00558nas a2200109 4500008004100000245009500041210006900136260009100205100002000296700002000316856011200336 2017 eng d00aImplementation of QbD for development of a downstream process for a therapeutic biosimilar0 aImplementation of QbD for development of a downstream process fo aSan Francisco, CAbAMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USAcAPR 1 aRathore, Anurag1 aBhambure, Rahul uhttps://library.ncl.res.in/content/implementation-qbd-development-downstream-process-therapeutic-biosimilar02114nas a2200133 4500008004100000245008800041210006900129260002800198520157300226100002001799700002001819700002201839856011901861 2019 eng d00aPopulation balance-enabled model for batch and continuous crystallization processes0 aPopulation balanceenabled model for batch and continuous crystal bJohn Wiley & Sons, Inc.3 a

Crystallization is a critical unit operation in manufacturing of various active pharmaceutical ingredients and drugs. Irrespective of the large-scale use of crystallization in pharmaceutical manufacturing industry, crystallization is still considered as an art. Recently, quality by design (QbD) is gaining industry acceptance as an approach toward development and commercialization of the pharmaceutical drug molecules. In QbD, the unit operations and the process are designed and controlled to deliver specified quality attributes consistently. The proposed chapter is motivated by the necessity of the prediction of crystallization events like onset of the crystallization, nucleation, and growth during crystallization of active pharmaceutical ingredients. The chapter demonstrates the integrated use of population balance modeling framework for developing a fundamental understanding of the kinetic aspects of crystallization and correlating the developed understanding for scale-up and design for the crystallization processes. Efforts are made to discuss the impact of various critical process parameters like metastable zone width, scale effects, and residence time on crystal size during batch and continuous modes with the help of couple of case studies. The presented model and approach will be useful for implementing QbD for crystallizers.

1 aPandit, Ajinkya1 aBhambure, Rahul1 aRanade, Vivek, V. uhttps://library.ncl.res.in/content/population-balance-enabled-model-batch-and-continuous-crystallization-processes02022nas a2200253 4500008004100000022001400041245010800055210006900163260000900232300001100241490000700252520118300259653002301442653002901465653001501494653002601509653001401535100002001549700001701569700001901586700002001605700002001625856012301645 2020 eng d a0734-975000aEngineering staphylococcal protein A for high-throughput affinity purification of monoclonal antibodies0 aEngineering staphylococcal protein A for highthroughput affinity cNOV  a1076320 v443 a

Protein A chromatography is one of the most widely used purification steps in the manufacturing of the various classes of recombinant and non-recombinant antibodies. Due to the higher cost, lower binding capacity, and limited life cycle of Protein A ligand, this affinity-based purification step is often one of the most significant contributors to the cost of manufacturing of monoclonal antibody (mAb) products. In the last decade, there has been significant progress in improving the Protein A chromatography throughput by designing new engineered Staphylococcal Protein A (SPA) variants with higher dynamic binding capacity, considerable alkaline tolerance, and mild acidic elution pH. This review aims at summarizing the various protein engineering approaches used for improving the throughput of the Protein A-based affinity purification of various immunoglobulins. With biopharmaceutical producers operating under ever-increasing pressure towards reducing the cost of manufacturing, these advances in engineered protein A variants will help in processing larger cell culture volumes with high throughput and thereby significantly lower the cost of raw materials.

