
To remove precipitated proteins 1. Inject 1 CV of pepsin (1 mg/mL in 500 mM NaCl, 100 mM acetic acid). Leave
Use the recommended flow rate for cleaning of the media and column, see Chapter 3. When guanidine hydrochloride is overnight at room temperature or for 1 h at 37°C.
used, the flow rate should be lowered to half of this flow rate to avoid overpressure.
2. R
 inse with at least 2 CV of distilled water until the UV-baseline and the
eluent pH are stable.
3. W
 ash with at least 4 CV of start buffer or storage buffer, until eluent pH and
conductivity have reached the required values.

152
To remove lipids, hydrophobically bound proteins, or lipoproteins
Organic solvents or detergents might be required to completely remove contaminants of this type. 1. Wash with 4 CV of up to 70% ethanol or 30% isopropanol.

Before using organic solvents, wash the medium with at least 4 CV of distilled water to avoid salts precipitating 2. R
 inse with at least 2 CV of distilled water until the UV-baseline and eluent


on the column. pH are stable.

When applying organic solvents or solutions it might be necessary to reduce the flow rate considerably to avoid 3. Wash immediately with 3 CV of start buffer.


overpressuring the column. Alternatively,

Use cleaning solutions such as up to 30% isopropanol, up to 100% methanol, up to 100% acetonitrile, up to 2 M NaOH, 1. Wash with 2 CV of detergent in a basic or acidic solution, for example,
up to 75% acetic acid, up to 100% ethanol, ionic or nonionic detergents. 0.1% to 0.5% nonionic detergent in 100 mM acetic acid.
 When cleaning larger columns, allow a contact time of 1 to 2 h for any solution that is used as an initial cleaning step. 2. Rinse with 5 CV 70% ethanol to remove residual detergent.
Always check for solvent compatibility in the instructions supplied with the medium 3. R
 inse with at least 2 CV of distilled water until the UV-baseline and the
or column. eluent pH are stable.
Avoid anionic detergents with Q, DEAE, and ANX charged groups. Avoid cationic detergents with S, SP, and 4. Wash with 3 CV of start buffer.
CM charged groups.

Extended cleaning procedures

Use the recommended flow rate and cleaning procedure for the media and column, see Chapter 3. If this is not sufficient,
extended cleaning procedures can be tested. When organic solvents, such as 30% isopropanol or 70% ethanol are used,
the flow rate should be lowered to half of this flow rate to avoid overpressure.

153
Appendix 11
Media selection
Using prepacked small columns such as HiTrap during the early stages of development saves time, solvents and START CONDITIONS Run 1 Run 2 Run 3 Run 4 Run 5 Run 6 Run 7
sample. HiTrap IEX Selection Kit allows quick and efficient screening for the most suitable charge group and enables pH 7 6.5 6 5.5 5 4.5 4
development of the basic method. The following are descriptions of different screening methods for selection of media
and optimal conditions.
80.0
Selection of media for automated purification 250
Users of ÄKTA systems with automatic buffer preparation functionality can select from a range of buffer recipes to test
different media over a range of pH values and other elution conditions. See ÄKTA Laboratory-scale Chromatography
Systems: Instrument Management Handbook, 29010831 for details of how to vary flow rate and gradient slope in order to 200 60.0

Conductivity (mS/cm)
optimize the separation.
Note that the condition of the sample is very important in order to achieve the most effective separations. Samples pH 4.0

A 280 nm


should preferably have the same conditions as the start buffer (see Buffer exchange and desalting in Appendix 1 150
pH 4.5
for details). When working with small volumes during screening and scouting, it might be sufficient to dilute the
40.0
sample in start buffer in order to lower the ionic strength and adjust the pH to a value similar to that of the start buffer. pH 5.0
100
pH 5.5

1. Scout for optimum pH by testing a range of pH values within which the proteins of interest are known to be pH 6.0
stable. If the pI of the target protein is known, then begin with a narrower pH range, for example, 0.5 to 1.0 50 20.0
pH 6.5
pH unit away from the pI. Typical results from an automatic pH scouting run are shown in Figure A11.1.
2. If required, scout for optimum selectivity (testing strong or weak exchangers) using automatic media scouting. pH 7.0
0
3. Scout for the steepest gradient that gives acceptable resolution at the selected pH. 2.0 4.0 6.0 8.0 Time (min)
4. S
 cout for the highest flow rate that maintains resolution and minimizes separation time. Check recommended
Column: RESOURCE S, 6 mL
flow rates for the specific medium. Sample: Fab fraction from HIC separation, 20 mL
5. S
 cout for the maximum sample load that can be applied while maintaining satisfactory resolution. In general, Eluents: Automatic BufferPrep with 60 mM sodium acetate, 30 mM sodium phosphate,
loading 20% to 30% of the total binding capacity of the column gives optimal resolution with gradient elution. 30 mM sodium formate 100 mM HCI and 2 M NaCl
Gradient: 20 column volumes, to 1 M NaCl
Flow rate: 60 mL/min
Reduce separation time and buffer consumption by transfering to a step elution when optimized separation Curves: A280nm, from top: pH 4.0; 4.5; 5.0; 5.5; 6.0; 6.5; 7.0


