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Antibody Purification

Biointron 2024-01-23 Read time: 4 mins
Affinity chromatography. Image credit: DOI: 10.1021/acsomega.2c03530img

Antibody purification is a process used to isolate and obtain highly purified antibodies from a mixture of various proteins and molecules. Monoclonal antibodies (mAbs), polyclonal antibodies, recombinant antibodies, and antibody fragments each have unique applications, and require different purification techniques. This can be carried out with chromatographic or non-chromatographic techniques.1,2

Chromatographic methods typically involve isolating antibodies by binding or passing them through a solid phase, which can be materials like silica resin, beads, monolithic columns, or cellulose membranes. An example that can be used is mixed-mode (multimodal) chromatography, in which “bind/elute” or “flowthrough” techniques can be employed. Other chromatographic purifications include affinity-tag binding, ion-exchange, size-exclusion, and immunoaffinity chromatography.2,3

One of the most popular techniques is Protein A affinity chromatography. Protein A is an ideal bioaffinity ligand, as it is a bacterial cell wall protein that binds specifically to the Fc region of immunoglobulin G (IgG) antibodies.4 After allowing the antibody-containing sample to flow through the resin in which Protein A is immobilized, the antibodies are captured through binding, washing, and then elution. Similarly, Protein G and Protein L can be used instead of Protein A, although their binding properties differ. This method is highly effective for purifying IgG antibodies, which constitute the majority of therapeutic and research antibodies. 

As an alternative to the relatively high cost of large-scale production using chromatographic purification, non-chromatographic techniques have been developed which do not rely on differential migration through a column. These methods include precipitation, filtration, affinity binding, flocculation, and crystallization.2 Unlike affinity chromatography, precipitation can also use Protein A, G, or L by covalently immobilizing them on resins or beads and subsequent antibody collection and separation through techniques like centrifugation or filtration.  

Another example of a non-chromatographic method is aqueous two-phase partitioning (ATPS), which involves the formation of two immiscible liquid phases. The system is based on the partitioning property of the antibody’s charge, hydrophilicity, and hydrophobicity, as well as on the physicochemical properties of the two liquid phases. Because of this, partition behavior is complex and can be difficult to predict. However, ATPS’s ease of use, low cost, biocompatibility, and scale-up potential gives it plenty of advantages over traditional chromatography methods, in addition to high recovery yield and being environmentally friendly.5

At Biointron, we are dedicated to accelerating your antibody discovery, optimization, and production needs. Our team of experts can provide customized solutions that meet your specific research needs. Contact us to learn more about our services and how we can help accelerate your research and drug development projects. 


  1. Liu, H. F., Ma, J., Winter, C., & Bayer, R. (2010). Recovery and purification process development for monoclonal antibody production. MAbs, 2(5), 480-499.

  2. O’Kennedy, R., Murphy, C., & Devine, T. (2016). Technology advancements in antibody purification. Antibody Technology Journal, Volume 6, 17–32.

  3. Kallberg, K., Johansson, O., & Bulow, L. (2012). Multimodal chromatography: An efficient tool in downstream processing of proteins. Biotechnology Journal, 7(12), 1485-1495.

  4. Hober, S., Nord, K., & Linhult, M. (2007). Protein A chromatography for antibody purification. Journal of Chromatography B, 848(1), 40-47.

  5. Iqbal, M., Tao, Y., Xie, S. et al. Aqueous two-phase system (ATPS): an overview and advances in its applications. Biol Proced Online 18, 18 (2016).

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