Real time monitoring of commercial biocatalysis processes using near and mid infrared spectroscopies

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The aim of this proposal is to investigate the use of Near and Mid Infrared Spectroscopy (NIRS and MIRS) in monitoring commercial biocatalysis processes. Both IR spectroscopies have been shown to offer improved monitoring capabilities in bioprocessing, especially in bioprocess(fermentation) monitoring. In fermentations, the practical utility of NIR/MIR for monitoring analyte levels in (near) real time is clear, but few studies focus on such spectroscopic techniques in biocatalysis. Those that do, concentrate on qualitative methods to characterise products rather than quantify them or monitor reactions of limited commercial interest. This is surprising, since fermentation fluids are often complex, the recent advances in applying spectroscopic techniques to these challenging processes clearly points to the potential of such techniques in biocatalysis where the matrix tends to be simpler. The use of quantitative IR in biocatalysis could allow development of real time analysis for such reactions, permitting in-process control. Scientific Case Ingenza has been developing amine and amino acid manufacture using biocatalysis from technology established at Edinburgh University. This technology represents a powerful approach to manufacture enantiopure unnatural chiral amines and amino acids, which are high value pharmaceutical intermediates. However, one limiting aspect of the technology development is the analysis of the biocatalytic process, as no current method allows real time monitoring, thus, process improvements are slow using current analytical methods. IR offers significant improvement in biocatalytic processes via enhanced monitoring and in-process control. Initial feasibility studies carried out by Ingenza and Strathclyde University have shown the considerable potential of NIR/MIR in monitoring a robust, economical manufacturing process for L-aminobutyric acid ( L-ABA). The process comprises of a kinetic resolution in which a racemic mixture of DL-ABA is converted to L-ABA and ketobutyric acid (KBA). The unwanted D-enantiomer of DL-ABA is oxidised to imino-butyric acid by a D-amino acid oxidase, subsequently imino-butyric acid rapidly hydrolyses to KBA and ammonia. The L-ABA is easily isolated from the reaction mixture in high yield and excellent entantiomeric excess (e.e). The disappearance and appearance of the two key components (ABA and KBA), is vital to understanding the reaction kinetics, chemical efficiency, and volumetric productivity in this bioprocess. Distinct spectral signatures for each analyte could readily be detected in both IRS. On this basis, the formulation of models capable of predicting the concentrations of ABA and KBA should be possible. Since enantioselective enzymatic oxidation is a route of manufacture for major classes of chemicals, namely amines, amino acids and alcohols IR monitoring is likely to be broad reaching in its application. In addition, all of these classes of compounds are likely to have a strong IR absorbance, due to strong dipole moments that are apparent from the structure. This means in-situ IR monitoring has far reaching potential for biocatalysis monitoring. Accordingly, we wish to investigate the use of such techniques further in industrially important biocatalytic processes, including the amino acid oxidase type reactions described above. The investigation has the potential to be wide ranging in process application since Ingenza operate the kinetic resolution and deracemisation processes as a platform technology across a broad range of amino acids. It will enhance process development by examining what effect critical reaction parameters (e.g. temp, pH, substrate/enzyme loading, etc) have on the overall efficiency and productivity.