We have employed an inverse engineering strategy based on quantitative proteome analysis to identify changes in intracellular protein abundance that correlate with increased specific recombinant monoclonal antibody production (qMab) by engineered murine myeloma (NS0) cells. Four homogeneous NS0 cell lines differing in qMab were isolated from a pool of primary transfectants. The proteome of each stably transfected cell line was analyzed at mid-exponential growth phase by two-dimensional gel electrophoresis (2D-PAGE) and individual protein spot volume data derived from digitized gel images were compared statistically. To identify changes in protein abundance associated with qMab datasets were screened for proteins that exhibited either a linear correlation with cell line qMab or a conserved change in abundance specific only to the cell line with highest qMab. Several proteins with altered abundance were identified by mass spectrometry. Proteins exhibiting a significant increase in abundance with increasing qMab included molecular chaperones known to interact directly with nascent immunoglobulins during their folding and assembly (e.g., BiP, endoplasmin, protein disulfide isomerase). 2D-PAGE analysis showed that in all cell lines Mab light chain was more abundant than heavy chain, indicating that this is a likely prerequisite for efficient Mab production. In summary, these data reveal both the adaptive responses and molecular mechanisms enabling mammalian cells in culture to achieve high-level recombinant monoclonal antibody production.

We previously compared changes in individual protein abundance between the proteomes of GS-NS0 cell lines with varying rates of cell-specific recombinant monoclonal antibody production (qMab). Here we extend analyses of our proteomic dataset to statistically determine if particular cell lines have distinct functional capabilities that facilitate production of secreted recombinant Mab. We categorized 79 proteins identified by mass spectrometry according to their biological function or location in the cell and statistically compared the relative abundance of proteins in each category between GS-NS0 cell lines with varying qMab. We found that the relative abundance of proteins in ER chaperone, non-ER chaperone, cytoskeletal, cell signaling, metabolic, and mitochondrial categories were significantly increased with qMab. As the GS-NS0 cell line with highest qMab also had an increased intracellular abundance of unassembled Mab heavy chain (HC), we tested the hypothesis that the increased ER chaperone content was caused by induction of an unfolded protein response (UPR) signaling pathway. Immunoblot analyses revealed that spliced X-box binding protein 1 (XBP1), a marker for UPR induction, was not detectable in the GS-NS0 cells with elevated qMab, although it was induced by chemical inhibitors of protein folding. These data suggest that qMab is functionally related to the abundance of specific categories of proteins that together facilitate recombinant protein production. We infer that individual cells within parental populations are more functionally equipped for high-level recombinant protein production than others and that this bias could be used to select cells that are more likely to achieve high qMab.

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