Excellent removal of knob-into-hole bispecific antibody byproducts and impurities in a single-capture chromatography

Abstract

Bispecific antibodies (bsAbs) are therapeutically promising due to their ability to bind to two different antigens. However, the bsAb byproducts and impurities, including mispaired homodimers, half-antibodies, light chain mispairings, antibody fragments and high levels of high molecular weight (HMW) species, all pose unique challenges to their downstream processing. Here, using two knob-into-hole (KiH) constructs of bsAbs as model molecules, we demonstrate the excellent removal of bsAb byproducts and impurities in a single Protein A chromatography under optimized conditions, including hole–hole homodimer mispaired products which are physicochemically very similar to the target bsAbs and still present even with the use of the KiH format, though at reduced levels. The removal occurs through the incorporation of an intermediate low-pH wash step and optimal elution conditions, achieving ~ 60% monomeric purity increase in a single Protein A step, without the introduction of sequence-specific bsAb modifications to specifically induce differential Protein A binding. Our results also suggest that the higher aggregation propensity of bsAbs may cause aggregation during the column process, hence an optimization of the appropriate loading amount, which may be lower than that of monoclonal antibodies (mAbs), is required. With the use of loading at 50% of 10% breakthrough (QB10) at 6-min residence time, we show that an overall high monomer purity of 92.1–93.2% can be achieved with good recovery of 78.4–90.6% within one capture step, which is a significant improvement from a monomer purity of ~ 30% in the cell culture supernatant (CCS). The results presented here would be an insightful guidance to all researchers working on the purification process development to produce bispecific antibodies, especially for knob-into-hole bispecific antibodies.

Keywords

Bispecific antibody / Knob-into-hole / Chromatography purification / Low-pH wash / Product-related impurities / Hole–hole homodimer / Process-related impurities / Aggregation propensity

References

[1] Andrade C, Arnold L, Motabar D, Aspelund M, Tang A, Hunter A, . An integrated approach to aggregate control for therapeutic bispecific antibodies using an improved three column mAb platform-like purification process. Biotechnol Prog, 2019, 35(1): e2720.

[2] Baeuerle PA, Reinhardt C. Bispecific T-cell engaging antibodies for cancer therapy. Can Res, 2009, 69(12): 4941-4944.

[3] Brinkmann U, Kontermann R. The making of bispecific antibodies. Mabs, 2017, 9(2): 182-212.

[4] Chames P, Baty D. Bispecific antibodies for cancer therapy: the light at the end of the tunnel. Mabs, 2009, 1(6): 539-547.

[5] Chen T, Han J, Guo G, Wang Q, Wang Y, Li Y. Monitoring removal of hole-hole homodimer by analytical hydrophobic interaction chromatography in purifying a bispecific antibody. Protein Expr Purif, 2019, 164: 105457.

[6] Chen X, Wang Y, Li Y. Removing half antibody byproduct by Protein A chromatography during the purification of a bispecific antibody. Protein Expr Purif, 2020, 172: 105635.

[7] Chen WS, Zhang W. Current trends and challenges in the downstream purification of bispecific antibodies. Antib Ther, 2021, 4(2): 73-88.

[8] Cytiva (2020a). Capacity and performance of MabSelect PrismA protein A chromatography resin. https://cdn.cytivalifesciences.com/api/public/content/digi-26524-original#:~:text=Dynamic%20binding%20capacity,-As%20shown%20in&text=At%206%20min%20residence%20time,human%20IgG%2FmL%20resin ) Accessed 2 May 2022.

[9] Cytiva (2020b). MabSelect PrismA affinity chromatography 29262586 AE. https://cytiva-delivery.sitecorecontenthub.cloud/api/public/content/digi-31662-original Accessed 18 June 2022.

[10] Cytiva (2021). Guide to transition from MabSelect SuRe^TM based resins to next-generation MabSelectTM PrismA resin. https://cdn.cytivalifesciences.com/api/public/content/digi-28317-pdf Accessed 2 May 2022.

[11] Cytiva (2022). Efficient protein A chromatography for bispecific antibodies. https://www.cytivalifesciences.com/en/us/solutions/bioprocessing/knowledge-center/purifying-bispecific-antibodies-in-a-single-step Accessed 2 May 2022.

[12] Fischer N, Elson G, Magistrelli G, Dheilly E, Fouque N, Laurendon A, . Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG. Nat Commun, 2015, 6: 6113.

[13] Garber K. Bispecific antibodies rise again. Nat Rev Drug Discov, 2014, 13(11): 799-801.

[14] Gökbuget N, Dombret H, Bonifacio M, Reichle A, Graux C, Faul C, . Blinatumomab for minimal residual disease in adults with B-cell precursor acute lymphoblastic leukemia [correction (2019) Blood 133(24):2625]. Blood, 2018, 131(14): 1522-1531.

[15] Jakobsen BK, Harwood N, Liddy NR (2011) T cell receptors. WO2011001152A1, 2011

[16] Kantarjian H, Stein A, Gökbuget N, Fielding AK, Schuh AC, Ribera J-M, . Blinatumomab versus chemotherapy for advanced acute lymphoblastic leukemia. N Engl J Med, 2017, 376(9): 836-847.

