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Biochromatography:

Affinity Chromatography

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Contents Page

1 Principles of Affinity Chromatography 2-5


2 Affinity Chromatography Stationary Phases 6-8
3 Monoclonal Antibody Titer Determination 9-11
4 Carrying Out A mAb Titer Determination 12-13
5 Clean-In-Place Protocol 14
6 References 15-16

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1. Principles of Affinity Chromatography

Affinity chromatography separates proteins based on reversible interactions between the proteins
and a specific ligand bound to the chromatographic stationary phase. Affinity chromatography is
highly specific, providing very good resolution, and considerable capacity for analytes of interest. It
is the only technique that enables protein purification on the basis of its biological function or
individual chemical structure. Active proteins can be separated from denatured or functionally
different forms, allowing isolation of pure substances present at low concentrations in large volumes
of crude samples, or the removal of specific contaminants.

Figure 1: Affinity Chromatography Process.

1: Equilibration

The affinity chromatography media is equilibrated using a binding buffer.

Binding buffers used are typically at physiological pH (7.0-7.4) and ionic strength. This is especially
true when antibody-antigen or native protein-protein interactions are the basis for the affinity
purification. Binding and elution buffers are specific for each affinity medium since it is their
influence on the interaction between the target molecule and the ligand that facilitates the affinity
based separation. Some affinity media may also require a specific buffer in order to re-equilibrate
the medium.

Common buffers and additives include (this list is not exhaustive):

 Sodium phosphate
 Sodium chloride
 Imidazole
 Sodium citrate
 Ethylenediaminetetraacetic acid (EDTA)
 Potassium sulfate

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 Phosphate buffered saline (PBS)
 Potassium chloride
 Potassium phosphate
 Urea
 Guanidine hydrochloride
 Tris-HCl

An example of a binding buffer for a protein A column is, 50 mM sodium phosphate, pH 7.4 (this can
be optimized between pH 7 and 8).

2: Sample Application and Wash

The sample is applied under conditions that favor the specific binding of a protein to the ligand
bound to the stationary phase (i.e. pH, ionic strength etc.). The protein of interest will bind
specifically, but reversibly, to the ligand, while unbound material washes through the column.

Binding efficiency is related to the strength and kinetics of the interaction, which are dependent on
the amount of ligand bound to the stationary phase support, the concentration of the sample, and
the flow rate used for application. A higher flow rate may reduce binding efficiency, especially if the
protein/ligand interaction is weak.

Optimum binding occurs under physiological conditions (pH 7.0-7.4). If required, proteins bound by
non-specific interactions can be removed by washing. An increase in ionic strength (increased salt
concentration) or change in pH will reduce ionic interactions, while hydrophobic interactions can be
reduced by decreasing salt concentration, altering pH, or adding surfactants. Contaminants with
weak affinity for the ligand or solid support can be removed using competitive reagents.
Appropriate flow rates and volumes of wash buffer will maximize contaminant removal while
minimizing loss of target analyte.1

3: Elution

The bound protein is eluted by changing the eluent conditions to disrupt the protein/stationary
phase interaction. This can be achieved specifically using a competitive ligand, or non-specifically, by
changing the pH, ionic strength, or polarity. For example, the use of low pH (2.0-2.5) will disrupt
ionic interactions and hydrogen bonds between an antibody and antigen.2

The ionic strength of the buffer can be altered in a linear or step gradient (NaCl is normally used). It
is also possible to use selective eluents to elute molecules from group specific media, or when the
protein/stationary phase interaction is high; the eluting agent competes for binding with either the
protein of interest or the stationary phase bound ligand. If the eluent polarity is lowered it can
promote elution without inactivating the protein; dioxane (up to 10%) or ethylene glycol (up to 50%)
are often used for this purpose. Denaturing agents can promote protein/ligand desorption via
protein unfolding, whereas, chaotropic salts or reagents (sodium thiocyanate, magnesium chloride,
guanidine hydrochloride, or urea) disrupt water molecules around the protein/ligand interaction.3
Denaturing agents and chaotropic salts can damage labile proteins, resulting in low yields, and may
also damage the column itself. As an alternative, displacer agents can be used to compete for
binding with the surface ligand; this method is specific and mild, however, large molar excesses of
the displacer agent are required, elution can be slow, and often broad peaks are observed.4

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Gradient Elution

Gradient elution is used when working with unknown samples, and for development of purification
methods. The retention time of eluted peaks provide information regarding optimal binding and
elution conditions, which can then be applied in step elution.

Step Elution

Step elution is practical for less complex samples, or after conditions from a gradient elution have
been optimized. Step elution decreases separation time and reduces buffer consumption. Proteins
of interest can also be concentrated and rapidly removed from unwanted interferences using step
elution.

