Process development and characterisation of transient expression technology in CHO cells

Giuseppe Codamo (2010). Process development and characterisation of transient expression technology in CHO cells PhD Thesis, Australian Institute for Bioengineering & Nanotechnology, The University of Queensland.

       
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Author Giuseppe Codamo
Thesis Title Process development and characterisation of transient expression technology in CHO cells
School, Centre or Institute Australian Institute for Bioengineering & Nanotechnology
Institution The University of Queensland
Publication date 2010-12
Thesis type PhD Thesis
Supervisor Professor Peter Gray
Dr Trent Munro
Total pages 227
Total colour pages 19
Total black and white pages 2
Subjects 10 Technology
Abstract/Summary Recombinant protein-based biologics are a rapidly growing segment of the pharmaceutical marketplace. Transient gene expression (TGE) systems in mammalian cells provide a rapid way to produce significant amounts of recombinant protein biologics for pre-clinical assessment. Human embryonic kidney (HEK293) cells have traditionally been used for TGE mainly due to the high transfection efficiencies observed with the cell line which have delivered high yields of recombinant protein. There is now demand from industry for efficient, high-producing TGE systems that utilise Chinese hamster ovary (CHO) cells as they are the predominant host for clinical manufacturing. To facilitate improved recombinant protein yields with CHO cell-based TGE, the Epi-CHO transient expression system was developed (Kunaparaju et al, 2005). This system enables elevated and prolonged recombinant protein expression through the utilisation of elements from the Polyoma (PyV) and Epstein-Barr (EBV) viruses to promote the replication and maintenance of plasmid DNA in transfected CHO cells. While the Epi-CHO system as described has been successful in the transient production of recombinant proteins, further understanding of key aspects of the system and the need to drive further improvements in titre are essential in realising the maximum potential of the system. This project aimed to improve the industrial relevance of the Epi-CHO system through a combination of strategies. Elevated transgene mRNA and protein levels of two monoclonal antibodies (mAbs) and a fluorescent protein is demonstrated with the Epi-CHO system compared to cell lines deficient in plasmid replication and/or retention. Secondly, through the examination of variables critical to the success of a TGE process, novel TGE platforms for the Epi-CHO system in both serum-free and chemically defined (CD) media were established. Parameters that were examined, optimised and incorporated included the transfection reagent, the conditions for the preparation of transfection complexes, the determination of the optimal heavy chain and light chain plasmid DNA ratios for transient mAb expression and the incorporation of mild hypothermia. Through the implementation of these optimised parameters, mAb titres of 140 mg/L were obtained in serum-free conditions, representing a 64% increase over the previously highest reported mAb titre in CHO cells (90 mg/L). A 5-fold improvement in mAb volumetric productivity was also achieved in CD media with the new platform (80 mg/L) compared to titres achieved prior to optimisation of the process (15 mg/L). Furthermore, optimising growth conditions with the Epi-CHO system facilitated a 6-fold expansion in culture volume post-transfection, generating 400% more mAb/μg of plasmid DNA when compared to a non-episomal system. Finally, studies evaluating plasmid size, histone deacetylase inhibitor supplementation and the overexpression of intracellular factors such as human EBNA-1 binding protein 2 (EBP2) and X-box binding protein 1 (XBP-1) were performed to augment plasmid maintenance, transgene mRNA levels and protein secretion in transfected cells. Co-expression of XBP-1 provided for a 37% improvement in recombinant mAb titre under mild hypothermic conditions. The results presented in this thesis highlight the capacity for the Epi-CHO system to be employed for the rapid and cost-efficient production of candidate biologics during pre-clinical development.
Keyword Biologic
CHO
Episomal
Mammalian Cell Culture
Mild Hypothermia
Monoclonal antibody
Transfection
Transient Gene Expression
Additional Notes Colour pages: 22,46,47,52,67,69,72,73,74,81,88,102,105,107,114,118,119,181, 202 Landscape pages: 44,45

 
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Created: Mon, 27 Jun 2011, 01:47:00 EST by Mr Giuseppe Codamo on behalf of Library - Information Access Service