What is affinity chromatography?

Affinity chromatography is a purification technique that typically offers purities >95% in one step.

It makes use of a specific native or added property of the target molecule to isolate it from all contaminants in the sample.

Binding sites of receptors and antibodies or active sites of enzymes are examples of very specific properties that can be used for affinity chromatography. Prosthetic groups like polysaccharides are also used, but then for group separations. Group separations can also be accomplished by using ligands with broader specificity.

Common for all types of affinity chromatography is that a ligand (affinity ligand) specific for the binding site of the target molecule, is coupled to an inert chromatography matrix. Under binding conditions this specific ligand on the chromatography matrix will bind molecules according to its specificity only. All other sample components will pass through the chromatography medium unbound. After a wash step the bound molecules are released and eluted by changing the conditions towards dissociation or by adding an excess of a substance that displaces the target molecule from the affinity ligand (competitive elution).

With recombinant produced proteins affinity chromatography can be applied thanks to gene fusion techniques to produce tagged recombinant proteins.Special vectors are used to introduce an affinity tag into the target protein by gene fusion. Very often a cleavage recognition site is also introduced between the target protein and the affinity tag. When necessary the affinity tag can then be removed by the aid of a protease specific for this site when bound to the chromatography medium or in batch after purification. Gradient elution is normally not necessary in affinity chromatography, instead one-step elution with very simple equipment like a syringe or a peristaltic pump can be used.

Basic principles in affinity chromatography

The affinity chromatography separation mechanism

For all types of affinity chromatography, an affinity ligand specific for a binding site on the target molecule, is coupled to an inert chromatography matrix. Under suitable binding conditions this affinity chromatography medium will bind molecules according to its specificity only. All other sample components will pass through the medium unbound (Fig.1.1).


Fig 1.1 Affinity chromatography relies upon a reversible highly specific binding reaction.

After a wash step the bound molecules are released and eluted by changing the conditions towards dissociation or by
adding an excess of a substance that displaces the target molecule from the affinity ligand (competitive elution).

As for all reversible binding in chromatography, desorption curves (Fig. 1.2) can be used to describe the partitioning of the
sample between the mobile phase (the "buffer") and the stationary phase(the affinity matrix).




Fig 1.2 Since only the target protein binds, step elution will
not co-elute other sample components.

However, since no other sample components bind within the partition zone, a simple "on-off" mode of chromatography
can be applied by switching abruptly from full binding to complete release conditions (one-step elution).
Thus affinity chromatography fishes out the target molecule by way of highly specific binding and release, rather than
removings contaminants by fine-tuned isocratic or gradient elution techniques.

The development of an affinity chromatography method boils down to:

1. Finding a ligand specific enough to allow step elution.
2. Finding conditions for safe binding and release within the stability window of the target molecule and the ligand.

Note: When purifiying e.g. histidine-tagged recombinant proteins on Ni Sepharose chromatography media also
naturally proteins with exposed surfaced histidines may bind to the Ni Sepharose and optimization is needed to
increase purity.

The affinity chromatography experiment

The typical affinity chromatography experiment consists of three phases as illustrated below:
Symbolic representation of a section of an affinity chromatography bead surface.

Explanation to the symbols

Affinity ligand

Sample molecules with no affinity for the ligand

Target sample molecule with full affinity for the ligand

1. Equilibration The column is conditioned to promote binding of the target molecule by equilibrating it with binding buffer.	2. Sample application and wash The sample is applied under binding conditions. The target molecule binds specifically to the affinity ligands, while all other sample components are washed through.	3. Elution The target molecule is desorbed and eluted by switching to elution buffer.

Types of target molecular properties

Three groups of properties of the target molecule are used in affinity chromatography:

1. Specific binding properties based on biological activity such as:
- Enzyme active sites
- Receptor binding sites
- Antibody binding sites etc.
These are used together with the natural ligand or an analogue of it. Sometimes the analogue has a broader
specificity and can be used for group separations.

2. Naturally occurring prosthetic groups such as: polysaccharides etc.
Such properties normally allow group separations only.