If a protein is very tightly bound to the affinity chromatography surface, the flow of eluent can be
stopped after application (10 min. to 2 h is common) before continuing elution, allowing more time
for dissociation to occur, which can improve recovery.5

The flow rates used in affinity chromatography vary widely due to the different dissociation rates of
protein/ligand interactions; commercially available media is provided with recommended usage
instructions (flow rates, pH ranges, pressure maxima etc.).

Optimization of flow rates can determine optimal rates for effective binding as well as optimal
elution to maximize recovery. Flow rates can also be optimized for column re-equilibration to
minimize run times. Sharp elution profiles with maximum recovery and minimum dilution should be
achieved using the lowest acceptable flow rate.

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Binding and elution buffers are specific for each affinity medium since it is their influence on the
interaction between the target molecule and the ligand that facilitates the affinity based separation.
Some affinity media may also require a specific buffer in order to re-equilibrate the medium.

Condition Examples
Low pH 100 mM glycine-HCl, pH 2.5-3.0
100 mM citric acid, pH 3.0
High pH 50-100 mM triethylamine or triethanolamine, pH 11.5
150 mM ammonium hydroxide, pH 10.5
0.1M glycine-NaOH, pH 10.0
Ionic strength (and chaotropic effects) 5 M lithium chloride
3.5 M magnesium or potassium chloride
3.0 M potassium chloride
2.5 M sodium or potassium iodide
0.2-3.0 M sodium thiocyanate
0.1 M Tris-acetate with 2.0 M NaCl, pH 7.7
Denaturing 2-6 M guanidine HCl (also classed as chaotropic)
2-8 M urea (also classed as chaotropic)
1.0 M ammonium thiocyanate
1% sodium deoxycholate
1% sodium dodecyl sulfate (SDS)
Organic 10% dioxane
50% ethylene glycol, pH 8.0-11.5 also classed as
chaotropic)
Competitor >0.1 M counter ligand or analog
Table 1: Examples of affinity chromatography eluents.

A widely used elution buffer for affinity purification based on protein interactions is 0.1 M glycine-
HCl, pH 2.5-3.0. This buffer effectively dissociates most protein-protein and antibody-antigen binding
interactions without permanently affecting protein structure.

However, some antibodies and proteins are damaged by low pH, so eluted protein fractions are best
neutralized immediately by addition of 1/10th volume of alkaline buffer such as 1 M Tris-HCl, pH 8.5.

An example of an elution buffer for a protein A column is, 100 mM citric acid, with 500 mM acetic
acid (pH 2.6), 100 mM glycine HCl (pH 2.8), and 12 mM HCl (pH 1.9) also being suitable.

4: Re-equilibration

The affinity chromatography column is re-equilibrated using binding buffer.

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2. Affinity Chromatography Stationary Phases

Figure 1: Affinity chromatography stationary phase.

Stationary Phase Support

The stationary phase support material should have the following properties:

 Chemically and physically inert - the support and ligand should not react with solvents used
in the purification process
 Extremely low non-specific adsorption
 Surface groups for easy attachment of ligands
 Open pore structure to ensure high capacity binding, even for large biomolecules
 High surface area to volume ratio
 Uniform particle to pore size ratio
 Good flow properties for rapid separation
 Stability under a range of chromatographic conditions such as extremes of pH, detergents,
and dissociating agents

Materials based on carbohydrates (agarose, dextrose, or cellulose), synthetic organic supports such
as poly(glycidyl methacrylate-co-ethylene dimethacrylate) or other polymethacrylate derivatives,
polyacrylamide-based polymers, polyethersulfone matrices, inorganic materials silica and zirconia or
magnetic beads (silanized surface over an iron oxide core) are often employed as the stationary
phase support (Figure 2).

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Figure 2: Affinity chromatography stationary phase support materials.

A compromise should be reached between the pore size and surface area. Materials containing small
pores do have a large surface area, but much of this may not be available for ligand binding or be
accessible to large biomolecules. Materials with large pores will not suffer from problems with
accessibility, however, due to the low surface area the ligand binding capacity will be reduced (this
will in turn affect biomolecule binding capacity).1

Ligand

The ligand is the moiety attached to the solid support which is responsible for reversibly binding to
the biomolecule of interest. The ligand must show specific and reversible binding affinity for the
target analyte, and it must have chemically modifiable groups to allow attachment to the solid
support without destroying the binding activity. The dissociation constant (kD) for the ligand-
biomolecule complex should be in the range 10-4 to 10-8 M in free solution - values outside this range
may require alteration of the elution conditions to attain successful chromatography. Ligands can be
immobilized or coupled directly to solid support materials through formation of covalent chemical
bonds between particular functional groups on the ligand (e.g. primary amines, sulfhydryls,
carboxylic acids, or aldehydes) and reactive groups on the support surface.

