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The Use of Immobilized Cytochrome P4502C9 in PMMA-Based Plug Flow Bioreactors for the Production of Drug Metabolites

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Abstract

Cytochrome P450 enzymes play a key role in the metabolism of pharmaceutical agents. To determine metabolite toxicity, it is necessary to obtain P450 metabolites from various pharmaceutical agents. Here, we describe a bioreactor that is made by immobilizing cytochrome P450 2C9 (CYP2C9) to a poly(methyl methacrylate) surface and, as an alternative to traditional chemical synthesis, can be used to biosynthesize P450 metabolites in a plug flow bioreactor. As part of the development of the CYP2C9 bioreactor, we have studied two different methods of attachment: (1) coupling via the N-terminus using N-hydroxysulfosuccinimide 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and (2) using the Ni(II) chelator 1-acetato-4-benzyl-triazacyclononane to coordinate the enzyme to the surface using a C-terminal histidine tag. Additionally, the propensity for metabolite production of the CYP2C9 proof-of-concept bioreactors as a function of enzyme attachment conditions (e.g., time and enzyme concentration) was examined. Our results show that the immobilization of CYP2C9 enzymes to a PMMA surface represents a viable and alternative approach to the preparation of CYP2C9 metabolites for toxicity testing. Furthermore, the basic approach can be adapted to any cytochrome P450 enzyme and in a high-throughput, automated process.

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Acknowledgments

Financial support was provided through NIH GM081348 as well as the WVEPSCOR (HEPC.dsr.09.013) graduate student fellowships in STEM to JK and LW.

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Authors and Affiliations

  1. Basic Pharmaceutical Sciences, West Virginia University, 1 Medical Center Drive, P.O. Box 9530, Morgantown, WV, 26506, USA

    Lance A. Wollenberg, Jarod L. Kabulski & Peter M. Gannett

  2. Protea Biosciences, 955 Hartman Run Road, Morgantown, WV, 26505, USA

    Matthew J. Powell & Jifeng Chen

  3. College of Pharmacy, University of Minnesota, 308 Harvard Street SE, Minneapolis, MN, 55455, USA

    Darcy R. Flora

  4. College of Pharmacy, University of Kentucky, 789 S. Limestone, Lexington, KY, 40536, USA

    Timothy S. Tracy

  5. 1 Medical Center Drive, P.O. Box 9530, Morgantown, WV, 26505, USA

    Peter M. Gannett

Authors
  1. Lance A. Wollenberg
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  2. Jarod L. Kabulski
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  3. Matthew J. Powell
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  4. Jifeng Chen
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  5. Darcy R. Flora
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  6. Timothy S. Tracy
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  7. Peter M. Gannett
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Correspondence to Peter M. Gannett.

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Wollenberg, L.A., Kabulski, J.L., Powell, M.J. et al. The Use of Immobilized Cytochrome P4502C9 in PMMA-Based Plug Flow Bioreactors for the Production of Drug Metabolites. Appl Biochem Biotechnol 172, 1293–1306 (2014). https://doi.org/10.1007/s12010-013-0537-z

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  • DOI: https://doi.org/10.1007/s12010-013-0537-z

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