GM CSF epitope analysis using a panel of monoclonal antibodies

OGGERO EBERHARDT, M.; FRANK, R.; KRATJE, R. B.; ETCHEVERRIGARAY, M.

Hannover - Braunschweig. Alemania

Simposio; More Quality of Life by means of Biotechnology. International Symposium on the Bioconversion of Renewable Raw Materials; 2000

GBF – Gesellschaft für Biotechnologische Forschung mbH. CDG – Carl Duisberg Gesellschaft e.V

The production of recombinant secretory proteins in mammalian cells is affected by a large variety of genetic factors, growth media and culture conditions. Apart from achieving high expression levels, product quality is of paramount importance. Post-translational modifications can impose significant problems for in vivo applications of the product. Cellular or viral promoters can be used in different cell lines for high level protein expression. In stable transfections, individual clones express recombinant proteins at highly variable levels, and only a small fraction stably express high amounts of the desired product protein. Native human Granulocyte-Macrophage Colony Stimulating Factor (hGM-CSF) is a glycoprotein and many of its proposed uses center around its potential ability to augment current therapeutic regimens for cancer therapy and bone marrow transplantation. One of the major questions in any recombinant glycoprotein production is the selection of the cell lines and the appropiate vector system. For this reason we have produced hGM-CSF by recombinant techniques in different cell lines. To evaluate the vector integrity COS.7 cells were transfected with p91023-GM, which has an sv40 origin and enhancer segment. Incubation medium was collected 3 days after transfection and the gene expression was monitored by a specific enzyme linked immunosorbent assay and functionally validated by a cell proliferation test. To select the promoter which gives the highest expression, two plasmids which are able to express hGM-CSF were tested in CHO.K1 and BHK.21 cell lines: - pCI-Neo-GM: which contains the human cytomegalovirus immediate-early enhancer/promoter region; - pkG4-GM: which has the simian virus 40 early promoter. Stable gene expression was evaluated by ELISA and bioassay. In order to determine the type of carbohydrate moieties, deglycosylation of the rhGM-CSF was performed by using N-glycosidase F and the resulting digests were analized on SDS-PAGE and subsequent Western-Blot. The rhGM-CSF expressed by different systems in 72 hours were: Sample ELISA (ng/ml) Biological Activity (U/ml) Specific Biological Activity (U/ng) COS.7/p91023-GM 422.00 2297.20 5.4 CHO.K1/pkG4-GM 376.40 5120.00 1.3 CHO.K1/pCI-Neo-GM 380.40 7988.00 21.0 BHK.21/pkG4-GM 103.40 98.00 11.0 BHK.21/pCI-Neo-GM 14.60 19.80 1.4 Finally we selected CHO.K1/pCI-Neo-GM system to express the cytokine for biotechnological purpose. The Western-Blot pattern of this cytokine showed highly glycosylated isoforms and thus it could be expected that this molecule bear a longer serum half life and an increased in vivo activity.