Scarr, James Richard Hadley; (2008) Pharmaceutical development processes. Doctoral thesis , UCL (University College London).

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Abstract

The process of developing pharmaceuticals requires expertise from numerous different scientific areas. Four separate studies have been undertaken on Pharmaceuticals Testing, Process Development, Business Strategy and Process Validation within this industry. New pharmaceuticals generally require multi-step reactions, which increasingly feature the involvement of biological synthesis to improve the optical purity and thus efficacy and safety of the drug. Two of the problems with employing biological synthesis are the high level of inhibition observed and the potential difficulty with which these batch based reactions are combined with semi-continuous chemical synthesis. The first study characterises the inhibition of CHMO, a promising oxygenase enzyme, with the aid of flow cytometry using different systems: o A range of substrates, based around the natural substrate cyclohexanone but with differing ring size and increasing chain length. o Different CHMO catalysed reactions - isolated enzyme, free cell and immobilised whole cell. As expected, reactions with CHMO expressed in E. coli TOP 10 [pQR239] in their immobilised form reduced the observed reaction rate. Unexpectedly, for the more rapidly converted substrates (generally those closest to cyclohexanone), immobilisation was found to increase the inhibition observed. It has been postulated that this is due to an oxygen shortage for maintaining cell metabolism and a time based inhibitory effect. Advantages of immobilised cells are that they can be rapidly removed from the reaction broth allowing greater integration with other processes and can be recycled for multiple re-use. To facilitate their industrial use, the large scale production of immobilised whole cells is required. Whilst immobilised cell reactions are industrially employed, how such large quantities of immobilised cells are produced is yet to be reported. The feasibility of immobilisation of oxygenase expressing cells has been assessed in this first study, using the flow cytometry as a tool for assessing cell damage in the key step of cell separation. Within the pharmaceutical development process drug molecules are rigorously tested in clinical trials. However the metabolites likely to be produced in the body, which may be active (and preferable drug candidates to the parent molecule) or toxic (and thus responsible for the failure of the drug in the final stages of clinical trials) are often ignored. Within the human body the oxygenase enzymes Cytochrome P450s (CYPs) are responsible for the primary metabolism of more than 90% of drugs. The second study assesses different methods of identifying the CYP responsible for metabolism and discusses the importance of being able to produce gram scale quantities of metabolites. This study indicated that the best currently feasible option of CYP identification is the employment of Bactosome (individual CYP enzymes expressed in bacteria) with a selective inhibitor pre-screen. The scientific complexity of the pharmaceutical development process makes effective strategic planning and decision making difficult. Whilst the necessity of business plans to enable companies to secure finance has helped scientists to gain an understanding of their market and associated business risks, business decisions such as when to invest and how much, often rely solely on the company's tolerance of risk, collective intuition and experience. The third study investigates the business strategy of the pharmaceutical development process. StrategyDynamics modelling has been employed to create a living model of a start-up contract research organisation. The model demonstrates the advantages of being able to predict key resource bottlenecks, contrast different business decisions such as growth strategy and plan for future events and changes in technology and markets. This modelling can potentially save companies from expensive trial and error approaches and help to manage risk. Regulatory pressure within the pharmaceutical development industry and the importance of validation is increasing. In the fourth study the application of Process Validation to the areas of pharmaceutical development process in the first three studies are investigated. For CHMO biocatalysis the reproducibility of immobilised experiments was assessed, for drug metabolite production the importance of change validation, i.e. assay robustness, was determined and for the Strategy Dynamics modelling an approach to validating the model has been detailed.

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