Abstract
Ammonia is a toxic byproduct of CHO cell metabolism, which inhibits cell growth, reduces cell viability, alters glycosylation, and decreases recombinant protein productivity. In an attempt to minimize the ammonium accumulation in cell culture media, different amino acids were added individually to the culture medium before the production phase to alleviate the negative effects of ammonium on cell culture performance. Among all the amino acids examined in this study, valine showed the most positive impact on CHO cell culture performance. When the cultured CHO cells were fed with 5 mM valine, EPO titer was increased by 25% compared to the control medium, and ammonium and lactate production were decreased by 23 and 26%, respectively, relative to the control culture. Moreover, the sialic acid content of the EPO protein in valine-fed culture was higher than in the control culture, most likely because of the lower ammonium concentration. Flux balance analysis (FBA) results demonstrated that the citric acid cycle was enriched by valine feeding. The measurement of TCA cycle activity supported this finding. The analysis revealed that there might be a link between promoting tricarboxylic acid (TCA) cycle metabolism in valine-fed culture and reduction in lactate and ammonia accumulation. Furthermore, in valine-fed culture, FBA outcomes showed that alanine was excreted into the medium as the primary mechanism for reducing ammonium concentration. It was predicted that the elevated TCA cycle metabolism was concurrent with an increment in recombinant protein production. Taken together, our data demonstrate that valine addition could be an effective strategy for mitigating the negative impacts of ammonium and enhancing glycoprotein production in both quality and quantity.
Key points
• Valine feeding can mitigate the negative impacts of ammonia on CHO cell growth.
• Valine addition assists the ammonia removal mechanism by enriching the TCA cycle.
• Ammonia is removed from the media through alanine excretion in valine-fed culture.
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References
Aghamohseni H, Ohadi K, Spearman M, Krahn N, Moo-Young M, Scharer JM, Butler M, Budman HM (2014) Effects of nutrient levels and average culture pH on the glycosylation pattern of camelid-humanized monoclonal antibody. J Biotechnol 186:98–109. https://doi.org/10.1016/j.jbiotec.2014.05.024
Altamirano C, Illanes A, Casablancas A, Gámez X, Cairó JJ, Gòdia C (2001) Analysis of CHO cells metabolic redistribution in a glutamate-based defined medium in continuous culture. Biotechnol Prog 17(6):1032–1041. https://doi.org/10.1021/bp0100981
Bulté DB, Palomares LA, Parra CG, Martínez JA, Contreras MA, Noriega LG, Ramírez OT (2020) Overexpression of the mitochondrial pyruvate carrier reduces lactate production and increases recombinant protein productivity in CHO cells. Biotechnol Bioeng 117(9):2633–2647. https://doi.org/10.1002/bit.27439
Byrne B, Donohoe GG, O’Kennedy R (2007) Sialic acids: carbohydrate moieties that influence the biological and physical properties of biopharmaceutical proteins and living cells. Drug Discovery Today 12(7):319–326. https://doi.org/10.1016/j.drudis.2007.02.010
Chee Furng Wong D, Tin Kam Wong K, Tang Goh L, KiatHeng 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 P, Harcum SW (2005) Effects of amino acid additions on ammonium stressed CHO cells. J Biotechnol 117(3):277–286. https://doi.org/10.1016/j.jbiotec.2005.02.003
