<?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%">Bhambure, Rahul</style></author><author><style face="normal" font="default" size="100%">Deulgaonkar, Prashant</style></author><author><style face="normal" font="default" size="100%">Mehta, Deepa</style></author><author><style face="normal" font="default" size="100%">Kamble, Manoj</style></author></authors></contributors><titles><title><style face="normal" font="default" size="100%">Understanding unfolding and refolding of the antibody fragment (Fab). I. In-vitro study</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%">In-vitro refolding</style></keyword><keyword><style  face="normal" font="default" size="100%">Refolding kinetics</style></keyword><keyword><style  face="normal" font="default" size="100%">rHu Ranibizumab</style></keyword><keyword><style  face="normal" font="default" size="100%">Two-state and Three-state models</style></keyword></keywords><dates><year><style  face="normal" font="default" size="100%">2020</style></year><pub-dates><date><style  face="normal" font="default" size="100%">DEC</style></date></pub-dates></dates><volume><style face="normal" font="default" size="100%">164</style></volume><pages><style face="normal" font="default" size="100%">107764</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 protein refolding is a major rate-limiting step in the large scale production of antibody fragments expressed using a microbial source like E. coli. This investigation is focused on understanding the in-vitro unfolding and refolding of the multi-domain protein involving inter-domain disulfide linkage, like antibody fragment (Fab). Solubilization behavior of the inclusion bodies and unfolding events of Fab fragment (Biosimilar rHu Ranibizumab) were studied using nano-differential scanning fluorimetry (nano-DSF). Fab unfolding behavior was studied by fitting experimental data with the two-state and three-state thermodynamic model. Based on the Fab unfolding understanding, a two-stage design of experiment (DoE) strategy was used for the optimization of the in-vitro refolding condition of a Fab fragment. Refolding yield of 56.03 +/- 1.15 % was achieved using the optimized oxidative refolding conditions maintained by appropriate dilution factor and redox reagent ratio. Refolding kinetics of the rHu Ranibizumab was analyzed using a three-parameter kinetic model showing rate constant k(1) :7.05e(-6) l/mg.min, k(2) :0.57 l/mg.min, and k(3) :310.19 l/mg.min. Based on observed refolding kinetics, it was concluded that the Fab refolding follows a three-state mechanism with the refolding intermediate/(s) formation from light and heavy chain of the Fab fragment as an overall rate-limiting step. The method described here is a useful tool to identify high-yield scalable refolding conditions for multi-domain proteins involving inter-domain disulfide bonds.&lt;/p&gt;
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