@article {44421, title = {Zinc supplementation decreases galactosylation of recombinant IgG in CHO cells}, journal = {Applied Microbiology and Biotechnology}, year = {2018}, month = {MAY}, pages = {1-11}, type = {Journal Article}, abstract = {

Trace element composition of culture medium can be altered to modulate glycoform of recombinant glycoproteins. In this study, we show that Zn2+ supplementation at or above 100 μM decreases galactosylation of recombinant IgG expressed in Chinese Hamster Ovary cells. This decrease in galactosylation is not due to reduced galactosyltransferase expression. This effect persists upon supplementation of galactose and uridine to the culture, indicating that it may not be due to reduced UDP-Gal availability. Measurements of galactosyltransferase activity in the cell lysate show that activity decreases with increasing Zn2+/Mn2+ ratio. This suggests that one possible explanation of the effect of Zn2+ may be reduced intracellular galactosyltransferase activity due to increase in Zn2+/Mn2+ ratio. Consistent with this, the decrease in galactosylation of IgG could be reversed by supplementation of Mn2+ (a cofactor of galactosyltransferase) which increases intracellular Mn2+ content. Measurement of total intracellular Zn2+ content, however, indicates no significant upregulation of total intracellular Zn2+ content and no significant downregulation of intracellular Mn2+ content with Zn2+ supplementation. One possible explanation could be that cellular detoxification response to higher extracellular Zn2+ concentration might lead to changes in intracellular distribution of Mn2+. In this case, Zn2+ supplementation would be expected to interfere with other known effects of Mn2+. Indeed, the previously reported increase in high mannose glycans upon Mn2+ supplementation in the absence of glucose is reversed by Zn2+ supplementation. This study also suggests the use of Mn2+ supplementation as a strategy to overcome the effect of lot-to-lot variability in trace element concentrations on galactosylation.

}, keywords = {CHO cells, glycosylation, manganese, Trace metals, zinc}, issn = {1757598}, doi = {10.1007/s00253-018-9064-8}, author = {Prabhu, Anuja}, editor = {Gadre, Ramchandra} } @article {46354, title = {Superfluous glutamine synthetase activity in Chinese Hamster Ovary cells selected under glutamine limitation is growth limiting in glutamine-replete conditions and can be inhibited by serine}, journal = {Biotechnology Progress}, volume = {35}, year = {2019}, month = {SEP-OCT}, pages = {UNSP e2856}, type = {Article}, abstract = {

Passaging and expansion of animal cells in lean maintenance medium could result in periods of limitation of some nutrients. Over time, such stresses could possibly result in selection of cells with metabolic changes and contribute to heterogeneity. Here, we investigate whether selection of Chinese Hamster Ovary (CHO) cells under glutamine limitation results in changes in growth under glutamine-replete conditions. In glutamine-limiting medium, compared to control cells passaged in glutamine-rich medium, the selected cells showed higher glutamine synthetase (GS) activity and attained a higher peak viable cell density (PVCD). Surprisingly, in glutamine-replete conditions, selected cells still showed a higher GS activity but a lower PVCD. We show that in glutamine-replete medium, PVCD of selected cells was restored on (a) inhibition of GS activity with methionine sulfoximine, (b) supplementation of aspartate-without affecting GS activity, and (c) supplementation of serine, which is reported to inhibit GS in vitro. Consistent with the reported effect of serine, inhibition of GS activity was observed upon serine supplementation along with reduced growth of cells under glutamine-limiting conditions. The latter observation is important for the design of glutamine-free culture medium and feed used for GS-CHO and GS-NS0. In summary, we show that CHO cells selected under glutamine limitation have superfluous GS activity in glutamine-replete medium, which negatively affects their PVCD. This may be due to its effect on availability of aspartate which was the limiting nutrient for the growth of selected cells in glutamine-replete conditions.