conditions have been established. Sample loads can often be increased when using a step elution.
Fig A11.1. Automatic pH scouting on an ÄKTA system.

154
Selection of media for manual purification Screening for ionic strength conditions
HiTrap columns are well-suited to manual media screening, method development, and method optimization since they
can be used with a syringe or peristaltic pump as well as an automated chromatography system.
1. U
 sing the selected medium, start buffer and pH from the previous
 Note that the condition of the sample is very important to achieve the most effective separations. Samples should protocol, set up a series of elution buffers at the same pH, but vary the
preferably have the same conditions as the start buffer (see Appendix 1, Sample preparation for details). When salt concentration from 0 to 500 mM with intervals of 50 to 100 mM salt
working with small volumes during screening it might be sufficient to dilute the sample in start buffer in order to between each buffer.
lower the ionic strength and adjust the pH to a value similar to that of the start buffer.
2. Repeat steps 3 to 8 from the previous protocol for each salt concentration.
Scout for optimum pH by testing a range of pH values within which the proteins of interest are known to be stable.

3. D
 etermine the maximum ionic strength which permits binding of the protein(s)
If the pI of the target protein is known, then begin with a narrower pH range, for example, 0.5 to 1.0 pH unit away
of interest and the minimum ionic strength required for complete elution.
from the pI. The methods here are optimized for use with 1 mL HiTrap columns and should be adjusted if other
column volumes are used.
Screening for IEX medium and pH conditions Further optimization

1. S
 tart buffers: set up a series of buffers with pH values in the range 4.0 to 8.0 (SP, CM) or 5.0 to 9.0 (Q, DEAE, 1. I f gradient making equipment is available, determine the steepest gradient
ANX) and with 0.5 to 1.0 pH unit intervals between each buffer. See Appendix 2 for recommended buffers. that gives acceptable resolution at the selected pH. Begin with a gradient
of 10 CV over an ionic strength range based on the maximum and minimum
2. Elution buffers: set up a second series of buffers with the same pH values, but including 1 M NaCl.
values determined when screening. Alternatively, begin with a gradient
3. Equilibrate the column(s) with 5 mL start buffer at 1 mL/min. Wash with 5 mL elution buffer. of 0% to 50% elution buffer that contains 1 M NaCl and a gradient volume
4. Re-equilibrate with 5 to 10 mL start buffer. of 10 to 20 CV.

5. A
 djust the sample to the pH of the start buffer and apply a known amount of the sample at 1 mL/min. Collect 2. D
 etermine the highest flow rate that maintains resolution and minimizes
eluate. separation time. Check recommended flow rates for the specific medium.

6. Wash with at least 5 mL of start buffer or until no material appears in eluent. Collect eluate. 3. D
 etermine the maximum sample load that can be applied while maintaining
satisfactory resolution. In general, loading 20% to 30% of the total binding
7. E
 lute bound material with elution buffer (3 to 5 mL is usually sufficient, but other volumes might be required capacity of the column gives optimal resolution with gradient elution.
dependent on the exact experimental conditions). Collect eluate. Sample loads can often be increased if resolution is satisfactory or when
8. Analyze all eluates (for example by an activity assay) and determine purity and the amount bound to the using a step elution.
column.
9. Perform steps 3 to 8 for the next buffer pH.
10. Select medium and pH: the most suitable pH should allow the protein(s) of interest to bind, but should be as
close to their point of release as possible.

