A Comprehensive Guide for Affinity Chromatography of Antibodies

How To Guide 1 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. A COMPREHENSIVE GUIDE FOR AFFINITY CHROMATOGRAPHY OF ANTIBODIES 3 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. A COMPREHENSIVE GUIDE FOR AFFINITY CHROMATOGRAPHY OF ANTIBODIES 4 How To Guide 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 81 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