Presentation Information
[P01-137]Production of Fab antibodies in Escherichia coli and development of an automated refolding process
○Tung Anh Hoang1, Hideo Nakano1, Teruyo Ojima-Kato1 (1. Nagoya University (Japan))
Keywords:
antibody production,protein refolding,E. coli,translation-enhancing peptide,industrial
[Purpose]
Monoclonal antibodies (mAbs) play a crucial role in therapeutic, diagnostic, and research applications. Traditional mAb production primarily relies on mammalian expression systems, which are costly and time-consuming. In contrast, E. coli expression system offers a cost-effective alternative for producing mAb fragments although it’s still challenging in proper protein folding and solubility. To address these challenges, this study aims to develop a large-scale mAb production platform using E. coli, incorporating an optimized and scalable refolding strategy to improve yield and activity.
[Method]
Fab and FabLZ (Fab fused with leucine zippers tag) antibody formats previously obtained from mouse were expressed in E. coli Nico21(DE3) strain using the pET system. Two refolding strategies, conventional stepwise dialysis and an automated refolding method using a continuous dilution system (Hoang et al., 2026), were compared to determine their efficiency in recovering functional mAbs. Purification was performed by Immobilized Metal Affinity Chromatography followed by Size Exclusion Chromatography. The binding activity of refolded mAbs were analyzed by ELISA and Bio-Layer Interferometry assay.
[Results]
Obtained results indicate that the automated refolding method is capable of generating mAbs with functional performance comparable to those produced through the conventional stepwise refolding method, while markedly enhancing process efficiency and output. In particular, the automated refolding method produced waste equivalent to only 69% of that generated by the modified stepwise refolding method, used just 47% of the denaturant, and reduced processing time to 40% of the conventional modified stepwise refolding method. These considerable savings in time and materials not only support greater scalability and lower production costs but also contribute to a more environmentally sustainable strategy for recombinant mAb manufacturing in E. coli.
[Conclusion]
This study establishes a scalable, robust mAb production system in E. coli, by combining accumulation of inclusion bodied and refolding strategy. The apparatus is simple to set up and operate, making the platform practical from laboratory to industrial scales and providing a cost-effective, environmentally sustainable alternative for recombinant antibody fragment production without compromising functional quality.
[Reference]
Tung Anh Hoang, Hideo Nakano, Teruyo Ojima-Kato, A strategy for scalable antibody production: the combination of Escherichia coli expression, translation-enhancing peptide and automated refolding system, Bioscience, Biotechnology, and Biochemistry, Volume 90, Issue 3, March 2026, Pages 429–437, https://doi.org/10.1093/bbb/zbaf197.
Monoclonal antibodies (mAbs) play a crucial role in therapeutic, diagnostic, and research applications. Traditional mAb production primarily relies on mammalian expression systems, which are costly and time-consuming. In contrast, E. coli expression system offers a cost-effective alternative for producing mAb fragments although it’s still challenging in proper protein folding and solubility. To address these challenges, this study aims to develop a large-scale mAb production platform using E. coli, incorporating an optimized and scalable refolding strategy to improve yield and activity.
[Method]
Fab and FabLZ (Fab fused with leucine zippers tag) antibody formats previously obtained from mouse were expressed in E. coli Nico21(DE3) strain using the pET system. Two refolding strategies, conventional stepwise dialysis and an automated refolding method using a continuous dilution system (Hoang et al., 2026), were compared to determine their efficiency in recovering functional mAbs. Purification was performed by Immobilized Metal Affinity Chromatography followed by Size Exclusion Chromatography. The binding activity of refolded mAbs were analyzed by ELISA and Bio-Layer Interferometry assay.
[Results]
Obtained results indicate that the automated refolding method is capable of generating mAbs with functional performance comparable to those produced through the conventional stepwise refolding method, while markedly enhancing process efficiency and output. In particular, the automated refolding method produced waste equivalent to only 69% of that generated by the modified stepwise refolding method, used just 47% of the denaturant, and reduced processing time to 40% of the conventional modified stepwise refolding method. These considerable savings in time and materials not only support greater scalability and lower production costs but also contribute to a more environmentally sustainable strategy for recombinant mAb manufacturing in E. coli.
[Conclusion]
This study establishes a scalable, robust mAb production system in E. coli, by combining accumulation of inclusion bodied and refolding strategy. The apparatus is simple to set up and operate, making the platform practical from laboratory to industrial scales and providing a cost-effective, environmentally sustainable alternative for recombinant antibody fragment production without compromising functional quality.
[Reference]
Tung Anh Hoang, Hideo Nakano, Teruyo Ojima-Kato, A strategy for scalable antibody production: the combination of Escherichia coli expression, translation-enhancing peptide and automated refolding system, Bioscience, Biotechnology, and Biochemistry, Volume 90, Issue 3, March 2026, Pages 429–437, https://doi.org/10.1093/bbb/zbaf197.
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