@article {48728,
	title = {Chromatography assisted in-vitro refolding and purification of recombinant peptibody: recombinant romiplostim a case study},
	journal = {International Journal of Biological Macromolecules},
	volume = {249},
	year = {2023},
	month = {SEP },
	pages = {126037},
	type = {Article},
	abstract = {<p>
	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 \&amp; PLUSMN; 1.5 \% and a purity of over 79.73 \&amp; 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{\textquoteright}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.</p>
},
	keywords = {CE-SDS, Disulfide bonds, In-vitro refolding, Peptibody, Romiplostim},
	issn = {0141-8130},
	doi = {10.1016/j.ijbiomac.2023.126037},
	author = {Rana, Sunil and Ughade, Santosh and Kumthekar, Rupali and Bhambure, Rahul}
}
@article {49810,
	title = {Characterization of S-homocysteinylation of Human Insulin and Its Implications in Diabetes},
	journal = {Protein Journal},
	volume = {44},
	year = {2025},
	month = {OCT},
	pages = {598-610},
	type = {Article},
	abstract = {<p>
	Homocysteine thiolactone is a reactive thiol known for its interaction with various proteins. Nevertheless, there exists a paucity of information concerning the interaction between homocysteine thiolactone and human insulin, particularly regarding the mechanism by which homocysteine facilitates the reduction of disulfide bonds within insulin. In the present study, we have elucidated the binding sites of homocysteine to the cysteine residues (A6-B7 and A20-B19) that are implicated in the formation of intermolecular disulfide bonds in insulin through an in vitro reaction analyzed via LC-ESI MS/MS. This results in a reduction of disulfide bonds linking the A and B chains, which was corroborated by MALDI-TOF-MS and ESI-MS analysis. The secondary structure of insulin is affected by this modification, as evidenced by circular dichroism spectroscopy. In-silico studies also show that homocysteine affects the insulin structure. A glucose uptake assay conducted in Chinese hamster ovary (CHO) cells that stably express the insulin receptor revealed that HC-modified insulin is less effective in inducing glucose uptake compared to native insulin, suggesting that HC-induced structural modifications in insulin influence functional activity. This study provides insight into the HC-induced structural and functional changes in insulin and discusses the consequent implications for diabetes.</p>
},
	keywords = {Disulfide bonds, Homocysteinylation, insulin, Mass spectrometry},
	issn = {1572-3887},
	doi = {10.1007/s10930-025-10282-9},
	author = {Sheikh, Amreen B. and Jathar, Swaraj M. and Tammara, Vaishnavi and Das, Atanu and Kulkarni, Mahesh J.}
}
