Affinity chromatography remains one of the main methods for purifying antibodies, enabling high specificity and yield. Whether working with recombinant monoclonal antibodies or polyclonal antibodies from serum, the right approach is critical to avoid contamination, degradation or unwanted aggregates. Selecting the right resins, buffers and purification method can make all the difference.
This guide highlights key strategies for optimizing affinity chromatography and explores how to maximize purity, efficiency and reproducibility while minimizing common pitfalls.
Download this guide to discover:
- Best practices for resin selection, buffer preparation and process optimization
- Step-by-step workflows for gravity columns and FPLC-based purification
- Useful troubleshooting tips to improve yield and antibody integrity
How To Guide
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A Comprehensive Guide for Affinity
Chromatography of Antibodies
Aron Gyorgypal, PhD
Affinity chromatography is the gold standard for selective purification of antibodies, capitalizing on
the specific interactions between the antibody and their corresponding antigen or by an affinity ligand.
Affinity chromatography has broad utility in the purification of recombinant monoclonal antibodies
produced by cell culture or even the purification of polyclonal antibodies from serum for diagnostics.1
Understanding and adapting a method for antibody purification will enable the production of highly
pure and functional antibodies for downstream applications. However, incorrect purification can cause
contamination, degradation or production of dimers/trimers that may affect the efficacy of the proteins.
Here, we will break down each step involved in antibody purification using column-based affinity chromatography,
using either gravity or fast protein liquid chromatography (FPLC), and give tips for better
purification practices.
Figure 1: Gravity column- (A) and FPLC-based (B) affinity chromatography of antibodies.
Credit: Aron Gyorgypal, PhD.
Setting the stage: Resins, buffers and flow dynamics
Methodology: Sample preparation is key for successful antibody purification. It is important to understand
the nuances between gravity columns and FPLC (Figure 1). While both methods use affinity
resins, gravity columns offer simplicity and cost efficiency and are ideal for small-scale purification.
Using an FPLC system enables automation and precision when it comes to purification. This methodology
allows for proper control of the chromatography process, such as controlling the flow rate, solvent
A COMPREHENSIVE GUIDE FOR AFFINITY CHROMATOGRAPHY OF ANTIBODIES 2
How To Guide
gradients for washing and elution and ultraviolet (UV)-spectroscopy-based monitoring. FPLC is preferred
for larger-scale purification or when reproducibility in purification is required.
Affinity resins: The resin you chose is crucial for high purity and loading onto the column. The gold
standard resins are affinity ligands that bind to antibodies; these include protein A and protein G, which
are bacterial proteins that bind to multiple epitopes of an antibody fragment crystallizable (Fc) region.
Regarding human antibodies, protein A is the primary resin used for the therapeutic immunoglobulin
G1 (IgG1) antibody subclass (produced as biologics); this resin also has capabilities in binding to other
subclasses such as IgG2 and IgG4. Protein G, however, can bind to a broader range of IgG subclasses
compared to protein A and to various other species, such as mouse IgGs, for labs that work on translational
immunological work. Protein L is also worth mentioning as it binds to the variable kappa light
chain of antibodies and single-chain antibody fragments, making it a practical alternative when protein
A/G is not a suitable application.2
Buffers: The buffer used to apply and wash the affinity column is known as the binding buffer. This
buffer maintains a neutral pH to preserve antibody stability and promote optimal binding conditions.
Typical buffers include phosphate buffer, phosphate-buffered saline (PBS) and tris-buffered saline
(TBS) near a physiological pH of 7.4. Once the antibody is bound to the column and washed of contaminants,
it is time to remove the antibodies from the column, which is done using an elution buffer. The
elution buffer of choice is a low pH buffer, disrupting the antibody–ligand interaction and allowing the
antibody to come off the column. This elution buffer is typically glycine-HCl, citric acid, at a pH around
2.5–3 and molarity around 100 mM. Other nonionic solutions, such as formic or acetic acid, can also be
used. The protein is then directly neutralized back to a tolerable pH to mitigate degradation from the
low pH using a neutralizing buffer such as 1M tris-HCl at a pH of 8–9.
Sample preparation pro tips:
Always check the compatibility of your antibody to the specific resin; it may be worthwhile to do a
small-scale purification or try multiple types of resins to check resin compatibility and efficiency.
Always check the resin storage conditions. Some resins must be stored in 20% ethanol, while others
may require special instructions from the resin manufacturer.
Filter sterilize all buffers and your samples through a 0.22-micron filter to remove any particulates
that may block the pores of the resin.
Column set up 101 and loading your antibody
Gravity columns: Resin is usually purchased independently and packed into a chromatography
column unless purchased prepacked. It is essential to wash the column thoroughly with binding buffer
to remove stabilizers, such as ethanol, azide or surfactants. This involves 3–5 column volume washes
(volume with respect to resin volume used). Avoid air bubbles that can disrupt the flow through the
column and ensure that the column resin is settled before applying the sample. The sample can be
mixed 1:1 with binding buffer to improve binding, and ensure the sample is added to the preconditioned
column slowly to facilitate the flow rate. Maximizing the column’s contact time (residence time) with
the sample is essential to achieve maximum binding efficiency.
