<?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%">Gani, Kayanat</style></author><author><style face="normal" font="default" size="100%">Chirmade, Tejas</style></author><author><style face="normal" font="default" size="100%">Ughade, Santosh</style></author><author><style face="normal" font="default" size="100%">Thulasiram, Hirekodathakallu</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%">Understanding unfolding and refolding of the antibody fragment (Fab) III: mapping covalent and non-covalent interactions during in-vitro refolding of light chain, heavy chain, and Fab.</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%">Antibody fragment</style></keyword><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%">Light and heavy chain</style></keyword><keyword><style  face="normal" font="default" size="100%">rHu Ranibizumab</style></keyword></keywords><dates><year><style  face="normal" font="default" size="100%">2022</style></year><pub-dates><date><style  face="normal" font="default" size="100%">NOV</style></date></pub-dates></dates><volume><style face="normal" font="default" size="100%">187</style></volume><pages><style face="normal" font="default" size="100%">108644</style></pages><language><style face="normal" font="default" size="100%">eng</style></language><abstract><style face="normal" font="default" size="100%">&lt;p&gt;
	In-vitro refolding of the antibody fragments from inclusion bodies is a critical manufacturing bottleneck. We have previously reported that Fab refolding involves the formation of an intermediate. To further decode the refolding pathway, we have mapped the covalent and non-covalent interactions during in-vitro refolding of individual light chain (LC), heavy chain (HC), and intact Fab. Intrinsic fluorescence analysis was performed to map the non -covalent interactions, whereas time-dependent disulfide mapping was performed using LC-MS/MS analysis under non-reducing and reducing atmospheres. Refolding of purified LC, HC, and intact Fab (Biosimilar rHu Ranibi-zumab) was investigated in the study. Under the reducing condition, LC, HC, and intact Fab required around 96, 30, and 70 h, respectively, whereas, under non-reducing conditions, they required only 48, 10, and 48 h, respectively, to acquire the native conformation. The disulfide bond mapping revealed that the bond between LC_Cys23/Cys88 is more unstable. The peptides mapped with the inter-chain disulfide bond were identified at 48 h during the refolding and can be considered as the rate-limiting step in (Fab) refolding. We have also studied a new refolding strategy involving assembly of separately refolded LC and HC into Fab molecule. In this case the Fab refolding yield was low (18.0 +/- 1.36%), indicating inefficient assembly of refolded LC and HC chain into native Fab molecule.&lt;/p&gt;
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	Foreign&lt;/p&gt;
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	4.446&lt;/p&gt;
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