Biosynthesis of Drug Metabolites Using Microbes in Hollow Fiber Cartridge Reactors: Case Study of Diclofenac Metabolism by Actinoplanes Species

Fungal and bacterial microbes are known to mimic mammalian cytochrome P450 metabolism. Traditionally, microbial biotransformation screening and small scale-ups (<1 liter) are performed in shake-flask reactors. An alternative approach is the use of hollow fiber cartridge (HFC) reactors. The performance of HFC reactors is compared with shake-flask reactors using diclofenac as a model substrate. Actinoplanes sp. (American Type Culture Collection 53771) in a shake-flask reactor hydroxylated diclofenac (50 μM) with 100% turnover in less than 5 h. A scaled-up production resulted in the formation of 4′-hydroxy (169 mg, 54% yield), 5-hydroxy (42 mg, 13% yield), and 4′,5-dihydroxy (25 mg, 7.7% yield) metabolites. HFC reactors with Teflon, polysulfone, and cellulose membranes were screened for nonspecific binding of diclofenac. Concentration-time profiles for turnover of 50 to 2000 μM diclofenac by Actinoplanes sp. were then determined at 22 and 30°C in an HFC reactor. Cellulose-based HFC reactors exhibited the lowest nonspecific binding (87% of 50 μM diclofenac remaining after 5 h) and offered the best conditions for its biotransformation (100% conversion; < 5 h at 30°C at 50 μM; 25 h at 500 μM). The time profile for substrate turnover was equivalent in both a cellulose membrane HFC reactor and shake-flask reactor. Two cellulose membrane HFC reactors were also tested to evaluate the reusability of the cartridges for diclofenac metabolism (50 μM, 22°C, 15 h; 500 μM, 30°C, 36 h). Up to seven reaction cycles with intermediate wash cycles were tested. At least 98% conversion was observed in each reaction cycle at both diclofenac concentrations.