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

QR Microbiology;QR180 Immunology

Chalmers, Fiona ; Biotechnology and Biological Sciences Research Council (BBSRC) ; Bulleid, Neil

University:University of Glasgow

Department:Institute of Molecular Cell and Systems Biology

关键词: Antibodies, bioprocessing, unfolded protein response, protein folding, ER biochemistry, Chinese Hamster Ovary cells.;   

Others  :  http://theses.gla.ac.uk/7666/7/2016ChalmersPhD.pdf

来源: University of Glasgow

【 摘 要 】

Monoclonal antibodies are a class of therapeutic that is an expanding area of thelucrative biopharmaceutical industry. These complex proteins are predominantlyproduced from large cultures of mammalian cells; the industry standard cell line beingChinese Hamster Ovary (CHO) cells. A number of optimisation strategies have led toantibody titres from CHO cells increasing by a hundred-fold, and it has been proposedthat a further bottleneck in biosynthesis is in protein folding and assembly within thesecretory pathway. To alleviate this bottleneck, a CHO-derived host cell line wasgenerated by researchers at the pharmaceutical company UCB that stably overexpressedtwo critical genes: XBP1, a transcription factor capable of expanding the endoplasmicreticulum and upregulating protein chaperones; and Ero1α, an oxidase that replenishesthe machinery of disulphide bond formation. This host cell line, named CHO-S XE, wasconfirmed to have a high yield of secreted antibody.The work presented in this thesis further characterises CHO-S XE, with the aim of usingthe information gained to lead the generation of novel host cell lines with more optimalcharacteristics than CHO-S XE. In addition to antibodies, it was found that CHO-S XEhad improved production of two other secreted proteins: one with a simple tertiarystructure and one complex multi-domain protein; and higher levels of a number ofendogenous protein chaperones. As a more controlled system of gene expression tounravel 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 theUnfolded Protein Response, GADD34, were generated. From these cell lines, it wasshown that more antibody was secreted by cells with induced overexpression of XBP1;however, Ero1α and GADD34 overexpression did not improve antibody yield. Furtherinvestigation revealed that endogenous XBP1 splicing was downregulated in thepresence of an abundance of the active form of XBP1. This result indicated a novelaspect of the regulation of the activity of IRE1, the stress-induced endoribonucleaseresponsible for XBP1 splicing. Overall, the work described in this thesis confirms thatthe overexpression of XBP1 has an enhancing effect on the secretory properties of CHOcells; information which could contribute to the development of host cells with agreater capacity for antibody production.