While enzyme libraries and scope continue to grow over time, there are several key challenges that can provide complexity for implementation of biocatalytic processes, even when suitable enzymes exist. This includes: enzyme loading, enzyme costs, and efficient separation of the enzyme from the reaction product solution. In parallel, tangential flow filtration (TFF) is a very well-established unit operation in pharmaceutical production, and it is used routinely in manufacture of monoclonal antibodies (mAbs) and antibody drug conjugates (ADCs). This unit operation is highly efficient at separations of small and large molecules using membranes that may be selected for 1 kDa to 300 kDa molecular weight cutoff. When these membranes are run in a tangential flow configuration they can maintain high membrane flux due to high convection at the membrane surface, limiting low permeability gel layer formation.[2] In this configuration, polymers, mAbs, ADCs, or enzymes can be retained while small molecules permeate through. By integrating TFF with enzymatic processes, it is possible to achieve continuous separation and re-cycling of enzymes to the biocatalysis continuous stirred tank reactor while removing reaction products via the permeate. Further, the enzyme is also separated completely from the product mixture, avoiding more complex enzyme removal steps to purify the resulting API or intermediate, therefore making it ideal for efficient implementation of biocatalysis in pharma.
We will discuss how we have implemented custom-built and highly automated laboratory TFF systems through Ignition that incorporates feedback control on temperature, transmembrane pressure, and flowrates while logging all sensor data to efficiently scale such processes. In the laboratory demonstration of this configuration, we have shown that the biocatalysis-CSTR reactor itself can reduce overall enzyme loading to processes through continuous recycling of the biocatalyst. Furthermore, we have shown that enzyme activity can be maintained without deactivation by running many reactor turnovers at low enzyme loading, and therefore low conversion (<30%), to probe any reduction of catalyst efficacy over time. During this time, the enzyme levels in the permeate remained below detectable levels and conversion remained stable. Exploration of batch enzyme kinetics, application to TFF system design, and implementation into an overall system will be discussed. Finally, by operating for extended duration, high conversion will be shown at increased scale, all while keeping overall enzyme loading low through extended recycle in a CSTR configuration.
Overall, the integration of tangential flow filtration with enzymatic reactions offers a robust and sustainable solution for biocatalysis. This strategy enables efficient recycling and reuse of enzymes while improving productivity and advancing green chemistry approaches by minimization of material intensive work-ups to separate enzymes from the reaction solution.