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<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-non02482nas a2200253   4500008004100000022001400041245012400055210006900179260000800248300001100256490000800267520160400275653002901879653002401908653002701932653002201959653001401981653002301995100002202018700001602040700002002056700002002076856013202096       2023                        eng d  a1369-703X00aMapping time dependent disulfide bond formation during in-vitro refolding of recombinant peptibody: a Fc-fusion protein0 aMapping time dependent disulfide bond formation during invitro r  cAUG  a1089690 v1973 a<p>
	Disulfide bonds are commonly found in covalent interactions, which play a vital role in establishing the three-dimensional structure of proteins and maintaining their biological activity. This investigation is focused on time dependant mapping of intra and inter-disulfide bonds during in-vitro refolding of recombinant peptibody using LC-ESI-MS/MS. The selected recombinant peptibody is a homodimeric, aglycosylated Fc-fusion protein expressed in E. coli. The amino acid sequence in the disulfide bond containing peptides was confirmed at MS/MS level using the CID-based fragmentation approach. During in-vitro refolding of peptibody, a peptide with inter -chain disulfide bond is observed post 4 h, with low area intensity, and it reached a maximum in 96 h. How-ever, maximum area intensity was reached for the intra-chain disulfide bond in 72 h. Since cysteine residues responsible for intra-chain disulfide bonds are present in complex part of the Fc domain, formation of intra-chain disulfide bonds may be an overall rate-limiting step in recombinant Romiplostim refolding. Proper folding and contributions by non-covalent interactions will be required to form intra-chain disulfide bond. Monitoring time -based formation of such non-covalent interactions was carried out using intrinsic fluorescence during protein refolding. Observations from intrinsic fluorescence show that 4-6 h is most crucial time for domain formation. Observations from this study will help characterize structural integrity and quality during process and product development for consistent product quality.</p>
10aDisulfide bond formation10aIn -vitro refolding10aIntrinsic fluorescence10aMass spectroscopy10aPeptibody10aTime based mapping1 aKumthekar, Rupali1 aRana, Sunil1 aUghade, Santosh1 aBhambure, Rahul  uhttps://library.ncl.res.in/content/mapping-time-dependent-disulfide-bond-formation-during-vitro-refolding-recombinant-peptibody00762nas a2200205   4500008004100000022001400041245010100055210006900156260000800225300001200233490000800245653002700253653002900280653003500309100002400344700002100368700002200389700002000411856012500431       2024                        eng d  a1359-511300aUnderstanding in-vivo refolding of antibody fragments (Fab): Biosimilar Ranibizumab a case study0 aUnderstanding invivo refolding of antibody fragments Fab Biosimi  cNOV  a484-4970 v14610aCHERICHIA-COLI STRAINS10aDisulfide bond formation10aSOLUBLE RECOMBINANT PROTEINSES1 aTungekar, Aatir, A.1 aFulewar, Padmaja1 aKumthekar, Rupali1 aBhambure, Rahul  uhttps://library.ncl.res.in/content/understanding-vivo-refolding-antibody-fragments-fab-biosimilar-ranibizumab-case-study