Chitwood DG, Wang Q, Elliott K, Bullock A, Jordana D, Li Z, Wu C, Harcum SW, Saski CA (2021) Characterization of metabolic responses, genetic variations, and microsatellite instability in ammonia-stressed CHO cells grown in fed-batch cultures. BMC Biotechnol 21(1):4. https://doi.org/10.1186/s12896-020-00667-2
Dam G, Aamann L, Vistrup H, Gluud LL (2018) The role of branched chain amino acids in the treatment of hepatic encephalopathy. J Clin Exp Hepatol 8(4):448–451. https://doi.org/10.1016/j.jceh.2018.06.004
Dean J, Reddy P (2013) Metabolic analysis of antibody producing CHO cells in fed-batch production. Biotechnol Bioeng 110(6):1735–1747. https://doi.org/10.1002/bit.24826
Duarte TM, Carinhas N, Barreiro LC, Carrondo MJT, Alves PM, Teixeira AP (2014) Metabolic responses of CHO cells to limitation of key amino acids. Biotechnol Bioeng 111(10):2095–2106. https://doi.org/10.1002/bit.25266
Fan Y, Jimenez Del Val I, Müller C, Wagtberg Sen J, Rasmussen SK, Kontoravdi C, Weilguny D, Andersen MR (2015) Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation. Biotechnol Bioeng 112(3):521–535. https://doi.org/10.1002/bit.25450
Fukuda MN, Sasaki H, Lopez L, Fukuda M (1989) Survival of recombinant erythropoietin in the circulation: the role of carbohydrates. Blood 73(1):84–89. https://doi.org/10.1182/blood.V73.1.84.84
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%3c637::AID-BIT6%3e3.0.CO;2-C
Geoghegan D, Arnall C, Hatton D, Noble-Longster J, Sellick C, Senussi T, James DC (2018) Control of amino acid transport into Chinese hamster ovary cells. Biotechnol Bioeng 115(12):2908–2929. https://doi.org/10.1002/bit.26794
Ghafuri-Esfahani A, Shokri R, Sharifi A, Shafiee L, Khosravi R, Kaghazian H, Khalili M (2020) Optimization of parameters affecting on CHO cell culture producing recombinant erythropoietin. Prep Biochem Biotechnol 50(8):834–841. https://doi.org/10.1080/10826068.2020.1753072
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
Hansen HA, Emborg C (1994) Influence of ammonium on growth, metabolism, and productivity of a continuous suspension Chinese hamster ovary cell culture. Biotechnol Prog 10(1):121–124. https://doi.org/10.1021/bp00025a014
Hartley F, Walker T, Chung V, Morten K (2018) Mechanisms driving the lactate switch in Chinese hamster ovary cells. Biotechnol Bioeng 115(8):1890–1903. https://doi.org/10.1002/bit.26603
Hayashi M, Ohnishi H, Kawade Y, Muto Y, Takahashi Y (1981) Augmented utilization of branched-chain amino acids by skeletal muscle in decompensated liver cirrhosis in special relation to ammonia detoxication. Gastroenterol Jpn 16(1):64–70. https://doi.org/10.1007/BF02820426
Hefzi H, Ang KS, Hanscho M, Bordbar A, Ruckerbauer D, Lakshmanan M, Orellana CA, Baycin-Hizal D, Huang Y, Ley D, Martinez VS, Kyriakopoulos S, Jiménez NE, Zielinski DC, Quek L-E, Wulff T, Arnsdorf J, Li S, Lee JS, Paglia G, Loira N, Spahn PN, Pedersen LE, Gutierrez JM, King ZA, Lund AM, Nagarajan H, Thomas A, Abdel-Haleem AM, Zanghellini J, Kildegaard HF, Voldborg BG, Gerdtzen ZP, Betenbaugh MJ, Palsson BO, Andersen MR, Nielsen LK, Borth N, Lee D-Y, Lewis NE (2016) A Consensus Genome-scale Reconstruction of Chinese Hamster Ovary Cell Metabolism. Cell Syst 3(5):434-443.e8. https://doi.org/10.1016/j.cels.2016.10.020
Heirendt L, Arreckx S, Pfau T, Mendoza SN, Richelle A, Heinken A, Haraldsdóttir HS, Wachowiak J, Keating SM, Vlasov V, Magnusdóttir S, Ng CY, Preciat G, Žagare A, Chan SHJ, Aurich MK, Clancy CM, Modamio J, Sauls JT, Noronha A, Bordbar A, Cousins B, El Assal DC, Valcarcel LV, Apaolaza I, Ghaderi S, Ahookhosh M, Ben Guebila M, Kostromins A, Sompairac N, Le HM, Ma D, Sun Y, Wang L, Yurkovich JT, Oliveira MAP, Vuong PT, El Assal LP, Kuperstein I, Zinovyev A, Hinton HS, Bryant WA, Aragón Artacho FJ, Planes FJ, Stalidzans E, Maass A, Vempala S, Hucka M, Saunders MA, Maranas CD, Lewis NE, Sauter T, Palsson BØ, Thiele I, Fleming RMT (2019) Creation and analysis of biochemical constraint-based models using the COBRA Toolbox v.3.0. Nat Protoc 14(3):639–702. https://doi.org/10.1038/s41596-018-0098-2
