@article {48081,
	title = {Understanding 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.},
	journal = {Biochemical Engineering Journal},
	volume = {187},
	year = {2022},
	month = {NOV},
	pages = {108644},
	type = {Article},
	abstract = {<p>
	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.</p>
},
	keywords = {Antibody fragment, Disulfide bond formation, In-vitro refolding, Light and heavy chain, rHu Ranibizumab},
	issn = {1369-703X},
	doi = {10.1016/j.bej.2022.108644},
	author = {Gani, Kayanat and Chirmade, Tejas and Ughade, Santosh and Thulasiram, Hirekodathakallu 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}
}
