Optimization of Methods for the Production and Refolding of Biologically Active Disulfide Bond-Rich Antibody Fragments in Microbial Hosts.

Bhupal Ban,Bhupal Ban,Maya Sharma,Jagathpala Shetty

doi:10.3390/antib9030039

Bhupal Ban, Bhupal Ban + Show 2 more

Open Access

https://doi.org/10.3390/antib9030039

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Abstract

Antibodies have been used for basic research, clinical diagnostics, and therapeutic applications. Escherichia coli is one of the organisms of choice for the production of recombinant antibodies. Variable antibody genes have canonical and non-canonical disulfide bonds that are formed by the oxidation of a pair of cysteines. However, the high-level expression of an antibody is an inherent problem to the process of disulfide bond formation, ultimately leading to mispairing of cysteines which can cause misfolding and aggregation as inclusion bodies (IBs). This study demonstrated that fragment antibodies are either secreted to the periplasm as soluble proteins or expressed in the cytoplasm as insoluble inclusion bodies when expressed using engineered bacterial host strains with optimal culture conditions. It was observed that moderate-solubilization and an in vitro matrix that associated refolding strategies with redox pairing more correctly folded, structured, and yielded functionally active antibody fragments than the one achieved by a direct dilution method in the absence of a redox pair. However, natural antibodies have canonical and non-canonical disulfide bonds that need a more elaborate refolding process in the presence of optimal concentrations of chaotropic denaturants and redox agents to obtain correctly folded disulfide bonds and high yield antibodies that retain biological activity.

Highlights

Monoclonal antibodies are key biologics for several applications such as basic science research, therapeutics protein, and clinical diagnostics assays such as immunoassays, immunohistochemistry, flow cytometry, and immuno-PET scanning
The genes of the variable heavy chain and variable light chain regions from human-derived anti-Ebola (HumscFv), rabbit derived anti-peptide specific antibody (RabscFv), and llama derived anti-human serum albumin (HSA) were obtained from different sources as described in method
The refolded and purified antibody microbial hosts, as it results in the formation of inclusion bodies [31]

Summary

Introduction

Monoclonal antibodies (mAbs) are key biologics for several applications such as basic science research, therapeutics protein, and clinical diagnostics assays such as immunoassays, immunohistochemistry, flow cytometry, and immuno-PET scanning. Advancements in the area of immunoinformatic and recombinant antibodies have changed the applications of domain antibodies such as the single chain fragment variable (scFv), the antigen binding fragment (Fab), and single domain of variable heavy homodimers (VHH) are very popular reagents for basic scientific and clinical applications [2]. The nature of the antibody sequence and structural variability of Abs is necessity to obtain efficient reagents that are possibly simple to use. It implies the necessity of developing customized expression and purification procedures to obtain enough yield.

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