title: Development of at-line assay to monitor charge variants of MAbs during production.
abstract: One major challenge currently facing the biopharmaceutical industry is to understand how MAb microheterogeneity affects therapeutic efficacy, potency, immunogenicity, and clearance. MAb micro-heterogeneity can result from post-translational modifications such as sialylation, galactosylation, C-terminal lysine cleavage, glycine amidation, and tryptophan oxidation, each of which can generate MAb charge variants; such heterogeneity can affect pharmacokinetics (PK) considerably. Implementation of appropriate on-line quality control strategies may help to regulate bioprocesses, thus enabling more homogenous material with desired post-translational modifications and PK behavior. However, one major restriction to implementation of quality control strategies is the availability of techniques for obtaining on-line or at-line measurements of these attributes. In this work, we describe the development of an at-line assay to separate MAb charge variants in near real-time, which could ultimately be used to implement on-line quality control strategies for MAb production. The assay consists of a 2D-HPLC method with sequential in-line Protein A and WCX-10 HPLC column steps. To perform the 2D-HPLC assay at-line, the two columns steps were integrated into a single method using a novel system configuration that allowed parallel flow over column 1 or column 2 or sequential flow from column 1 to column 2. A bioreactor system was also developed such that media samples could be removed automatically from bioreactor vessels during production and delivered to the 2D-HPLC for analysis. With this at-line HPLC assay, we have demonstrated that MAb microheterogeneity occurs throughout the cell cycle whether the host cell line is grown under different or the same nominal culture conditions.
authors.affiliation: Dept. of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, 19716.
authors.forename: M M
authors.initials: MM
authors.lastname: St Amand
authors.forename: B A
authors.initials: BA
authors.lastname: Ogunnaike
authors.forename: A S
authors.initials: AS
authors.lastname: Robinson
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D019149:Bioreactors; D001709:Biotechnology / Q000295:instrumentation / Q000379:methods; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D011994:Recombinant Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins
keywords: HPLC; at-line monitoring; bioreactor; charge variants; monoclonal antibody