Abstract
Increasing recombinant protein production while ensuring a high and consistent protein quality remains a challenge in mammalian cell culture process development. In this work, we combined a nutrient substitution approach with a metabolic engineering strategy that improves glucose utilization efficiency. This combination allowed us to tackle both lactate and ammonia accumulation and investigate on potential synergistic effects on protein production and quality. To this end, HEK293 cells overexpressing the pyruvate yeast carboxylase (PYC2) and their parental cells, both stably producing the therapeutic glycoprotein interferon α2b (IFNα2b), were cultured in media deprived of glutamine but containing chosen substitutes. Among the tested substitutes, pyruvate led to the best improvement in growth (integral of viable cell density) for both cell lines in batch cultures, whereas the culture of PYC2 cells without neither glutamine nor any substitute displayed surprisingly enhanced IFNα2b production. The drastic reduction in both lactate and ammonia in the cultures translated into extended high viability conditions and an increase in recombinant protein titer by up to 47% for the parental cells and the PYC2 cells. Product characterization performed by surface plasmon resonance biosensing using Sambucus nigra (SNA) lectin revealed that the increase in yield was however accompanied by a reduction in the degree of sialylation of the product. Supplementing cultures with glycosylation precursors and a cofactor were effective at counterbalancing the lack of glutamine and allowed improvement in IFNα2b quality as evaluated by lectin affinity. Our study provides a strategy to reconcile protein productivity and quality and highlights the advantages of PYC2-overexpressing cells in glutamine-free conditions.
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References
Altamirano C, Illanes A, Becerra S, Cairo JJ, Godia F (2006) Considerations on the lactate consumption by CHO cells in the presence of galactose. J Biotechnol 125(4):547–556. https://doi.org/10.1016/j.jbiotec.2006.03.023
Altamirano C, Paredes C, Cairo JJ, Godia F (2000) Improvement of CHO cell culture medium formulation: simultaneous substitution of glucose and glutamine. Biotechnol Prog 16(1):69–75. https://doi.org/10.1021/bp990124j
Andersen DC, Goochee CF (1995) The effect of ammonia on the O-linked glycosylation of granulocyte colony-stimulating factor produced by Chinese hamster ovary cells. Biotechnol Bioeng 47(1):96–105. https://doi.org/10.1002/bit.260470112
Ashwell G, Harford J (1982) Carbohydrate-specific receptors of the liver. Annu Rev Biochem 51(1):531–554. https://doi.org/10.1146/annurev.bi.51.070182.002531
Baker KN, Rendall MH, Hills AE, Hoare M, Freedman RB, James DC (2001) Metabolic control of recombinant protein N-glycan processing in NS0 and CHO cells. Biotechnol Bioeng 73(3):188–202. https://doi.org/10.1002/bit.1051
Bruhlmann D, Jordan M, Hemberger J, Sauer M, Stettler M, Broly H (2015) Tailoring recombinant protein quality by rational media design. Biotechnol Prog 31(3):615–629. https://doi.org/10.1002/btpr.2089
Chee Furng Wong D, Tin Kam Wong K, Tang Goh L, Kiat Heng C, Gek Sim Yap M (2005) Impact of dynamic online fed-batch strategies on metabolism, productivity and N-glycosylation quality in CHO cell cultures. Biotechnol Bioeng 89(2):164–177. https://doi.org/10.1002/bit.20317
Chen SC, Huang CH, Lai SJ, Yang CS, Hsiao TH, Lin CH, Fu PK, Ko TP, Chen Y (2016) Mechanism and inhibition of human UDP-GlcNAc 2-epimerase, the key enzyme in sialic acid biosynthesis. Sci Rep 6:23274. https://doi.org/10.1038/srep23274
Cheng T, Sudderth J, Yang C, Mullen AR, Jin ES, Matés JM, DeBerardinis RJ (2011) Pyruvate carboxylase is required for glutamine-independent growth of tumor cells. Proc Natl Acad Sci U S A 108(21):8674–8679. https://doi.org/10.1073/pnas.1016627108