3. Molecules equipped with an affinity tag such as:
- Glutathione-S-Transferase (GST)
- Oligo histidine
- MBP (Maltose Binding Tag)
- Strep-tag™ II
This group of properties is used almost exclusively for recombinant tagged proteins.

Mono-specific separations Antigens Antibodies Hormones Receptors Enzymes Tagged recombinant proteins Group-specific separations Glycoproteins Antibodies Enzymes Proteins/peptides with accessible histidine

Types of affinity chromatography ligands Ligands used for mono-specific affinity chromatography are structurally and biologically closely related to the target molecule to be purified. This makes the selection of the ligand specific for each case. This also makes it commercially difficult to produce affinity media for mono-specific separations. However, media ready for a variety of coupling chemistries (see table below) are commercially available for the benefit of the user, who intends to run mono-specific affinity chromatography. Groups used for coupling ligands Chemistry Coupling group N-hydroxysuccinimide (NHS) Cyanogen bromide (CNBr) Carbodiimide Epoxide Thiol exchange -NH2 -NH2 -NH2; -COOH -SH; --NH2; OH; -SH; With small ligands (<1000) there is a risk for steric interference like binding between matrix and target molecule or low accessibility of the ligand. Introducing a spacer arm will minimize these risks (Fig 3.2). Principle of preparing affinity chromatography media Fig 3.1. The sequence of events used to prepare affinity chromatography media. Pre-activated media are commercially available. Spacer arm principle Fig 3.2. A spacer is sometimes used to ensure full accessibility of the affinity ligand.

Ligands used for group-specific affinity chromatography have a much wider applicability and affinity media for this purpose are consequently commercially available. The table below lists examples of commonly used ligands of this type. Group-specific ligand Specificity Protein A Protein G Concanavalin A Cibacron Blue Procion Red Lysine Arginine Bezamidine Calmodulin Heparin Fc region of IgG Fc region of IgG Glucopyranosyl and Mannopyranosyl groups Broad range of enzymes, serum albumin NADP+ dependent enzymes Plasminogen, ribosomal RNA Serine proteases Serine proteases Proteins regulated by calmodulin Coagulation factors, lipoproteins, lipases, hormones, steroid receptors, protein synthesis factors, nucleic acid-binding enzymes Transition metal ions (Ni2+, Co2+, Cu2+,Zn2+, etc.) Proteins and peptides that contain accessible histidine, cysteine, etc.

Suitable conditions for binding/elution The problem of finding a suitable ligand in affinity chromatography is not restricted to just specificity, but also concerns the binding strength and the kinetics of the ligand-target molecule reaction. Ideally the binding should be strong enough to avoid leakage during the sample application and wash, while the target should be completely released during the elution phase. In other words, one has to find a ligand specific enough, but with a binding strength that allows safe and mild elution to secure the biological archiving of the target molecule. Ideal affinity chromatography conditions also require the kinetics of the binding and elution reactions to be fast enough to ensure complete reaction under normal flow rates. Expected results under ideal conditions Fig 4.1. No leakage during sample application and wash. Target molecule elutes as a narrow peak.

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Binding Reversible reactions such as the reaction between the target molecule and the affinity ligand are characterized by their equilibrium dissociation constants (KD). The smaller the value of KD the stronger the binding. KD values for good binding are typically in the range of 10-4 - 10-6 M. Fig 4.2. KD values for good binding are typically in the range of 10-4 - 10-6 M. The KD value can be influenced by changing conditions like pH, ionic strength, temperature, polar properties etc. KD values > 10-4 provide too weak a binding and the target molecule may "leak" as a diluted broad zone during sample application and wash. Figure 4.3 Leakage of target due to an excessively high KD: When KD is to low, the target molecule elutes spontaneously and is diluted during sample application. If a ligand binds too strongly, it will be difficult to elute the target molecule without introducing harsh conditions. Under such conditions there is always a risk of abolishing the biological activity of the target molecule or finding it “irreversibly” bound to the affinity chromatography medium.

