title: Charge heterogeneity: Basic antibody charge variants with increased binding to Fc receptors.
abstract: We identified active isoforms of the chimeric anti-GD2 antibody, ch14.18, a recombinant antibody produced in Chinese hamster ovary cells, which is already used in clinical trials.1,2,3We separated the antibody by high resolution ion-exchange chromatography with linear pH gradient elution into acidic, main and basic charge variants on a preparative scale yielding enough material for an in-depth study of the sources and the effects of microheterogeneity. The binding affinity of the charge variants toward the antigen and various cell surface receptors was studied by Biacore. Effector functions were evaluated using cellular assays for antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. Basic charge variants showed increased binding to cell surface receptor FcγRIIIa, which plays a major role in regulating effector functions. Furthermore, increased binding of the basic fractions to the neonatal receptor was observed. As this receptor mediates the prolonged half-life of IgG in human serum, this data may well hint at an increased serum half-life of these basic variants compared to their more acidic counterparts. Different glycoform patterns, C-terminal lysine clipping and N-terminal pyroglutamate formation were identified as the main structural sources for the observed isoform pattern. Potential differences in structural stability between individual charge variant fractions by nano differential scanning calorimetry could not been detected. Our in-vitro data suggests that the connection between microheterogeneity and the biological activity of recombinant antibody therapeutics deserves more attention than commonly accepted.
authors: a Department of Biotechnology , University of Natural Resources and Life Sciences, Vienna , Vienna , Austria.
authors: Beate
authors: B
authors: Hintersteiner
authors: a Department of Biotechnology , University of Natural Resources and Life Sciences, Vienna , Vienna , Austria.
authors: Nico
authors: N
authors: Lingg
authors: b Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR) , Centros , Singapore.
authors: Peiqing
authors: P
authors: Zhang
authors: b Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR) , Centros , Singapore.
authors: Susanto
authors: S
authors: Woen
authors: b Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR) , Centros , Singapore.
authors: Kong Meng
authors: KM
authors: Hoi
authors: c Apeiron Biologics AG, Campus-Vienna-Biocenter , Vienna , Austria.
authors: Stefan
authors: S
authors: Stranner
authors: c Apeiron Biologics AG, Campus-Vienna-Biocenter , Vienna , Austria.
authors: Susanne
authors: S
authors: Wiederkum
authors: c Apeiron Biologics AG, Campus-Vienna-Biocenter , Vienna , Austria.
authors: Oliver
authors: O
authors: Mutschlechner
authors: c Apeiron Biologics AG, Campus-Vienna-Biocenter , Vienna , Austria.
authors: Manfred
authors: M
authors: Schuster
authors: c Apeiron Biologics AG, Campus-Vienna-Biocenter , Vienna , Austria.
authors: Hans
authors: H
authors: Loibner
authors: a Department of Biotechnology , University of Natural Resources and Life Sciences, Vienna , Vienna , Austria.
authors: Alois
authors: A
authors: Jungbauer
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism*; D000915:Antibody Affinity / Q000276:immunology; D000920:Antibody-Dependent Cell Cytotoxicity / Q000276:immunology; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D020033:Protein Isoforms / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D055550:Protein Stability; D017452:Receptors, IgG / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; C496124:FCGR3A protein, human; D020033:Protein Isoforms; D017452:Receptors, IgG; D011994:Recombinant Proteins; C112746:dinutuximab
keywords: Biosimilar; Microheterogeneity; glycoforms; immunoglobulin; isoforms; linear pH gradient; monoclonal antibody