The interaction between the protein and stationary phase bound ligand is governed by electrostatic
or hydrophobic interactions, van der Waals forces, and/or hydrogen bonding.6 A variety of different
ligands can be used to purify proteins:

Type of Column Target Analyte


Enzyme Substrate analogue, cofactor, inhibitor
Immunoaffinity Antigen, virus, cell
Lectin Polysaccharides, glycoproteins, cell surface
receptor, cell
Nucleic acid Complementary base sequence, histones, nucleic
acid polymerase, nucleic acid binding protein
Hormone, vitamin Receptor, carrier protein
Glutathione Glutathione-S-transferase, GST-tagged proteins
Immobilized metal ion affinity chromatography Histidine-(his)-tagged proteins, native proteins
(IMAC) with histidine, cysteine, or tryptophan residues
on their surfaces
Table 2: Affinity chromatography stationary phase types and target analytes.

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Spacer Molecule

Protein binding sites are often located deep within the molecule; therefore, sterics play an
important factor in affinity chromatography. If a small ligand is attached to the solid support this
may result in low binding capacity, as a large biomolecule cannot easily access and bind correctly to
it. Therefore, spacer molecules are sometimes bound to the solid support and subsequently to the
ligand to overcome steric effects and facilitate successful binding (Figure 3).

Figure 3: Effect of spacer molecule on analyte binding.

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3. Monoclonal Antibody Titer Determination

During development of monoclonal antibodies (mAbs) for biotherapeutics, a large number of


harvested cell culture samples must be screened for an immunoglobulin G (IgG) titer. Protein A and
G affinity chromatography are used to determine the mAb concentration for selection of high yield
clones, as well as for purification for downstream aggregate and charge variant analysis.7

Accurate mAb titer quantitation is essential during the early stages of development when the cell
line is selected, and also during manufacture when the amount of mAb in the cell-culture
supernatant determines the optimum harvest time. Affinity chromatography has several advantages
in that it is an easy, fast, and selective method for capturing proteins.

Staphylococcal protein A (SPA), and smaller ligands derived thereof, are the most commonly used
ligands for the separation of antibodies via affinity chromatography. SPA was one of the first native
interactions to be explored for affinity chromatography protein purification due to its specific
interaction with the Fc domain of immunoglobulin G (IgG) from a variety of mammals. 7,8 SPA is
capable of binding to the Fc domain of IgG1, IgG2, and IgG4 with an estimated affinity constant (KA) of
108 M-1, but shows only weak interaction with IgG3.9

Streptococcal protein G (SPG) is also commonly used for IgG purification. Protein A and G have high
affinity for antibodies, therefore, they bind only to antibodies in cell-culture supernatants. However,
they have different selectivity, with protein G showing preferential binding for the human IgG
subclass III (Table 1).

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Species Subclass Protein A Protein G
Human IgG1 **** ****
IgG2 **** ****
IgG3 - ****
IgG4 **** ****
IgA ** -
IgD ** -
IgE ** -
IgM ** -
Avian egg yolk IgY - -
Cow ** ****
Dog ** *
Goat - **
Guinea pig IgG1 **** **
IgG2 **** **
Hamster * **
Horse ** ****
Koala - *
Llama - *
Monkey (rhesus) **** ****
Mouse IgG1 * **
IgG2a **** ****
IgG2b **** ***
IgG3 * ***
IgM */- -
Pig *** ***
Rabbit No distinction **** ***
Rat IgG1 - *
IgG2a - ****
IgG2b - **
IgG3 * **
Sheep */- **
Table 3: Binding affinity of Protein A and Protein G bio-monolith columns for different IgG
subclasses. **** = strong affinity, *** = moderate affinity, ** = weak affinity, * = slight affinity, and -
= no affinity.10-12

In mAb titer determination it is important to assess absolute mAb concentration. This is achieved by
comparing the peak areas measured in cell-culture supernatants with an external calibration curve
constructed by diluting a mAb standard (Figure 1).

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Figure 1: UV 280 nm Protein A chromatogram showing supernatant of a mAb producing Chinese


hamster ovary (CHO) clone. Inset: Calibration curved produced by dilution of a mAb standard.

Protein A and G affinity media are relatively expensive and exhibit some chemical instability under
the high pH conditions used during clean-in-place (CIP) protocols. Hence, development of
alternative ligands, based on genetically engineered Z-domain mutants of Protein A,13,14 triazine
dyes,15,16 4-mercaptoethylpyridine (MEP),15,17-20 substituted pyridinylsulfanylethylamines (PSEAs),21-24
4’-aminoethylsulfanyl-2,2’:6’,2”-terpyridine (terpy),21,25,26 and peptides have also been developed for
antibody purification (Figure 2).27

Figure 2: Alternative ligands for protein purification.