10aAlkaline tolerance10aDynamic binding capacity10aElution pH10aMonoclonal antibodies10aProtein A1 aAmritkar, Vinod1 aAdat, Satish1 aTejwani, Vijay1 aRathore, Anurag1 aBhambure, Rahul uhttps://library.ncl.res.in/content/engineering-staphylococcal-protein-high-throughput-affinity-purification-monoclonal02423nas 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-study02419nas a2200253 4500008004100000022001400041245015400055210006900209260000800278300001100286490000800297520148800305653002501793653002901818653003501847653003101882653002501913100001701938700002001955700002401975700001801999700002002017856012802037 2021 eng d a1369-703X00aCloning and expression of antibody fragment (Fab) I: effect of expression construct and induction strategies on light and heavy chain gene expression0 aCloning and expression of antibody fragment Fab I effect of expr cDEC a1081890 v1763 aDual promoter expression constructs offer time and cost-effective alternatives to produce multi-domain proteins like antibody fragments. This investigation is focused on understanding the effect of expression construct (dual promoter vs. co-transformation strategy), codon optimization, and induction strategies on yield and expression stoichiometry of LC and HC genes of antibody fragment at shake-flask and bioreactor scale. rHu biosimilar Ranibizumab was selected as a model protein for the study. Expression stoichiometry of HC and LC gene at mRNA level was studied using RTqPCR, whereas protein expression level was studied quantitatively using RP-HPLC and SDS-PAGE analysis. In the case of dual promoter expression construct, it was observed that LC gene cloned in the MCS1 of the duet vectors has > 2-fold expression than the HC gene, cloned in the MCS2. Transcript abundance profile of the HC and LC genes determined at different time intervals post-induction shows a difference in the gene expression at the transcriptional level. Comparative analysis of dual promoter and co-transformation strategy shows better stoichiometry in co-transformation (1:1.3), whereas higher protein yield in a dual expression system (>2.4 fold). The use of lactose and galactose as inducers show higher Fab yield of 2.30 +/- 0.03 g/L and 2.81 +/- 0.06 g/L with expression stoichiometry of 1:1.9 and 1:2 (HC: LC) respectively than IPTG-based induction with a protein yield of 1.40 +/- 0.02 g/L.10aDual promoter vector10aExpression stoichiometry10aHigh cell density fermentation10arHu biosimilar Ranibizumab10aTranscript abundance1 aMehta, Deepa1 aChirmade, Tejas1 aTungekar, Aatir, A.1 aGani, Kayanat1 aBhambure, Rahul uhttps://library.ncl.res.in/content/cloning-and-expression-antibody-fragment-fab-i-effect-expression-construct-and-induction02178nas a2200217 4500008004100000245011100041210006900152260000800221300001200229490000800241520143400249100001801683700001501701700002001716700001801736700002101754700001701775700002001792700002001812856012801832 2022 eng d00aDimerization of SARS-CoV-2 nucleocapsid protein affects sensitivity of ELISA based diagnostics of COVID-190 aDimerization of SARSCoV2 nucleocapsid protein affects sensitivit cMAR a428-4370 v2003 a

Nucleocapsid protein (N protein) is the primary antigen of the virus for development of sensitive diagnostic assays of COVID-19. In this paper, we demonstrate the significant impact of dimerization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N-protein on sensitivity of enzyme-linked immunosorbent assay (ELISA) based diagnostics. The expressed purified protein from E. coli is composed of dimeric and monomeric forms, which have been further characterized using biophysical and immunological techniques. Indirect ELISA indicated elevated susceptibility of the dimeric form of the nucleocapsid protein for identification of protein-specific monoclonal antibody as compared to the monomeric form. This finding also confirmed with the modelled structure of monomeric and dimeric nucleocapsid protein via HHPred software and its solvent accessible surface area, which indicates higher stability and antigenicity of the dimeric type as compared to the monomeric form. The sensitivity and specificity of the ELISA at 95% CI are 99.0% (94.5-99.9) and 95.0% (83.0-99.4), respectively, for the highest purified dimeric form of the N protein. As a result, using the highest purified dimeric form will improve the sensitivity of the current nucleocapsid-dependent ELISA for COVID-19 diagnosis, and manufacturers should monitor and maintain the monomer-dimer composition for accurate and robust diagnostics.

1 aKhan, Wajihul1 aKhan, Nida1 aMishra, Avinash1 aGupta, Surbhi1 aBansode, Vikrant1 aMehta, Deepa1 aBhambure, Rahul1 aRathore, Anurag uhttps://library.ncl.res.in/content/dimerization-sars-cov-2-nucleocapsid-protein-affects-sensitivity-elisa-based-diagnostics02685nas a2200229 4500008004100000022001400041245016600055210006900221260000800290300001600298490000600314520183500320100001802155700002402173700002002197700002302217700002002240700002002260700002002280700002502300856013002325 2022 eng d a2470-134300aInvestigation of the captopril-insulin interaction by mass spectrometry and computational approaches reveals that captopril induces structural changes in insulin0 aInvestigation of the captoprilinsulin interaction by mass spectr cJUL a23115-231260 v73 a

