<?xml version="1.0" encoding="UTF-8"?><xml><records><record><source-app name="Biblio" version="7.x">Drupal-Biblio</source-app><ref-type>17</ref-type><contributors><authors><author><style face="normal" font="default" size="100%">Kumthekar, Rupali</style></author><author><style face="normal" font="default" size="100%">Rana, Sunil</style></author><author><style face="normal" font="default" size="100%">Ughade, Santosh</style></author><author><style face="normal" font="default" size="100%">Bhambure, Rahul</style></author></authors></contributors><titles><title><style face="normal" font="default" size="100%">Mapping time dependent disulfide bond formation during in-vitro refolding of recombinant peptibody: a Fc-fusion protein</style></title><secondary-title><style face="normal" font="default" size="100%">Biochemical Engineering Journal</style></secondary-title></titles><keywords><keyword><style  face="normal" font="default" size="100%">Disulfide bond formation</style></keyword><keyword><style  face="normal" font="default" size="100%">In -vitro refolding</style></keyword><keyword><style  face="normal" font="default" size="100%">Intrinsic fluorescence</style></keyword><keyword><style  face="normal" font="default" size="100%">Mass spectroscopy</style></keyword><keyword><style  face="normal" font="default" size="100%">Peptibody</style></keyword><keyword><style  face="normal" font="default" size="100%">Time based mapping</style></keyword></keywords><dates><year><style  face="normal" font="default" size="100%">2023</style></year><pub-dates><date><style  face="normal" font="default" size="100%">AUG</style></date></pub-dates></dates><volume><style face="normal" font="default" size="100%">197</style></volume><pages><style face="normal" font="default" size="100%">108969</style></pages><language><style face="normal" font="default" size="100%">eng</style></language><abstract><style face="normal" font="default" size="100%">&lt;p&gt;
	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.&lt;/p&gt;
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	Foreign&lt;/p&gt;
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	3.9&lt;/p&gt;
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