155
Using PD-10 columns for media selection and method development Ionic strength selection
If an assay is available to detect the target protein(s), PD-10 columns can be packed with various media and used to find
the most suitable separation conditions. With basic information on the requirements for pH and ionic strength, a suitable 1. S
 et up a series of 10 × PD-10 columns, each containing 5 mL of the chosen
column can be packed in order to begin optimization. IEX medium.
Note that the condition of the sample is very important in order to achieve the most effective separations. Samples 2. E
 quilibrate the column by washing (5 × 5 mL) with buffer (500 mM) at the
should preferably have the same conditions as the start buffer (see Appendix 1, Sample preparation for details). selected starting pH.
When working with small volumes during screening and scouting, it might be sufficient to dilute the sample in start
buffer in order to lower the ionic strength and adjust the pH to a value similar to that of the start buffer. 3. E
 quilibrate the columns at different ionic strengths, but constant pH,
ranging from 10 to 300 mM NaCl by washing (5 × 5 mL). Intervals of 50 mM
pH selection NaCl are sufficient.
4. Apply sample while collecting the eluent.
1. Set up a series of 10 × PD-10 columns for each medium to be tested and thoroughly resuspend the medium 5. A
 ssay the eluent to determine the maximum ionic strength which permits
in its storage solution. binding of the target protein and the minimum ionic strength required for
2. P
 our medium slurry containing 5 mL medium into the PD-10 column, allowing the medium to settle as the complete elution. The highest ionic strength which permits binding and
column fills. Do not allow the column to dry out. the lowest ionic strength for elution are used as start and elution buffers,
respectively, during subsequent gradient elution.
3. E
 quilibrate each column to a different pH by washing (5 × 5 mL) with buffer (500 mM) using buffers between
pH 5.0 to 9.0 for anion exchangers or pH 4.0 to 8.0 for cation exchangers and with 0.5 pH unit intervals between
columns (see Appendix 2 for buffer recommendations).
4. Equilibrate each column at a lower ionic strength: wash with 5 × 5 mL of buffer (20 to 50 mM) at the same pH.
5. Load a known constant amount of sample to each column while collecting the eluent.
6. A
 ssay the eluent for the protein of interest. The most suitable medium and pH should allow the protein to bind
(protein is absent from the eluent), but should be as close to the point of release as possible (the first pH at
which the protein appears in the eluent).

156
Product index
A C HiScreen Capto SP ImpRes 100 Mini S PC 3.2/3 49-51
ÄKTA avant 144–145 Capto DEAE 98–100, 107, 122-123 HiTrap ANX FF (high sub) 77, 79 Mono Q 10/100 GL 56
ÄKTAexplorer 145 Capto Q 98–100, 107, 117, 122, 124 HiTrap Capto DEAE 100 Mono Q 4.6/100 PE 56
ÄKTAfplc 145 Capto Q ImpRes 98-100, 107, 122, 124 HiTrap Capto Q 100 Mono Q 5/50 GL 29, 56-58
ÄKTAprime plus 144–145 Capto S 98–100, 107, 122–124 HiTrap Capto Q ImpRes 100 Mono Q HR 16/10 56
ÄKTApurifier 145 Capto S ImpAct 23, 98–100, 104, 107-108, 117, 122, 125 HiTrap Capto S 100 Mono S 10/100 GL 56
ÄKTA pure 48, 144 Capto SP ImpRes 98–100, 103, 107, 122, 125 HiTrap Capto S ImpAct 100 Mono S 4.6/100 PE 56
ÄKTA start 144–145 CM Sepharose Fast Flow 19, 78-80, 86, 122 HiTrap Capto SP ImpRes 100 Mono S 5/50 GL 56-58, 118
ÄKTAxpress 144–145 D HiTrap CM FF 80–81, 89 Mono S HR 16/10 56
Amersham ECL 150 DEAE Sepharose Fast Flow 19, 78-79, 91, 122-123 HiTrap DEAE FF 77, 79 P
Amersham ECL Plex 150 F HiTrap Desalting 132, 136-137 PD MiniTrap G-25 136
Amersham ECL Prime 150 FineLINE 100 column 64–66, 126 HiTrap Heparin HP 57 PD-10 Desalting column 73
Amersham ECL Select 150 FineLINE 800 column 126 HiTrap IEX Selection Kit 24, 88, 115, 141, 154 PhastGel 57
Homogeneous – 12.5
Amersham Hybond LFP 150 FineLine Pilot 35 column 126 HiTrap Q FF 77, 79, 83
PhastSystem 57
Amersham Hybond P 150 H HiTrap Q HP 36, 71, 73 electrophoresis unit
Amersham Protran Premium 150 HiPrep CM FF 16/10 80 HiTrap Q XL 77, 88-89 PlusOne Coomassie Tablets, 149
Amersham WB 150 HiPrep DEAE FF 16/10 79, 82 HiTrap SP FF 80-81, 89 PhastGel Blue R-350
(Western blotting) Cy5 PlusOne Silver Staining Kit, 149
HiPrep Q FF 16/10 79, 82-83 HiTrap SP HP 71-73
Amersham WB system 150 Protein
HiPrep Q XL 16/10 88, 115 HiTrap SP XL 81, 88-89
ANX Sepharose 4 Fast Flow 19, 77–79, 122 PrimeView software 144
(high sub) HiPrep SP FF 16/10 80 I
Puradisc 4 Syringe Filter, 130
Axichrom 1000 column 101 HiPrep SP HP 16/10 71-72, 80 INdEX 70 column 90 0.2 µm, PVDF