Christie A, Butler M (1994) Glutamine-based dipeptides are utilized in mammalian cell culture by extracellular hydrolysis catalyzed by a specific peptidase. J Biotechnol 37(3):277–290. https://doi.org/10.1016/0168-1656(94)90134-1
Crowell CK, Grampp GE, Rogers GN, Miller J, Scheinman RI (2007) Amino acid and manganese supplementation modulates the glycosylation state of erythropoietin in a CHO culture system. Biotechnol Bioeng 96(3):538–549. https://doi.org/10.1002/bit.21141
Cruz HJ, Freitas CM, Alves PM, Moreira JL, Carrondo MJ (2000) Effects of ammonia and lactate on growth, metabolism, and productivity of BHK cells. Enzym Microb Technol 27(1–2):43–52. https://doi.org/10.1002/bit.260390408
Davidson SM, Papagiannakopoulos T, Olenchock BA, Heyman JE, Keibler MA, Luengo A, Bauer MR, Jha AK, O'Brien JP, Pierce KA, Gui DY, Sullivan LB, Wasylenko TM, Subbaraj L, Chin CR, Stephanopolous G, Mott BT, Jacks T, Clish CB, Vander Heiden MG (2016) Environment impacts the metabolic dependencies of Ras-driven non-small cell lung cancer. Cell Metab 23(3):517–528. https://doi.org/10.1016/j.cmet.2016.01.007
Diers AR, Broniowska KA, Chang CF, Hogg N (2012) Pyruvate fuels mitochondrial respiration and proliferation of breast cancer cells: effect of monocarboxylate transporter inhibition. Biochem J 444(3):561–571. https://doi.org/10.1042/BJ20120294
Elias CB, Carpentier E, Durocher Y, Bisson L, Wagner R, Kamen A (2003) Improving glucose and glutamine metabolism of human HEK 293 and trichoplusiani insect cells engineered to express a cytosolic pyruvate carboxylase enzyme. Biotechnol Prog 19(1):90–97. https://doi.org/10.1021/bp025572x
Erbayraktar S, Grasso G, Sfacteria A, Xie Q-w, Coleman T, Kreilgaard M, Torup L, Sager T, Erbayraktar Z, Gokmen N (2003) Asialoerythropoietin is a nonerythropoietic cytokine with broad neuroprotective activity in vivo. Proc Natl Acad Sci U S A 100(11):6741–6746. https://doi.org/10.1073/pnas.1031753100
Ferreira T, Ferreira A, Carrondo M, Alves P (2005) Effect of refeed strategies and non-ammoniagenic medium on adenovirus production at high cell densities. J Biotechnol 119(3):272–280. https://doi.org/10.1016/j.jbiotec.2005.03.009
Fischer MJ (2010) Amine coupling through EDC/NHS: a practical approach. In: De Mol NJ, Fischer MJE (eds) Surface plasmon resonance: methods and protocols. Methods in Molecular Biology. Humana Press, pp 55-73
Gawlitzek M, Ryll T, Lofgren J, Sliwkowski MB (2000) Ammonium alters N-glycan structures of recombinant TNFR-IgG: degradative versus biosynthetic mechanisms. Biotechnol Bioeng 68(6):637–646. https://doi.org/10.1002/(SICI)1097-0290(20000620)68:6<637::AID-BIT6>3.0.CO;2-C
Gawlitzek M, Valley U, Wagner R (1998) Ammonium ion and glucosamine dependent increases of oligosaccharide complexity in recombinant glycoproteins secreted from cultivated BHK-21 cells. Biotechnol Bioeng 57(5):518–528. https://doi.org/10.1002/(SICI)1097-0290(19980305)57:5<518::AID-BIT3>3.0.CO;2-J
Genzel Y, Ritter JB, Konig S, Alt R, Reichl U (2005) Substitution of glutamine by pyruvate to reduce ammonia formation and growth inhibition of mammalian cells. Biotechnol Prog 21(1):58–69. https://doi.org/10.1021/bp049827d
Ghaderi D, Zhang M, Hurtado-Ziola N, Varki A (2012) Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation. Biotechnol Genet Eng Rev 28(1):147–175. https://doi.org/10.5661/bger-28-147
Gillmeister MP, Tomiya N, Jacobia SJ, Lee YC, Gorfien SF, Betenbaugh MJ (2009) An HPLC-MALDI MS method for N-glycan analyses using smaller size samples: application to monitor glycan modulation by medium conditions. Glycoconj J 26(9):1135–1149. https://doi.org/10.1007/s10719-009-9235-z
Gramer MJ, Eckblad JJ, Donahue R, Brown J, Shultz C, Vickerman K, Priem P, van den Bremer ET, Gerritsen J, van Berkel PH (2011) Modulation of antibody galactosylation through feeding of uridine, manganese chloride, and galactose. Biotechnol Bioeng 108(7):1591–1602. https://doi.org/10.1002/bit.23075
