02508nas 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-inter02165nas a2200229 4500008004100000022001400041245012600055210006900181260000800250490000800258520133400266653002401600653002901624653002201653653003101675653002501706100001701731700002001748700002001768700002001788856012701808 2023 eng d a1369-703X00aCloning and expression of antibody fragment (Fab) II: Effect of expression hosts on light and heavy chain gene expression0 aCloning and expression of antibody fragment Fab II Effect of exp cNOV0 v2003 a

Protein expression stoichiometry of individual subunits plays a critical role in the formation of multi-domain functional therapeutic proteins like antibody fragments. In this study, we have evaluated light to heavy chain ratio (LC: HC) of rHu Ranibizumab expressed using duet expression vector in six E. coli strains. The LC: HC ratio at the protein level was determined using RP-HPLC whereas transcript abundance of LC and HC at the mRNA level was determined using RTqPCR. Protein level study as well as the transcript abundance profiles of the LC and HC genes were observed to be differentially expressed across the E. coli strains. BL21 STAR (DE3) and Origami 2 strains resulted in a nearly equal expression ratio of LC and HC genes. Untargeted shotgun proteomics analysis was performed to identify the differentially expressed proteins. Total 1083 proteins were identified in the spectral IDA library, while 244 and 149 proteins were differentially expressed in Origami 2 and BL21 STAR (DE3) respectively. These proteins are associated with translation and nucleotide biosynthesis pathways. Ob-tained results provide insights into the impact of different E.coli host strains on LC: HC ratio, the probable reasons behind the unbalanced gene ratio, and provide useful transcriptomic and proteomics analysis protocols.

10aE.coli host strains10aExpression stoichiometry10aProteome analysis10arHu biosimilar Ranibizumab10aTranscript abundance1 aMehta, Deepa1 aChirmade, Tejas1 aNainwal, Neeraj1 aBhambure, Rahul uhttps://library.ncl.res.in/content/cloning-and-expression-antibody-fragment-fab-ii-effect-expression-hosts-light-and-heavy