title: Residual on column host cell protein analysis during lifetime studies of protein A chromatography.
abstract: Capacity reduction in protein A affinity chromatography with extended cycling during therapeutic antibody manufacture is well documented. Identification of which residual proteins remain from previous cycles during the lifetime of these adsorbent materials is required to understand their role in this ageing process, but represents a significant metrological challenge. Scanning electron microscopy (SEM) and liquid chromatography mass spectrometry (LC-MS/MS) are combined to detect and map this phenomenon of protein carry-over. We show that there is a morphological change at the surface of the agarose resin, revealing deposits on the polymer fibres increasing with cycle number. The amount of residual host cell proteins (HCPs) by LC-MS/MS present on the resin is shown to increase 10-fold between 50 and 100 cycles. During this same period the functional class of the predominant HCPs associated with the resin increased in diversity, with number of proteins identified increasing 5-fold. This ageing is observed in the context of the product quality of the eluate HCP and protein A leachate concentration remaining constant with cycle number.
authors.affiliation: Department of Biochemical Engineering, University College London, Bernard Katz Building, Gordon Street, London WC1H 0AH, United Kingdom.
authors.forename: Katherine
authors.initials: K
authors.lastname: Lintern
authors.affiliation: Department of Chemical Engineering, IIT Delhi, Hauz Khas, New Delhi, Delhi 110016, India.
authors.forename: Mili
authors.initials: M
authors.lastname: Pathak
authors.affiliation: Centre for Industrial Biotechnology, School of Biosciences, University of Kent, Canterbury, CT2 7NJ, United Kingdom.
authors.forename: C Mark
authors.initials: CM
authors.lastname: Smales
authors.affiliation: Centre for Industrial Biotechnology, School of Biosciences, University of Kent, Canterbury, CT2 7NJ, United Kingdom.
authors.forename: Kevin
authors.initials: K
authors.lastname: Howland
authors.affiliation: Department of Chemical Engineering, IIT Delhi, Hauz Khas, New Delhi, Delhi 110016, India.
authors.forename: Anurag
authors.initials: A
authors.lastname: Rathore
authors.affiliation: Department of Biochemical Engineering, University College London, Bernard Katz Building, Gordon Street, London WC1H 0AH, United Kingdom. Electronic address: d.bracewell@ucl.ac.uk.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Bracewell
mesh_terms: D000818:Animals; D016466:CHO Cells; D002853:Chromatography, Liquid / Q000379:methods; D003412:Cricetulus; D011506:Proteins / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013205:Staphylococcal Protein A / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D011506:Proteins; D013205:Staphylococcal Protein A
keywords: Bioprocessing; Host cell proteins; LC–MS/MS; Process-related impurities; Protein A affinity chromatography; Resin ageing