02297nas a2200217 4500008004100000022001400041245012800055210006900183260000800252300001400260490000700274520150600281100003701787700002401824700002201848700002401870700001801894700001701912700001901929856013101948 2018 eng d a0268-257500a In situ base release for pH maintenance can allow shake flasks to better mimic bioreactor performance for CHO cell culture0 aIn situ base release for pH maintenance can allow shake flasks t cOCT a2842-28500 v933 aBACKGROUNDShake flasks are widely used for evaluating mammalian cells in suspension. Lack of pH control can contribute to differences in culture performance between them and bioreactors. This study evaluates whether a previously reported in situ base releasing hydrogel (pHmH) to counter pH decrease can enable shake flask cultures to better mimic bioreactor cultures. RESULTSCompared with bioreactor culture, fed-batch cultures of a recombinant Chinese hamster ovary (CHO) cell-line in shake flasks without pHmH showed a decrease in pH to 6.6, accompanied by 40, 60 and 22% lower peak cell density, lactate accumulation, and immunoglobulin G (IgG) titer, respectively. Use of pHmH allowed shake flasks to maintain pH above 6.8 and reduced this difference to 20, 30, and 15%, respectively, thus enabling culture performance in shake flasks to better mimic the bioreactor. IgG glycosylation profiles were similar in identically fed cultures across all three platforms. Application of pHmH hydrogel during clone screening was evaluated by comparing correlation between titers for five recombinant CHO clones in bioreactors and shake flasks with and without pHmH; a higher correlation was found in shake flasks with pHmH than without. CONCLUSIONIn situ base release through hydrogel can allow identically fed fed-batch cultures in shake flasks to better mimic cell growth, lactate accumulation and IgG titers in bioreactors, without additional infrastructure. (c) 2018 Society of Chemical Industry.1 aMaralingannavar, Vishwanathgouda1 aShenoy, Bharath, R.1 aHazarika, Jahnabi1 aUnnikrishnan, Divya1 aPrabhu, Anuja1 aMaity, Sunit1 aGadgil, Mugdha uhttps://library.ncl.res.in/content/situ-base-release-ph-maintenance-can-allow-shake-flasks-better-mimic-bioreactor-performance02503nas 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-cells02045nas 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-er02199nas a2200241 4500008004100000022001400041245009100055210006900146260000800215300001200223490000800235520139800243653003201641653001801673653002301691653002401714653002001738653002501758653001701783100001801800700001901818856012001837 2021 eng d a1359-511300aTrace metals in cellular metabolism and their impact on recombinant protein production0 aTrace metals in cellular metabolism and their impact on recombin cNOV a251-2620 v1103 aReplacement of serum and increasing use of chemically defined media demands optimisation of trace metal components for biomanufacturing applications. Trace metal availability can impact culture performance, productivity and product quality. Several trace metals are cofactors of metabolic and other enzymes, and thus their availability regulates cellular metabolism. Additionally, they can also affect the availability of other trace metals and stability of some medium components. Such factors also need to be considered while formulating trace metal concentrations in the culture medium. Due to their very low concentrations, these components are susceptible to substantial variability arising from contaminants from other raw material and leaching from process equipment and can contribute to process variability. Understanding the role and impact of trace metals will help develop strategies to achieve targeted process parameters and increase process robustness vis-`a-vis any lot-to-lot variability in trace metal concentration in culture medium. This review describes the role of trace metals, particularly manganese, copper and zinc, in central carbon metabolism to aid in understanding the basis of metal-mediated effects on culture performance and provides a comprehensive review of the reported impact of trace metals on CHO cell culture performance and recombinant protein quality.10aChinese hamster ovary cells10aglycosylation10aMedium formulation10aProcess variability10aProduct quality10aRecombinant proteins10aTrace metals1 aPrabhu, Anuja1 aGadgil, Mugdha uhttps://library.ncl.res.in/content/trace-metals-cellular-metabolism-and-their-impact-recombinant-protein-production03197nas a2200277 4500008004100000022001400041245015400055210006900209260000800278300001000286490000700296520225700303653001302560653002102573653001702594653000702611653002002618653000902638653001802647653003102665653002902696100001802725700002802743700001902771856012902790 2022 eng d a1096-717600aEngineering nucleotide sugar synthesis pathways for independent and simultaneous modulation of N-glycan galactosylation and fucosylation in CHO cells0 aEngineering nucleotide sugar synthesis pathways for independent  cNOV a61-710 v743 a

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 `-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.

10aAntibody10aCell engineering10aFucosylation10aFx10aGalactosylation10aGale10aglycosylation10aNucleotide sugar synthesis10aRecombinant therapeutics1 aPrabhu, Anuja1 aShanmugam, Dhanasekaran1 aGadgil, Mugdha uhttps://library.ncl.res.in/content/engineering-nucleotide-sugar-synthesis-pathways-independent-and-simultaneous-modulation-n