The functional competence of animal cells in culture: the NSO cell proteome

Mammalian cells are routinely utilised in industry for the production of therapeutic proteins. However, the adaptations that enable enhanced cellular productivity are poorly understood and improvements to date have been largely achieved by empirical optimisation of the cell culture environment and the use of enhanced expression systems. We have utilised an optimised proteomic platform, specifically two-dimensional gel electrophoresis (2-D PAGE) to investigate the alterations in functional gene expression that enable murine myeloma NSO cells in culture to maintain high-level recombinant monoclonal antibody production. We have addressed the following fundamental questions; (1) when do cells in bioreactor culture perceive stress and how do associated changes in the pattern of protein synthesis during batch culture relate to secretory productivity and cell viability in vitrol and (2) are there conserved changes in gene expression which permit higher specific monoclonal antibody production (qMAb)? The protein complement of NSO cells harvested at lag, exponential and death phase of batch culture was separated by high-resolution large format 2-D PAGE. Nascent polypeptide synthesis was also concurrently assessed by labelling with [35S] methionine followed by 2- D PAGE. Analysis of the resultant digital images confirmed that although many proteins were constitutively expressed (and were therefore critical for the growth and survival of NSO cells throughout culture), approximately 50% of the total proteins detected exhibited a change in the level of protein and polypeptide expression during batch culture. Changes in the level of polypeptide synthesis preceded a change in the level of protein expression. Proteins of interest were identified by mass spectrometry in order to characterise proteins (or groups of proteins) exhibiting alterations in expression causally related to changes in cellular activity during culture. The identified proteins could be separated into three major groups of proteins; (1) chaperones, (2) glycolytic proteins and (3) structural proteins. Closer analysis identified conserved, ‘time specific’ and productivity related changes in protein expression. These results suggest that although it is difficult to pinpoint exactly when NSO cells in culture perceive stress it appears that NSO cells recognised stress early in exponential phase. However, changes in the pattern of protein expression could not be directly attributable to the stress response or rlgG accumulation even though changes in protein expression correlated with the production of rlgG by GS-NSO cells. n A series of NSO cell transfectants expressing differing levels of antibody B72.3 productivity were also prepared. The cell lines were sequentially weaned off serum containing media into 1% serum. A selection of the resulting cell lines were cultured under controlled conditions and extracted at mid-exponential phase for whole NSO cell proteome analysis. During 4 L bioreactor culture the cell lines lost productivity and were therefore used to investigate clonal variation between a series of essentially non-producing cell lines derived from the same parental NSO cell host. The results clearly show that many cell line specific changes in the global NSO cell proteome were apparent at exponential phase. This cell specific ‘bias’ implies that engineering strategies which involve the overexpression of one or two proteins will not unlock the full potential of engineering cells for desired functional phenotypes.