Gu X, Wang DI (1998) Improvement of interferon-g sialylation in Chinese hamster ovary cell culture by feeding of N-acetylmannosamine. Biotechnol Bioeng 58(6):642–648. https://doi.org/10.1002/(SICI)1097-0290(19980620)58:6<642::AID-BIT10>3.0.CO;2-9
Ha TK, Lee GM (2014) Effect of glutamine substitution by TCA cycle intermediates on the production and sialylation of Fc-fusion protein in Chinese hamster ovary cell culture. J Biotechnol 180:23–29. https://doi.org/10.1016/j.jbiotec.2014.04.002
Ha TK, Lee GM (2015) Glutamine substitution: the role it can play to enhance therapeutic protein production. Pharm Bioprocess 3(3):249–261. https://doi.org/10.4155/pbp.15.6
Haseley SR, Talaga P, Kamerling JP, Vliegenthart JF (1999) Characterization of the carbohydrate binding specificity and kinetic parameters of lectins by using surface plasmon resonance. Anal Biochem 274(2):203–210. https://doi.org/10.1006/abio.1999.4277
Hassell T, Gleave S, Butler M (1991) Growth inhibition in animal cell culture. The effect of lactate and ammonia. Appl Biochem Biotechnol 30(1):29–41. https://doi.org/10.1007/BF02922022
Hayter PM, Curling EM, Baines AJ, Jenkins N, Salmon I, Strange PG, Tong JM, Bull AT (1992) Glucose-limited chemostat culture of Chinese hamster ovary cells producing recombinant human interferon-gamma. Biotechnol Bioeng 39(3):327–335. https://doi.org/10.1002/bit.260390311
Henry O, Durocher Y (2011) Enhanced glycoprotein production in HEK-293 cells expressing pyruvate carboxylase. Metab Eng 13(5):499–507. https://doi.org/10.1016/j.ymben.2011.05.004
Hills AE, Patel A, Boyd P, James DC (2001) Metabolic control of recombinant monoclonal antibody N-glycosylation in GS-NS0 cells. Biotechnol Bioeng 75(2):239–251. https://doi.org/10.1002/bit.10022
Hinderlich S, Weidemann W, Yardeni T, Horstkorte R, Huizing M (2013) UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE): a master regulator of sialic acid synthesis. In: Gerardy-Schahn R, Delannoy P, Von Itzstein M (eds) SialoGlyco chemistry and biology I: biosynthesis, structural diversity and sialoglycopathologies. Topics in Current Chemistry. Springer-Verlag, Berlin Heidelberg, pp 97–137
Hong JK, Cho SM, Yoon SK (2010) Substitution of glutamine by glutamate enhances production and galactosylation of recombinant IgG in Chinese hamster ovary cells. Appl Microbiol Biotechnol 88(4):869–876. https://doi.org/10.1007/s00253-010-2790-1
Hossler P, Khattak SF, Li ZJ (2009) Optimal and consistent protein glycosylation in mammalian cell culture. Glycobiology 19(9):936–949. https://doi.org/10.1093/glycob/cwp079
Irani N, Wirth M, van Den Heuvel J, Wagner R (1999) Improvement of the primary metabolism of cell cultures by introducing a new cytoplasmic pyruvate carboxylase reaction. Biotechnol Bioeng 66(4):238–246. https://doi.org/10.1002/(SICI)1097-0290(1999)66:4<238::AID-BIT5>3.0.CO;2-6
Karengera E, Robotham A, Kelly J, Durocher Y, De Crescenzo G, Henry O (2017) Altering the central carbon metabolism of HEK293 cells: impact on recombinant glycoprotein quality. J Biotechnol 242:73–82. https://doi.org/10.1016/j.jbiotec.2016.12.003
Kaufman RJ, Swaroop M, Murtha-Riel P (1994) Depletion of manganese within the secretory pathway inhibits O-linked glycosylation in mammalian cells. Biochemistry 33(33):9813–9819. https://doi.org/10.1021/bi00199a001
Kildegaard HF, Fan Y, Sen JW, Larsen B, Andersen MR (2016) Glycoprofiling effects of media additives on IgG produced by CHO cells in fed-batch bioreactors. Biotechnol Bioeng 113(2):359–366. https://doi.org/10.1002/bit.25715
Kim DY, Chaudhry MA, Kennard ML, Jardon MA, Braasch K, Dionne B, Butler M, Piret JM (2013) Fed-batch CHO cell t-PA production and feed glutamine replacement to reduce ammonia production. Biotechnol Prog 29(1):165–175. https://doi.org/10.1002/btpr.1658
Kornfeld S, Kornfeld R, Neufeld EF, O'Brien PJ (1964) The feedback control of sugar nucleotide biosynthesis in liver. Proc Natl Acad Sci U S A 52(2):371–379