}, keywords = {C-13 tracer analysis, CHO cells, glutamine limitation, Glutamine synthetase, GS-CHO, GS-NS0, selection under nutrient limitation, serine}, issn = {8756-7938}, doi = {10.1002/btpr.2856}, author = {Maralingannavar, Vishwanathgouda and Parmar, Dharmeshkumar and Panchagnula, Venkateswarlu and Gadgil, Mugdha} } @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 = {

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.

}, 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 {46957, title = {Towards in situ continuous feeding via controlled release of complete nutrients for fed-batch culture of animal cells}, journal = {Biochemical Engineering Journal}, volume = {154}, year = {2020}, month = {FEB }, pages = {107436}, type = {Article}, abstract = {

Small-scale culture of animal cells in suspension is of importance for many applications. At a small-scale, fed-batch is achieved either by manual bolus feeding or the use of liquid handling robots. In this study, we report an alternate application of a hydrogel for in situ continuous delivery of a nutrient feed comprising 18 amino acids, vitamins, antioxidants, and trace elements. We show that amino acid release is sustained for at least seven days. Importantly, release rates of individual amino acids can be independently modulated by changing their loading. We demonstrate the application of this hydrogel for complete in situ feeding of nutrients to a suspension adapted CHO cell line expressing IgG leading to 2.7-fold and 4-fold improvement in integral viable cell density (IVCD) and volumetric productivity respectively. This is similar to improvements obtained by bolus liquid feeding. Further, supplying glucose from the same hydrogel to eliminate manual feeding led to a 1.8-fold increase in IVCD accompanied by a 3-fold increase in volumetric productivity as compared to batch culture. In summary, this study provides a proof-of-concept that hydrogels can enable completely closed in situ feeding for mammalian cell culture requiring no external intervention. Such continuous in situ delivery can potentially enable closed culture systems maintaining nutrients at low levels mimicking physiological concentrations.

}, keywords = {Amino acids, CHO cells, Closed system, Fed-batch, Hydrogel for continuous feeding, In situ nutrient release}, issn = {1369-703X}, doi = {10.1016/j.bej.2019.107436}, author = {Rawat, Jyoti and Gadgil, Mugdha} } @article {48439, title = {Amino acid abundance and composition in cell culture medium affects trace metal tolerance and cholesterol synthesis}, journal = {Biotechnology Progress}, volume = {39}, year = {2023}, month = {JAN}, type = {Article}, abstract = {

Amino acid compositions of cell culture media are empirically designed to enhance cell growth and productivity and vary both across media formulations and over the course of culture due to imbalance in supply and consumption. The interconnected nature of the amino acid transporters and metabolism suggests that changes in amino acid composition can affect cell physiology. In this study, we explore the effect of a step change in amino acid composition from a DMEM: F12-based medium to a formulation varying in relative abundances of all amino acids, evaluated at two amino acid concentrations (lean LAA vs. rich HAA). Cell growth was inhibited in LAA but not HAA. In addition to the expected effects on expression of the cell cycle, amino acid response and mTOR pathway genes in LAA, we observed an unanticipated effect on zinc uptake and efflux genes. This was accompanied by a lower tolerance to zinc supplementation in LAA but not in the other formulations. Histidine was sufficient but not necessary to prevent such zinc toxicity. Additionally, an unanticipated downregulation of genes in the cholesterol synthesis pathway was observed in HAA, accompanied by an increase in cellular cholesterol content, which may depend on the relative abundances of glutamine and other amino acids. This study shows that changes in the amino acid composition without any evident effect on growth may have profound effects on metabolism. Such analyses can help rationalize the designing of medium and feed formulations for bioprocess applications beyond replenishment of consumed components.

}, keywords = {Amino acids, cell culture medium, CHO cells, Cholesterol, zinc}, issn = {8756-7938}, doi = {10.1002/btpr.3298}, author = {Rawat, Jyoti and Bhambri, Aksheev and Pandey, Ujjiti and Banerjee, Sanchita and Pillai, Beena and Gadgil, Mugdha} }