01739nas a2200253 4500008004100000022001400041245010700055210006900162260009900231300001400330490000800344520081300352653001801165653002101183653001901204653001801223653001801241653000901259100002401268700001901292700002201311700002001333856013201353 2011 eng d a0008-621500aGold catalyzed glycosidations for the synthesis of sugar acrylate/acrylamide hybrids and their utility0 aGold catalyzed glycosidations for the synthesis of sugar acrylat aTHE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, OXON, ENGLANDbELSEVIER SCI LTDcSEP a1511-15180 v3463 a

Propargyl glyco 1,2-orthoesters were exploited for the efficient synthesis of interesting glycomonomers such as glyco-acrylates and acrylamides using gold catalysts. It was observed that propargyl glyco 1,2-orthoesters with hydroxyethyl acrylates gives very good yield of the corresponding glyco-acrylates in a single step in the presence of catalytic amount of gold(III) catalyst; whereas, gold catalyzed glycosidation reaction on hydroxyethyl acrylamides was found to yield the corresponding acrylamidoyl 1,2-orthoester which was then converted to the corresponding glycol-acrylamide in the presence of catalytic amount of TMSOTf. Synthesized glyco-acrylate/acrylamide monomers are shown to undergo thiolate addition as well as free radical polymerization (C) 2011 Elsevier Ltd. All rights reserved.

10acarbohydrates10aGlycoacrylamides10aGlycoacrylates10aGlycopolymers10aglycosylation10aGold1 aThadke, Shivaji, A.1 aKar, Mritunjoy1 aGupta, Sayam, Sen1 aHotha, Srinivas uhttps://library.ncl.res.in/content/gold-catalyzed-glycosidations-synthesis-sugar-acrylateacrylamide-hybrids-and-their-utility-002540nas a2200277 4500008004100000022001400041245011000055210006900165260007900234300001200313490000800325520151400333653001401847653001801861653002401879653002301903653005501926653002301981100001802004700002802022700002602050700002702076700000902103700002702112856012302139 2013 eng d a0273-228900aFunctional characterization of a flavonoid glycosyltransferase gene from withania somnifera (Ashwagandha)0 aFunctional characterization of a flavonoid glycosyltransferase g a999 RIVERVIEW DRIVE SUITE 208, TOTOWA, NJ 07512 USAbHUMANA PRESS INCcJUN a729-7410 v1703 a

Glycosylation of flavonoids is mediated by family 1 uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs). Until date, there are few reports on functionally characterized flavonoid glycosyltransferases from Withania somnifera. In this study, we cloned the glycosyltransferase gene from W. somnifera (UGT73A16) showing 85-92 % homology with UGTs from other plants. UGT73A16 was expressed as a His(6)-tagged fusion protein in Escherichia coli. Several compounds, including flavonoids, were screened as potential substrates for UGT73A16. HPLC analysis and hypsochromic shift indicated that UGT73A16 transfers a glucose molecule to several different flavonoids. Based on kinetic parameters, UGT73A16 shows more catalytic efficiency towards naringenin. Here, we explored UGT73A16 of W. somnifera as whole cell catalyst in E. coli. We used flavonoids (genistein, apigenin, kaempferol, naringenin, biochanin A, and daidzein) as substrates for this study. More than 95 % of the glucoside products were released into the medium, facilitating their isolation. Glycosylation of substrates occurred on the 7- and 3-hydroxyl group of the aglycone. UGT73A16 also displayed regiospecific glucosyl transfer activity towards 3-hydroxy flavone compound, which is the backbone of all flavonols and also for a chemically synthesized compound, not found naturally. The present study generates essential knowledge and molecular as well as biochemical tools that allow the verification of UGT73A16 in glycosylation.

10aFlavonoid10aglycosylation10aGlycosyltransferase10aHypsochromic shift10aPlant secondary product glycosyltransferase (PSPG)10aWithania somnifera1 aSingh, Somesh1 aVishwakarma, Rishi., K.1 aKumar, R., J. Santosh1 aSonawane, Prashant, D.1 aRuby1 aKhan, Bashir, Mohammad uhttps://library.ncl.res.in/content/functional-characterization-flavonoid-glycosyltransferase-gene-withania-somnifera-002344nas a2200241 4500008004100000022001400041245016400055210006900219260007100288300001200359490000700371520145300378653000801831653001301839653001401852653001201866653001801878653002401896653001401920100001801934700001901952856013101971 2015 eng d a8756-793800aManganese increases high mannose glycoform on monoclonal antibody expressed in cho when glucose is absent or limiting: implications for use of alternate sugars0 aManganese increases high mannose glycoform on monoclonal antibod a111 RIVER ST, HOBOKEN 07030-5774, NJ USAbWILEY-BLACKWELLcMAR-APR a460-4670 v313 a

Alternate sugars such as galactose and fructose are metabolized at a slower rate than glucose and result in lower accumulation of lactate. While low lactate accumulation is desirable, we report that complete substitution of glucose with these sugars results in an increase in M5 high mannose glycans. Surprisingly, this increase is much higher when the culture is supplemented with manganese: for example, when cells are cultured with galactose, M5 high mannose glycan content increased from 5% at 1 nM Mn2+ in the basal medium to 32% with 16 mu M Mn2+ supplementation. When galactose is supplemented with glucose maintained at low concentrations, a small reduction in high mannose glycans is seen. In control cultures with glucose, the high mannose content was however <2% in this range of Mn2+ concentration. By varying Mn2+ and glucose supplementation levels, with or without galactose, we systematically demonstrate that Mn2+ concentration and glucose availability, together, significantly affect the high mannose glycan content. To our knowledge, this is the first report that shows that the effect of Mn2+ on high mannose glycan content depends on glucose availability. At each Mn2+ supplementation level evaluated, galactosylation percentages were highest for cultures where galactose was supplemented with glucose at non-limiting concentration. (c) 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:460-467, 2015