ABSTRACT: Post-translational modifications remarkably regulate proteins' biological function. Small molecules such as reactive thiols, metabolites, and drugs may covalently modify the proteins and cause structural changes. This study reports the covalent modification and noncovalent interaction of insulin and captopril, an FDA-approved antihypertensive drug, through mass spectrometric and computation-based approaches. Mass spectrometric analysis shows that captopril modifies intact insulin, reduces it into its ``A'' and ``B'' chains, and covalently modifies them by forming adducts. Since captopril has a reactive thiol group, it might reduce the insulin dimer or modify it by reacting with cysteine residues. This was proven with dithiothreitol treatment, which reduced the abundance of captopril adducts of insulin A and B chains and intact Insulin. Liquid chromatography tandem mass spectrometric analysis identified the modification of a total of four cysteine residues, two in each of the A and B chains of insulin. These modifications were identified to be Cys6 and Cys7 of the A chain and Cys7 and Cys19 of the B chain. Mass spectrometric analysis indicated that captopril may simultaneously modify the cysteine residues of intact insulin or its subunits A and B chains. Biophysical studies involving light scattering and thioflavin T assay suggested that the binding of captopril to the protein leads to the formation of aggregates. Docking and molecular dynamics studies provided insights into the noncovalent interactions and associated structural changes in insulin. This work is a maiden attempt to understand the detailed molecular interactions between captopril and insulin. These findings suggest that further investigations are required to understand the long-term effect of drugs like captopril.

1 aGhosh, Amrita1 aPawar, Aiswarya, B.1 aChirmade, Tejas1 aJathar, Swaraj, M.1 aBhambure, Rahul1 aSengupta, Durba1 aGiri, Ashok, P.1 aKulkarni, Mahesh, J. uhttps://library.ncl.res.in/content/investigation-captopril-insulin-interaction-mass-spectrometry-and-computational-approaches02210nas a2200265 4500008004100000022001400041245011100055210006900166260000800235300001400243490000700257520132700264653001501591653003001606653002601636653001401662653001501676100002601691700002001717700002501737700001801762700001901780700001701799856012801816 2022 eng d a0268-257500aMechanistic modeling of continuous capture step purification of biosimilar monoclonal antibody therapeutic0 aMechanistic modeling of continuous capture step purification of  cSEP a2404-24190 v973 a

{BACKGROUND Continuous multicolumn Protein A chromatography offers various advantages for capture stage purification of monoclonal antibody therapeutics, like higher productivity and resin capacity utilization, lower buffer consumption, small footprint, etc. Due to the complexity of the continuous process, experimental optimization is time-consuming and cost-intensive. This investigation proposes a hybrid process development approach integrating experimental and mechanistic modeling for time- and cost-effective development and optimization of continuous Protein A affinity chromatography. RESULTS Productivity and capacity utilization of the continuous CaptureSMB process under varying operating conditions were predicted using the Chromatography Analysis and Design Toolkit (CADET) framework and validated with experimental results. Effects of critical process parameters like feed concentration (c(0)), loading breakthrough (s) and residence time (RT) on productivity and capacity utilization were evaluated. Model predictions were validated using the experimental results proving the reliability and feasibility of the modeling approach. At 15.00 +/- 0.20 mg mL(-1) feed model mAb concentration, the model-based approach predicted the best performance giving 27.56 g L-1 h(-1) productivity (RT = 2 min

10aCaptureSMB10acontinuous chromatography10aMathematical modeling10aProtein A10asimulation1 aDeulgaonkar, Prashant1 aBhambure, Rahul1 aPrasad, Bhaskarjyoti1 aMishra, Ashok1 aTiwari, Sanjay1 aMody, Rustom uhttps://library.ncl.res.in/content/mechanistic-modeling-continuous-capture-step-purification-biosimilar-monoclonal-antibody02426nas 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-inter02732nas 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-recombinant02165nas 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-heavy02482nas 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-peptibody02312nas a2200217 4500008004100000022001400041245011500055210006900170260000800239490000700247520154500254653002501799653001901824653001401843653001401857100002901871700001901900700002001919700002701939856012801966 2023 eng d a1860-676800aPost-refolding stability considerations for optimization of in-vitro refolding: L-asparaginase as a case study0 aPostrefolding stability considerations for optimization of invit cAPR0 v183 a