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4. Carrying Out A mAb Titer Analysis

Example Protein A Methods

Sample Preparation:

Cell supernatants are appropriately diluted in binding buffer (i.e. 1:1 in 50 mM Na2HPO4).
Supernatants are centrifuged at 5,000 g for 5 minutes prior to injection to remove cells and debris.
Filtering the sample may be required to remove particulate material that could block the column
(use a 0.22-0.45 μm filter).

Conditions:28
Column: Protein A (4.95 x 5.2 mm)
Mobile phase: A) 50 mM phosphate, pH 7.4
B) 100 mM citric acid, pH 2.8 mM or acetic acid, pH 2.6
Gradient: Time (min) %B
0-0.5 0 (binding)
0.6-1.7 100 (elution)
1.8-3.5 0 (re-equilibration)
Flow rate: 1 mL/min.
Injection volume: Variable (should be optimized)
Detection: UV 280 nm

Conditions:29
Column: Protein A (4 x 35 mm)
Mobile phase: A) 50 mM sodium phosphate, 150 mM sodium chloride, 5% acetonitrile pH 7.5
B) 50 mM sodium phosphate, 150 mM sodium chloride, 5% acetonitrile pH 2.5
Gradient: Time (min) %B
0-0.2 0 (binding)
0.2-1.8 100 (elution)
0.8-2.0 0 (re-equilibration)
Flow rate: 2 mL/min.
Injection volume: Variable (should be optimized)
Detection: UV 280 nm
Temperature: 25 °C

Unbound material elutes during the flow-through while the mAb is retained at neutral pH (binding)
and is eluted when the pH is lowered during a step gradient (elution, Figure 1).

When developing a new method using a Protein A column, both binding and elution buffers should
be optimized. For binding, 50 mM sodium phosphate, pH 7.4 is a good starting buffer and can be
optimized between pH 7 and 8. For elution, 100 mM citric acid is a good starting buffer, with 500
mM acetic acid (pH 2.6), 100 mM glycine HCl (pH 2.8), and 12 mM HCl (pH 1.9) also being suitable.

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Figure 1: UV 280 nm Protein A chromatogram showing supernatant of a mAb producing Chinese


hamster ovary (CHO) clone. Inset: Calibration curved produced by dilution of a mAb standard.

Example Protein G Method

Conditions:10
Column: Protein G (4.95 x 5.2 mm)
Mobile phase: A) 50 mM sodium phosphate, pH 7.4
B) 0.1 M citric acid, pH 2.0
Gradient: Time (min) %A
0 100
0.4 100
0.5 0
2.0 0
2.1 100
4.2 100
Flow rate: 1 mL/min.
Injection volume: Variable (should be optimized)
Detection: UV 280 nm
Temperature: 25 °C

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5. Clean-In-Place Protocol (CIP)

Clean-in-place (CIP) protocols during protein purification ensure product quality, improve column life
time, and reduce the cost of the purification processes, particularly when using expensive affinity
resins, such as Protein A.30

CIP protocols should be designed for each process based on the contaminants present in the
purification mixture. Sodium hydroxide (NaOH) in concentrations from 0.1 to 1 M is commonly used
for cleaning equipment, as well as resins, since it effectively removes tightly bound, precipitated, or
denatured proteins, lipids, and nucleic acids.

Furthermore, sodium hydroxide effectively inactivates most microorganisms including bacteria,


viruses, yeast, and endotoxins.7,31,32 Additionally, it is easy to remove, simple to monitor, and is low
cost.

Therefore, it is advantageous to have equipment and matrices that can tolerate high pH.

Example CIP (Agilent Bio-Monolith Protein A Column, 4.95 x 5.2 mm)28

1. Wash with 1-2 mL (10-20 CV) of 0.1 M NaOH (reverse flow direction) at 0.2-0.5 mL/min.
2. Wash with 1-2 mL (10-20 CV) of DI water at 0.5-1.0 mL/min.
3. Wash with 1-2 mL (10-20 CV) of concentrated buffer (0.1-0.5 M) to restore normal pH (7.0-
7.4)
4. Re-equilibrate with 5 ML (50 CV) of binding buffer

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12. References

1. Abi-Ghanem, D. A.; Berghman, L. R. Immunoaffinity Chromatography: A Review. In: S.


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4. Urh, M.; Simpson, D.; Zhao, K. Methods Enzymol. 2009, 463, 417-438.
5. GE Healthcare, Affnity Chromatography, Principles and Methods Handbook.
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pp. 21-37, Cold Spring Harbor Laboratory Press, New York, USA, 1999.
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10. Duong, P. T. Bio-monolith Protein G-column - More Options for mAb Titer Determination,
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27. Akiyama, Y.; Miyata, H.; Komiyama, M.; Nogami, M.; Ozawa, K.; Oshita, C.; Kume, A.;
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28. Dumont, E.; Vandenheede, I.; Sandra, P.; Sandra, K.; Martosella, J.; Duong, P.; Joseph, M.
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