title: Expression of difficult-to-remove host cell protein impurities during extended Chinese hamster ovary cell culture and their impact on continuous bioprocessing.
abstract: During biopharmaceutical manufacturing, Chinese hamster ovary (CHO) cells produce hundreds of extracellular host cell protein (HCP) impurities, which must be removed from the therapeutic product by downstream purification operations to ensure patient safety. A subset of 118 of these HCPs have been reported as exceptionally difficult to remove during downstream purification because they co-purify due to retention characteristics on chromatographic media and/or product-association through strongly attractive interactions to the therapeutic protein. As the biopharmaceutical industry moves towards continuous bioprocessing, it is important to consider the impact of extended culture of CHO cells on the expression of extracellular HCP impurities, especially those HCPs known to challenge downstream purification. Two complementary proteomic techniques, two-dimensional electrophoresis (2DE) and shotgun, were applied to detect variations in the extracellular CHO HCP profile over 500 days of culture. In total, 92 HCPs exhibited up to 48-fold changes in expression, with 34 of these HCPs previously reported as difficult to purify. Each proteomic technique detected differential expression by a distinct set of HCPs, with 10 proteins exhibiting significant variable expression by both methods. This study presents the impact of cell age on the extracellular CHO HCP impurity profile and identifies HCPs with variable expression levels, which warrant further investigation to facilitate their clearance in downstream purification.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, 19716, Delaware; Delaware Biotechnology Institute, University of Delaware, Newark, 19711, Delaware.
authors.forename: Kristin N
authors.initials: KN
authors.lastname: Valente
authors.forename: Abraham M
authors.initials: AM
authors.lastname: Lenhoff
authors.forename: Kelvin H
authors.initials: KH
authors.lastname: Lee
mesh_terms: D000818:Animals; D001688:Biological Products / Q000302:isolation & purification* / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods; D016466:CHO Cells / Q000378:metabolism*; D018929:Cell Culture Techniques; D003412:Cricetulus; D004340:Drug Contamination; D005260:Female; D015870:Gene Expression; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D013678:Technology, Pharmaceutical / Q000379:methods
chemical_list: D001688:Biological Products; D020543:Proteome; D011994:Recombinant Proteins
keywords: Chinese hamster ovary (CHO) cells; biotechnology; continuous bioprocessing; extended cell culture; host cell proteins; proteomics