Rapid Purification of Human Bispecific Antibodies via Selective Modulation of Protein A Binding

• Published in: Scientific reports Vol. 7; no. 1; pp. 15521 - 11
• Main Authors: Zwolak, Adam, Leettola, Catherine N., Tam, Susan H., Goulet, Dennis R., Derebe, Mehabaw G., Pardinas, Jose R., Zheng, Songmao, Decker, Rose, Emmell, Eva, Chiu, Mark L.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 14.11.2017; Nature Publishing Group
• Subjects: 13/1; 631/154/51; 64/60; 692/4017; 82/1; 82/80; 82/83; Bispecific antibodies; Humanities and Social Sciences; Immunoglobulin G; Monoclonal antibodies; multidisciplinary; Mutation; pH effects; Protein A; Protein purification; Proteins; Science; Science (multidisciplinary); Thermal stability
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-017-15748-0

Methods to rapidly generate high quality bispecific antibodies (BsAb) having normal half-lives are critical for therapeutic programs. Here, we identify 3 mutations (T307P, L309Q, and Q311R or “TLQ”) in the Fc region of human IgG1 which disrupt interaction with protein A while enhancing interaction with FcRn. The mutations are shown to incrementally alter the pH at which a mAb elutes from protein A affinity resin. A BsAb comprised of a TLQ mutant and a wild-type IgG1 can be efficiently separated from contaminating parental mAbs by differential protein A elution starting from either a) purified parental mAbs, b) in-supernatant crossed parental mAbs, or c) co-transfected mAbs. We show that the Q311R mutation confers enhanced FcRn interaction in vitro , and Abs harboring either the Q311R or TLQ mutations have serum half-lives as long as wild-type human IgG1. The mutant Abs have normal thermal stability and Fcγ receptor interactions. Together, the results lead to a method for high-throughput generation of BsAbs suitable for in vivo studies.