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Reduction of N-terminal methionylation while increasing titer by lowering metabolic and protein production rates in E. coli auto-induced fed-batch fermentation

  • Fermentation, Cell Culture and Bioengineering
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

A standard fed-batch fermentation process using 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) induction at 37 °C in complex batch and feed media had been developed for manufacturing of a therapeutic protein (TP) expressed in inclusion bodies (IBs) by E. coli BL21 (DE3) driven by T7 promoter. Six unauthentic TP N-terminal variants were identified, of which methionylated TP (Met-TP) ratio was predominant. We hypothesized that lowering metabolic and protein production rates would reduce the Met-TP ratio while improving TP titer. The standard process was surprisingly auto-induced without added IPTG due to galactose in the complex media. Without changing either the clone or the batch medium, a new process was developed using lower feed rates and auto-induction at 29 °C after glucose depletion while increasing induction duration. In comparison to the standard process, the new process reduced the unauthentic Met-TP ratio from 23.6 to 9.6 %, increased the TP titer by 85 %, and the specific production yield from 210 to 330 mg TP per gram of dry cell weight. Furthermore, the TP recovery yield in the purified IBs was improved by ~20 %. Adding together, ~105 % more TP recovered in the purified IBs from per liter of fermentation broth for the new process than the standard process. The basic principles of lowering metabolic and production rates should be applicable to other recombinant protein production in IBs by fed-batch fermentations.

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Acknowledgments

We would like to thank Michael Stevens and Leonard Bettinger for their help on fermentor operation, Dr. Kern Chang and John Devincentis for analytical support, Dr. Michael Borys for critical review of the manuscript, and John L. Hickey and Dr. Shu-Jen Chiang for their support on the project.

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Authors and Affiliations

  1. Biologics Process Science, Global Manufacturing and Supply, Bristol-Myers Squibb, 6000 Thompson Road, Syracuse, NY, 13057, USA

    Jianlin Xu, Yueming Qian, Paul M. Skonezny, Li You, Zizhuo Xing, David S. Meyers, Robert J. Stankavage, Shih-Hsie Pan & Zheng Jian Li

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  1. Jianlin Xu
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  2. Yueming Qian
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Correspondence to Zheng Jian Li.

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Xu, J., Qian, Y., Skonezny, P.M. et al. Reduction of N-terminal methionylation while increasing titer by lowering metabolic and protein production rates in E. coli auto-induced fed-batch fermentation. J Ind Microbiol Biotechnol 39, 1199–1208 (2012). https://doi.org/10.1007/s10295-012-1127-8

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  • DOI: https://doi.org/10.1007/s10295-012-1127-8

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