FPLC: Typically, columns for FPLC systems are bought prepacked for batch consistency. It is important
to ensure the buffers used are degassed before use to mitigate issues with the pump and protect the
system’s integrity. After the column is installed, the flow rate of the binding buffer is adjusted, and the
binding buffer is flowed through until the column reaches a baseline on the UV detector. The sample is
injected through a sample port, mixed with binding buffer and loaded onto the column. The UV detector
monitors the loading and the removal of contaminants. This real-time UV monitoring facilitates more
stringent purification control than the gravity column and ensures the system is not overloaded.
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How To Guide
Column preparation and sample application pro tips:
When using a gravity column, plan to scale the size of the column to the volume of resin so as not to
produce too much back pressure, leading to slow or complete loss of flow.
For FPLC, decreasing the flow rate will increase contact time, which can increase binding efficiency
between the antibody and ligand. However, this may not be suitable for large-volume purification.
Binding (your antibody) washing (off impurities) and eluting (your
pure protein)!
Washing off impurities while binding your antibody: The binding buffer is typically the same as
the wash buffer. After loading the antibody, an additional amount of buffer is added to remove the contaminants
while the bound target antibody remains on the column. The gravity column is washed with
multiple volumes to remove impurities thoroughly. FLPC can confirm that all unbound impurities are
washed away through UV spectroscopy in real time, providing continuous loading feedback and ensuring
the column is ready for the target antibody to be eluted.
Elution of target antibody: Pure protein is achieved after washing and subsequent elution with 3–4
column volumes of elution buffer at a low pH. In the gravity column, it is best to add the neutralization
buffer to the eluent immediately after elution to maintain protein integrity.3 When using FPLC, gradient
elution can be used to elute different antibodies based on their affinities to the ligand, and the elutions
are collected by a fraction collector based on the UV absorbance peaks. Immediate neutralization is
also critical to protect antibodies from denaturation due to exposure to low pH.
Antibody binding pro-tips:
While using PBS and TBS is advised, sometimes the binding buffer needs to be fine-tuned, such as
by adjusting the pH and the ionic strength of the buffer, as this will significantly alter antibody–ligand
interactions. For example, adding 0.1% Tween-20 can reduce hydrophobic interactions that cause
non-specific binding, and increasing the salt concentration of the wash buffer can disrupt ionic interactions
that contribute to non-specific binding.
Especially for temperature-sensitive antibodies, performing all steps at 4 °C can prevent degradation.
Post-elution steps
Checking the eluent: After the protein is eluted and neutralized from a gravity column, a UV spectrophotometer
can be used to determine protein concentration and purity, or alternative methods such as
Bradford or bicinchoninic acid (BCA) assays can also be used. Afterward, it is advisable to run sodium
dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to assess protein size and purity.
Buffer exchange and concentration: Buffer exchange and concentration are performed to prepare
proteins for downstream applications. This can be done through dialysis or using a desalting column.
The antibody can then be concentrated using a centrifugation column with an appropriate molecular
weight cut-off.
Storage: For short-term storage, the antibody can be kept in a 4 °C fridge with an appropriate preservative
agent such as 0.02% sodium azide. For long-term storage, proteins can be aliquoted and frozen
at either -20 °C or -80 °C to prevent degradation and to avoid repeated freeze–thaw cycles.4
Post elution pro tips:
Running a reducing and non-reducing SDS-PAGE can provide insights into the antibody’s structural
integrity.
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Running western blots will confirm antibody identity and integrity if specific detection is necessary.
To maintain protein integrity, it may be advisable to add stabilizers such as glycerol (10–50% v/v) as a
cryoprotectant, sucrose that can protect against dehydration, arginine ( 50–500 mM), which can prevent
aggregation or carrier protein such as bovine serum albumin (BSA) (0.1–1% w/v), which can prevent
aggregation and protein adsorption to container walls.5
Did you get it right? Some final thoughts
Like any experimental procedure, affinity chromatography requires some art in its optimization, as
every antibody acts differently during purification. Although the methods are similar for every antibody,
fine-tuning may be necessary to purify the target antibody efficiently.
References:
1. Arora S, Saxena V, Ayyar BV. Affinity chromatography: A versatile technique for antibody purification. Methods.
2017;116:84-94. doi:10.1016/j.ymeth.2016.12.010
2. Goulet DR, Atkins WM. Considerations for the design of antibody-based therapeutics. J Pharm Sci. 2020;109(1):74-103.
doi:10.1016/j.xphs.2019.05.031
3. Ayyar BV, Arora S, Murphy C, O’Kennedy R. Affinity chromatography as a tool for antibody purification. Methods.
2012;56(2):116-129. doi:10.1016/j.ymeth.2011.10.007
4. Kaur H. Stability testing in monoclonal antibodies. Crit Rev Biotechnol. 2021;41(5):692-714. doi:10.1080/07388551.2021.18742
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5. Mueller M, Loh MQT, Tee DHY, Yang Y, Jungbauer A. Liquid formulations for long-term storage of monoclonal IgGs. Appl
Biochem Biotechnol. 2013;169(4):1431-1448. doi:10.1007/s12010-012-0084-z