10aCHO10afructose10agalactose10aglucose10aglycosylation10ahigh-mannose glycan10amanganese1 aSurve, Tanaya1 aGadgil, Mugdha uhttps://library.ncl.res.in/content/manganese-increases-high-mannose-glycoform-monoclonal-antibody-expressed-cho-when-glucose-001496nas a2200217 4500008004100000022001400041245007700055210006900132260008600201300001000287490000700297520071900304653001701023653002201040653001801062653001601080653003001096100002201126700002501148856010501173 2016 eng d a0732-830300aSulfonated graphene oxide as highly efficient catalyst for glycosylation0 aSulfonated graphene oxide as highly efficient catalyst for glyco a530 WALNUT STREET, STE 850, PHILADELPHIA, PA 19106 USAbTAYLOR & FRANCIS INCcJAN a57-680 v353 a

Heterogeneous sulfonated graphene oxide for the first time has been used as a green and efficient catalyst for atom-economic glycosylation of unprotected, unactivated glycosyl donors or 2,3,4,6-tetra-O-acetylglycosyltrichloroacetimidate with various acceptors basically in the absence of solvent. The unprotected, unactivated glycosyl donors afforded mixtures of alpha- and beta-glycosides, while the 2,3,4,6-tetra-O-acetylglycosyltrichloroacetimidate afforded beta-glycosylated products with high yields and selectivity. The main advantages of this methodology are easy catalyst preparation, no need for dry reagents and reaction conditions, easy catalyst separation and recycling, and high product yields.

10aglycol donor10aglycosyl acceptor10aglycosylation10areusability10aSulfonated graphene oxide1 aThombal, Raju, S.1 aJadhav, Vrushali, H. uhttps://library.ncl.res.in/content/sulfonated-graphene-oxide-highly-efficient-catalyst-glycosylation01110nas a2200205 4500008004100000022001400041245006300055210006300118260000900181300001400190490000800204520048100212653002200693653002600715653000800741653001800749653002400767100001900791856009400810 2017 eng d a0011-389100aCell culture processes for biopharmaceutical manufacturing0 aCell culture processes for biopharmaceutical manufacturing cAPR  a1478-14880 v1123 a

Recombinant proteins manufactured using animal cell culture processes comprise a significant fraction of biopharmaceuticals. With the expiry of patents on this class of therapeutics, there is also a significant interest in manufacture of biosimilar versions of such therapeutics. This article provides a birds-eye view of upstream process development for animal cell culture processes, with a focus on advances pertinent to the development of processes for biosimilars.

10aBiopharmaceutical10aCell line development10aCHO10aglycosylation10amonoclonal antibody1 aGadgil, Mugdha uhttps://library.ncl.res.in/content/cell-culture-processes-biopharmaceutical-manufacturing02503nas 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-cells02290nas a2200229 4500008004100000022001400041245008800055210006900143260000800212300001000220490000700230520157600237653001101813653002001824653001801844653001101862653002401873653001701897100001501914700001501929856011601944 2019 eng d a0966-084400aNickel and cobalt affect galactosylation of recombinant IgG expressed in CHO cells.0 aNickel and cobalt affect galactosylation of recombinant IgG expr cOCT a11-190 v323 a

Glycosylation is an important product quality attribute of antibody biopharmaceuticals. It involves enzymatic addition of oligosaccharides on proteins by sequential action of glycosyltransferases and glycosidases in the endoplasmic reticulum and golgi. Some of these enzymes like galactosyltransferase and N-acetylglucosaminyltransferase-I require trace metal cofactors. Variations in trace metal availability during production can thus affect glycosylation of recombinant glycoproteins such as monoclonal antibodies. Variability in trace metal concentrations can be introduced at multiple stages during production such as due to impurities in raw materials for culture medium and leachables from bioreactors. Knowledge of the effect of various trace metals on glycosylation can help in root-cause analysis of unintended variability in glycosylation. In this study, we investigated the effect of nickel and cobalt on glycosylation of recombinant IgG expressed in Chinese hamster ovary cells. Nickel concentrations below 500 µM did not affect glycosylation, but above 500 µM it significantly decreases galactosylation of IgG. Cobalt at 50 µM concentration causes slight increase in G1F glycans (mono galactosylated) as previously reported. However, higher concentrations result in a small increase in G0F (non galactosylated) glycans. This effect of nickel and cobalt on galactosylation of recombinant IgG can be reversed by supplementation of uridine and galactose which are precursors to UDP-Galactose, a substrate for the enzymatic galactosylation reaction.

10acobalt10aGalactosylation10aglycosylation10aNickel10aProcess variability10aTrace metals1 aPrabhu, A.1 aGadgil, M. uhttps://library.ncl.res.in/content/nickel-and-cobalt-affect-galactosylation-recombinant-igg-expressed-cho-cells02199nas 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