(12a) Development of Multi-Enzyme Immobilized Biocatalytic Processes for Synthesis of Small Molecule APIs

Biocatalysis is gaining popularity in pharmaceutical manufacturing, specifically because it enables chemical transformations that may be difficult or lengthy to perform with traditional synthetic chemistry. This provides a substantial advantage for some reactions, however, removal of enzyme and host cell proteins from the final product is often a concern. One solution to this issue is enzyme immobilization, which allows these enzymes to be bound to a solid resin bead and separated from the liquid reaction stream via filtration. Immobilization can also improve enzyme stability, activity and catalyst loading, and decrease side reactions driven by host cell proteins. If necessary for the chemical synthesis, it may be useful to immobilize multiple enzymes that work together in an enzymatic reaction cascade. This introduces process development challenges that need to be addressed, such as balancing the correct loading ratio to push the reaction to completion while minimizing byproducts. This type of development is typically very time consuming and testing may only be performed on a few select conditions due to resource limitations. Here we discuss the data-rich methods we have designed and implemented to efficiently develop immobilized processes involving multiple enzymes. We have adapted existing technologies originally intended for crystallization screens to screen enzymes at a significantly smaller scale. This approach provides sufficient information to allow the scientist to make a well-informed choice on the best conditions, and then proceed directly to scale up for confirmation. This talk describes the techniques and workflow we have developed at Merck to enable rapid tailoring of conditions in multi-enzyme biocatalytic processes for small molecule active pharmaceutical ingredient (API) synthesis.