Production of Recombinant Protein-based Pharmaceuticals in Mammalian Cells

OGGERO EBERHARDT, M.; BOLLATI FOGOLÍN M. R.; FRANK, R.; ETCHEVERRIGARAY, M.; WAGNER, R.; KRATJE, R. B.

Buenos Aires. Argentina

Workshop; German-Argentinean Workshop on Biotechnology: “From the idea to the market: Innovation in Bioproducts”; 2001

Secretaría para la Tecnología, la Ciencia y la Innovación Productiva – Ministerio de Educación. Argentina; Bundesministerium für Bildung und Forschung. Alemania

One of the major goals in the application of biotechnology towards human therapeutics is the discovery and development of protein-based pharmaceuticals using recombinant techniques. These recombinant proteins are often used for the augmentation or replacement of naturally occuring proteins in the treatment of numerous pathological conditions. The majority of proteins secreted by mammalian cells, including many proteins of pharmacological importance, are glycoproteins with different carbohydrate content. It is becoming increasingly clear that polypeptides which are glycosylated in their native state require glycosylation for full in vivo efficacy. Furthermore, the therapeutic profile of an administered polypeptide may be profoundly influenced by its glycosylation. Therefore it is essential in designing a human therapeutic protein that the structure is as close as possible to the naturally occuring product, and so it is imperative that correct glycosylation is achieved. To date, several therapeutically significant glycoproteins have been expressed in mammalian cells and many products are now commercially available. Unfortunately, continuous mammalian cell lines have the metabolic disadvantage of being unable to completely oxidize glucose to CO2. Hence, most of the glucose ends up in pyruvate and finally in lactate which causes acidification of cell culture media. This can be attributed to a very poor activity of the enzymes connecting glycolysis with the tricarbonic acid (TCA) cycle. Thus, mammalian cells consume high amounts of glutamine to cover their energy requirement. A byproduct of glutaminolysis is ammonia which causes growth inhibition and cell death and also affects glycosylation. To reduce the production costs of pharmaceuticals it is desirable to improve the exploitation of glucose to form ATP. This can be done by increasing the flux of glucose into the oxidative phosphorylation pathway. One strategy to improve the exploitation of glucose to form ATP can be performed by increasing the flux of glucose into the TCA cycle by introducing a new enzymatic way. Experiments performed by the german research group led by Dr. Roland Wagner have shown that introducing a cytosolic pyruvate carboxylase (PYC2) derived from the yeast Saccharomyces cerevisiae into some mammalian cells enabled them to transfer glycolysis derived pyruvate into malate, which then entered the TCA cycle for complete oxidation. As a result, higher yield of the recombinant protein were achieved using PYC2-expressing cells. The research group in Argentina has successfully obtained different recombinant mammalian cells with high-level expression for a variety of human proteins as erythropoeitin (rhEPO), granulocyte-macrophage colony stimulating factor (rhGM CSF), granulocyte colony stimulating factor (rhG CSF) and interferon  (rhIFN ). In this presentation, we will focuse our studies on human GM CSF production in recombinant baby hamster kidney (BHK) cells and chinese hamster ovary (CHO) cells. Briefly, human GM CSF is a glycoprotein which regulates the proliferation, differentiation and function of neutrophilic and eosinophilic granulocytes as well as monocytic cell lineages. There is much clinical interest in this lymphokine for its ability to stimulate granulocyte and macrophage production in patients that are immunosuppressed, either from disease or from receiving chemotherapy or radiation therapy. Due to variable glycosylation, the molecular mass of the mature protein, which comprises 127 amino acids, ranges from 14 to 35 kD. Bioactive human GM CSF was expressed in distinct host systems, as bacteria, yeast and mammalian cells. Despite all of them have biological activity, patients receiving the non glycosylated molecule produced in bacteria or the partially glycosylated one obtained in yeast may develop antibodies that compromise the clinical efficacy of the trial. Therefore, cultured mammalian cells are becoming more frequently used to produce this cytokine. In this presentation, different studies related to the human GM CSF molecule and its production will be shown. On one hand, we evaluated the GM CSF epitopes involved in the bioactivity of the cytokine and on the other, we studied the impact on the metabolism of CHO cells producing rhGM CSF, which also expressed the PYC2 enzyme.