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<p>Production of Fab (fragment antigen-binding) molecules using <i>Escherichia coli</i> as a host presents a significant challenge due to low protein expression and the resulting poor yields. In this study, recombinant Ranibizumab was expressed in <i>E. coli</i> as inclusion bodies (IB) and optimization of lysis parameters, IB recovery, and IB washing conditions was performed to achieve optimal product yield and purity. Design of experiments (DOE) was employed to explore the interaction between variables and to facilitate optimization of buffer composition. Optimization of lysis buffer resulted in a yield of 0.069&#xa0;g protein/g IB, 61% IB purity, and 87% lysis efficiency. Optimization of homogenization conditions, using two passes at 1000&#xa0;bar, resulted in a 93.5% lysis efficiency with 60% IB purity. Additionally, optimizing the IB washing steps with 1% Triton X-100 and 2&#xa0;M urea for 30&#xa0;min at room temperature offered 84.53% IB recovery and 75% IB purity. Further, the impact of IB quality on refolding yield has been examined. Overall, the process optimization translated into a significant improvement in refolding yield, which increased from 18% under unoptimized conditions to 29% post-optimization and it has been demonstrated that optimization of lysis and washing steps can significantly enhance refolding yield, a key hurdle when expressing Fabs in <i>E. coli</i>.</p>

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Lysis and IB washing procedures significantly impact the recovery of rHumanized ranibizumab from inclusion bodies expressed in Escherichia coli

  • 期刊论文

  • 发表日期：

    2025年10月17日

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  • Rucha S. Patil,
  • Nida Khan,
  • Anurag S. Rathore

摘要

Production of Fab (fragment antigen-binding) molecules using Escherichia coli as a host presents a significant challenge due to low protein expression and the resulting poor yields. In this study, recombinant Ranibizumab was expressed in E. coli as inclusion bodies (IB) and optimization of lysis parameters, IB recovery, and IB washing conditions was performed to achieve optimal product yield and purity. Design of experiments (DOE) was employed to explore the interaction between variables and to facilitate optimization of buffer composition. Optimization of lysis buffer resulted in a yield of 0.069 g protein/g IB, 61% IB purity, and 87% lysis efficiency. Optimization of homogenization conditions, using two passes at 1000 bar, resulted in a 93.5% lysis efficiency with 60% IB purity. Additionally, optimizing the IB washing steps with 1% Triton X-100 and 2 M urea for 30 min at room temperature offered 84.53% IB recovery and 75% IB purity. Further, the impact of IB quality on refolding yield has been examined. Overall, the process optimization translated into a significant improvement in refolding yield, which increased from 18% under unoptimized conditions to 29% post-optimization and it has been demonstrated that optimization of lysis and washing steps can significantly enhance refolding yield, a key hurdle when expressing Fabs in E. coli.