A study to define the critical operating parameters for a new CCC bioreactor tool

The primary objective of the project is to define the operational parameters and design criteria for a new bioreactor. This bioreactor will function as an extremely useful tool to make and isolate a range of important compounds required by industry more rapidly and efficiently than before by using catalysts to perform chemical reactions in countercurrent chromatography (CCC) centrifuges. The project combines CCC with biotransformations using the experience of the Brunel Institute for Bioengineering (BIB) in separating molecules by CCC and industrially relevant biocatalysts and biotransformations developed by Ingenza. Chromatography separates components of a mixture by their different affinities for mobile and stationary phases held in columns. In CCC physical forces e.g. centrifugal, hold one liquid phase stationary and permit mobile phase to flow over it, with many mixing and settling steps. Component molecules distribute differently between the two immiscible phases so that they exit the machine at different times and are separated. BIB are world leaders in design, manufacture and use of hydrodynamic CCC-centrifuges and the scale up of separations made on small, lab machines to larger, production scale machines. Biotransformation is the process of transforming one molecule (substrate) into another (product) by a biocatalyst (enzyme). Ingenza has developed catalysts that convert cheap, readily available starting materials to high value products - particularly chiral compounds which have different left- and right-handed forms. Frequently, only one form is biologically active or safe and can therefore gain regulatory approval by e.g. the FDA. The chemical synthesis of chirally pure materials can be expensive and difficult; biotransformations often provide elegant and cost-effective manufacture. Preliminary work undertaken by BIB/Ingenza strongly indicates that their CCC equipment is superior to existing bioreactors as it selectively removes the biotransformation product continuously in the mobile phase (CCC being a chromatographic process) with the starting material and biocatalyst being kept in the stationary phase. This drives the reaction more quickly and efficiently to completion. With such a rapid and continuous process, large-scale manufacture can be achieved by a relatively small footprint machine, which is of considerable economic benefit to industry. The process is environmentally friendly ('Green') with low energy needs, renewable substrates, minimal waste and solvent use, and has the potential to replace wasteful chemical processes. We will compare two types of CCC machines which work on different principles to assess their relative merits: hydrodynamic CCC, which is BIB's speciality, and hydrostatic CCC. Two of Ingenza's well characterised biotransformations will be used to manufacture a polar and a non-polar molecule and thereby establish the broad application of the bioreactor tool. Molecules of such differing polarities need very different phases which differ in physical properties and thus require very different operating conditions. In biotransformation 1, the CCC bioreactor will perform the biotransformation and simultaneously separate product and starting material. Continuous flow reactions are highly desirable for industry, facilitating higher process throughputs and productivity, lower capital costs, considerable benefits in consistency of product quality and reproducibility and obviating many of the complex regulatory issues inherent in batch processing. This reaction uses non-polar materials. . In biotransformation 2, by contrast, highly water-soluble material will be used. High value unnatural L-amino acids will be made from available low cost racemic mixtures of L- and D-amino acids (different chiral forms), by converting the D-form with a specific enzyme to a reaction-product leaving the high value L-amino acids, which will be separated from the by-product in the CCC-bioreactor.

Our preliminary investigations strongly suggest that biotransformations can be carried out in a Counter Current Chromatography (CCC) system, whose inherent features (high substrate loadings; thousands of mixing steps/minute; fast selective product removal) render it a more efficient means of carrying out biotransformations than current bioreactors. We propose to compare the performance of our CCC bioreactor with conventional bioreactors in two representative, industry-standard biotransformations: one for nonpolar starting materials and products and the other for water-soluble materials. This 'proof of principle' research will give an understanding of the critical engineering techniques and operational methods needed for operating CCC bioreactor systems with a view subsequently to optimizing these and thereby laying the foundations for successful industrialization. Biotransformation 1. CCC as a reactor/separator: resolution of racemic a-methyl benzylamine using a monoamine oxidase enzyme. A problem for industrial scale resolution processes is the need for downstream processing to separate product and starting material. As a CCC-bioreactor can elute these separately, it should have a clear technical advantage over conventional bioreactors. Aqueous-organic phase systems are required for these non polar materials. Biotransformation 2. CCC bioreactor with highly water-soluble substrates and products: resolution of DL-Norvaline. Amino acids have industrial importance as starting materials for high-value chiral pharmaceutical intermediates. Because of their low organic phase solubility, aqueous-organic systems (as in 1) are precluded; robust aqueous two-phase systems (ATPS) will be developed for the CCC bioreactor. We will also study the mode of mixing in the bioreactors by comparing our hydrodynamic CCC (dynamic extraction) which has wave mixing, with hydrostatic CCC (centrifugal partition chromatography - CPC) which has cascade mixing.