Chapter 5

Scale-Up of Protein Purification: Downstream Processing

Issues
John Joseph Milne
Abstract
Large-scale chromatography operations continue to occupy a key position in the overall strategy for the
downstream processing and purification of protein products for therapeutic use. Increasing product titres
from mammalian cell culture has resulted in a trend to identify ways of improving the economics of
product recovery and purification processes. In commercial manufacturing, a requirement exists to
increase the scale of the chromatography operations, which are typically developed and optimised in
small-scale experiments. This short review discusses the key factors in the chromatography process that
need to be considered as the scale of the purification step is increased in order to maintain the purity and
integrity of the product purified at smaller scale.
Key words: Protein purification, Downstream processing, Chromatography, Scale-up

1. Introduction
The market for biopharmaceutical products represents an increasingly significant proportion of total pharmaceutical annual sales
resulting mainly from the considerable interest in monoclonal
antibodies as therapeutic agents (1). The system of choice for the
production of complex recombinant proteins remains the mammalian cell with expression in Chinese Hamster Ovary (CHO)
cells grown in suspension culture being the most popular choice
to date for manufacturers (2, 3). Expression of recombinant proteins in mammalian cells offers advantages in terms of protein
folding and post-translational modifications when compared with
other systems (4). Tremendous developments have taken place in
mammalian cell culture in the areas of cell-line development, new
media formulations and bioreactor design (5) which has led to a

Dermot Walls and Sinad T. Loughran (eds.), Protein Chromatography: Methods and Protocols, Methods in Molecular
Biology, vol. 681, DOI 10.1007/978-1-60761-913-0_5, Springer Science+Business Media, LLC 2011

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concomitant increase in cell titres with industrial claims of product

titres in the range 510g/L. Due to these advances in industrial
cell-culture processes, the focus for biopharmaceutical manufacturers has shifted to those unit operations downstream of the bioreactor where new innovations in process design will be required
in order to improve process capacity, speed, and economics (6).
Downstream operations are typically carried out at a scale that is
dependent on the amount of product required and in order to
avoid increased capital investment and optimise facility usage,
process improvements, and new innovations will be required in
the future (7).
Production of a licenced biopharmaceutical therapeutic
product involves several disciplines with downstream processing
occupying a central position in the overall process of ensuring the
ultimate safety of the target product. Downstream processing can
be defined as a series of separation and purification activities that
together can produce a protein product fit for its intended use.
Protein purification using chromatography is currently the central
enabling technology in all biopharmaceutical downstream processing (8). Large-scale process chromatography has played a key
role in the purification of therapeutic proteins and although alternative techniques such as the use of membrane adsorption are
currently receiving attention preparative chromatography will
most likely remain the primary purification platform within the
biopharmaceutical industry (7, 9). However, due to the increased
volumes of cell-culture harvests (>10,000L) and associated high
levels of protein expression (>10g/L) that are predicted to occur
in the near future, increased productivity will be required to alleviate the real bottleneck in downstream processing that will exist
over the next few years (10, 11).
1.1. Protein
Purification Using
Chromatography

Following cell-culture expression, the purification of proteins

proceeds through a series of sequential unit operations downstream of the bioreactor to achieve solidliquid fractionation after
harvesting. One such operation is chromatography in which a
sample dissolved in a mobile phase is passed through a stationary
phase held in a column shell. In preparative chromatography, the
stationary phase is selected on the basis of its ability to purify the
target molecule from a panel of contaminating proteins. In the
case of non-affinity-based capture chromatography, product concentration using ultrafiltration and diafiltration is used to condition the feed material produced in the bioreactor prior to
separation. The purification phase of protein manufacture is
designed to specifically take the target molecule from the upstream
stage to the final formulation stage, achieving the required level
of purity and recovery and to do so safely, reliably, and economically. In order to achieve a product licence, regulatory agencies
require that process characterisation, risk mitigation, and validation

