title: Controlled Fab-arm exchange for the generation of stable bispecific IgG1.
abstract: The generation of bispecific antibodies (bsAbs) with natural IgG architecture in a practical and efficient manner has been a longstanding challenge. Here we describe controlled Fab-arm exchange (cFAE), which is an easy-to-use method to generate bispecific IgG1 (bsIgG1). The protocol involves the following: (i) separate expression of two parental IgG1s containing single matching point mutations in the CH3 domain; (ii) mixing of parental IgG1s under permissive redox conditions in vitro to enable recombination of half-molecules; (iii) removal of the reductant to allow reoxidation of interchain disulfide bonds; and (iv) analysis of exchange efficiency and final product using chromatography-based or mass spectrometry (MS)-based methods. The protocol generates bsAbs with regular IgG architecture, characteristics and quality attributes both at bench scale (micrograms to milligrams) and at a mini-bioreactor scale (milligrams to grams) that is designed to model large-scale manufacturing (kilograms). Starting from good-quality purified proteins, exchange efficiencies of ≥95% can routinely be obtained within 2-3 d (including quality control).
authors.affiliation: Genmab, Utrecht, the Netherlands.
authors.forename: Aran F
authors.initials: AF
authors.lastname: Labrijn
authors.affiliation: Genmab, Utrecht, the Netherlands.
authors.forename: Joyce I
authors.initials: JI
authors.lastname: Meesters
authors.affiliation: Genmab, Utrecht, the Netherlands.
authors.forename: Patrick
authors.initials: P
authors.lastname: Priem
authors.affiliation: Genmab, Utrecht, the Netherlands.
authors.forename: Rob N
authors.identifier: 0000000293006633
authors.initials: RN
authors.lastname: de Jong
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, Utrecht, the Netherlands.
authors.forename: Arnout F
authors.initials: AF
authors.lastname: Gerritsen
authors.affiliation: Genmab, Utrecht, the Netherlands.
authors.forename: Janine
authors.identifier: 0000000297389926
authors.initials: J
authors.lastname: Schuurman
authors.affiliation: Genmab, Utrecht, the Netherlands.
authors.forename: Paul W H I
authors.initials: PW
authors.lastname: Parren
mesh_terms: D018033:Antibodies, Bispecific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019149:Bioreactors; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D010084:Oxidation-Reduction; D017354:Point Mutation; D015202:Protein Engineering / Q000295:instrumentation / Q000379:methods*; D011786:Quality Control
chemical_list: D018033:Antibodies, Bispecific; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G