@article {46601,
	title = {Understanding unfolding and refolding of the antibody fragment (Fab). I. In-vitro study},
	journal = {Biochemical Engineering Journal},
	volume = {164},
	year = {2020},
	month = {DEC},
	pages = {107764},
	type = {Article},
	abstract = {<p>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.</p>
},
	keywords = {Antibody fragment, In-vitro refolding, Refolding kinetics, rHu Ranibizumab, Two-state and Three-state models},
	issn = {1369-703X},
	doi = {10.1016/j.bej.2020.107764},
	author = {Gani, Kayanat and Bhambure, Rahul and Deulgaonkar, Prashant and Mehta, Deepa and Kamble, Manoj}
}
@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}
}
