A novel strategy for efficient expression of an antibody fragment in Escherichia coli: ranibizumab as a case study

Abstract

BACKGROUND: Complex biotherapeutics such as non-glycosylated antibody fragments can be easily manufactured in Escherichia coli due to advancements in recombinant DNA technology. Ranibizumab is one such example of a complex, nonglycosylated protein used to treat age-related macular degeneration and macular oedema. Ranibizumab, a humanized monoclonal antibody fragment, is often expressed as inclusion bodies using the E. coli expression system. In this study, we propose a novel strategy for achieving efficient expression of antibody fragments in E. coli BL21 (DE3). Here, the whole antibody fragment (heavy chain + light chain of ranibizumab) has been engineered and cloned in a pET series vector under single promoter belonging to a prokaryotic host along with an extra ribosome binding site to allow equal expression of the light and heavy chains.RESULTS: We demonstrate heterologous expression of ranibizumab with a protein concentration of 0.4 ± 0.019 mg mL -1 , confirmed using reversed-phase high-performance liquid chromatography with the double copy clone. The reduced and refolded antibody fragment have been analytically characterized using liquid chromatography-mass spectrometry, where masses of the in-house clone were in correspondence with marketed product. Native polyacrylamide gel electrophoresis and surface plasmon resonance (SPR) analyses were performed to confirm the formation of purified and active recombinant product. Binding kinetics to the target analyte vascular endothelial growth factor of refolded in-house product was found to be similar to the marketed product as per SPR (12.7 nmol L −1 vs. 10.4 nmol L −1 ).CONCLUSION: Cloning and process optimization have been performed to enhance expression yield. The proposed strategy offers an efficient approach for enhanced production of antibody fragments in microbial hosts.

Cole‐Cole modeling of real‐time capacitance data for estimation of cell physiological properties in recombinant Escherichia coli cultivation

“…The batch was harvested (approximately around 12th h), and the therapeutic protein was obtained in the form of inclusion bodies which were subjected to further downstream processing. The details regarding the cloning and expression of the therapeutic product, media, and operating conditions have been explained elsewhere (Priyanka et al, 2022; Priyanka & Rathore, 2021; Swaminathan et al, 2020).…”