title: Effects of lysosomal biotherapeutic recombinant protein expression on cell stress and protease and general host cell protein release in Chinese hamster ovary cells.
abstract: Recombinant human Acid Alpha Glucosidase (GAA) is the therapeutic enzyme used for the treatment of Pompe disease, a rare genetic disorder characterized by GAA deficiency in the cell lysosomes (Raben et al., Curr Mol Med. 2002; 2:145-166). The manufacturing process for GAA can be challenging, in part due to protease degradation. The overall goal of this study was to understand the effects of GAA overexpression on cell lysosomal phenotype and host cell protein (HCP) release, and any resultant consequences for protease levels and ease of manufacture. To do this we first generated a human recombinant GAA producing stable CHO cell line and designed the capture chromatographic step anion exchange (IEX). We then collected images of cell lysosomes via transmission electron microscopy (TEM) and compared the resulting data with that from a null CHO cell line. TEM imaging revealed 72% of all lysosomes in the GAA cell line were engorged indicating extensive cell stress; by comparison only 8% of lysosomes in the null CHO had a similar phenotype. Furthermore, comparison of the HCP profile among cell lines (GAA, mAb, and Null) capture eluates, showed that while most HCPs released were common across them, some were unique to the GAA producer, implying that cell stress caused by overexpression of GAA has a molecule specific effect on HCP release. Protease analysis via zymograms showed an overall reduction in proteolytic activity after the capture step but also revealed the presence of co-eluting proteases at approximately 80 KDa, which MS analysis putatively identified as dipeptidyl peptidase 3 and prolyl endopeptidase. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:666-676, 2017.
authors.affiliation: The Advanced Centre of Biochemical Engineering, Dept. of Biochemical Engineering, University College London, Bernard Katz Building, London, WC1E 6BT, United Kingdom.
authors.forename: Damiano
authors.identifier: http://orcid.org/0000-0001-6601-3443
authors.initials: D
authors.lastname: Migani
authors.affiliation: Industrial Biotechnology Centre and School of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, United Kingdom.
authors.forename: C Mark
authors.initials: CM
authors.lastname: Smales
authors.affiliation: The Advanced Centre of Biochemical Engineering, Dept. of Biochemical Engineering, University College London, Bernard Katz Building, London, WC1E 6BT, United Kingdom.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Bracewell
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D008247:Lysosomes / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000520:alpha-Glucosidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D000520:alpha-Glucosidases; D010447:Peptide Hydrolases
keywords: Chinese hamster ovary cells; acid alpha glucosidase; biotherapeutic recombinant protein overexpression; downstream purification; host cell protein; proteases; proteomics