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

Authors
  • Ban, Bhupal1, 2, 3
  • Sharma, Maya
  • Shetty, Jagathpala1, 4
  • 1 Antibody Engineering and Technology Core, University of Virginia, Charlottesville, VA 22904, USA
  • 2 Department of Cell Biology, University of Virginia, Charlottesville, VA 22904, USA
  • 3 Pharmaceutical Biotechnology Center, Indiana Biosciences Research Institutes (IBRI), Indianapolis, IN 46202, USA
  • 4 Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22904, USA
Type
Published Article
Journal
Antibodies
Publisher
MDPI
Publication Date
Aug 05, 2020
Volume
9
Issue
3
Identifiers
DOI: 10.3390/antib9030039
PMID: 32764309
PMCID: PMC7551518
Source
PubMed Central
Keywords
Disciplines
  • Article
License
Green

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.

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