title: Biosynthesis of drug metabolites using microbes in hollow fiber cartridge reactors: case study of diclofenac metabolism by Actinoplanes species.
abstract: 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 microM) 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 microM diclofenac by Actinoplanes sp. were then determined at 22 and 30 degrees C in an HFC reactor. Cellulose-based HFC reactors exhibited the lowest nonspecific binding (87% of 50 microM diclofenac remaining after 5 h) and offered the best conditions for its biotransformation (100% conversion; < 5 h at 30 degrees C at 50 microM; 25 h at 500 microM). 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 microM, 22 degrees C, 15 h; 500 microM, 30 degrees 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.
authors.affiliation: Pharmacokinetics and Drug Metabolism, Amgen Inc., Thousand Oaks, California 91320, USA.
authors.forename: Antonio
authors.initials: A
authors.lastname: Osorio-Lozada
authors.forename: Sekhar
authors.initials: S
authors.lastname: Surapaneni
authors.forename: Gary L
authors.initials: GL
authors.lastname: Skiles
authors.forename: Raju
authors.initials: R
authors.lastname: Subramanian
mesh_terms: D039903:Actinobacteria / Q000378:metabolism*; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism*; D019149:Bioreactors; D001711:Biotransformation; D004008:Diclofenac / Q000378:metabolism*; D005658:Fungi / Q000378:metabolism; D006900:Hydroxylation
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D004008:Diclofenac