Improving protein yield from mammalian cells by manipulation of stress response pathways

Chalmers, Fiona (2016) Improving protein yield from mammalian cells by manipulation of stress response pathways. PhD thesis, University of Glasgow.

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Abstract

Monoclonal antibodies are a class of therapeutic that is an expanding area of the
lucrative biopharmaceutical industry. These complex proteins are predominantly
produced from large cultures of mammalian cells; the industry standard cell line being
Chinese Hamster Ovary (CHO) cells. A number of optimisation strategies have led to
antibody titres from CHO cells increasing by a hundred-fold, and it has been proposed
that a further bottleneck in biosynthesis is in protein folding and assembly within the
secretory pathway. To alleviate this bottleneck, a CHO-derived host cell line was
generated by researchers at the pharmaceutical company UCB that stably overexpressed
two critical genes: XBP1, a transcription factor capable of expanding the endoplasmic
reticulum and upregulating protein chaperones; and Ero1α, an oxidase that replenishes
the machinery of disulphide bond formation. This host cell line, named CHO-S XE, was
confirmed to have a high yield of secreted antibody.
The work presented in this thesis further characterises CHO-S XE, with the aim of using
the information gained to lead the generation of novel host cell lines with more optimal
characteristics than CHO-S XE. In addition to antibodies, it was found that CHO-S XE
had improved production of two other secreted proteins: one with a simple tertiary
structure and one complex multi-domain protein; and higher levels of a number of
endogenous protein chaperones. As a more controlled system of gene expression to
unravel the specific roles of XBP1 and Ero1α in the secretory properties of CHO-S XE,
CHO cells with inducible overexpression of XBP1, Ero1α, or a third gene involved in the
Unfolded Protein Response, GADD34, were generated. From these cell lines, it was
shown that more antibody was secreted by cells with induced overexpression of XBP1;
however, Ero1α and GADD34 overexpression did not improve antibody yield. Further
investigation revealed that endogenous XBP1 splicing was downregulated in the
presence of an abundance of the active form of XBP1. This result indicated a novel
aspect of the regulation of the activity of IRE1, the stress-induced endoribonuclease
responsible for XBP1 splicing. Overall, the work described in this thesis confirms that
the overexpression of XBP1 has an enhancing effect on the secretory properties of CHO
cells; information which could contribute to the development of host cells with a
greater capacity for antibody production.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Antibodies, bioprocessing, unfolded protein response, protein folding, ER biochemistry, Chinese Hamster Ovary cells.
Subjects: Q Science > QR Microbiology
Q Science > QR Microbiology > QR180 Immunology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Funder's Name: Biotechnology and Biological Sciences Research Council (BBSRC)
Supervisor's Name: Bulleid, Prof. Neil and Cain, Dr. Katharine
Date of Award: 2016
Depositing User: Ms Fiona Chalmers
Unique ID: glathesis:2016-7666
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 20 Oct 2016 08:30
Last Modified: 09 Nov 2016 08:14
URI: http://theses.gla.ac.uk/id/eprint/7666

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