@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 {48707,
	title = {Mapping time dependent disulfide bond formation during in-vitro refolding of recombinant peptibody: a Fc-fusion protein},
	journal = {Biochemical Engineering Journal},
	volume = {197},
	year = {2023},
	month = {AUG},
	pages = {108969},
	type = {Article},
	abstract = {<p>
	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.</p>
},
	keywords = {Disulfide bond formation, In -vitro refolding, Intrinsic fluorescence, Mass spectroscopy, Peptibody, Time based mapping},
	issn = {1369-703X},
	doi = {10.1016/j.bej.2023.108969},
	author = {Kumthekar, Rupali and Rana, Sunil and Ughade, Santosh and Bhambure, Rahul}
}