Le H, Kabbur S, Pollastrini L, Sun Z, Mills K, Johnson K, Karypis G, Hu W-S (2012) Multivariate analysis of cell culture bioprocess data—lactate consumption as process indicator. J Biotechnol 162(2):210–223. https://doi.org/10.1016/j.jbiotec.2012.08.021
Liste-Calleja L, Lecina M, Lopez-Repullo J, Albiol J, Solà C, Cairó JJ (2015) Lactate and glucose concomitant consumption as a self-regulated pH detoxification mechanism in HEK293 cell cultures. Appl Microbiol Biotechnol 99(23):9951–9960. https://doi.org/10.1007/s00253-015-6855-z
Liu B, Spearman M, Doering J, Lattova E, Perreault H, Butler M (2014) The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody. J Biotechnol 170:17–27. https://doi.org/10.1016/j.jbiotec.2013.11.007
Liu B, Villacres-Barragan C, Lattova E, Spearman M, Butler M (2013) Differential affects of low glucose on the macroheterogeneity and microheterogeneity of glycosylation in CHO-EG2 camelid monoclonal antibodies. BMC Proc 7(6):1. https://doi.org/10.1186/1753-6561-7-S6-P112
Loignon M, Perret S, Kelly J, Boulais D, Cass B, Bisson L, Afkhamizarreh F, Durocher Y (2008) Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells. BMC Biotechnol 8:65. https://doi.org/10.1186/1472-6750-8-65
Luchansky SJ, Yarema KJ, Takahashi S, Bertozzi CR (2003) GlcNAc 2-epimerase can serve a catabolic role in sialic acid metabolism. J Biol Chem 278(10):8035–8042. https://doi.org/10.1074/jbc.M212127200
Luo J, Vijayasankaran N, Autsen J, Santuray R, Hudson T, Amanullah A, Li F (2012) Comparative metabolite analysis to understand lactate metabolism shift in Chinese hamster ovary cell culture process. Biotechnol Bioeng 109(1):146–156. https://doi.org/10.1002/bit.23291
McAtee AG, Templeton N, Young JD (2014) Role of Chinese hamster ovary central carbon metabolism in controlling the quality of secreted biotherapeutic proteins. Pharm Bioprocess 2(1):63–74. https://doi.org/10.4155/pbp.13.65
Morell AG, Gregoriadis G, Scheinberg IH, Hickman J, Ashwell G (1971) The role of sialic acid in determining the survival of glycoproteins in the circulation. J Biol Chem 246(5):1461–1467
Nadeau I, Sabatie J, Koehl M, Perrier M, Kamen A (2000) Human 293 cell metabolism in low glutamine-supplied culture: interpretation of metabolic changes through metabolic flux analysis. Metab Eng 2(4):277–292. https://doi.org/10.1006/mben.2000.0152
Noh SM, Lee GM (2016) Limitations to the development of recombinant human embryonic kidney 293E cells using glutamine synthetase-mediated gene amplification: methionine sulfoximine resistance. J Biotechnol. https://doi.org/10.1016/j.jbiotec.2016.06.003
Nyberg GB, Balcarcel RR, Follstad BD, Stephanopoulos G, Wang DI (1999) Metabolic effects on recombinant interferon-gamma glycosylation in continuous culture of Chinese hamster ovary cells. Biotechnol Bioeng 62(3):336–347. https://doi.org/10.1002/(SICI)1097-0290(19990205)62:3<336::AID-BIT10>3.0.CO;2-N
Paredes C, Prats E, Cairo J, Azorin F, Cornudella L, Godia F (1999) Modification of glucose and glutamine metabolism in hybridoma cells through metabolic engineering. Cytotechnology 30(1–3):85–93. https://doi.org/10.1023/A:1008012518961
Raymond C, Robotham A, Spearman M, Butler M, Kelly J, Durocher Y (2015) Production of α2, 6-sialylated IgG1 in CHO cells. MAbs 7(3):571–583. https://doi.org/10.1080/19420862.2015.1029215
Rijcken WP, Overdijk B, Van den Eijnden D, Ferwerda W (1995) The effect of increasing nucleotide-sugar concentrations on the incorporation of sugars into glycoconjugates in rat hepatocytes. Biochem J 305(3):865–870. https://doi.org/10.1042/bj3050865
Safina G, Duran Iu B, Alasel M, Danielsson B (2011) Surface plasmon resonance for real-time study of lectin-carbohydrate interactions for the differentiation and identification of glycoproteins. Talanta 84(5):1284–1290. https://doi.org/10.1016/j.talanta.2011.01.030
