@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 {49232,
	title = {Understanding in-vivo refolding of antibody fragments (Fab): Biosimilar Ranibizumab a case study},
	journal = {PROCESS BIOCHEMISTRY},
	volume = {146},
	year = {2024},
	month = {NOV},
	pages = {484-497},
	type = {Journal Article},
	keywords = {CHERICHIA-COLI STRAINS, Disulfide bond formation, SOLUBLE RECOMBINANT PROTEINSES},
	issn = {1359-5113},
	doi = {10.1016/j.procbio.2024.09.027},
	author = {Tungekar, Aatir A. and Fulewar, Padmaja and Kumthekar, Rupali and Bhambure, Rahul}
}
