Disulfide bonding in recombinant protein therapeutics have significant impact on integrity and biological activity of the drug product. Mapping the disulfide bond position provide insights into the structural integrity of the therapeutic protein. Disulfide bond formation is the key rate limiting step in <i>in-vitro</i> refolding and overall manufacturing of antibody fragments. Inappropriate disulfide bond formation lead to aggregation and loss of the biological activity for therapeutic proteins. In this study we have investigated the formation of inter-chain and intra-chain  disulfide bond formation in two antibody fragments: rHu biosimilar Ranibizumab and rHu biosimilar Certolizumab. Both Fabs were expressed in E. coli as inclusion bodies, and subsequently extracted and in vitro refolded to obtain the Fabs. Unfolding and refolding of the purified Fabs revealed the dynamic nature of the disulfide make-and-break. LC-MS/MS analysis of the time-course experiment to map the disulfide bonds during the refolding of the Fabs reveal the inter-chain disulfie bond formation as the rate limiting step to form native Fab.

Mapping disulfide bond formation during in vitro refolding of antibody fragments

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.