B HiPrep SP XL 16/10 88 L Puradisc 13 Syringe Filter, 130

0.2 µm, PVDF
Benzamidine Sepharose 4 45 HiPrep 26/10 Desalting 118, 136-137 LMW-SDS Marker Kit 57
Fast Flow (high sub) Puradisc 25 Syringe Filter, 130
HiScreen Capto DEAE 100 M
0.2 µm, PVDF
BPG 300 column 94 HiScreen Capto Q 100, 124 Mini Q 4.6/50 PE 49-50
Puradisc 4 Syringe Filter, 130
BPG 450 column 101 HiScreen Capto Q ImpRes 100, 124 Mini Q PC 3.2/3 49, 51 0.45 µm, PVDF

HiScreen Capto S 100, 125 Mini S 4.6/50 PE 49-50 Puradisc 13 Syringe Filter, 130
0.45 µm, PVDF
HiScreen Capto S ImpAct 100, 125

157
Puradisc 25 Syringe Filter, 130 SPARTAN 13 mm 130
0.45 µm, PVDF HPLC-Certified Syringe Filter,
RC, 0.45 µm
Puradisc FP 30 Syringe Filter, 130
0.2 µm, CA SPARTAN 30 mm 130
HPLC-Certified Syringe Filter,
Puradisc FP 30 Syringe Filter, 130 RA, 0.2 µm
0.45 µm, CA
SPARTAN 30 mm 130
Q HPLC-Certified Syringe Filter,
Q Sepharose Big Beads 95, 97, 122 RA, 0.45 µm

Superdex 75 prep grade 119

Q Sepharose Fast Flow 13, 18, 23, 77-79, 83, 122-123
Superdex 200 Increase 104
Q Sepharose High 23, 70-71, 76, 124
10/300 GL
Performance
T
Q Sepharose XL 23, 87-88, 90, 93, 122-123
Tricorn 10/100 63, 72, 81, 88
R
Tricorn 10/150 63, 72, 81, 88
RESOURCE Q 23, 61, 63-66
Tricorn 10/200 63, 72, 81, 88
RESOURCE S 63-65, 126, 154
Tricorn 5/100 102, 104, 117
S
Tricorn 5/50 103, 146
Sephadex G-25 134-136
U
SOURCE 15Q 57, 62-63, 66, 122, 126
UNICORN start software 144
SOURCE 15Q 4.6/100 PE 57, 63, 66
UNICORN 5 software 144
SOURCE 15S 62-64, 122, 126
UNICORN 6 software 144
SOURCE 15S 4.6/100 PE 63
X
SOURCE 30Q 23, 30, 62-63, 65, 122, 126
XK 16/20 column 63, 72, 81, 88, 90, 95, 144
SOURCE 30S 33, 62-63, 66, 122, 126
XK 16/40 column 102
SP Sepharose Big Beads 94-95, 97, 122
XK 16/60 column 119
SP Sepharose Fast Flow 18, 78, 80, 86, 122-123
XK 16/70 column 146
SP Sepharose High 23, 70-71, 76, 98, 125
Performance XK 26/20 column 63, 72, 81, 88, 95
SP Sepharose XL 87-88, 93, 122-123 XK 26/40 column 63, 72, 81, 88, 95

