02426nas 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

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.

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