Mammalian Cell Technology:

This involves the industrial application of animal cells to the large scale commercial production of biologicals such as monoclonal antibodies, recombinant proteins and viral vaccines. The industrially important cell lines include hybridomas capable of secreting monoclonal antibodies, transfected cells (eg: CHO) for specific protein expression and Vero (or MRC-5) cells grown on microcarriers which are important for vaccine production. The focus of the research activity is to characterise and control the cell culture process so that product synthesis is maximised. This can be achieved by enhancement of cell specific protein expression or by achieving high cell densities. Post-translational modification of glycoproteins is important to ensure the biological activity of products and is an extremely important process in mammalian cells. The scope of the present work can be summarised as follows :-

Current projects

Glycosylation. Secreted mammalian cell products are usually glycoproteins which can vary in the type and extent of glycosylation. The culture parameters that give rise to variability of the carbohydrate group are being studied with respect to Mab-secreting hybridomas and recombinant glycoproteins from CHO cells. This involves the identification and control of culture parameters that perturb protein glycosylation. It has been established that the molecular heterogeneity of glycoforms changes in cell culture in response to the micro-environment. Changes in substrate availability, product accumulation, pH and dissolved oxygen are all factors that can cause changes in glycosylation. For an industrial bioprocess it is important that at least the batch to batch variation of glycosylation is minimised. Ideally the most active glycoforms predominate in the final product. These parameters are being studied in cultured of CHO cells transfected with either the human erythropoietin or the human beta interferon gene. Both are therapeutic products for which appropriate glycosylation is essential for biological activity. Glycoforms are being analysed by sequential enzymic degradation using HPLC – novel method developed at Oxford. Structural confirmation is by mass spectrometry.

Bioprocess development. Culture conditions and media formulations are being studied to optimise the production of recombinant proteins. A process for human erythropoietin production has been implemented in pilot scale production by Cangene Inc. The process involves a unique media formulation which has been commercialized by Biogro Technologies, for which MB is a major shareholder. These principles of bioprocess development are presently being applied to the production of recombinant beta interferon.:Alternative methods of culture such as fed-batch, perfusion or chemostat are being studied to maximise cell yields and product yields (process intensification). Continuous culture offers the possibility of a constant product supply from the effluent of the bioreactor. Chemostat cultures are particularly valuable in the study of culture parameters because the cells can be maintained in a growth equilibrium for long periods. Perfusion cultures have been particularly successful in a project leading to high densities of Vero cells on microcarriers (solid or porous). Also, a perfused fluidized-bed bioreactor containing macroporous carriers has been developed for the continuous production of erythropoietin.

Metabolic management/ engineering: Biochemical techniques are being used to study the metabolism of cells with the aim of understanding the intracellular requirements during protein production. The metabolic flux associated with substrate utilisation is being studied by use of radioactively-labeled compounds. Specific nucleotide pool ratios have been shown to be good indicators and predictors of growth and productivity. This has allowed the establishment of culture control strategies whereby cells at relatively high densities can be switched to sub-optimal growth conditions (eg; with respect to temperature, osmolarity or chemical addition) for maximisation of product expression. Such control strategies can maximize cell and product yield. Much of the necessary optimization studies can be performed on a laboratory scale (2-5 litre fermenters) but this can lead to significant gains that may be applied to a larger scale. Limitations to cell yield have been related to nutrient depletion or by-product accumulation. Strategies are being developed to reduce the effect of these limitations. For example, the use of glutamine analogues (eg; glu or gln-peptides) can reduce the accumulation of ammonia which is growth inhibitory. Nutrient supply can be maintained by strategic supplementation of specific compounds during growth. This may lead to strategies for the introduction of genes for key enzymes into producer cell lines.

Cell robustness Mammalian cells are generally considered to be fragile in agitated bioreactors. However, there are a number of procedures that we have identified to improve the robustness of these cells. A metabolic approach that we

have implemented is to provide a suitable cocktail of lipids that enhances the unsaturated fatty acid content of the cell membrane. Over 2 or 3 passages this has been shown to enhance significantly the survival and productivity of cells at high agitation. The use of a non-ionic surfactant (Pluronic F-68) has been studied and shown to reduce the hydrophobicity of the cell membrane. This causes a significant enhancement improvement in membrane strength. However, there are significant differences in response between cells suggesting the need to develop cell line-specific strategies.

Viral production.  Reovirus production from Vero and MRC-5 cells is being developed in a microcarrier culture process. Reovirus has shown potential as an oncolytic agent that selectively causes the lysis of ras-activated cells and therefore may be used as an anti-cancer therapeutic . We have shown high productivity of the virus in a serum-free medium that we have designed for Vero cells. The basis for the apparent difference in viral productivity between the serum-based and serum-free media formulations is presently being studied. A technology has been developed for dengue virus production as a potential vaccine candidate.