Downstream Processing:

Downstream processing refers to the recovery and purification of biosynthetic products,

particularly pharmaceuticals, from natural sources such as animal or plant tissue or
fermentation broth, including the recycling of salvageable components and the proper
treatment and disposal of waste. It is an essential step in the manufacture of pharmaceuticals
such as antibiotics, hormones (e.g. insulin and human growth hormone), antibodies (e.g.
infliximab and abciximab) and vaccines; antibodies and enzymes used in diagnostics;
industrial enzymes; and natural fragrance and flavor compounds. Downstream processing is
usually considered a specialized field in biochemical engineering, itself a specialization
within chemical engineering, though many of the key technologies were developed by
chemists and biologists for laboratory-scale separation of biological products.
Downstream processing and analytical bioseparation both refer to the separation or
purification of biological products, but at different scales of operation and for different
purposes. Downstream processing implies manufacture of a purified product fit for a specific
use, generally in marketable quantities, while analytical bioseparation refers to purification
for the sole purpose of measuring a component or components of a mixture, and may deal
with sample sizes as small as a single cell.

Stages of the process:

A widely recognized heuristic for categorizing downstream processing operations divides
them into four groups which are applied in order to bring a product from its natural state as a
component of a tissue, cell or fermentation broth through progressive improvements in purity
and concentration.
1. Removal of insolubles is the first step and involves the capture of the product as a
solute in a particulate-free liquid, for example the separation of cells, cell debris or
other particulate matter from fermentation broth containing an antibiotic. Typical
operations to achieve this are filtration, centrifugation, sedimentation, flocculation,
electro-precipitation, and gravity settling. Additional operations such as grinding,
homogenization, or leaching, required to recover products from solid sources such as
plant and animal tissues, are usually included in this group.
2. Product Isolation is the removal of those components whose properties vary
markedly from that of the desired product. For most products, water is the chief
impurity and isolation steps are designed to remove most of it, reducing the volume of
material to be handled and concentrating the product. Solvent extraction, adsorption,
ultrafiltration, and precipitation are some of the unit operations involved.
3. Product Purification is done to separate those contaminants that resemble the
product very closely in physical and chemical properties. Consequently steps in this
stage are expensive to carry out and require sensitive and sophisticated equipment.
This stage contributes a significant fraction of the entire downstream processing
expenditure. Examples of operations include affinity, size exclusion, reversed phase
chromatography, crystallization and fractional precipitation.

4. Product Polishing describes the final processing steps which end with packaging of
the product in a form that is stable, easily transportable and convenient.
Crystallization, desiccation, lyophilization and spray drying are typical unit
operations. Depending on the product and its intended use, polishing may also include
operations to sterilize the product and remove or deactivate trace contaminants which
might compromise product safety. Such operations might include the removal of
viruses or depyrogenation.
A few product recovery methods may be considered to combine two or more stages. For
example, A.expanded bed adsorption accomplishes removal of insolubles and product
isolation in a single step. B.Affinity chromatography often isolates and purifies in a single
step.
A. Expanded bed adsorption (EBA) is a preparative chromatographic technique which
makes processing of viscous and particulate liquids possible. Where classical column
chromatography uses a solid phase made by a packed bed, EBA uses particles in a
fluidized state. Expanded bed adsorption is, however, different from fluidised bed
chormatography in essentially two ways: one, the EBA resin contains particles of
varying size and density which results in a gradient of particle size when expanded;
and two, when the bed is in its expanded state, local loops are formed. Particles such
as whole cells or cell debris, which would clog a packed bed column, readily pass
through a fluidized bed.[1] EBA can therefore be used on crude culture broths or
slurries of broken cells, thereby bypassing initial clearing steps such as centrifugation
and filtration, which is mandatory when packed beds are used. The feed flow rate is
kept low enough that the solid packing remains stratified and does not fluidize
completely. Hence EBA can be modeled as frontal adsorption in a packed bed, rather
than as a well mixed continuous-flow adsorber.
The protein binding principles in EBA are the same as in classical column
chromatography and the common ion-exchange, hydrophobic interaction and affinity
chromatography ligands can be used. After the adsorption step is complete, the fluidized bed
is washed to flush out any remaining particulates. Elution of the adsorbed proteins is
commonly performed with the eluent flow in the reverse direction, that is, as a conventional
packed bed, in order to recover the adsorbed solutes in a smaller volume of eluent.
EBA may be considered to combine both the "Removal of Insolubles" and the
"Isolation" steps of the 4-step downstream processing heuristic.
B. Affinity chromatography is a method of separating biochemical mixtures, based on
a highly specific biological interaction such as that between antigen and antibody,
enzyme and substrate, or receptor and ligand. Affinity chromatography combines the
size fractionation capability of gel permeation chromatography with the ability to
design a stationary phase that reversibly binds to a known subset of molecules. The
method was invented in 1968 by Pedro Cuatrecasas and Meir Wilchek for which the
Wolf Prize in Medicine was awarded in 1987. Due to its interdisciplinary nature,
affinity chromatography has been the means by which many scientists from different
disciplines have been introduced to the exciting fields of modern biology.
Affinity chromatography can be used to:

