Molecular analysis of successful cell line selection in transfected GS-NS0 myeloma cells.

The production of recombinant proteins from mammalian cells is now an essential part of biotechnology. However, despite this importance, the detailed characteristics of good producing cell lines remain largely unknown. The industrially important GS-NS0 mammalian expression system is able to produce large amounts of protein from relatively few copies of recombinant genes. This makes GS-NS0 cell lines ideal candidates to study the consequence of recombinant plasmid transfection in mammalian cells. This study investigated the molecular features of a panel of 17 randomly chosen GS-NS0 cell lines engineered to produce a recombinant antibody. The research analysed antibody production via enzyme-linked immunosorbent assay (ELISA), and investigated the molecular features of the transfectants by Northern, Southern and copy number analysis. The cell lines generated produced a range of antibody concentrations. In addition, for transfectants defined as producers of recombinant antibody there was a positive correlation between specific productivity and heavy chain mRNA expression. The use of Northern and Southern analysis allowed determination of the functional integrity of the transfected plasmid. Over 50% of the transfectants studied had molecular defects at the level of mRNA and/or cDNA. Cell lines were identified with suspected defects in the regulatory regions of transfected genes in addition to cell lines which lacked recombinant genes. Also, "false-positive" cell lines were generated which were able to overcome the GS selection pressure without producing any recombinant antibody. This article discusses these findings in relation to vector design.

Molecular definition of predictive indicators of stable protein expression in recombinant NS0 myeloma cells.

We have generated a molecular description of the loci at which stability/instability of expression of a monoclonal antibody (MAb) (anti-CD38) occurs within the GS-NS0 expression system. Critically, these data show that, in the absence of changes to copy number for the recombinant gene sequences, all cell lines examined exhibit a progressive loss (instability) in expression of mRNA during prolonged culture. However, not all cell lines express instability at the level of MAb protein production. The molecular distinction between stable and unstable production at the protein level is a reflection of the cellular amount of recombinant mRNA encoding MAb. Our data indicate a threshold level, a putative saturation point for utilisation of mRNA in translational/secretory events, that defines stability or instability of protein production. Above this level of recombinant mRNA expression, cell lines are stable, whereas below this level cell lines will show instability of protein production. Our studies indicate that absolute levels of expression of recombinant mRNA encoding for MAb in the GS-NS0 expression system offer a potential predictive indicator for the selection of stable cell lines for scale-up. These studies identify molecular facets of host cell biology of generic interest for gene regulation and expression and define techniques and approaches for enhancement of recombinant protein expression and process development.

Stability of recombinant protein production in the GS-NS0 expression system is unaffected by cryopreservation.

Mammalian cells form a very important part of the repertoire of production systems available to scientists involved in the production of recombinant proteins. During the production of therapeutic proteins it is vital for regulatory approval of products that no phenotypic or genetic changes are observed in the cell line or product. As part of the generation and development of therapeutic protein production, cell lines have to be frozen at various stages to create cell banks. If cryopreservation and revival of frozen stocks were to give rise to any phenotypic changes in the cells, this would again be detrimental to the further development of that particular cell line. This study uses one of the most industrially important expression systems, the GS-NS0 expression system, to examine the effect of cryopreservation on the growth and productivity profile of cell lines that exhibit differential degrees of stability during prolonged (production) culture periods. Results show that cryopreservation and revival procedures do not alter the stability characteristics of cell lines. This type of information is of great value in definition of protocols for cell line development.

Stability of protein production from recombinant mammalian cells.

One of the most important criteria for successful generation of a therapeutic protein from a recombinant cell is to obtain a cell line that maintains stability of production. If this is not achieved it can generate problems for process yields, effective use of time and money, and for regulatory approval of products. However, selection of a cell line that sustains stability of production over the required time period may be difficult to achieve during development of a therapeutic protein. There are several studies in the literature that have reported on the instability of protein production from recombinant cell lines. The causes of instability of production are varied and, in many cases, the exact molecular mechanisms are unknown. The production of proteins by cells is modulated by molecular events at levels ranging from transcription, posttranscriptional processing, translation, posttranslational processing, to secretion. There is potential for regulation of stability of protein production at many or all of these stages. In this study we review published information on stability of protein production for three industrially important cell lines: hybridoma, Chinese hamster ovary (CHO), and nonsecreting (NS0) myeloma cell lines. We highlight the most likely molecular loci at which instability may be engendered and indicate other areas of protein production that may affect stability from mammalian cells. We also outline approaches that could help to overcome the problems associated with unpredictable expression levels and maximized production, and indicate the consequences these might have for stability of production.