Format

Send to

Choose Destination
J Chromatogr A. 2016 Aug 26;1461:70-7. doi: 10.1016/j.chroma.2016.07.055. Epub 2016 Jul 21.

Residual on column host cell protein analysis during lifetime studies of protein A chromatography.

Author information

1
Department of Biochemical Engineering, University College London, Bernard Katz Building, Gordon Street, London WC1H 0AH, United Kingdom.
2
Department of Chemical Engineering, IIT Delhi, Hauz Khas, New Delhi, Delhi 110016, India.
3
Centre for Industrial Biotechnology, School of Biosciences, University of Kent, Canterbury, CT2 7NJ, United Kingdom.
4
Department of Biochemical Engineering, University College London, Bernard Katz Building, Gordon Street, London WC1H 0AH, United Kingdom. Electronic address: d.bracewell@ucl.ac.uk.

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.

KEYWORDS:

Bioprocessing; Host cell proteins; LC–MS/MS; Process-related impurities; Protein A affinity chromatography; Resin ageing

PMID:
27473513
DOI:
10.1016/j.chroma.2016.07.055
[Indexed for MEDLINE]
Free full text

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center