SPARTAN 13 mm 130 XK 50/20 column 81, 88, 95

HPLC-Certified Syringe Filter,
RC, 0.2 µm XK 50/30 column 81, 88, 95

158
Related literature
Code number

Purification handbooks

Affinity Chromatography 18102229

Antibody Purification 18103746

Hydrophobic Interaction and Reversed Phase Chromatography 11001269

Multimodal Chromatography 29054808

Protein Sample Preparation 28988741

Purifying Challenging Proteins 28909531

Recombinant Protein Purification 18114275

Size Exclusion Chromatography 18102218

Strategies for Protein Purification 28983331

ÄKTA Laboratory-scale Chromatography Systems 29010831

Protein analysis handbooks

Biacore Assay 29019400

Biacore Sensor Surface BR100571

Western Blotting 28999897

Selection guides and multimedia

Ion exchange columns and media, Selection guide 18112731

Prepacked chromatography columns for ÄKTA systems, Selection guide 28931778

159
Ordering information
Ion exchange
Product Quantity Code number Product Quantity Code number Product Quantity Code number
Prepacked columns HiTrap SP HP 1 × 1 mL 29051324
MiniBeads
RESOURCE Q 1 × 1 mL 17117701 HiTrap SP HP 5 × 1 mL 17115101
Prepacked columns
17068601 RESOURCE Q 1 × 6 mL 17117901 HiTrap SP HP 5 × 5 mL 17115201
Mini Q PC 3.2/3 1 × 0.24 mL
17068701 SOURCE 15Q 4.6/100 PE 1 × 1.7 mL 17518101 HiScreen SP HP 1 × 4.7 mL 28950515
Mini S PC 3.2/3 1 × 0.24 mL
17517701 RESOURCE S 1 × 1 mL 17117801 HiPrep SP HP 16/10 1 × 20 mL 29018183
Mini Q 4.6/50 PE 1 × 0.8 mL
17517801 RESOURCE S 1 × 6 mL 17118001
Mini S 4.6/50 PE 1 × 0.8 mL Sepharose Fast Flow
SOURCE 15S 4.6/100 PE 1 × 1.7 mL 17518201
Chromatography media packs
MonoBeads
SOURCE 30 Q Sepharose Fast Flow 25 mL 17051010
Prepacked columns
17516601 Chromatography media packs Q Sepharose Fast Flow 300 mL 17051001
Mono Q 5/50 GL 1 × 1 mL
17516701 SOURCE 30Q 10 mL 17127510 SP Sepharose Fast Flow 25 mL 17072910
Mono Q 10/100 GL 1 × 8 mL
17517901 SOURCE 30Q 50 mL 17127501 SP Sepharose Fast Flow 300 mL 17072901
Mono Q 4.6/100 PE 1 × 1.7 mL
17050601 SOURCE 30Q 200 mL 17127505 DEAE Sepharose Fast Flow 25 mL 17070910
Mono Q HR 16/10 1 × 20 mL
17516801 SOURCE 30S 10 mL 17127320 DEAE Sepharose Fast Flow 500 mL 17070901
Mono S 5/50 GL 1 × 1 mL
17516901 SOURCE 30S 50 mL 17127301 CM Sepharose Fast Flow 25 mL 17071910
Mono S 10/100 GL 1 × 8 mL
17518001 SOURCE 30S 200 mL 17127302 CM Sepharose Fast Flow 500 mL 17071901
Mono S 4.6/100 PE 1 × 1.7 mL
17050701 ANX Sepharose 4 Fast Flow (high sub) 25 mL 17128710
Mono S HR 16/10 1 × 20 mL Sepharose High Performance
ANX Sepharose 4 Fast Flow (high sub) 500 mL 17128701
Chromatography media packs
SOURCE 15
17108701 Prepacked columns
Chromatography media packs SP Sepharose High Performance 75 mL
HiTrap IEX Selection Kit 7 × 1 mL 17600233
17094720 Q Sepharose High Performance 75 mL 17101401
SOURCE 15Q 10 mL
Kit contains seven HiTrap columns prepacked with Fast Flow (FF) media: Q Sepharose FF, DEAE Sepharose FF, SP Sepharose FF,
SOURCE 15Q 50 mL 17094701 Prepacked columns CM Sepharose FF, ANX Sepharose FF(high sub), Q Sepharose XL, and SP Sepharose XL

