title: An efficient process of generating bispecific antibodies via controlled Fab-arm exchange using culture supernatants.
abstract: Bispecific antibody generation is actively pursued for therapeutic and research antibody development. Although there are multiple strategies for generating bispecific antibodies (bsAbs); the common challenge is to develop a scalable method to prepare bsAbs with high purity and yield. The controlled Fab-arm exchange (cFAE) method combines two parental monoclonal antibodies (mAbs), each with a matched point mutation, F405L and K409R in the respective CH3 domains. The conventional process employs two steps: the purification of two parental mAbs from culture supernatants followed by cFAE. Following a reduction/oxidation reaction, the bispecific mAb is formed with greater than 95% heterodimerization efficiency. In this study, cFAE was initiated in culture supernatants expressing the two parental mAbs, thereby eliminating the need to first purify the parental mAbs. The bsAbs formed in culture supernatant was then purified using a Protein A affinity chromatography. The BsAbs generated in this manner had efficiency comparable to the conventional method using purified parental mAbs. BsAbs prepared by two different routes showed indistinguishable characteristics by SDS capillary electrophoresis, analytical size exclusion, and cation exchange chromatography. This alternative method significantly shortened timelines and reduced resources required for bsAb generation, providing an improved process with potential benefits in large-scale bsAb preparation, as well as for HTP small-scale bsAb matrix selection.
authors.affiliation: Biologics Research, Biotechnology Center of Excellence, Janssen Research and Development, LLC., 1400 McKean Road, Spring House, PA 19477, USA.
authors.forename: Suparna
authors.initials: S
authors.lastname: Paul
authors.affiliation: Biologics Research, Biotechnology Center of Excellence, Janssen Research and Development, LLC., 3210 Merryfield Row, San Diego, CA 92121, USA.
authors.forename: Judy
authors.initials: J
authors.lastname: Connor
authors.affiliation: Biologics Research, Biotechnology Center of Excellence, Janssen Research and Development, LLC., 1400 McKean Road, Spring House, PA 19477, USA.
authors.forename: Tom
authors.initials: T
authors.lastname: Nesspor
authors.affiliation: Biologics Research, Biotechnology Center of Excellence, Janssen Research and Development, LLC., 1400 McKean Road, Spring House, PA 19477, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Haytko
authors.affiliation: Biologics Research, Biotechnology Center of Excellence, Janssen Research and Development, LLC., 1400 McKean Road, Spring House, PA 19477, USA.
authors.forename: Ken
authors.initials: K
authors.lastname: Boakye
authors.affiliation: Biologics Research, Biotechnology Center of Excellence, Janssen Research and Development, LLC., 1400 McKean Road, Spring House, PA 19477, USA.
authors.forename: Mark L
authors.initials: ML
authors.lastname: Chiu
authors.affiliation: Biologics Research, Biotechnology Center of Excellence, Janssen Research and Development, LLC., 1400 McKean Road, Spring House, PA 19477, USA. Electronic address: Hjiang1@its.jnj.com.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Jiang
mesh_terms: D018033:Antibodies, Bispecific / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D002845:Chromatography; D003470:Culture Media; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D017354:Point Mutation
chemical_list: D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D003470:Culture Media; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G
keywords: Affinity purification; Bispecific antibody; Controlled Fab-arm exchange; Monoclonal antibody; Protein A; Protein culture supernatant