02732nas a2200241 4500008004100000022001400041245012700055210006900182260000900251300001100260490000800271520191900279653001102198653002002209653002302229653001402252653001602266100001602282700002002298700002202318700002002340856013002360 2023 eng d a0141-813000aChromatography assisted in-vitro refolding and purification of recombinant peptibody: recombinant romiplostim a case study0 aChromatography assisted invitro refolding and purification of re cSEP  a1260370 v2493 a

In-vitro protein refolding is one of the key rate-limiting unit operations in manufacturing of fusion proteins such as peptibodies expressed using E. coli. Dilution-assisted refolding is the most commonly used industrial practice to achieve the soluble, native functional form of the recombinant protein from the inclusion bodies. This study is focused on developing a chromatography-assisted in-vitro refolding platform to produce the biologically active, native form of recombinant peptibody. Recombinant Romiplostim was selected as a model protein for the study. A plug flow tubular reactor was connected in series with capture step affinity chromatography to achieve simultaneous in-vitro refolding and capture step purification of recombinant Romiplostim. Effect of various critical process parameters like fold dilution, temperature, residence time, and Cysteine: DTT ratio was studied using a central composite based design of experiment strategy to achieve a maximum refolding yield of selected peptibody. Under optimum refolding conditions, the maximum refolding yield of 57.0 & PLUSMN; 1.5 % and a purity of over 79.73 & PLUSMN; 3.4 % were achieved at 25-fold dilution, 15 degrees C temperature, 6 h residence time with 6 mM and 10 mM of cysteine and DTT, respectively. The formation of native peptibody structure was examined using various orthogonal analytical tools to study the protein's primary, secondary, and tertiary structure. The amino acid sequence for the disulfide-linked peptide was mapped using collision-induced dissociation (CID) to confirm the formation of interchain disulfide bonds between Cys7-Cys7 and Cys10-Cys10 similarly for intra-chain disulfide bonds between Cys42-Cys102, and Cys148-Cys206. The developed protocol here is a valuable tool to identify high-yield scalable refolding conditions for multi-domain proteins involving inter-domain disulfide bonds.

10aCE-SDS10aDisulfide bonds10aIn-vitro refolding10aPeptibody10aRomiplostim1 aRana, Sunil1 aUghade, Santosh1 aKumthekar, Rupali1 aBhambure, Rahul uhttps://library.ncl.res.in/content/chromatography-assisted-vitro-refolding-and-purification-recombinant-peptibody-recombinant02482nas 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

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.

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-peptibody02683nas a2200205 4500008004100000022001400041245013800055210006900193260000900262300001400271490000600285520194300291100002002234700001602254700001702270700002202287700002102309700002002330856012702350 2024 eng d a2470-134300aMechanistic modeling of size exclusion chromatography-assisted in vitro refolding of the recombinant biosimilar teriparatide (PTH-34)0 aMechanistic modeling of size exclusion chromatographyassisted in cJAN  a3204-32160 v93 a

In vitro protein refolding is one of the critical unit operations in manufacturing recombinant peptides expressed using Escherichia coli as host cells. This study is focused on designing size exclusion chromatography-assisted in vitro refolding process for biosimilar recombinant parathyroid hormone. Inclusion bodies (IBs) of recombinant parathyroid hormone were solubilized at higher pH, and in vitro refolding was performed using size exclusion chromatography. In the first part of the investigation, DoE-based empirical optimization was performed to achieve a higher refolding yield for a biosimilar recombinant parathyroid hormone. The effect of solubilized inclusion body (IB) feed volume, concentration of IBs, and residence time on in vitro refolding of recombinant teriparatide was studied using the Box-Behnken design. Size exclusion chromatography (SEC)-assisted in vitro refolding was performed at 8 C-degrees at pH 10.5 by using 20 mM Tris buffer. The maximum refolding yield of 98.12% was achieved at feed volume (12.5% of CV) and 20 mg/mL inclusion body (IB) concentration with a residence time of 50 min and a purity of 66.1% based on densitometric analysis using SDS-PAGE. In the latter part of the investigation, the general rate mechanistic model framework for size exclusion chromatography was developed and validated with the experimental results. The developed model helped in the accurate prediction of the elution volumes and product yield. The developed model also helps to predict the elution performance of a scalable column a priori. Post in vitro refolding, the formation of the native peptide structure was examined using various orthogonal analytical tools to study the protein's primary, secondary, and tertiary structures. The developed hybrid process development approach is a valuable tool toachieve high-yield, scalable refolding conditions for recombinant proteins without disulfide bonds.

1 aUghade, Santosh1 aRana, Sunil1 aNadeem, Mohd1 aKumthekar, Rupali1 aMahajani, Sanjay1 aBhambure, Rahul uhttps://library.ncl.res.in/content/mechanistic-modeling-size-exclusion-chromatography-assisted-vitro-refolding-recombinant00762nas 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