title: Analysis and purification of IgG4 bispecific antibodies by a mixed-mode chromatography.
abstract: Therapeutic non-hinge-modified IgG4 molecules form bispecific hybrid antibodies with endogenous human IgG4 molecules via a process known as Fab-arm exchange (or called half molecule exchange). Analysis of the bispecific hybrids is critical for studies of half molecule exchange. A number of analytical methods are available to detect IgG4 hybrids. These methods mostly necessitate labeling or alteration of the model IgG4 molecules, or rely on time-consuming immunoassays and mass spectrometry. In addition, these methods do not allow isolation of hybrid antibodies. We report here the only analytical method to date that relies on chromatographic separation for detection of hybrids formed from intact antibodies in their native forms using pembrolizumab as an example. This method employs a mixed-mode chromatography using a Sepax Zenix SEC-300 column to separate a bispecific hybrid from the parental antibodies. The simultaneous quantitative monitoring of the newly formed hybrid and parental antibodies was achieved by UV absorption and/or protein fluorescence. The bispecific hybrid antibodies were purified with the same method for further biochemical characterization. The method has allowed monitoring of half molecule exchange between a human serum IgG4 and a tested IgG4 molecule, and has been implemented for the analysis of in vitro as well as in vivo samples.
authors.affiliation: Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ 07033, USA. Electronic address: xiaoyu.yang@merck.com.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Yang
authors.affiliation: Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ 07033, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ 07033, USA.
authors.forename: Fengqiang
authors.initials: F
authors.lastname: Wang
authors.affiliation: Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ 07033, USA.
authors.forename: Larry Jin
authors.initials: LJ
authors.lastname: Wang
authors.affiliation: Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ 07033, USA.
authors.forename: Daisy
authors.initials: D
authors.lastname: Richardson
authors.affiliation: Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ 07033, USA.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Shameem
authors.affiliation: Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ 07033, USA. Electronic address: alexandre.ambrogelly@gilead.com.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Ambrogelly
mesh_terms: D018033:Antibodies, Bispecific / Q000276:immunology* / Q000302:isolation & purification*; D002845:Chromatography / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D007700:Kinetics
chemical_list: D018033:Antibodies, Bispecific; D007074:Immunoglobulin G
keywords: Bispecific antibodies; Fab-arm exchange; Half molecule exchange; IgG4; Mixed-mode chromatography