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

本提案的目的是研究近红外和中红外光谱（NIRS和MIRS）在监测商业生物催化过程中的应用。两种红外光谱已被证明在生物加工中提供改进的监测能力，特别是在生物过程（发酵）监测中。在发酵过程中，NIR/MIR用于（近）实时监测分析物水平的实际用途是明确的，但很少有研究关注这种光谱技术在生物催化中的应用。那些采用定性方法来描述产品的特征，而不是对其进行量化，或监测有限商业利益的反应。这是令人惊讶的，因为发酵液通常是复杂的，最近在将光谱技术应用于这些具有挑战性的过程中的进展清楚地指出了这种技术在基质趋于简单的生物催化中的潜力。在生物催化中使用定量IR可以对这些反应进行实时分析，从而实现过程控制。Ingenza一直在利用爱丁堡大学建立的生物催化技术开发胺和氨基酸制造。该技术代表了一种强大的方法来制造对映纯非天然手性胺和氨基酸，这是高价值的医药中间体。然而，技术发展的一个限制方面是对生物催化过程的分析，因为目前没有方法允许实时监控，因此，使用当前的分析方法改进过程是缓慢的。IR通过加强监测和过程控制，显著改善了生物催化过程。Ingenza和Strathclyde大学进行的初步可行性研究表明，NIR/MIR在监测l -氨基丁酸（L-ABA）稳健、经济的生产过程方面具有相当大的潜力。该过程包括动力学分解，其中DL-ABA的外消旋混合物转化为L-ABA和酮丁酸（KBA）。DL-ABA不需要的d -对映体被d -氨基酸氧化酶氧化成亚氨基丁酸，随后亚氨基丁酸迅速水解成KBA和氨。L-ABA很容易从反应混合物中分离出来，产率高，对映体过量（e.e）。两种关键成分（ABA和KBA）的消失和出现对于理解该生物过程中的反应动力学、化学效率和体积生产力至关重要。每个分析物的不同光谱特征可以很容易地在两个IRS中检测到。在此基础上，建立能够预测ABA和KBA浓度的模型是可能的。由于对映选择性酶氧化是制造主要类别的化学品，即胺，氨基酸和醇的途径，红外监测在其应用中可能具有广泛的意义。此外，所有这些类型的化合物都可能具有很强的红外吸光度，因为从结构上可以明显看出强偶极矩。这意味着原位红外监测在生物催化监测方面具有深远的潜力。因此，我们希望进一步研究这些技术在工业上重要的生物催化过程中的应用，包括上述氨基酸氧化酶类型的反应。该研究具有广泛的工艺应用潜力，因为Ingenza将动力学分辨率和脱碳过程作为一种跨多种氨基酸的平台技术进行操作。它将通过检查关键反应参数（如温度、pH值、底物/酶负载等）对整体效率和生产率的影响来加强工艺开发。

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.