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Title: Proteomics Based process and cell line development applied to a mammalian therapeutic enzyme
Author: Migani, D.
ISNI:       0000 0004 7225 3066
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2017
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Recombinant human Acid Alpha Glucosidase (GAA) is the therapeutic enzyme used for the treatment of Pompe disease, a rare genetic disorder characterised by GAA deficiency in the cell lysosomes. The manufacturing process for GAA can be challenging, in part due to protease degradation. The overall goal of this project 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 clonal cell line and then developed a two-step bioprocess based on capture chromatographic step anion exchange (IEX) and intermediate hydrophobic interaction (HIC). The purity of GAA after HIC was determined via LC/MSMS to be above 80%. 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.
Supervisor: Bracewell, D. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available