Schell JC, Rutter J (2013) The long and winding road to the mitochondrial pyruvate carrier. Cancer Metab 1(1):6. https://doi.org/10.1186/2049-3002-1-6
Sellers K, Fox MP, Bousamra M, Slone SP, Higashi RM, Miller DM, Wang Y, Yan J, Yuneva MO, Deshpande R (2015) Pyruvate carboxylase is critical for non-small-cell lung cancer proliferation. J Clin Invest 125(2):687–698. https://doi.org/10.1172/JCI72873
Sinclair AM, Elliott S (2005) Glycoengineering: the effect of glycosylation on the properties of therapeutic proteins. J Pharm Sci 94(8):1626–1635. https://doi.org/10.1002/jps.20319
Sola RJ, Griebenow K (2010) Glycosylation of therapeutic proteins: an effective strategy to optimize efficacy. BioDrugs 24(1):9–21. https://doi.org/10.2165/11530550-000000000-00000
Swiech K, Picanco-Castro V, Covas DT (2012) Human cells: new platform for recombinant therapeutic protein production. Protein Expr Purif 84(1):147–153. https://doi.org/10.1016/j.pep.2012.04.023
Toussaint C, Henry O, Durocher Y (2016) Metabolic engineering of CHO cells to alter lactate metabolism during fed-batch cultures. J Biotechnol 217:122–131. https://doi.org/10.1016/j.jbiotec.2015.11.010
Vallee C, Durocher Y, Henry O (2014) Exploiting the metabolism of PYC expressing HEK293 cells in fed-batch cultures. J Biotechnol 169:63–70. https://doi.org/10.1016/j.jbiotec.2013.11.002
Wong DC, Wong NS, Goh JS, May LM, Yap MG (2010a) Profiling of N-glycosylation gene expression in CHO cell fed-batch cultures. Biotechnol Bioeng 107(3):516–528. https://doi.org/10.1002/bit.22828
Wong NS, Wati L, Nissom PM, Feng HT, Lee MM, Yap MG (2010b) An investigation of intracellular glycosylation activities in CHO cells: effects of nucleotide sugar precursor feeding. Biotechnol Bioeng 107(2):321–336. https://doi.org/10.1002/bit.22812
Wong NS, Yap MG, Wang DI (2006) Enhancing recombinant glycoprotein sialylation through CMP-sialic acid transporter over expression in Chinese hamster ovary cells. Biotechnol Bioeng 93(5):1005–1016. https://doi.org/10.1002/bit.20815
Yang M, Butler M (2000) Effects of ammonia on CHO cell growth, erythropoietin production, and glycosylation. Biotechnol Bioeng 68(4):370–380. https://doi.org/10.1002/(SICI)1097-0290(20000520)68:43.0.CO;2-K
Yu DY, Noh SM, Lee GM (2016) Limitations to the development of recombinant human embryonic kidney 293E cells using glutamine synthetase-mediated gene amplification: methionine sulfoximine resistance. J Biotechnol 231:136–140. https://doi.org/10.1016/j.jbiotec.2016.06.003
Zagari F, Jordan M, Stettler M, Broly H, Wurm FM (2013) Lactate metabolism shift in CHO cell culture: the role of mitochondrial oxidative activity. New Biotechnol 30(2):238–245. https://doi.org/10.1016/j.nbt.2012.05.021
Zhang F, Sun X, Yi X, Zhang Y (2006) Metabolic characteristics of recombinant Chinese hamster ovary cells expressing glutamine synthetase in presence and absence of glutamine. Cytotechnology 51(1):21–28. https://doi.org/10.1007/s10616-006-9010-y
Zhou M, Crawford Y, Ng D, Tung J, Pynn AF, Meier A, Yuk IH, Vijayasankaran N, Leach K, Joly J, Snedecor B, Shen A (2011) Decreasing lactate level and increasing antibody production in Chinese hamster ovary cells (CHO) by reducing the expression of lactate dehydrogenase and pyruvate dehydrogenase kinases. J Biotechnol 153(1–2):27–34. https://doi.org/10.1016/j.jbiotec.2011.03.003
Funding
This work was supported by Fonds de Recherche du Québec—Nature et Technologies (FRQNT, grant number 175187). Eric Karengera received a scholarship from Wallonia-Brussels International (WBI). This is NRC publication NRC-HHT_53347.
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Karengera, E., Durocher, Y., De Crescenzo, G. et al. Combining metabolic and process engineering strategies to improve recombinant glycoprotein production and quality. Appl Microbiol Biotechnol 101, 7837–7851 (2017). https://doi.org/10.1007/s00253-017-8513-0
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DOI: https://doi.org/10.1007/s00253-017-8513-0