Purify and concentrate a substance from a mixture into a buffering solution

Reduce the amount of a substance in a mixture

Discern what biological compounds bind to a particular substance, such as drugs

Purify and concentrate an enzyme solution.

Affinity chromatography can be used in a number of applications, including nucleic

acid purification, protein purification from cell free extracts, and antibody purification
from blood serum.

Antibody affinity

Another use for the procedure is the affinity purification of antibodies from
blood serum. If serum is known to contain antibodies against a specific antigen (for
example if the serum comes from an organism immunized against the antigen
concerned) then it can be used for the affinity purification of that antigen. This is also
known as Immunoaffinity Chromatography. For example if an organism is immunised
against a GST-fusion protein it will produce antibodies against the fusion-protein, and
possibly antibodies against the GST tag as well. The protein can then be covalently
coupled to a solid support such as agarose.
For thoroughness the GST protein and the GST-fusion protein can each be coupled
separately. The serum is initially allowed to bind to the GST affinity matrix. This will
remove antibodies against the GST part of the fusion protein. The serum is then
separated from the solid support and allowed to bind to the GST-fusion protein
matrix. This allows any antibodies that recognize the antigen to be captured on the
solid support. Elution of the antibodies of interest is most often achieved using a low
pH buffer such as glycine pH 2.8. The eluate is collected into a neutral tris or
phosphate buffer, to neutralize the low pH elution buffer and halt any degradation of
the antibody's activity. This is a nice example as affinity purification is used to purify
the initial GST-fusion protein, to remove the undesirable anti-GST antibodies from the
serum and to purify the target antibody.
A simplified strategy is often employed to purify antibodies generated against peptide
antigens. When the peptide antigens are produced synthetically, a terminal cysteine
residue is added at either the N- or C-terminus of the peptide. This cysteine residue
contains a sulfhydryl functional group which allows the peptide to be easily
conjugated to a carrier protein (e.g. KLH). The same cysteine-containing peptide is
also immobilized onto an agarose resin through the cysteine residue and is then used
to purify the antibody. A review of this process is available here [1].

Immobilized metal ion affinity chromatography

Immobilized metal ion affinity chromatography (IMAC) is based on the
specific coordinate covalent binding of amino acids to metals, particularly histidine.
This technique works by allowing proteins with an affinity for metal ions to be
retained in a column containing immobilized metal ions, such as cobalt, nickel,
copper, zinc, or iron ions. Many naturally occurring proteins do not have an affinity
for metal ions, therefore recombinant DNA techniques can be used to introduce this
property into a protein of interest. Methods used to elute the protein of interest include
changing the pH, or adding a competitive molecule, such as imidazole.

Recombinant proteins
Possibly the most common use of affinity chromatography is for the
purification of recombinant proteins. Proteins with a known affinity are tagged in

order to aid their purification. The protein may have been genetically modified so as
to allow it to be selected for affinity binding, this is known as a fusion protein. Tags
include His-tags and GST (glutathione-S-transferase) tags. His6-tags have an affinity
for nickel or cobalt ions which are coordinated with a chelator for the purposes of
solid medium entrapment. For elution, an excess amount of a compound able to act as
a metal ion ligand, such as imidazole, is used. GST has an affinity for glutathione commercially available immobilized as glutathione agarose. For elution, excess
glutathione is used to displace the tagged protein.

Lectins
Lectin affinity chromatography is a form of affinity chromatography where
lectins are used to separate components within the sample. Lectins, such as
concanavalin A, are proteins which can bind specific carbohydrate (sugar) molecules.
The most common application is to separate proteins based on their glycan groups.

Use of immobilized ligands in other methods

Immobilized ligands may be used in electrophoretic systems for estimation of
binding constants, as for instance in lectin affinity electrophoresis or characterization
of molecules with specific features like glycan content or ligand binding.