@article {46929,
	title = {Enhancing titers and productivity of rCHO clones with a combination of an optimized fed-batch process and ER-stress adaptation},
	journal = {Journal of Biotechnology},
	volume = {311},
	year = {2020},
	month = {MAR},
	pages = {49-58},
	type = {Article},
	abstract = {<p>To increase the productivity of rCHO cells, many cell engineering approaches have been demonstrated that over-express or knockout a specific gene to achieve increased titers. In this work, we present an alternate approach, based on the concept of evolutionary adaptation, to achieve cells with higher titers. rCHO cells, producing a monoclonal antibody, are adapted to ER-stress, by continuous culturing under increasing concentration of tunicamycin. A sustained higher productivity of at-least 2-fold was achieved in all the clones, in a concentration-dependent manner. Similarly, a 1.5-2 fold increase in final titers was also achieved in the batch culture. Based on metabolic analysis of the adapted cells, a fed-batch process was designed where significantly higher titersare achieved as compared to control. Metabolic flux analysis is employed in addition with gene expression analysis of key genes to understand the basis of increased performance of the adapted cells. Overall, this work illustrates how process modifications and cellular adaptation can be used in synergy to drive up product titers.</p>
},
	keywords = {Adaptive laboratory evolution, CHO cells, ER-stress, Fed-batch, Increased productivity and titers, Recombinant protein production, UPR pathway},
	issn = {0168-1656},
	doi = {10.1016/j.jbiotec.2020.02.008},
	author = {Chandrawanshi, Vikas and Kulkarni, Rohan and Prabhu, Anuja and Mehra, Sarika}
}
@article {48348,
	title = {Engineering nucleotide sugar synthesis pathways for independent and simultaneous modulation of N-glycan galactosylation and fucosylation in CHO cells},
	journal = {Metabolic Engineering},
	volume = {74},
	year = {2022},
	month = {NOV},
	pages = {61-71},
	type = {Article},
	abstract = {<p>
	Glycosylation of recombinant therapeutics like monoclonal antibodies (mAbs) is a critical quality attribute. N-glycans in mAbs are known to affect various effector functions, and thereby therapeutic use of such glycoproteins can depend on a particular glycoform profile to achieve desired efficacy. However, there are currently limited options for modulating the glycoform profile, which depend mainly on over-expression or knock-out of glyco-syltransferase enzymes that can introduce or eliminate specific glycans but do not allow predictable glycoform modulation over a range of values. In this study, we demonstrate the ability to predictably modulate the gly-coform profile of recombinant IgG. Using CRISPR/Cas9, we have engineered nucleotide sugar synthesis pathways in CHO cells expressing recombinant IgG for combinatorial modulation of galactosylation and fucosylation. Knocking out the enzymes UDP-galactose 4 {\textquoteleft}-epimerase (Gale) and GDP-L-fucose synthase (Fx) resulted in ablation of de novo synthesis of UDP-Gal and GDP-Fuc. With Gale knock-out, the array of N-glycans on recom-binantly expressed IgG is narrowed to agalactosylated glycans, mainly A2F glycan (89\%). In the Gale and Fx double knock-out cell line, agalactosylated and afucosylated A2 glycan is predominant (88\%). In the double knock-out cell line, galactosylation and fucosylation was entirely dependent on the salvage pathway, which allowed for modulation of UDP-Gal and GDP-Fuc synthesis and intracellular nucleotide sugar availability by controlling the availability of extracellular galactose and fucose. We demonstrate that the glycoform profile of recombinant IgG can be modulated from containing predominantly agalactosylated and afucosylated glycans to up to 42\% and 96\% galactosylation and fucosylation, respectively, by extracellular feeding of sugars in a dose-dependent manner. By simply varying the availability of extracellular galactose and/or fucose, galactosylation and fucosylation levels can be simultaneously and independently modulated. In addition to achieving the pro-duction of tailored glycoforms, this engineered CHO host platform can cater to the rapid synthesis of variably glycoengineered proteins for evaluation of biological activity.</p>
},
	keywords = {Antibody, Cell engineering, Fucosylation, Fx, Galactosylation, Gale, glycosylation, Nucleotide sugar synthesis, Recombinant therapeutics},
	issn = {1096-7176},
	doi = {10.1016/j.ymben.2022.09.003},
	author = {Prabhu, Anuja and Shanmugam, Dhanasekaran and Gadgil, Mugdha}
}
