Recent advances in understanding of the apoptotic death (Apoptosis) response of cells and of the underlying mechanisms controlling survival, growth and cell proliferation will be applied to the development of cell lines and culture strategies for the quantitative improvement of cellular and biologic product productivity. Specifically, the intention will be to use metabolic engineering to alter advantageously the survival and proliferative capacity of cells in intensive cell culture environments via the expression of bc1-2 and other genes known to be involved in the regulation of apoptosis, as well as of cyclin E which is primarily involved in Gl to S progression. Simultaneous overexpression of anti-apoptotic gene and cell cycle gene may well have synergistic positive effects in averting apoptosis. The suppression of apoptotic response will be particularly important in cell growth and adaptation in nutrient limited conditions and protein-free medium and as an aid to the specific production of proteins such as chimeric antibodies of improved fidelity under controlled but intensive culture conditions. Also, it should be possible to enhance viral production by blocking the apoptotic response known to be induced on viral infection, thereby reducing the rate of cell death and prolonging and increasing viral replication. The resolution of the primary metabolic pathways associated with apoptosis and the influence of anti-apoptotic gene activity on cellular metabolic fluxes during intensified bioreaction processes are major undertaking of this project. A major additional objective of this project is to increase interactions and collaborations between European research laboratories on one hand and between these laboratories and industry on the other. It is anticipated that the outcome of this project will be to enhance the competitiveness of the European pharmaceutical-industry by improving the efficiency and productivity of animal cell culture processes as well as meeting future process and product safety targets. In order to optimise use of the skills involved, the work detailed in this application will be managed in an integrated manner. The first aim of this proposal is to use genetic engineering to generate productive cell lines with enhanced survivability, a task which will be accomplished by Labs Cotter, Piacentini, Bailey and Al-Rubeai-Emery. Lab Bailey will produce and isolate cell lines that are able to survive in serum free medium and in suboptimal bioreaction conditions as well as in conditions of restricted or no growth. The other theme of this proposal is the determination of the culture conditions which are optimal for growth and survival of cells by inhibiting apoptosis using media additives. Labs Cotter and Al-Rubeai-Emery w ill determine the ability of a number of compounds such as anti-oxidants and zinc ions to inhibit apoptosis and the influence of anti-apoptotic genes on media composition and utilization. These labs will develop a new media formulation with anti- apoptotic characteristics. The role of apoptosis in productive cell lines and its relation to productivity will be determined by Labs AlRubeai and Merten- Perrin. Labs Merten-Perrin will be specifically involved with apoptosis in cell lines used for vaccine production and the effect of viral infection. All labs participating in this project will be involved in the assessment of the mode of action of anti-apoptotic genes and their role in process engineering. Lab Bailey will determine the interaction between bc1-2 or v-abl and cyclin E and E2F in myeloma, BHK, hybridoma and Vero cells. Lab Brindle will use their expertise to determine changes in flux in the energy generating pathways of the cell associated with the process of apoptotic cell death and also to determine the influence of anti-apoptotic genes on these fluxes. Lab Portner will investigate the relationship between growth and death and medium perfusion rate in high density fixed bed immobilised cell culture, where relatively high death rate is of a particular concern, with particular regard to the influence of anti- apoptotic genes. Labs Portner and Al-Rubeai will develop a kinelic model to describe the growth and death processes in intensified perfusion cultures. Lab Brindle will use NMR microimaging to examine such processes in high cell density hollow fibre bioreactors while Lab Al-Rubeai-Emery will use flow cytometry to study the influence of these genes on cell proliferation and cell cycle in spin- filter bioreactor. The new genetically modified cell lines generated, selected and tested at the above labs will be grown in different bioreactor configurations to assess their bioprocess characteristics. Celltech (Birch) will be involved in an advisory role in process scale up and testing product consistency of industrially important cell lines such as the myeloma and CHO cells producing chimeric antibody. The results generated by this project will benefit the European community in two ways; there will be generic benefit to the scientific and technical community at large; and there will be specific commercial benefit through exploitation by European companies involved in the Industrial Platform.
Funding SchemeCSC - Cost-sharing contracts
CB2 1GA Cambridge