Development of an integrated platform for production of recombinant protein biopharmaceuticals using disposable processing technology

An ever increasing number of the new drugs currently under development are based upon proteins rather than traditional small molecules (e.g. antibiotics). These protein drugs are produced for the treatment of diseases such as cancer by bacterial, yeast or mammalian cells kept in culture under defined conditions. Many of the new antibody based drugs are produced from in vitro cultured mammalian cells in order to produce correctly folded and assembled and post-translationally modified products, these processes being essential to the bioactivity of these drug molecules. However, one problem with this approach is that the cells used to make such proteins secrete not only the target protein into the medium in which the cells grow, but other proteins from the cell as well, called host cell proteins (HCPs), and product impurities (incorrectly processed products e.g. half-antibodies), whilst product aggregates can also accumulate in the medium. Further, cell breakage during fermentation or downstream handling (e.g. centrifugation) can result in the release of intracellular protein material and product aggregates. To complicate things further, what these HCPs are (cellular the contaminants) and how they change throughout cell fermentation and with target products is not known. What this means is that the target drug must be purified from the rest of the material in the medium and other product related contaminants before it is deemed safe for use and this is referred to as downstream bioprocessing. During purification it is not usually known what HCPs or product impurities are removed by any given step and this makes the development of new or novel approaches particularly challenging. Almost all approaches to date also bind and elute the target protein, and there is scope for investigating disposable technology that binds the contaminants. Thus, whilst improvements in product yield are economically beneficial, this does not directly correlate with downstream processing and there is little economy of scale. This is largely due to the reliance of downstream bioprocessing approaches on multiple chromatography steps that are often complex and low yielding and it is the total mass of the product that determines the amount of chromatography resin required and therefore cost. Downstream bioprocessing is now a major part (>40%) of the total cost of manufacturing such drugs and as such improvements in this area would be of major benefit to both manufacturers and the NHS. This project will begin to address these areas and investigate the development of an integrated platform (upstream and downstream) for the production of recombinant protein biopharmaceuticals using disposable processing technology. Specifically, we will test the hypothesis that identification/characterization of the major host cell protein contaminants (HCPs) and product impurities in CHO cell culture and their interaction with the downstream process will allow the development of alternative knowledge-based disposable purification strategies and the rational engineering of host cell lines to limit the levels of such problematic HCPs and product contaminants. To achieve this we will use state-of-the-art proteomic technology at Kent and standard purification procedures developed by Pall to determine the fate and relationship of product, product impurities, and HCPs throughout the downstream processing of model recombinant proteins expressed in mammalian cells (CHO expression systems). Typically, we will use traditional 2D-PAGE based and non-gel based LC-MS based approaches at Kent to monitor the protein profile throughout a standard Pall purification process of a monoclonal antibody. We will then compare this to alternative downstream processes using Pall based disposable technology. We expect that these approaches will identify both the cell HCP contaminants and identify the recombinant protein degradation products and the heterogeneity of the recombinant material.