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

Pharmacokinetics and Drug Metabolism, Amgen Inc., Thousand Oaks, California

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.