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Elution Ideal elution requires that target molecule desorbs (elutes) completely from the affinity ligand in elution buffer. Two possibilities exist: 1. Apply conditions (pH, ionic strength, temperature, polar properties etc.) that change KD from low to high. KD values suitable for elution are typically in the range of 10-1 - 10-2. Fig 4.4. Elution (desorption): KD values suitable for elution are typically in the range of 10-1 - 10-2. For KD values <10-2 retardation of the target molecule may occur during elution with severe peak broadening as a probable result. Fig 4.5. Retarded elution due to too low a KD value: Too strong binding may lead to retarded elution. 2a. Add a free ligand (or analogue) to displace the target molecule by competitive binding to the target. Example: Elution of NADP dependent enzymes from Blue Sepharose™ by adding NADPH Fig 4.6. Elution by displacement, free ligand: free ligand is added to displace the target from the matrix-bound ligand. 2b. Add a competitor to displace the target molecule by competitive binding to the matrix-bound ligand . Example: Elution of Histidine-tagged proteins from HisTrap™ columns by adding imidazole. Fig 4.7. Elution by displacement, free target analogue: an analogue is added to displace the target from the matrix-bound ligand.

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Binding/desorption kinetics Ideally affinity chromatography provides distinct and rapid binding and desorption of the target molecule and the expected results should look like those shown in Fig 5.1. Fig 5.1. Expected behavior, quick on/quick off: with sufficiently fast binding and elution kinetics the target molecule elutes as a sharp peak. If slow binding and/or desorption is experienced the best way out is to turn to another affinity system providing "fast on/fast off" kinetics, if available. Sometimes, however, slow kinetics cannot be avoided and special ways to bind and elute the target molecule have to be adopted or recovery may suffer. This may be seen when purifiying GST-tagged recombinant proteins. Slow Binding Leakage of target molecule during sample application and wash (Fig 5.2) indicates that the sample residence time is too short for complete binding. If such leakage escapes attention the results might be incorrectly interpreted as providing low recoveries. Fig 5.2. Slow binding requires enough residence time for complete binding. A way to extend the residence time is to inject the sample in small portions and stop the flow after each injection (Fig 5.3). The duration of the flow stop has to be worked out by trial and error. Fig 5.3. "Stopped flow" binding: increased residence time can be created by injecting the sample in small portions and stopping the flow between injections. Slow desorption The problem here is not the degree of purification obtained, but rather that the eluted target molecule may elute as a diluted long zone (Fig 5.4). Fig 5.4. Slow desorption may result in broad and dilute peaks. Even in this case an elution technique based on "stopped flow" may help. One column volume of elution buffer is pumped into the column and the flow is then stopped. This will allow desorbed target molecule to accumulate in a restricted volume of eluent before leaving the column and thus elute in more concentrated form. The stopped flow elution is then repeated until all target has been eluted (Fig 5.5.). Fig 5.5. "Stopped flow" elution: "Slow off" kinetics requires extra time for full desorption.

Affinity chromatography applied to recombinant proteins

The purification of recombinant proteins may be drastically simplified by fusing the gene for an affinity tag (or handle)
with the gene for the recombinant.
The host will then express the recombinant protein tagged with the affinity handle and affinity chromatography techniques
can be applied to isolate and purify this so-called fusion protein or tagged protein (Fig 6.1.). Though not always necessary, the affinity tag can
be removed by special cleave-off enzymes after the purification.




Fig 6.1. Production and purification of tagged proteins.
Note: Histidine-tagged can be purified directly on prepacked HisTrap FF crude columns without the need for the clarification step. This will decrease purification time and may increase the purity of the biologically active target protein as proteases are removed earlier and faster from the sample.