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are carried out prior to market approval. Each of these disciplines

presents distinct challenges and requires a thorough knowledge
of manufacturing capabilities (12).
Within industry, the primary method of protein purification
currently used is process chromatography which is typically performed on a large scale. Such chromatography must be robust
and reproducible to be capable of dealing with the variations
inherent in the use of cell-based protein expression systems. When
chromatography is selected as a method of purification, process
development will typically involve distinct phases of bench-scale
development, optimisation of chromatographic parameters, and
scale-up to the required final scale. Scale-up of chromatography
presents unique challenges in terms of both the media selected
and equipment used and is a critical issue for the successful commercialisation of biopharmaceuticals (13).
When practitioners set out to design a purification process,
the basis of each chromatography step in the overall scheme should
be assessed in small-scale laboratory experiments. Purification
schemes for currently licenced products typically involve at least
three orthogonal chromatography steps, each offering a different
selectivity and exploiting alternative attributes of the target product and/or contaminants in order to maximise the overall purity
of the product (14). It is important that as a purification process
is being developed at small scale, reference is made to the predicted final scale of the process, such that specific issues relating to
the scale-up can be fully appraised. This is particularly important,
if the protein is to be purified according to the guidelines of current good manufacturing practice (GMP) as defined by regulatory
authorities (15).
Chromatography is generally regarded as being composed of
three distinct phases namely capture, intermediate purification,
and final polishing. Each of these phases requires a different focus
and different challenges will be presented as the process is scaledup. The initial capture step can involve protein isolation from
crude feedstock and thus requires a medium with a high capacity
and high operating flow rate. The binding capacity of the medium
for the target protein in the presence of many impurities, rather
than resolution, is the most critical factor. As process streams can
be large, fast flow resins possessing a large bead size range are
used to improve overall processing times and thus stabilise the
protein product quickly. Economic constraints generally require
the resin to be recycled for a validated number of uses when largescale capture applications are involved in which the volume of
medium is large. Due to the crude nature of the starting material,
the ability to clean and sanitise media effectively is an important
issue to be considered. Resolution of the target protein from
host-cell contaminants becomes more important in intermediate
purification applications. To ensure productivity and process

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economics, the binding capacity of the medium for the target

protein is also important. Since bead size correlates with resolution, smaller bead sizes are often more appropriate at this stage of
purification. Speed is usually less critical in intermediate purification applications since impurities affecting stability of the target
will have been removed in a successfully optimised capture step.
In polishing chromatography, the main issue is resolution, to
ensure removal of trace contaminants and also structural variants
of the target that can lead to immunogenicity concerns following
therapeutic administration (16). High efficiency media with small
bead size are used in final polishing chromatography. As the use
of a smaller bead size will result in increased pressure, lower flow
rates are used and process times can be longer. In overall terms,
the performance criteria to be considered in developing an optimised process are speed, recovery, capacity, and resolution. The
priority given to these attributes will be dependent on the nature of
the chromatographic step and in particular whether the purification step is operating in capture, intermediate, or polishing mode.
1.2. Scale-Up
ofProtein
Chromatography

Following small-scale scouting trials, a variety of media will have

been screened in order to select a suitable medium that can provide a basis for separation of the target protein from impurities in
the sample feed material. Once a medium has been defined, the
next stage in the process is to design specific chromatographic
procedures that can optimise the dual requirements of product
recovery and purity, by defining the buffer solutions used during
sample conditioning, media equilibration, and product elution.
Typically, at this stage, the purification process will be capable of
producing milligram quantities of protein. The main result of
process optimisation is that product information and process
parameters will have been considered in detail and where possible
defined to ensure smooth technical transfer when the process is
ultimately scaled-up (see Table1). When the running conditions
and chromatography parameters have been determined for any
column purification step, the final issue to resolve is how the
chromatographic process can be scaled appropriately to a level
required for required large-scale production (17). Scaling from
laboratory to pilot plant can involve scale-up factors of 50- to
100-fold, while further increasing from pilot plant to commercial
production scale will involve another 10- to 50-fold scale-up (18).
Scale-up of chromatography should involve a detailed consideration of chromatographic, non-chromatographic, and equipmentrelated issues, and the role of these factors can play in successful
scale-up.
An increase in the scale of chromatographic purification is
most typically achieved by increasing the column diameter and
volumetric flow rate, while at the same time ensuring that the
media bed height and linear flow rate remain constant (19)

