Chinese hamster ovary (CHO) cell lines are frequently used as hosts for the production of recombinant therapeutics, such as monoclonal antibodies (MAbs), due to their ability to perform correct post-translational modifications. A major issue for use of CHO cells lines for the production of recombinant proteins is the selection of cell lines that do not retain stable protein expression during long-term culture (LTC). Instability of expression impairs process yields, effective usage of time and money, and regulatory approval. Protein production is complex and is influenced by cell growth, transcription, translation, protein folding and post-translational processing and secretory events, which may interact to determine stability of expression during prolonged culture. This thesis aims to identify features associated with stability/instability of recombinant protein expression and methods to improve protein production, with the addition of chemically defined (CD) feed and chemicals.Two exemplar CHO cell lines, which secrete the same recombinant antibody were characterised in response to LTC, feed and DMSO addition. Both cell lines (3.90 and 51.69) exhibited unstable protein production over LTC, with a loss in final antibody titres and specific productivity (Qp). The instability observed within the exemplar cell lines was not due to decreased recombinant gene copy numbers or mRNA expression but was associated with lower viable cell densities, increased ER stress (GADD153 and spliced XBP-1 [XBP-1(s)]) and enhanced rates of lactate utilisation (observed during the decline phase of batch culture). Improvement of recombinant protein expression in response to feed or DMSO addition was associated with lower expression of ER stress markers (ATF4, XBP-1(s) and GADD153 at mRNA level and GADD153 at protein level) and alterations to the metabolic activity of the cultures (prevention of alanine and lactate re-utilisation, and greater glucose utilisation between the stationary and decline phase of batch culture).Although feed or DMSO addition improved recombinant protein production, these additions did not reverse the appearance or progression of instability for cells after LTC. ER stress expression was not abolished as a consequence of feed or DMSO addition. Expression of stress markers at earlier time points may be the factor that limits antibody production and secretion. The consequences of the presence of feed and DMSO addition on ER stress markers and antibody production serves to highlight approaches that may be utilised for engineering more productive or stable protein production phenotypes in parental cell lines.
|Date of Award||1 Aug 2011|
- The University of Manchester
|Supervisor||Alan Dickson (Supervisor)|
- Chinese hamster ovary cells
- monoclonal antibodies