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-study02426nas a2200253   4500008004100000022001400041245018900055210006900244260000800313300001100321490000800332520147300340653002201813653002901835653002301864653002601887653002001913100001801933700002001951700002001971700003301991700002002024856012802044       2022                        eng d  a1369-703X00aUnderstanding 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.0 aUnderstanding unfolding and refolding of the antibody fragment F  cNOV  a1086440 v1873 a<p>
	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.</p>
10aAntibody fragment10aDisulfide bond formation10aIn-vitro refolding10aLight and heavy chain10arHu Ranibizumab1 aGani, Kayanat1 aChirmade, Tejas1 aUghade, Santosh1 aThulasiram, Hirekodathakallu1 aBhambure, Rahul  uhttps://library.ncl.res.in/content/understanding-unfolding-and-refolding-antibody-fragment-fab-iii-mapping-covalent-and-non