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Table1
Parameters to be defined during the optimisation phase
of development
Process parameters

Sample pre-conditioning required

Sample concentration (load/ml resin)
Sample volume (load/ml resin)
Product concentration (load/ml resin)
Resin bed height (cm)
Linear flow rates (cm/h)
Process volumes
Pressure drop
Packed column qualification
Buffer conditions (pH, conductivity)
Fractionation scheme if used
Stability of resin to cleaning and sanitisation agents

Product parameters Stability

Solubility in buffers
Storage conditions
Storage time

Table2
Status of various process parameters during chromatography
scale-up
Remain constant

Column bed height

Linear flow rates (cm/h) at all stages
Sample attributes (concentration and
conditioning)
Ratio of sample volume to media volume
Ratio of gradient volume to media volume
Buffer specifications

Increased

Column diameter
Volumetric flow rate (ml/min)
Sample volume proportionally
Gradient volume proportionally
Buffer volumes proportionally

Issues to be addressed

Decreased wall support as column

diameter increases
Quality of packing/bed instability
Increased pressure drop
Uneven flow distribution
Zone broadening

(see Table2). This ensures that the overall residence time of the
target molecule on the small-scale and large-scale columns remains
constant. In theory, the respective separations should be very
similar and therefore provide the basis for successful scale-up.

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However, as the column scale increases other factors such as

choice of buffers and their preparation, media packing, column
engineering, and process hygiene can present limitations and
often problems particularly at the scale of manufacturing required
in commercial biopharmaceutical manufacturing (20). Thus
proper scale-up is a crucial consideration in process design involving the optimisation of many factors in order to ensure robust
and reproducible purification of a protein therapeutic over the
lifetime of the product licence. The remainder of this review will
consider some of the main issues in chromatography scale-up
and the approaches taken by industry to provide acceptable
solutions.

2. Downstream
Processing Issues
Preserving the quality of resolution and the level of purification
determined at laboratory scale is the key to successful chromatography scale-up (21). Whenever a requirement exists to increase
the scale of a chromatography process from small-scale scouting
experiments through pilot scale and on to full commercial manufacturing adhering to the conditions of GMP, a series of key issues
need to be considered to determine their effect on the overall
scale-up process. Such issues can be categorised into those relating to the choice of medium itself and to the chromatographic
separation process. In commercial manufacture, equipment and
hardware issues can often present challenges due to the need to
use large columns and automated systems.
2.1. Chromatography
Media

In protein purification applications where chromatography is

utilised, it is the physical and chemical properties of the medium
used in the process that are the main route to achieving the
required purity. The rational selection of chromatography media
prior to screening experiments is critical for successful downstream processing for industrial chromatography applications
(21). Modern processes require media that are not only consistent and selective but also offer high-dynamic capacities and lowcycle times to be compatible with the increasing large volumes
and high expression levels of current cell-culture processes. The
main considerations when choosing a medium for inclusion in a
production process for therapeutic products are a route to scalable and robust processing, while at the same time ensuring that
procedures can be validated. As the volume of medium required
increases the available commercial supply of media with demonstrated lot-to-lot consistency and full regulatory support documentation is mandatory. Such issues should be noted prior to
early small-scale trials and if not resolved might dictate that a

