Utilizing a Novel Human P450 Biocatalytic System for the Multi‐Cycle Production of Metabolites

Abstract

The large‐scale preparation of drug metabolites is typically a lengthy and problematic step in pharmaceutical development. We have characterized a robust P450 Catalytic System (PCS) which contains unmodified recombinant human cytochrome P450 (CYP) and oxidoreductase enzymes. The engineered inclusion of cofactors and antioxidants within its semipermeable structure greatly stabilizes the PCS and increases its usable lifetime. The present study focused on evaluating the ability of PCS expressing different isoforms to absorb and sequester substrate and generate additional product in multiple reaction cycles. After incubation at 30°C with 500 µM substrate for an initial 4 hr. reaction cycle, the PCS was pelleted via low speed centrifugation and resuspended in fresh buffer containing glucose‐6‐phosphate but no additional substrate, and incubated for an additional 4 hr. (cycle 2). The substrate and product concentrations in the reaction supernatant and that sequestered within the PCS system after each reaction cycle were determined by reversed phase HPLC. The second cycle produced significant amounts of additional product. Isoform Substrate Cycle 1 Yield, µM Cycle 2 Yield, µM 1A2 Phenacetin 43 28 2C9 Diclofenac 68 308 2C19 Mephenytoin 43 95 2D6 Dextromethorphan 68 89 3A4 Testosterone 47 46 With vigorous agitation to provide sufficient oxygen, the PCS retained P450 activity for multiple reaction cycles performed at 30° ‐ 37°C and for substrate concentrations ranging from 200 – 1,000 µM. Similar results were obtained for reaction volumes ranging from 200 µL to 400 mL, with the larger scale reactions typically yielding several milligrams of product. After the first reaction cycle, the PCS could also be stored at 4°C overnight without significant loss of activity.