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Optimization of Process Parameters for Enhanced Production of Ranibizumab in Escherichia coli

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Abstract

Escherichia coli based systems, are favoured hosts for recombinant protein expression. However, Low Fab expression and high production costs are major obstacles for manufacturers. To enhance process economics, an efficient, well-defined upstream process is crucial. This paper presents a systematic approach of process optimization for production of Fab fragment, ranibizumab. A two-step design of experiments (DOE) approach has been used. Six variables were examined: post-induction temperature, postinduction time, inducer concentration, agitation speed, media pH, and cell density at induction time (induction OD). These parameters were evaluated for their effect on biomass concentration, protein titre, impurity level, and light to heavy chain ratio. First, screening DOE with fractional factorial design was performed to shortlist important process parameters. Next, response surface study based on central composite design was conducted for identifying the operating range for the significant process parameters. Induction OD, inducer concentration, pH, and temperature were identified as significant parameters with their respective optima at 1.2 OD, 1 mM, 7.4 pH, and 35°C, respectively. Operating under these conditions resulted in formation of 0.051 g protein/g inclusion bodies, 4.99 g/L biomass concentration, and light chain to heavy chain equal expression ratio (LC:HC=0.98), with 8.2% impurity. Additionally, based on the predictions, we found that temperature, induction OD, and their interactions significantly affect the LC:HC ratio and impurity level, and need to be tightly controlled. This study has highlighted the importance of fine-tuning of physical parameters for production of Fab fragments and to control impurity expression in microbial host systems.

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Ranibizumab periplasmic double copy under the Accession Number BankIt2302631 Seq2:MN931856.

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Acknowledgements

Author Rucha S. Patil would like to thank Department of Biotechnology, Ministry of Science and Technology, New Delhi, for providing DBT-JRF Fellowship grant during her PhD (Fellow number DBT/2017/IIT-D/964).

Funding

Authors would like to acknowledge funding support from Department of Biotechnology, Ministry of Science and Technology (File number BT/COE/34/SP15097/2015) and by Biotechnology Industry Research Assistance Council (Reference number BT/NBM0160/04/19).

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Authors and Affiliations

  1. Department of Chemical Engineering, Indian Institute of Technology, Hauz Khas, New Delhi, 110016, India

    Rucha S. Patil, Nidhi Upadhyay & Anurag S. Rathore

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  1. Rucha S. Patil
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  2. Nidhi Upadhyay
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  3. Anurag S. Rathore
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Contributions

Rucha S. Patil: Conceptualization, Methodology, Formal analysis, Validation, Investigation, Writing–original draft, reviewing and editing. Nidhi Upadhyay: Methodology, Formal analysis, Validation, Investigation. Anurag S. Rathore: Conceptualization, Resources, Writing–review & editing, Supervision, Project administration, Funding acquisition.

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Correspondence to Anurag S. Rathore.

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Patil, R.S., Upadhyay, N. & Rathore, A.S. Optimization of Process Parameters for Enhanced Production of Ranibizumab in Escherichia coli. Biotechnol Bioproc E 28, 386–397 (2023). https://doi.org/10.1007/s12257-022-0323-2

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  • DOI: https://doi.org/10.1007/s12257-022-0323-2

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