BackgroundL-Asparaginase is an essential enzyme for the food and biopharmaceutical industry. The stability, however, of L-asparaginase is widely known to be an issue. Commercial manufacturing of any biopharmaceutical involves hold-ups during processing, and can result in product loss if stability is an issue, as is the case with L-asparaginase. This interplay of product intermediate stability and process design is the focus of this investigation. Methods and ResultsIn this study, we propose a strategy to simultaneously increase the refolding yield and stability of refolded L-asparaginase so as to improve overall process yield. Using one variable at a time (OVAT) experiments, urea (6 M), solubilized inclusion bodies (15 mg/ml), refolding method (step dilution), and pH (8.6) were identified as significant process parameters. A design of experiment (DOE)-based optimization was then performed for the refolding step. The net outcome was more than a three-fold increase in enzyme recovery (i.e., 4.90 IU/ml) compared to unoptimized conditions (i.e., 1.26 IU/ml). Further, the L-asparaginase process intermediate was found to be stable for more than a week at room temperature and 2-8 degrees C, while the unoptimized sample was stable at 2-8 degrees C but did not show any activity at room temperature after 72 h. ConclusionsThe current study elucidates how process intermediate stability needs to be given due consideration during process optimization, particularly for products such as L-asparaginase which are labile.

10adesign of experiment10aL-asparaginase10arefolding10astability1 aMihooliya, Kanti, Nandan1 aNitika, Nitika1 aBhambure, Rahul1 aRathore, Anurag, S. S. uhttps://library.ncl.res.in/content/post-refolding-stability-considerations-optimization-vitro-refolding-l-asparaginase-case02683nas 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-recombinant02670nas a2200361 4500008004100000022001400041245023500055210007000290260000800360300001000368490000800378520138500386653002601771653002601797653001501823653002501838653001501863653001501878100001701893700001701910700002401927700002001951700001801971700001801989700002702007700003102034700003002065700002002095700001802115700001602133700002502149856013402174 2024 eng d a0168-165600aSustainable bioprocess technology for producing food-flavour (+)-γ-decalactone from castor oil-derived ricinoleic acid using enzymatic activity of Candida parapsilosis: Scale-up optimization and purification using novel composite0 aSustainable bioprocess technology for producing foodflavour γdec cSEP a17-300 v3933 a

Ricinoleic acid (RA) from castor oil was employed in biotransformation of peach-flavoured gamma-decalactone (GDL), using a Candida parapsilosis strain (MTCC13027) which was isolated from waste of pineapple crown base. Using four variables-pH, cell density, amount of RA, and temperature-the biotransformation parameters were optimized using RSM and BBD. Under optimized conditions (pH 6, 10 % of microbial cells, 10 g/L RA at 28 degrees C), the conversion was maximum and resulted to 80 % (+)-GDL (4.4 g/L/120 h) yield in shake flask (500 mL). Furthermore, optimization was achieved by adjusting the aeration and agitation parameters in a 3 L bioreactor, which were then replicated in a 10 L bioreactor to accurately determine the amount of (+)-GDL. In bioreactor condition, 4.7 g/L (>85 %) of (+)-GDL is produced with 20 % and 40 % dissolved oxygen (1.0 vvm) at 150 rpm in 72 h and 66 h, respectively. Further, a new Al-Mg-Ca-Si composite column-chromatography method is developed to purify enantiospecific (+)-GDL (99.9 %). This (+)-GDL is 100 % nature-identical as validated through 14C-radio-carbon dating. Thorough chemical investigation of enantiospecific (+)-GDL is authenticated for its use as flavour. This bioflavour has been developed through a cost-effective biotechnological process in response to the demand from the food industry on commercial scale.

10a(+)-gamma-Decalactone10aAl-Mg-Ca-Si composite10aBioflavour10aCandida parapsilosis10acastor oil10aFood-waste1 aSyed, Naziya1 aSingh, Suman1 aChaturvedi, Shivani1 aKumar, Prashant1 aKumar, Deepak1 aJain, Abhinav1 aSharma, Praveen, Kumar1 aNannaware, Ashween, Deepak1 aChanotiya, Chandan, Singh1 aBhambure, Rahul1 aKumar, Pankaj1 aKalra, Alok1 aRout, Prasant, Kumar uhttps://library.ncl.res.in/content/sustainable-bioprocess-technology-producing-food-flavour-%CE%B3-decalactone-castor-oil-derived00762nas 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