17094705 HiTrap Q HP 1 × 1 mL 29051325

SOURCE 15Q 200 mL HiTrap Q FF 5 × 1 mL 17505301

17094410 HiTrap Q HP 5 × 1 mL 17115301

SOURCE 15S 10 mL HiTrap Q FF 5 × 5 mL 17515601

17094401 HiTrap Q HP 5 × 5 mL 17115401

SOURCE 15S 50 mL HiPrep Q FF 16/10 1 × 20 mL 28936543

17094405 HiPrep Q HP 16/10 1 × 20 mL 29018182

SOURCE 15S 200 mL

160
Product Quantity Code number Product Quantity Code number Product Quantity Code number

HiTrap SP FF 5 × 1 mL 17505401
Capto Prepacked columns
HiTrap SP FF 5 × 5 mL 17515701
Chromatography media packs HiTrap Capto Q ImpRes 5 × 1 mL 17547051
HiPrep SP FF 16/10 1 × 20 mL 28936544
Capto Q 25 mL 17531610 HiTrap Capto Q ImpRes 5 × 5 mL 17547055
HiTrap DEAE FF 5 × 1 mL 17505501
Capto Q 100 mL 17531602 HiScreen Capto Q ImpRes 1 × 4.7 mL 17547015
HiTrap DEAE FF 5 × 5 mL 17515401
Capto S 25 mL 17544110 HiTrap Capto SP ImpRes 5 × 1 mL 17546851
HiPrep DEAE FF 16/10 1 × 20 mL 28936544
Capto S 100 mL 17544101 HiTrap Capto SP ImpRes 5 × 5 mL 17546855
HiTrap CM FF 5 × 1 mL 17505601
Capto DEAE 25 mL 17544310 HiScreen Capto SP ImpRes 4.7 mL 17546815
HiTrap CM FF 5 × 5 mL 17515501
Capto DEAE 100 mL 17544301
Capto ImpAct
HiPrep CM FF 16/10 1 × 20 mL 28936542
Prepacked columns
Chromatography media packs
HiTrap ANX FF (high sub) 5 × 1 mL 17516201
HiTrap Capto IEX Selection Kit 5 × 1 mL 28934388
Capto S ImpAct 25 mL 17371701
HiTrap ANX FF (high sub) 5 × 5 mL 17516301 Contains five HiTrap columns prepacked with: Capto Q, Capto S, Capto DEAE, Capto MMC, Capto adhere
Capto S ImpAct 100 mL 17371702
Sepharose XL HiTrap Capto Q 5 × 1 mL 11001302
Prepacked columns
Chromatography media packs HiTrap Capto Q 5 × 5 mL 11001303
HiTrap Capto S ImpAct 5 × 1 mL 17371751
Q Sepharose XL 300 mL 17507201 HiScreen Capto Q 1 × 4.7 mL 28926978
HiTrap Capto S ImpAct 5 × 5 mL 17371755
SP Sepharose XL 300 mL 17507301 HiTrap Capto S 5 × 1 mL 17544122
HiScreen Capto S ImpAct 1 × 4.7 mL 17371747
Prepacked columns HiTrap Capto S 5 × 5 mL 17544123 SOURCE, Sepharose High Performance, Sepharose Fast Flow, Sepharose XL, Sepharose Big Beads, and Capto are all available as
BioProcess media for large-scale production. Visit www.cytiva.com/bioprocess
HiTrap Q XL 5 × 1 mL 17515801 HiScreen Capto S 1 × 4.7 mL 28926979

HiTrap Q XL 5 × 5 mL 17515901 HiTrap Capto DEAE 5 × 1 mL 28916537

Prepacked desalting columns
HiPrep Q XL 16/10 1 × 20 mL 28936538 HiTrap Capto DEAE 5 × 5 mL 28916540
HiTrap Desalting 1 × 5 mL 29048684
HiTrap SP XL 5 × 1 mL 17516001 HiScreen Capto DEAE 1 × 4.7 mL 28926982
HiTrap Desalting 5 × 5 mL 17140801
HiTrap SP XL 5 × 5 mL 17516101 HiPrep 26/10 Desalting 1 × 53 mL 17508701
Capto ImpRes
HiPrep SP XL 16/10 1 × 20 mL 28936540 Chromatography media packs HiPrep 26/10 Desalting 4 × 53 mL 17508702

Sepharose Big Beads Capto Q ImpRes 25 mL 17547010 PD-10 Desalting Column 30 17085101

Chromatography media packs Capto Q ImpRes 100 mL 17547002

Q Sepharose Big Beads 1L 17098903 Capto SP ImpRes 25 mL 17546810

Empty columns
Tricorn 10/100 1 18116315
SP Sepharose Big Beads 1L 17065703 Capto SP ImpRes 100 mL 17546802
XK 16/20 1 18877301

XK 26/20 1 18100072

161
Product Quantity Code number

XK 50/20 1 18100071

Empty Disposable PD-10 Desalting columns 50/pk 17043501

LabMate PD-10 Buffer Reservoir 1 18321603

Accessories and spare parts

Packing Connector XK 16 1 18115344

Packing Connector XK 26 1 18115345

Packing equipment 10/100 (Tricorn) 1 18115325

Packing Connector 10-10 1 18115323

162
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