@article {47590,
	title = {Cloning and expression of antibody fragment (Fab) I: effect of expression construct and induction strategies on light and heavy chain gene expression},
	journal = {Biochemical Engineering Journal},
	volume = {176},
	year = {2021},
	month = {DEC},
	pages = {108189},
	type = {Article},
	abstract = {Dual 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.},
	keywords = {Dual promoter vector, Expression stoichiometry, High cell density fermentation, rHu biosimilar Ranibizumab, Transcript abundance},
	issn = {1369-703X},
	doi = {10.1016/j.bej.2021.108189},
	author = {Mehta, Deepa and Chirmade, Tejas and Tungekar, Aatir A. and Gani, Kayanat and Bhambure, Rahul}
}
@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 {48954,
	title = {Cloning and expression of antibody fragment (Fab) II: Effect of expression hosts on light and heavy chain gene expression},
	journal = {Biochemical Engineering Journal },
	volume = {200},
	year = {2023},
	month = {NOV},
	type = {Journal Article},
	abstract = {<p>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.</p>
},
	keywords = {E.coli host strains, Expression stoichiometry, Proteome analysis, rHu biosimilar Ranibizumab, Transcript abundance},
	issn = {1369-703X},
	doi = {10.1016/j.bej.2023.109094},
	author = {Mehta, Deepa and Chirmade, Tejas and Nainwal, Neeraj and Bhambure, Rahul}
}