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different media should be considered for use. There are many

commercial suppliers who offer a large portfolio of media with
differing functionalities that are compatible with the requirements
of industry and regulators alike.
Media and capital costs at large scale should not be underestimated, and cost analysis is frequently an important component
when considering an approach to scale-up. Affinity-based separations while offering clear advantages for overall effectiveness can
often be very expensive particularly as column volumes are
increased. Protein A, a protein naturally found in the cell wall of
Staphylococcus aureus has the ability to selectively interact with the
fraction-crystallizable (Fc) domain of immunoglobulins. A regularly cited limitation with the use of Protein A media for the purification of monoclonal antibodies is the cost of the media itself
which is an order of magnitude more expensive when compared
with media using non-proteinaceous ligands (22). The particle
size distribution of chromatography media impacts directly on
scalability due to its effect on column pressures. The particle size
chosen for any purification step will depend on the nature of the
feed sample and on the degree of resolution required. The desired
resolution will depend on whether the chromatography is operating in capture, intermediate, or final polishing mode.
The inherent stability of the medium chosen is an important
attribute in large-scale manufacture, which can be overlooked
during small-scale development studies. An ideal medium for
large-scale applications should exhibit good chemical and physical stability profiles. The chemical instability of packed column
beds can be caused by leakage of the ligand following repeated
purification cycles and from the deterioration in the quality
ofthe media following necessary treatments with harsh cleaning
agents. In large-scale manufacturing operations, resins are
re-cycled due to economic necessity for a validated number of
uses, often >50 times. A medium with low-ligand leakage that
can withstand harsh cleaning-in-place/regeneration protocols
will lower production costs. At the same time, assurance can be
given that contamination arising from the medium itself or from
carryover from the feed material will be minimised in the product
stream.
In order to satisfy regulatory scrutiny, concerning the important issue of process validation full documentation and technical
support is required for media used in a process. Column media
should be considered as a raw material and new batches must be
tested before being introduced into a production cycle. Currently
for therapeutic products, the use of media prepared using animalfree components and validated manufacturing technologies are
mandated by regulatory agencies. A guarantee of the supply of
the chosen media along with full and proper vendor certification
should also be ensured prior to specifying a medium for use.

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2.2. Column Packing

at Large Scale

Ensuring reproducibility of media packing as the diameter of the

column increases is often regarded as the most problematic aspect
in scale-up. It is not surprising that small-scale columns are easier
to pack reproducibly. One limitation in the approach to scale-up
by increasing the column diameter, while maintaining packed bed
height, is that commercial manufacturers typically supply column
diameters of a set defined size. From a practical viewpoint, the
packing of large-diameter columns becomes more difficult and
the stability of the resultant packed bed can also be reduced.
Instability in the media bed at large scale is due in the main to the
decreased physical support offered by the wall of the column as
the diameter increases. This can result in high pressures caused by
compression of the medium via drag forces exerted by fluid flow
through the bed. In general terms, decreased stability may result
in unpredictable flow distribution during processing leading to
hysteresis, edge effects, and media compression (11) all of which
will affect process reproducibility.
The packing procedure itself can become quite challenging
when large-diameter columns are used. To satisfy the need for
larger-production capacities, biopharmaceutical facilities will typically use columns with diameters in the range 12m for the initial
capture step. The size of column required will depend on the
volume of the feed material and the concentration of binding
protein contained therein. Insufficient quality in column packing
can cause channelling in the bed, which will lead to broadening
or splitting of peaks and an obvious decrease in the resultant resolution. Manual packing of chromatography columns is used
within industry and is often appropriate if the column diameter is
relatively small (<0.5m). It is performed by pouring the required
amount of prepared medium slurry into the column shell and
pumping an appropriate buffer through the column adaptor thus
inducing the medium bed to pack. This operation is obviously
quite manual and at stages is open to the surrounding environment. Thus media packed using this method is usually sanitised
with a caustic solution prior to equilibration and processing to
remove any potential bioburden that could have been introduced.
Automated packing-in-place systems can be utilised which allow
for more consistent and efficient packing operations when largediameter columns are used. Such systems also allow for packing
operations to be carried out in a more closed and controlled manner and are composed of diaphragm pumps and associated valving
that serves to direct the flow of buffers or resin slurries to appropriate valves on the column or slurry vessels, respectively (23).
All chromatography media will require different conditions
to achieve optimum packing and trials should take place in order
to develop robust and optimal packing procedures prior to scaleup (20). When a column is packed, the integrity of that packing
is usually assessed by estimating parameters such as the asymmetry

