02508nas a2200265   4500008004100000022001400041245014900055210006900204260000800273300001100281490000800292520154400300653002901844653003501873653003501908653003901943653002301982653001602005100002002021700002002041700001802061700001602079700002002095856012702115       2022                        eng d  a1369-703X00aUnderstanding unfolding and refolding of the antibody fragments (Fab). II. Mapping intra and inter-chain disulfide bonds using mass spectrometry0 aUnderstanding unfolding and refolding of the antibody fragments   cMAY  a1084390 v1823 a<p>
	Disulfide bond formation in recombinant protein therapeutics has a significant impact on the integrity and biological activity of the drug product. Formation of the disulfide linkage is the key rate-limiting step in in vitro refolding and overall manufacturing of the antibody fragments (Fab). This investigation is focused on mapping the intra, and inter-chain disulfide bonds in the in vitro refolded antibody fragments by using mass spectrometry (MS). Biosimilar rHu Ranibizumab and rHu Certolizumab expressed using E. coli were selected for the study. Both Fabs contain ten cysteine residues leading to two intra-chain disulfide bonds on each subunit and a single interchain disulfide linkage. rHu Certolizumab has an additional cysteine which is unpaired and used for pegylation. The amino acid sequence in the disulfide-bonded peptides was confirmed by Collision-induced dissociation (CID), Electron transfer dissociation (ETD) and High-energy collision dissociation (HCD). The light chain (LC) intra-chain disulfide is formed between Cys23-Cys88 and Cys134-Cys194 in both the Fabs. The heavy chain (HC) intra-chain disulfides are formed between Cys22-Cys96 and Cys150-Cys206 in rHu Ranibizumab. LC and HC subunits of rHu Ranibizumab are covalently linked by disulfide linkage formed between Cys214 of LC and Cys226 of HC. This study suggests that information from multiple MS platforms and orthogonal methods for peptide fragmentation can be effectively used to map disulfide linkages in biosimilar therapeutic proteins.</p>
10aAntibody fragments (Fab)10aCollision-induced dissociation10aElectron transfer dissociation10aHigh-energy collision dissociation10ain vitro refolding10aRanibizumab1 aNainwal, Neeraj1 aChirmade, Tejas1 aGani, Kayanat1 aRana, Sunil1 aBhambure, Rahul  uhttps://library.ncl.res.in/content/understanding-unfolding-and-refolding-antibody-fragments-fab-ii-mapping-intra-and-inter