title: Capillary size exclusion chromatography with picogram sensitivity for analysis of monoclonal antibodies purified from harvested cell culture fluid.
abstract: Size exclusion chromatography (SEC) is widely used in the characterization and quality control of therapeutic proteins to detect aggregates. Aggregation is a carefully monitored quality attribute from the earliest stages of clinical development owing to the possibility of eliciting an immunogenic response in the patient. During early stage molecule assessment for cell culture production, small-scale screening experiments are performed to permit rapid turn-around of results so as to not delay timelines. We report the development of a capillary SEC methodology for preliminary molecule assessment to support the evaluation of therapeutic candidates at an early stage of development. By making several key modifications to a commercially available liquid chromatography system, we demonstrate a platform process to perform capillary SEC with excellent precision, picogram sensitivity and good ruggedness. The limit of quantitation was determined to be approximately 15 pg; picogram sensitivity for SEC analysis of monoclonal antibodies had not been achieved prior to this work. In addition, we have developed a method to capture low levels of antibody (1 μg/mL) from harvested cell culture fluid (HCCF) for capillary SEC analysis. Up to 40% recovery efficiency was achieved using this micro-recovery method on 3 mL HCCF samples. Using early stage cell culture transient transfection samples, which typically have much lower titers than stable cell line samples, we demonstrate a consistent method for analyzing aggregates in low protein concentration HCCF samples for molecule assessment and early stage candidate screening.
authors.affiliation: Protein Analytical Chemistry, Genentech, Inc., South San Francisco, CA 94080, USA.
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Rea
authors.forename: G Tony
authors.initials: GT
authors.lastname: Moreno
authors.forename: Lisa
authors.initials: L
authors.lastname: Vampola
authors.forename: Yun
authors.initials: Y
authors.lastname: Lou
authors.forename: Bjorn
authors.initials: B
authors.lastname: van Haan
authors.forename: Guillaume
authors.initials: G
authors.lastname: Tremintin
authors.forename: Laura
authors.initials: L
authors.lastname: Simmons
authors.forename: Adrian
authors.initials: A
authors.lastname: Nava
authors.forename: Yajun Jennifer
authors.initials: YJ
authors.lastname: Wang
authors.forename: Dell
authors.initials: D
authors.lastname: Farnan
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000302:isolation & purification; D016466:CHO Cells; D002850:Chromatography, Gel / Q000295:instrumentation / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D057230:Limit of Detection; D015203:Reproducibility of Results
chemical_list: D000911:Antibodies, Monoclonal