Impact of feed addition on UPR-related gene expression in CHO

Abstract

Biopharmaceuticals are used therapeutically in a variety of human and animal conditions. Expression systems, such as Chinese Hamster Ovary (CHO) cell lines, are used in the production of recombinant proteins. Since the approval of the first FDA-approved therapeutic recombinant protein, tissue plasminogen activator (tPA), CHO cell lines have been the predominant expression systems used in the biopharmaceutical industry. There is a continual endeavour to optimise CHO expression systems to increase product yield and quality, through molecular and bioprocess engineering strategies. The aim of this report was to contribute to a better understanding of the molecular biology of the recombinant IgG1 CHO-S cell line for the improvement of manufacture capacity. To achieve this, the first objective was to study growth and productivity of the CHO-S cell line in batch and fed-batch culture, to assess the impact of feed addition on those parameters. Feed addition resulted in a 48hr extension in culture duration, an almost two-fold increase in peak cell density and an over three-fold increase in IgG1 titre. The specific productivity was not altered between the two culture methods. The second objective was to develop methods to assess the expression of proteins involved in the unfolded protein response (UPR). The UPR has been implicated in secretory bottlenecks in CHO expression systems. This resulted in the development of primers specific to BiP, GRP94, Xbp1s and CHOP. The third objective was to examine the mRNA expression of these UPR-relevant proteins in batch and fed-batch culture, which was done using RT-PCR, qRT-PCR techniques. This confirmed that the proteins were expressed in the IgG1 CHO-S line. In batch culture there was no significant difference in gene expression but in fed-batch culture CHOP was significantly up-regulated later in culture. Between days 4, 6 and 8 of batch and fed-batch the expression of Xbp1 was significantly different between the culture method. BiP and Xbp1 expression was investigated using western blot analysis. The relationship between the expression of the UPR-relevant proteins, the specific productivity of the cell line and the overall IgG1 yield in response to BC and FBC conditions was assessed in the final objective of the report, but proved in conclusive due to the lack of change in specific productivity between the two cultures. Further work to assess the impact of feed on CHO expression systems, factors that influence the extent of UPR activation, as well as factors that influence the specific productivity of cell lines is proposed.

Date of Award	1 Aug 2024
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Alan Dickson (Supervisor)