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factor (As) and the height equivalent to the theoretical plate

(HETP). Both of these factors are determined from an analysis of
the chromatogram that is generated when a small volume
(<0.025CV) of a low molecular weight tracer compound (NaCl
or acetone) is applied and eluted from the packed column (24).
Continued monitoring of asymmetry and HETP is useful and
may be used as a first indication of the deterioration of column
integrity as the number of processing cycles is increased during
the lifetime of a packed column.
2.3. Cleaning
of Chromatography
Media

The maintenance of proper sanitary conditions is crucial to current

biopharmaceutical processing operations. Whatever media and
column systems are chosen for a particular application at large
scale, the issue of cleaning and sanitisation is therefore of high
importance. If a chromatography medium is to be re-used, assessment of the cleaning regime employed between cycles will form
the major part of the required resin lifetime study (25). The
potential for carryover of impurities from one column run to the
next must be minimised and any cleaning regime must be capable
or removing residual contamination. Indeed a validated cleaning
strategy for packed chromatography columns and associated
equipment is a major component in the overall process validation
requirements requested by regulatory agencies.
Cleaning and sanitisation protocols are most usually performed after every each batch of material is processed on the column. These activities are designed to mitigate the risk by
minimising exposure of the medium to contaminating bioburden
thus prolonging the lifetime of the medium and reducing downtime due to the need to re-pack with fresh medium.
Notwithstanding the time involved, repacking of columns can
necessitate the use of large quantities of water and buffers and a
re-qualification of the packed bed quality should also be
performed.
Cleaning-in-place (CIP) strategies that are often performed
after every cycle must be designed having regard to the stability
of the medium used, the properties of the feed material applied
and the position of the step in the purification sequence. The
important decision to re-use a medium in a process can often
depend on the ability to clean the medium satisfactorily. Media
used in the first capture step can be more difficult to clean due to
the nature of the feedstock applied. However, at the capture
stage, media volumes are generally large and re-use presents the
only option, particularly given that the medium may be very
expensive. During polishing operations feed samples should be
much cleaner and hence column volumes will be generally be
smaller. However, at this late stage in manufacturing, carryover
and any potential ligand leaching could significantly compromise
product quality and a decision should be made as to the merits of

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a re-use strategy in this case (26). Sanitisation cycles typically use

alkali (0.51M NaOH) to reduce the build up of endotoxins and
bioburden. If the CIP protocol is carefully designed, it is possible
to re-use a medium many times without negative effects. In one
study, a Protein A medium was used for 150 cycles with cleaningin-place being performed after every cycle. It was demonstrated
in this study that the yield and purity were consistent after each
cycle. In addition, there was no detectable carryover and the
binding capacity for the target IgG1 antibody remained at a high
level (>85%) when compared with the initial cycle (10).
2.4. Production
Equipment

While in theory at least chromatographic processes can be scaled

efficiently by taking note of key chromatography parameters and
scaling accordingly, one aspect of large-scale downstream processing that will clearly be an issue is the nature of the equipment
used. Glass and acrylic columns are used as scale is increased but
for very large-diameter columns stainless steel is usually the material of construction. Column adaptors at large scale (0.52.0M)
are very heavy and contingency must exist within a facility for
packing and maintenance operations. This will invariably involve
the use of hydraulic lifting equipment to protect column hardware and to ensure operator safety. When a column is specified for
inclusion in a GMP process, the manufacture of all components
of the column and the materials of construction therein must be
fully traceable to minimise the risk associated with leaching, particularly given the harsh cleaning solutions that will be used during processing.
Large-scale chromatography systems are generally automated
systems that use a series of pipings, inlet and fraction valves, flow
meters, air detectors, air-traps, pressure sensors, and buffer mixing capability for application requiring gradient formation. Such
equipment may give rise to increased dead volumes, leading to
dilution, higher pressure drops, and peak broadening, which will
cause extra dilution of the product fraction or even loss of resolution if the application is sensitive to variations in plate number in
the system used. Chromatography systems are typically constructed from inert piping and are configured to minimise holdup volumes and to ensure that the fluid path can be properly
cleaned and sanitised to minimise contamination. Proper qualification of chromatography equipment can identify problems prior
to undertaking production activities (13). It is generally necessary
to further refine previously optimised parameters (e.g. flow rates)
upon scale-up as a direct result of differing equipment design.

