title: Production of stable bispecific IgG1 by controlled Fab-arm exchange: scalability from bench to large-scale manufacturing by application of standard approaches.
abstract: The manufacturing of bispecific antibodies can be challenging for a variety of reasons. For example, protein expression problems, stability issues, or the use of non-standard approaches for manufacturing can result in poor yield or poor facility fit. In this paper, we demonstrate the use of standard antibody platforms for large-scale manufacturing of bispecific IgG1 by controlled Fab-arm exchange. Two parental antibodies that each contain a single matched point mutation in the CH3 region were separately expressed in Chinese hamster ovary cells and manufactured at 1000 L scale using a platform fed-batch and purification process that was designed for standard antibody production. The bispecific antibody was generated by mixing the two parental molecules under controlled reducing conditions, resulting in efficient Fab-arm exchange of>95% at kg scale. The reductant was removed via diafiltration, resulting in spontaneous reoxidation of interchain disulfide bonds. Aside from the bispecific nature of the molecule, extensive characterization demonstrated that the IgG1 structural integrity was maintained, including function and stability. These results demonstrate the suitability of this bispecific IgG1 format for commercial-scale manufacturing using standard antibody manufacturing techniques.
authors.affiliation: Genmab; Brooklyn Park, MN USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Gramer
authors.affiliation: Genmab; Utrecht, the Netherlands.
authors.forename: Ewald T J
authors.initials: ET
authors.lastname: van den Bremer
authors.affiliation: Genmab; Utrecht, the Netherlands.
authors.forename: Muriel D
authors.initials: MD
authors.lastname: van Kampen
authors.affiliation: Genmab; Brooklyn Park, MN USA.
authors.forename: Amitava
authors.initials: A
authors.lastname: Kundu
authors.affiliation: Genmab; Brooklyn Park, MN USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Kopfmann
authors.affiliation: Genmab; Brooklyn Park, MN USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Etter
authors.affiliation: Genmab; Brooklyn Park, MN USA.
authors.forename: David
authors.initials: D
authors.lastname: Stinehelfer
authors.affiliation: Genmab; Brooklyn Park, MN USA.
authors.forename: Justin
authors.initials: J
authors.lastname: Long
authors.affiliation: Genmab; Brooklyn Park, MN USA.
authors.forename: Tom
authors.initials: T
authors.lastname: Lannom
authors.affiliation: Genmab; Utrecht, the Netherlands.
authors.forename: Esther H
authors.initials: EH
authors.lastname: Noordergraaf
authors.affiliation: Genmab; Utrecht, the Netherlands.
authors.forename: Jolanda
authors.initials: J
authors.lastname: Gerritsen
authors.affiliation: Genmab; Utrecht, the Netherlands.
authors.forename: Aran F
authors.initials: AF
authors.lastname: Labrijn
authors.affiliation: Genmab; Utrecht, the Netherlands.
authors.forename: Janine
authors.initials: J
authors.lastname: Schuurman
authors.affiliation: Genmab; Utrecht, the Netherlands.
authors.forename: Patrick H C
authors.initials: PH
authors.lastname: van Berkel
authors.affiliation: Genmab; Utrecht, the Netherlands.
authors.forename: Paul W H I
authors.initials: PW
authors.lastname: Parren
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis* / Q000235:genetics; D019149:Bioreactors; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D017354:Point Mutation; D015202:Protein Engineering; D055550:Protein Stability; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D018033:Antibodies, Bispecific
keywords: bispecific antibodies; cell culture; controlled Fab-arm exchange; large-scale manufacturing; purification; scale up