Therapeutic antibodies have become an important class of protein drugs for the treatment of numerous human diseases as well as uses in immunoassays. Nearly 100 monoclonal antibody-based drugs have been approved for the treatment of immunologic diseases and cancer. These therapeutic protein antibodies include hybridomas, recombinant antibodies (mAbs), humanized monoclonal antibodies (including chimeric antibodies), glyco-engineered antibodies, Fab fragments, single chain antibodies, single domain antibodies, bispecific/bifunctional antibodies, antibody-drug conjugates (ADC), “antigenized” antibodies, or antibody-fusion proteins. Development of robust, efficient, and cost-effective methods for large-scale purification of therapeutically active antibodies/proteins with homogeneous purity are inevitable for therapeutic antibodies.

Methods for the Purification of Therapeutic Antibodies

Selection of methods for the purification of therapeutic antibodies are dependent on the source of antibodies (hybridoma vs. cell culture), types of antibodies (full length vs. recombinant antibodies). Commonly used methods for the purification of various types of antibody-based protein drugs are a shown in Table 1 (for details refer to articles cited in the “Further reading” section). Specific protein purification methods need to be selected based on the types of antibody sources or expression systems and the genetic or protein nature of therapeutic antibodies.

Table A: Methods for purification of therapeutic antibodies

Strategies for the Purification of Antibodies with Homogeneous Purity and Therapeutic Function

Therapeutic antibodies must be highly pure proteins without contaminating proteins or non-protein moieties (e.g. endotoxin) and devoid of any structural or functional modifications, which include aggregation, fragmentation, protease susceptibility, aberrant post-translational modifications, or immunogenic domains. These issues, if it occurred, can be detected using biochemical or bioanalytical methods in combination with or in vitro (ELISA) or Cell Based Assays and addressed by optimization of cell culture conditions (recombinant expression), genetic modifications of recombinant antibodies, optimization of conditions for structural and functional integrity of recombinant antibodies during (stability) and after protein purification (storage conditions). Strategies for the purification of structurally and functionally active recombinant therapeutic antibodies are shown in Fig. 1.

Fig 1: Strategies for purification of therapeutic antibodies

Further reading

• Murphy et al (2016) Dovepress 6:17-32. https://www.dovepress.com/technology-advancements-in-antibody-purification-peer-reviewed-fulltext-article-ANTI
• Fishman and Berg (2019) Cold Spring Harbor Protocols http://cshprotocols.cshlp.org/content/2019/5/pdb.top099101.full
• Sifniotis, et al (2019) Antibodies (Basel). 8: 36. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6640721/
• Chen and Zhang (2021) Antibody Therapeutics. 4: 73–88 https://academic.oup.com/abt/article/4/2/73/6271369