02482nas a2200253   4500008004100000022001400041245012400055210006900179260000800248300001100256490000800267520160400275653002901879653002401908653002701932653002201959653001401981653002301995100002202018700001602040700002002056700002002076856013202096       2023                        eng d  a1369-703X00aMapping time dependent disulfide bond formation during in-vitro refolding of recombinant peptibody: a Fc-fusion protein0 aMapping time dependent disulfide bond formation during invitro r  cAUG  a1089690 v1973 a<p>
	Disulfide bonds are commonly found in covalent interactions, which play a vital role in establishing the three-dimensional structure of proteins and maintaining their biological activity. This investigation is focused on time dependant mapping of intra and inter-disulfide bonds during in-vitro refolding of recombinant peptibody using LC-ESI-MS/MS. The selected recombinant peptibody is a homodimeric, aglycosylated Fc-fusion protein expressed in E. coli. The amino acid sequence in the disulfide bond containing peptides was confirmed at MS/MS level using the CID-based fragmentation approach. During in-vitro refolding of peptibody, a peptide with inter -chain disulfide bond is observed post 4 h, with low area intensity, and it reached a maximum in 96 h. How-ever, maximum area intensity was reached for the intra-chain disulfide bond in 72 h. Since cysteine residues responsible for intra-chain disulfide bonds are present in complex part of the Fc domain, formation of intra-chain disulfide bonds may be an overall rate-limiting step in recombinant Romiplostim refolding. Proper folding and contributions by non-covalent interactions will be required to form intra-chain disulfide bond. Monitoring time -based formation of such non-covalent interactions was carried out using intrinsic fluorescence during protein refolding. Observations from intrinsic fluorescence show that 4-6 h is most crucial time for domain formation. Observations from this study will help characterize structural integrity and quality during process and product development for consistent product quality.</p>
10aDisulfide bond formation10aIn -vitro refolding10aIntrinsic fluorescence10aMass spectroscopy10aPeptibody10aTime based mapping1 aKumthekar, Rupali1 aRana, Sunil1 aUghade, Santosh1 aBhambure, Rahul  uhttps://library.ncl.res.in/content/mapping-time-dependent-disulfide-bond-formation-during-vitro-refolding-recombinant-peptibody