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

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.

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

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-non02508nas a2200265 4500008004100000022001400041245014900055210006900204260000800273300001100281490000800292520154400300653002901844653003501873653003501908653003901943653002301982653001602005100002002021700002002041700001802061700001602079700002002095856012702115 2022 eng d a1369-703X00aUnderstanding unfolding and refolding of the antibody fragments (Fab). II. Mapping intra and inter-chain disulfide bonds using mass spectrometry0 aUnderstanding unfolding and refolding of the antibody fragments  cMAY a1084390 v1823 a

Disulfide bond formation in recombinant protein therapeutics has a significant impact on the integrity and biological activity of the drug product. Formation of the disulfide linkage is the key rate-limiting step in in vitro refolding and overall manufacturing of the antibody fragments (Fab). This investigation is focused on mapping the intra, and inter-chain disulfide bonds in the in vitro refolded antibody fragments by using mass spectrometry (MS). Biosimilar rHu Ranibizumab and rHu Certolizumab expressed using E. coli were selected for the study. Both Fabs contain ten cysteine residues leading to two intra-chain disulfide bonds on each subunit and a single interchain disulfide linkage. rHu Certolizumab has an additional cysteine which is unpaired and used for pegylation. The amino acid sequence in the disulfide-bonded peptides was confirmed by Collision-induced dissociation (CID), Electron transfer dissociation (ETD) and High-energy collision dissociation (HCD). The light chain (LC) intra-chain disulfide is formed between Cys23-Cys88 and Cys134-Cys194 in both the Fabs. The heavy chain (HC) intra-chain disulfides are formed between Cys22-Cys96 and Cys150-Cys206 in rHu Ranibizumab. LC and HC subunits of rHu Ranibizumab are covalently linked by disulfide linkage formed between Cys214 of LC and Cys226 of HC. This study suggests that information from multiple MS platforms and orthogonal methods for peptide fragmentation can be effectively used to map disulfide linkages in biosimilar therapeutic proteins.

10aAntibody fragments (Fab)10aCollision-induced dissociation10aElectron transfer dissociation10aHigh-energy collision dissociation10ain vitro refolding10aRanibizumab1 aNainwal, Neeraj1 aChirmade, Tejas1 aGani, Kayanat1 aRana, Sunil1 aBhambure, Rahul uhttps://library.ncl.res.in/content/understanding-unfolding-and-refolding-antibody-fragments-fab-ii-mapping-intra-and-inter00762nas 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