Low-protein media for specialised mammalian cells

Specialised mammalian cells can be used to produce monoclonal antibodies and recombinant proteins that offer exciting prospects for the treatment of disease. The main objective of the presented studies was to develop low-protein culture media to produce clinical-grade proteins from the specialised mammalian cells used by the Wellcome Foundation Ltd.

This commentary contains six peer-reviewed papers and other experimental results, that describe the development of novel low-protein culture media for mammalian cells used for protein production. Monoclonal antibodies and other clinically important proteins can be produced from specialised mammalian cells by means that include hybridoma or recombinant gene technology. In order to grow in vitro, mammalian cells need to be cultured in a complex medium which provides them with the necessary nutrients within a suitable physical environment. Existing production media are generally sub-optimal and contain serum or other animal-derived proteins that cause considerable problems associated with cost, reproducibility of process, purification of product and regulatory issues. There is therefore a need to develop defined low-protein culture media that can be used to support large-scale production of functionally active protein. The aim of these studies was to develop such media for a number of commercially important mammalian cell lines used by the Wellcome Foundation Ltd. Most previous studies have developed serum-free medium by replacing serum with defined protein supplements. The composition of these media can subsequently be improved by screening for beneficial additives, or by trial and error. In the studies presented here a strategy was devised which combined screening experiments, addition of selected components, toxicity titrations, nutrient depletion studies and feeding experiments to produce improved low-protein media. This strategy linked to an appreciation of the metabolic requirements of the cell lines used, allowed a methodical approach to be used to develop defined low-protein media. This strategy successfully generated protein-free and low-protein media for a range of cell lines. The first medium developed, WCM5S, had a relatively simple composition and contained soy peptone. WCMS performed well with Chinese hamster ovary (CHO) cells, and CHO cells engineered using dihydrofolate reductase as a selectable marker, but was toxic to the other cells of interest. The other papers describe the development of a series of complex defined media that were either protein-free or contained insulin, and the application of these media for the growth of cell lines secreting proteins for therapeutic use. The first of the series W38 medium, which was completely protein-free, was developed for rat myeloma and hybridoma cells, and also supported growth of a number of other anchorage independent cell lines. This medium was used to produce monoclonal antibody from hybridomas, and interferon-alpha (IFNA) from the Namalwa human lymphoblastoid cell line. Insulin caused mitotic and phenotypic changes to CHO cells in WCMS5 medium and potentiated IFNA production from Namalwa cells in W38 medium, but had little measurable effect on YO or NSO cells. NSO mouse myeloma cells would not grow in W38 medium and were found to be cholesterol auxotrophs. Protein-free NSO cell growth was achieved when a novel lipid delivery system, utilising B-cyclodextrin and phosphatidylcholine/cholesterol vesicles, was added to W38 medium. A weaning process was devised which enabled NSO cells to be 'adapted' to protein-free growth without cholesterol. The 'adapted' NSO cell line (NSO.CF) is now routinely used for recombinant gene and hybridoma technology. W38 medium was enriched and modified to give WNSD medium, that is used for protein production from NSO cells engineered using glutamine synthetase as a selectable marker. Antibody produced from engineered CHO and NSO cells in lowprotein medium was found to be glycosylated and biologically active in vitro. The remainder of the commentary puts the results into context by examining the work as a single entity. This examination relates the results to metabolism, proliferation and survival of engineered and non-engineered mammalian cell lines in vitro.