A two-column process for bispecific antibody purification based on MabSelect VL resin’s strong byproduct removal capability
Background: Protein L-conjugated resins are affinity media that bind to the variable region of the kappa light chain (LC) and have been used for initial product capture in the downstream processing of full-length antibodies and antibody fragments. Previous studies, including ours, have demonstrated that Protein L chromatography effectively separated various byproducts generated during the production of bispecific antibodies (bsAbs), including half-antibody, homodimer, LC-missing species, and aggregates. Cytiva recently launched its second-generation Protein L resin, MabSelect VL, which offers significantly improved binding capacity compared to its predecessor, Capto L. Objective: This study aimed to explore the feasibility of developing a two-column process, which includes MabSelect VL capture step and a polishing step, for purification of complex antibody molecules. Methods: We employed two bsAb cases to demonstrate that MabSelect VL’s enhanced byproduct removal capability allows for a potential two-column purification process. Results: For both bsAbs, the developed two-column process yielded a product with quality attributes comparable to those obtained using the traditional three-column process. Conclusion: The MabSelect VL-based two-column process can be successfully applied to bsAb purification. In addition, it should also be feasible with regular monoclonal antibodies, whose purification is generally less challenging than that of bsAbs. By reducing the downstream process from three columns to two columns, significant savings in terms of time, labor, and materials can be achieved.
- Björck L. Protein L. A novel bacterial cell wall protein with affinity for Ig L chains. J Immunol. 1988;140(4):1194-1197.
- Kastern W, Sjöbring U, Björck L. Structure of peptostreptococcal protein L and identification of a repeated immunoglobulin light chain-binding domain. J Biol Chem. 1992;267(18):12820-12825.
- Murphy JP, Duggleby CJ, Atkinson MA, Trowern AR, Atkinson T, Goward CR. The functional units of a peptostreptococcal protein L. Mol Microbiol. 1994;12(6):911-920. doi: 10.1111/j.1365-2958.1994.tb01079.x
- Graille M, Stura EA, Housden NG, et al. Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural convergence in the interaction modes of Fab binding proteins. Structure. 2001;9(8):679-687. doi: 10.1016/s0969-2126(01)00630-x
- Paloni M, Cavallotti C. Molecular modeling of the interaction of protein L with antibodies. ACS Omega. 2017;2(10):6464-6472. doi: 10.1021/acsomega.7b01123
- Chen C, Wakabayashi T, Muraoka M, et al. Controlled conductivity at low pH in Protein L chromatography enables separation of bispecific and other antibody formats by their binding valency. MAbs. 2019;11(4):632-638. doi: 10.1080/19420862.2019.1583996
- Wang Y, Chen X, Wang Y, Li Y. Removing a difficult-to-separate byproduct by Capto L affinity chromatography during the purification of a WuXiBody-based bispecific antibody. Protein Expr Purif. 2020;175:105713. doi: 10.1016/j.pep.2020.105713
- Chen W, Zhang T, Wan Y, Li, Y. Assessing four subdomain-specific affinity resins’ capability to separate half-antibody from intact bispecific antibody. Protein Expr Purif. 2022;198:106124. doi: 10.1016/j.pep.2022.106124
- Chen X, Wang Y, Wang Y, Li Y. Protein L chromatography: A useful tool for monitoring/separating homodimers during the purification of IgG-like asymmetric bispecific antibodies. Protein Expr Purif. 2020;175:105711. doi: 10.1016/j.pep.2020.105711
- Chen SW, Tan D, Yang YS, Zhang W. Investigation of the effect of salt additives in Protein L affinity chromatography for the purification of tandem single-chain variable fragment bispecific antibodies. MAbs. 2020;12(1):1718440. doi: 10.1080/19420862.2020.1718440
- Cytiva. MabSelect VL Affinity Chromatography Resin, Data File, CY26149. Marlborough, MA: Cytiva; 2022.
- Aboulaich N, Chung WK, Thompson JH, Larkin C, Robbins D, Zhu M. A novel approach to monitor clearance of host cell proteins associated with monoclonal antibodies. Biotechnol Prog. 2014;30(5):1114-1124. doi: 10.1002/btpr.1948
- Chollangi S, Parker R, Singh N, Li Y, Borys M, Li Z. Development of robust antibody purification by optimizing protein-A chromatography in combination with precipitation methodologies. Biotechnol Bioeng. 2015;112(11):2292-2304. doi: 10.1002/bit.25639
- Cui T, Chi B, Thompson JH, Kasali T, Sellick C, Turner R. Cathepsin D: Removal strategy on protein A chromatography, near real time monitoring and characterisation during monoclonal antibody production. J Biotechnol. 2019;305:51-60.doi: 10.1016/j.jbiotec.2019.08.013
- Capto adhere ImpRes. Available from: https://cdn. cytivalifesciences.com/api/public/content/digi-16466-pdf
- Gao D, Wanga LL, Lin DQ, Yao SJ. Evaluating antibody monomer separation from associated aggregates using mixed-mode chromatography. J Chromatogr A. 2013;1294:70-75. doi: 10.1016/j.chroma.2013.04.018
- Chen SW, Hoi KM, Mahfut FB, Yang Y, Zhang W. Effective flow-through polishing strategies for knob-into-hole bispecific antibodies. Bioresour Bioprocess. 2022;9(1):98. doi: 10.1186/s40643-022-00590-8
- Cai K, Anderson J, Orchard JD, Afdahl CD, Dickson M, Li Y. Virus removal robustness of ion exchange chromatography. Biologicals. 2019;58:28-34. doi: 10.1016/j.biologicals.2019.01.004
- Strauss DM, Gorrell J, Plancarte M, Blank GS, Chen Q, Yang B. Anion exchange chromatography provides a robust, predictable process to ensure viral safety of biotechnology products. Biotechnol Bioeng. 2009;102(1):168-175. doi: 10.1002/bit.22051
- Connell-Crowley L, Nguyen T, Bach J, et al. Cation exchange chromatography provides effective retrovirus clearance for antibody purification processes. Biotechnol Bioeng. 2012;109(1):157-165. doi: 10.1002/bit.23300
- Miesegaes GR, Lute S, Strauss DM, et al. Monoclonal antibody capture and viral clearance by cation exchange chromatography. Biotechnol Bioeng. 2012;109(8):2048-2058. doi: 10.1002/bit.24480