02503nas a2200217 4500008004100000022001200041245008300053210006900136260000800205300000900213520182000222653001402042653001802056653001402074653001702088653000902105100001802114700002202132700001902154856011202173 2018 eng d a175759800aZinc supplementation decreases galactosylation of recombinant IgG in CHO cells0 aZinc supplementation decreases galactosylation of recombinant Ig cMAY a1-113 a

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.

10aCHO cells10aglycosylation10amanganese10aTrace metals10azinc1 aPrabhu, Anuja1 aGadre, Ramchandra1 aGadgil, Mugdha uhttps://library.ncl.res.in/content/zinc-supplementation-decreases-galactosylation-recombinant-igg-cho-cells02699nas a2200277 4500008004100000022001400041245019500055210006900250260001200319300001500331490000700346520166900353653002502022653001402047653002502061653002502086653001102111653001102122653004002133653001102173100003702184700002602221700003102247700001902278856012402297 2019 eng d a8756-793800aSuperfluous glutamine synthetase activity in Chinese Hamster Ovary cells selected under glutamine limitation is growth limiting in glutamine-replete conditions and can be inhibited by serine0 aSuperfluous glutamine synthetase activity in Chinese Hamster Ova cSEP-OCT aUNSP e28560 v353 a

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.

10aC-13 tracer analysis10aCHO cells10aglutamine limitation10aGlutamine synthetase10aGS-CHO10aGS-NS010aselection under nutrient limitation10aserine1 aMaralingannavar, Vishwanathgouda1 aParmar, Dharmeshkumar1 aPanchagnula, Venkateswarlu1 aGadgil, Mugdha uhttps://library.ncl.res.in/content/superfluous-glutamine-synthetase-activity-chinese-hamster-ovary-cells-selected-under02045nas a2200265 4500008004100000022001400041245013100055210006900186260000800255300001000263490000800273520112000281653003401401653001401435653001401449653001401463653003801477653003501515653001601550100002501566700002001591700001801611700001801629856013201647 2020 eng d a0168-165600aEnhancing titers and productivity of rCHO clones with a combination of an optimized fed-batch process and ER-stress adaptation0 aEnhancing titers and productivity of rCHO clones with a combinat cMAR a49-580 v3113 a

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.

10aAdaptive laboratory evolution10aCHO cells10aER-stress10aFed-batch10aIncreased productivity and titers10aRecombinant protein production10aUPR pathway1 aChandrawanshi, Vikas1 aKulkarni, Rohan1 aPrabhu, Anuja1 aMehra, Sarika uhttps://library.ncl.res.in/content/enhancing-titers-and-productivity-rcho-clones-combination-optimized-fed-batch-process-and-er02238nas a2200229 4500008004100000022001400041245012200055210006900177260000900246300001100255490000800266520144400274653001601718653001401734653001801748653001401766653003601780653002901816100001701845700001901862856012701881 2020 eng d a1369-703X00aTowards in situ continuous feeding via controlled release of complete nutrients for fed-batch culture of animal cells0 aTowards in situ continuous feeding via controlled release of com cFEB  a1074360 v1543 a

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.

10aAmino acids10aCHO cells10aClosed system10aFed-batch10aHydrogel for continuous feeding10aIn situ nutrient release1 aRawat, Jyoti1 aGadgil, Mugdha uhttps://library.ncl.res.in/content/towards-situ-continuous-feeding-controlled-release-complete-nutrients-fed-batch-culture02437nas a2200253 4500008004100000022001400041245012000055210006900175260000800244490000700252520159800259653001601857653002401873653001401897653001601911653000901927100001701936700002101953700001901974700002301993700001802016700001902034856013002053 2023 eng d a8756-793800aAmino acid abundance and composition in cell culture medium affects trace metal tolerance and cholesterol synthesis0 aAmino acid abundance and composition in cell culture medium affe cJAN0 v393 a

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.

10aAmino acids10acell culture medium10aCHO cells10aCholesterol10azinc1 aRawat, Jyoti1 aBhambri, Aksheev1 aPandey, Ujjiti1 aBanerjee, Sanchita1 aPillai, Beena1 aGadgil, Mugdha uhttps://library.ncl.res.in/content/amino-acid-abundance-and-composition-cell-culture-medium-affects-trace-metal-tolerance-and