[17] Klein C, Sustmann C, Thomas M, Stubenrauch K, Croasdale R, Schanzer J, . Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies. Mabs, 2012, 4(6): 653-663.

[18] Kontermann RE. Recombinant bispecific antibodies for cancer therapy. Acta Pharmacol Sin, 2005, 26(1): 1-9.

[19] Kontermann RE. Dual targeting strategies with bispecific antibodies. Mabs, 2012, 4(2): 182-197.

[20] Labrijn AF, Janmaat ML, Reichert JM, Parren PWHI. Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov, 2019, 18(8): 585-608.

[21] Lindhofer H, Mocikat R, Steipe B, Thierfelder S. Preferential species-restricted heavy/light chain pairing in rat/mouse quadromas Implications for a single-step purification of bispecific antibodies. J Immunol, 1995, 155(1): 219-225.

[22] Li Y, Wang Y, Shen P, Zhou W. Matte A. Chapter 8—A roadmap for IgG-like bispecific antibody purification. Approaches to the purification, analysis and characterization of antibody-based therapeutics, 2020, Amsterdam: Elsevier, 167-179.

[23] Merchant AM, Zhu Z, Yuan JQ, Goddard A, Adams CW, Presta LG, . An efficient route to human bispecific IgG. Nat Biotechnol, 1998, 16: 677-681.

[24] Michaelson JS, Demarest SJ, Miller B, Amatucci A, Snyder WB, Wu X, . Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2 and LTbetaR. Mabs, 2009, 1(2): 128-141.

[25] Neijssen J, Cardoso RMF, Chevalier KM, Wiegman L, Valerius T, Anderson GM, . Discovery of amivantamab (JNJ-61186372), a bispecific antibody targeting EGFR and MET. J Biol Chem, 2021, 296: 100641.

[26] Oldenburg J, Mahlangu JN, Kim B, Schmitt C, Callaghan MU, Young G, . Emicizumab prophylaxis in hemophilia A with inhibitors. N Engl J Med, 2017, 377(9): 809-818.

[27] Ollier R, Wassmann P, Monney T, Fecourt CR, Gn S, . Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development. Mabs, 2019, 11(8): 1464-1478.

[28] Qin T, Wang Y, Li Y. Separating antibody species containing one and two kappa light chain constant region by KappaSelect affinity chromatography. Protein Expr Purif, 2020, 171: 105618.

[29] Ridgway JB, Presta LG, Carter P. ‘Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng, 1996, 9(7): 617-621.

[30] Sasso EH, Silverman GJ, Mannik M. Human IgA and IgG F(ab')2 that bind to staphylococcal protein A belong to the VHIII subgroup. J Immunol, 1991, 147(6): 1877-1883.

[31] Sasso EH, Silverman GJ, Mannik M. Human IgM molecules that bind staphylococcal protein A contain VHIII H chains. J Immunol, 1989, 142(8): 2778-2783.

[32] Schanzer J, Jekle A, Nezu J, Lochner A, Croasdale R, Dioszegi M, . Development of tetravalent, bispecific CCR5 antibodies with antiviral activity against CCR5 monoclonal antibody-resistant HIV-1 strains. Antimicrob Agents Chemother, 2011, 55(5): 2369-2378.

[33] Skegro D, Stutz C, Ollier R, Svensson E, Wassmann P, Bourquin F, . Immunoglobulin domain interface exchange as a platform technology for the generation of Fc heterodimers and bispecific antibodies. J Biol Chem, 2017, 292(23): 9745-9759.

[34] Smith EJ, Olson K, Haber LJ, Varghese B, Duramad P, Tustian AD, . A novel, native-format bispecific antibody triggering T-cell killing of B-cells is robustly active in mouse tumor models and cynomolgus monkeys. Sci Rep, 2015, 5: 17943.

[35] Syed YY. Amivantamab: first approval. Drugs, 2021, 81(11): 1349-1353.

[36] Taki S, Kamada H, Inoue M, Nagano K, Mukai Y, Higashisaka K, . A novel bispecific antibody against human CD3 and ephrin receptor A10 for breast cancer therapy. PLoS ONE, 2015, 10(12): e0144712.

[37] Tustian AD, Endicott C, Adams B, Mattila J, Bak H. Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity. Mabs, 2016, 8(4): 828-838.

[38] Vallera DA, Miller JS (2017) Therapeutic compounds and methods. WO2017062604 A1, 2017

[39] Zhang T, Wan Y, Wang Y, Li Y. Removing a single-arm species by Fibro PrismA in purifying an asymmetric IgG-like bispecific antibody. Protein Expr Purif, 2021, 182: 105847.

[40] Zwolak A, Armstrong AA, Tam SH, Pardinas JR, Goulet DR, Zheng S, . Modulation of protein A binding allows single-step purification of mouse bispecific antibodies that retain FcRn binding. Mabs, 2017, 9(8): 1306-1316.

[41] Zwolak A, Leettola CN, Tam SH, Goulet DR, Derebe MG, Pardinas JR, . Rapid purification of human bispecific antibodies via selective modulation of Protein A binding. Sci Rep, 2017, 7(1): 15521.

Funding

Cytiva Life Sciences (Sweden)

the Agency for Science, Technology and Research (A*STAR), Singapore