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Monoclonal Antibody Production and Purification Methods

Monoclonal antibodies are widely used in biochemistry and medicine. The implementation of more accurate purification methods and the development of better technology for protein analysis have improved the production of these molecules. This has enabled them to become very important tools in diagnostic testing and in the treatment of many diseases, such as cancer.


Engineering the Perfect Clones

The Cloning and Expression of Human Monoclonal Antibodies: Implications for Allergen Immunotherapy

Monoclonal antibodies (mAbs) are proteins synthetized by plasma cells that are clones of a parent immune cell. mAbs are employed to detect or purify substances that they bind to specifically. This means that monoclonal antibodies recognize the same epitope of a substance and bind solely to that substance.While on the other hand, polyclonal antibodies, which are made by different plasma cell lineages, have polivalent affinity, and stick to multiple epitopes (1). 


In 1975, Georges Köhler and César Milstein began to experiment with myeloma cells and B cells. They managed to create hybridomas that were capable of producing monoclonal antibodies. Many years later, mAbs are undoubtedly still molecules of invaluable relevance in research and therapeutic drug development (2).


Antibody-based therapy of cancer, for example, consists of the use of mAbs that are specific to certain ligands, membrane antigens of T-lymphocyte surfaces, or proteins of cancer cells, in combination with cytotoxic regimens or kinase inhibitors (2). This therapy has become the most employed cancer treatment of the last decade, and its clinical efficacy has increased as technology has improved, enhancing the engineering of mAbs.


The Importance of Better Purification Methods

Recovery and purification process development for monoclonal antibody production

mAbs generally share homologous physico-chemical properties, which allows scientists to use similar purification processes in the recovery of these molecules. Chromatographic and non-chromatographic techniques are two of the methods used in the isolation of antibodies from complex mixtures. 


The chromatographic method involves a solid phase that works as a filter. The filter allows the antibodies to either bind or pass through, depending on the affinity of the components to the phase and the substance that carries the materials to be separated (3). Sometimes, though, this can be too complex to achieve on a large-scale. In this case non-chromatographic separation methods such as precipitation, flocculation, or crystallization are used instead. Non-chromatographic methods can often be less accurate, but fortunately today, new technology, allows scientists to rapidly determine the quality of the protein of interest and also to find out if they are using a viable sample (3). 


Current protein purification protocols and the development of modern equipment utilized to remove impurities and measure protein expression have contributed to the synthesis and the isolation of high quality mAbs. The functionality and the potential drug efficacy is based on the structural integrity of the mAbs (4). Moreover, small monovalent antibody fragments with high tumor penetration characteristics have been synthetized in the past two decades, thanks to new techniques. These new antibody fragments are exponentially gaining relevance in the realm of new treatment development due to their evident effectiveness (4). 


Perhaps the most important thing is that the consistent improvement of these molecules has finally brought into a new light is the treatment of cancer, autoimmune disorders, and inflammatory diseases. As these critical diseases continue to spread throughout our human environment, science becomes more eager to explore more “customized” mAb therapies (5). 



  1. Saaed A, Wang R, Ling S, Wang S. Antibody Engineering For Pursuing a Better Future. Front Microbiol. 2017; 8: 495. Published online 2017 Mar 28. doi:  10.3389/fmicb.2017.00495


  1. Carvalho Silvia et al. Immunotherapy of cancer: from monoclonal to oligoclonal cocktails of anti‐cancer antibodies: IUPHAR Review 18. Br J Pharmacol. 2016 May; 173(9): 1407–1424. Published online 2016 Mar 14. doi:  10.1111/bph.13450.


  1. Liu H, Ma J, Winter C, Bayer R. Recovery and purification process development for monoclonal antibody production. MAbs. 2010 Sep-Oct; 2(5): 480–499. doi:  10.4161/mabs.2.5.12645


  1. Nelson A. Antibody fragments. MAbs. 2010 Jan-Feb; 2(1): 77–83.


  1. Low D, O´LEary R, Pujar N. Future of antibody purification. J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Mar 15;848(1):48-63. Epub 2006 Nov 28.

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