The merits of tagged protein technique are: The ligand/affinity tag system can in principle be applied to any recombinant protein. The purification protocol is reduced to one fast step. Note: A second polishing gel filtration step is always recommended. Conditions used may become standardized. Very little optimization work is needed for tags like GST, MBP and Strep-tag™ II. Note that due to natural occuring proteins with surface histidines purification of histidine-tagged proteins may need some optimization to get high purity. Figures 6.2 and 6.3 show the workflow for purification of GST-tagged and (His)6-tagged fusion proteins: Fig 6.2. Schematic overview of GST-tagged protein purification using GSTrap™. The same applies for purification of a histidine-tagged protein using HisTrap™ columns. Note that the availability of HisTrap FF crude columns for direct loading of crude, unclarified cell lysate.

Affinity chromatography in practice

Use of Affinity Chromatography

Mono-specific affinity chromatography	Group-specific affinity chromatography	Tagged protein affinity chromatography
One-step purification of enzymes, receptors, antibodies, etc. employing active site-directed ligands.	One-step purification of proteins containing a more general binding site such as some antibody classes, glycoproteins, and NADP-dependent enzymes.	One-step purification of affinity tagged recombinant proteins. The affinity tag is introduced by gene fusion techniques.

Experimental affinity chromatography

		Mono-specific		Group-specific		Fusion protein
Affinity chromatography medium		Media for affinity chromatography should be highly porous to guarantee high coupling yields and full access to the affinity ligand. Not often commercially available. Pre-activated chromatography media available for preparing own mono-specific medium.		Media are commercially available in many formats such as prepacked columns, kits, lab packs, etc. to guarantee proper function.		Media are commercially available in prepacked columns and kits to guarantee proper function.
Column		Affinity chromatography is step-eluted. Columns are therefore typically "short and fat".		Affinity chromatography is in most cases step-eluted. Columns are therefore typically "short and fat".		Columns are typically "short and fat" prepacked in HiTrap™ and HiPrep™ formats. For parallel purification use Spin columns or prefilled 96-well plates, Multitrap™.
Column regeneration and storage .		Some affinity ligands are sensitive to proteases. Follow recommended regeneration and storage procedures to ensure full column lifetime. Re-use of columns should be restricted to identical proteins to prevent cross-contamination		Some affinity ligands are sensitive to proteases and/or low pH. Follow recommended regeneration and storage procedures to ensure full column lifetime. Re-use of columns should be restricted to identical proteins to prevent cross-contamination		Some affinity ligands are sensitive to proteases. Follow recommended regeneration and storage procedures to ensure column lifetime. Re-use of columns should be restricted to identical tagged proteins to prevent cross contamination.
Eluents		Harsh elution conditions, which may influence ligand lifetime are sometimes required. Always wash column after use.		Both mild and harsh elution conditions, which may influence ligand lifetime are sometimes required. Always wash column after use.		Often mild elution condition, but fast removal of proteases is always recommended.
Flow rates		Binding/desorption kinetics may restrict applicable flow rate.		Binding/desorption kinetics may restrict applicable flow rate.		For proper function follow recommended procedure.
Sample volume		Sample volume is generally of less importance, as long as the maximum load capacity of the medium is not violated Samples too dilute may hamper binding if KD is higher than optimal.		Sample volume is generally of less importance, as long as the maximum load capacity of the medium is not violated.		Sample is generally of less importance, as long as the maximum load capacity of the medium is not violated.

Affinity chromatography profile

Working principle	Separates molecules according to bio-specific affinity.
Used for	One step purification of target molecules from complex samples Purification of tagged recombinant proteins Group separations Removal of specific contaminants
Suitable stage in a purification protocol	Capture: Intermediate: Polishing: (Number of stars indicate suitability.)
Separation characteristics .	Resolution Very High Load capacity High . Speed Moderate . Gentleness High Mass yield. High
Main optimizing parameter	Choice of affinity ligand
Special features	Very simple purification protocols Requires simple equipment Excellent for tagged recombinant proteins Fast

Affinity chromatography

Selection guide

Afffinity Chromatography columns and media pdf

Handbooks

Afffinity Chromatography, Principles & Methods pdf
Protein Purification Handbook pdf
Recombinant Protein Purification: Principles and Methods pdf