2.5. Nonchromatographic
Factors Applicable
at Large Scale

Several factors not directly related to the chromatography process per se become an issue when the downstream process
increases to large scale. These factors can affect the purity and
yield achieved from a purification cycle when the resultant product

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83

fraction is characterised and compared with the corresponding

purification step performed at small scale. As the size of the cellculture bioreactor increases, both the concentration of the target
molecule and the distribution of the associated panel of contaminants present will change due to the natural variation that will
take place during cell culture. In general, during scale-up design,
culture feedstock should be supplied from reactor runs performed
close to the predicted final scale so that the effect of bioreactor
conditions on product quality can be determined. When a chromatography column step itself is up-scaled, there will be an obvious increase in the amount of buffer solutions required to achieve
purification. As volumes increase, the storage space taken up by
such solutions in a facility can be problematic. Reproducibility in
buffer preparation at large scale becomes very important and
realistic specifications for pH and conductivity must be set in
order to ensure that buffer manufacturers can effectively prepare
solutions that will perform robustly during processing. The use
of expensive organic solvents is often required to achieve the
desired level of purification in reverse-phase chromatography
applications. In addition, processing suites where such activities
take place within a facility will have to be designed carefully to
ensure that the room is properly vented and risk of explosion is
removed. This will ensure that operators and personnel entering
the area can be protected from the effects of large volumes of
concentrated solvents. Such requirements can add considerable
expense to a downstream process and will require that facilities
must be dedicated to such activities, which will reduce overall
flexibility.
If a purification step has been carefully optimised, the volumes of buffers required at each stage of the chromatography
should have been minimised. If scale-up is planned, it is more
appropriate to have the target product molecule elute in 12
column volumes rather than in 510 column volumes. This feature will decrease the volume of buffers required, reduce the
resultant storage required, and lower accompanying material
costs. A model study has shown the effect of increasing elution
volume per cycle as column volume is increased for the affinity
purification of a monoclonal antibody (20). Furthermore, if the
volume of a bioreactor is increased and the associated capture
chromatography step has to be scaled up by increasing the
number of individual cycles rather than the diameter of the column, the additional volumes of buffer required will necessitate
higher costs. Membrane adsorption now offers the potential of
a cost-effective alternative to conventional chromatography,
for flow through and some polishing applications due to the
lower consumption of buffers that results and the obvious cost
benefit that accrues when compared with the chromatography
process (11).

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3. Summary
Due to the recent developments in cell-culture technology, there
is an increasing demand on biopharmaceutical manufacturers to
develop robust and reproducible downstream processes to cater
for the increased product titres that are now routinely achieved.
Scaling-up of chromatography operations represents an increasingly important issue in the successful commercial manufacture of
therapeutic proteins. Only when the rational design of a chromatography process has taken place can effective scale-up of that
purification process be realised. Such design must have regard to
all factors that can affect scale-up from media properties, packing
operations, column hardware, ancillary equipment, and facility
issues. This review has described some of the key issues that
can present themselves whenever a decision is made to increase
the scale of a purification step involving chromatography. Due to
the central role chromatography plays in the manufacturing of the
current pipeline of high dose protein products in development,
the impetus to find new innovations in large-scale bioprocessing
will remain a real and necessary challenge.
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