02423nas a2200253   4500008004100000022001400041245009200055210006900147260000800216300001100224490000800235520158900243653002201832653002301854653002301877653002001900653003701920100001801957700002001975700002601995700001702021700001802038856011302056       2020                        eng d  a1369-703X00aUnderstanding unfolding and refolding of the antibody fragment (Fab). I. In-vitro study0 aUnderstanding unfolding and refolding of the antibody fragment F  cDEC  a1077640 v1643 a<p>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.</p>
10aAntibody fragment10aIn-vitro refolding10aRefolding kinetics10arHu Ranibizumab10aTwo-state and Three-state models1 aGani, Kayanat1 aBhambure, Rahul1 aDeulgaonkar, Prashant1 aMehta, Deepa1 aKamble, Manoj  uhttps://library.ncl.res.in/content/understanding-unfolding-and-refolding-antibody-fragment-fab-i-vitro-study