title: Characterization of monoclonal antibody size variants containing extra light chains.
abstract: Size exclusion chromatography (SEC) is the most commonly used method to separate and quantify monoclonal antibody (mAb) size variants. MAb-A is an IgG1 subtype humanized monoclonal antibody recombinantly produced in Chinese hamster ovary (CHO) cells. SEC analysis of MAb-A resolved a peak, named Peak 1, which elutes between monomer and dimer peaks. MAb-A lots produced from different clones and production scales all have 0.2-0.3% of SEC Peak 1. Electron spray ionization--time of flight mass spectrometry (ESI-TOF MS), microfluidics capillary electrophoresis and sodium dodecyl sulfate-PAGE (SDS PAGE) results demonstrated that SEC Peak 1 contains two structural variants: MAb-A with one extra light chain (2H3L) and MAb-A with two extra light chains (2H4L). The C-terminal Cys of the extra light chain in Peak 1 variants is either a free thiol, capped by glutathione, cysteine, or another light chain. Both electrophoresis and LC/MS analyses of non-reduced and reduced samples suggested that the extra light chains are linked to the MAb-A light chain through disulfide bonds. Isolated SEC Peak 1 fraction had a potency of 50% relative to MAb-A reference material. The 50% potency loss may result from the reduced accessibility to the antigen-binding site caused by the extra light chain(s)' steric hindrance.
authors.affiliation: Protein Analytical Chemistry, Genentech Inc., South San Francisco, CA, USA. lu.xiuzhen@gene.com
authors.forename: Connie
authors.initials: C
authors.lastname: Lu
authors.forename: Dandan
authors.initials: D
authors.lastname: Liu
authors.forename: Hongbin
authors.initials: H
authors.lastname: Liu
authors.forename: Paul
authors.initials: P
authors.lastname: Motchnik
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016466:CHO Cells; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006224:Cricetinae; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D010449:Peptide Mapping / Q000379:methods
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007147:Immunoglobulin Light Chains