Chinese hamster ovary (CHO) cells are frequently used for the commercial expression of recombinant therapeutic antibodies due to their ability to perform appropriate post-translational modifications and therefore generate an accurate rendition of natural products. As a consequence of their initial derivation by mutagenesis and the divergence into distinct cell lines, clonally-derived cell lines are phenotypically distinct. Molecular understanding of the features that determine the properties of a CHO clone is fundamental to the optimisation of cell environment and, potentially, engineering or selection of CHO clones with the "best" phenotype.The profile of the endoplasmic reticulum (ER) environment (with a specific complement of chaperones, co-chaperones, and sensors) is important for cell growth and maximum recombinant protein secretion. I have addressed how the modulation of two components in the ER, XBP1(s) and ERO1L alpha, influence CHO cell function. XBP1(s) is generated by a novel mRNA splicing mechanism in response to ER stress and is the key regulator factor for the development of professional secretory cells. ERO1L alpha plays a critical role in setting the redox state of foldases (such as PDI) and is also known to be induced by ER stress. In this study, CHO S cells were doubly transfected with human XBP1(s) and human ERO1L alpha constructs to generate a series of CHO cell lines that overexpressed each gene. The engineered cell lines exhibited a series of improvements in terms of desirable phenotypes compared to the non-engineered CHO S cell line. These improvements included up-regulation of chaperone expression, alteration in growth profile and associated glucose consumption and lactate production, increased antibody titres, and improved recovery from an oxidative stress. My interpretation is that engineering cells to over-express XBP1(s) and ERO1L alpha generated a more favourable phenotype for cells to handle the stresses that result from protein transit in the ER. Whether this is a direct effect of XBP1(s) and ERO1L alpha or due to a secondary consequence of their over-expression on the ER chaperone complement remains unclear. However, this study identified important combinations of regulatory factors that influence ER function and, consequently, the ability to define improved CHO cell phenotypes for expression of different types of protein products.
|Date of Award||1 Aug 2013|
- The University of Manchester
|Supervisor||Alan Dickson (Supervisor) & Raymond Boot-Handford (Supervisor)|