Hennicke J, Reinhart D, Altmann F, Kunert R (2019) Impact of temperature and pH on recombinant human IgM quality attributes and productivity. New Biotechnol 50:20–26. https://doi.org/10.1016/j.nbt.2019.01.001
Hiller GW, Clark DS, Blanch HW (1994) Transient responses of hybridoma cells in continuous culture to step changes in amino acid and vitamin concentrations. Biotechnol Bioeng 44(3):303–321. https://doi.org/10.1002/bit.260440308
Holecek M (2015) Ammonia and amino acid profiles in liver cirrhosis: Effects of variables leading to hepatic encephalopathy. Nutrition 31(1):14–20. https://doi.org/10.1016/j.nut.2014.03.016
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
Horvat J, Narat M, Spadiut O (2020) The effect of amino acid supplementation in an industrial Chinese Hamster Ovary process. Biotechnol Prog 36(5):e3001. https://doi.org/10.1002/btpr.3001
Huang Z, Xu J, Yongky A, Morris CS, Polanco AL, Reily M, Borys MC, Li ZJ, Yoon S (2020) CHO cell productivity improvement by genome-scale modeling and pathway analysis: Application to feed supplements. Biochem Eng J 160:107638. https://doi.org/10.1016/j.bej.2020.107638
Huang Z, Yoon S (2020) Identifying metabolic features and engineering targets for productivity improvement in CHO cells by integrated transcriptomics and genome-scale metabolic model. Biochem Eng J 159:107624. https://doi.org/10.1016/j.bej.2020.107624
Jiang H, Horwitz AA, Wright C, Tai A, Znameroski EA, Tsegaye Y, Warbington H, Bower BS, Alves C, Co C, Jonnalagadda K, Platt D, Walter JM, Natarajan V, Ubersax JA, Cherry JR, Love JC (2019) Challenging the workhorse: comparative analysis of eukaryotic micro-organisms for expressing monoclonal antibodies. Biotechnol Bioeng 116(6):1449–1462. https://doi.org/10.1002/bit.26951
Kanehisa M, Goto S (2000) KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res 28(1):27–30. https://doi.org/10.1093/nar/28.1.27
Kastelic M, Kopač D, Novak U, Likozar B (2019) Dynamic metabolic network modeling of mammalian Chinese hamster ovary (CHO) cell cultures with continuous phase kinetics transitions. Biochem Eng J 142:124–134. https://doi.org/10.1016/j.bej.2018.11.015
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
Kishishita S, Katayama S, Kodaira K, Takagi Y, Matsuda H, Okamoto H, Takuma S, Hirashima C, Aoyagi H (2015) Optimization of chemically defined feed media for monoclonal antibody production in Chinese hamster ovary cells. J Biosci Bioeng 120(1):78–84. https://doi.org/10.1016/j.jbiosc.2014.11.022
Kochanowski N, Blanchard F, Cacan R, Chirat F, Guedon E, Marc A, Goergen JL (2008) Influence of intracellular nucleotide and nucleotide sugar contents on recombinant interferon-γ glycosylation during batch and fed-batch cultures of CHO cells. Biotechnol Bioeng 100(4):721–733. https://doi.org/10.1002/bit.21816
Lalonde M-E, Durocher Y (2017) Therapeutic glycoprotein production in mammalian cells. J Biotechnol 251:128–140. https://doi.org/10.1016/j.jbiotec.2017.04.028
Lao M-S, Toth D (1997) Effects of ammonium and lactate on growth and metabolism of a recombinant Chinese hamster ovary cell culture. Biotechnol Prog 13(5):688–691. https://doi.org/10.1021/bp9602360
Larsen S, Nielsen J, Hansen CN, Nielsen LB, Wibrand F, Stride N, Schroder HD, Boushel R, Helge JW, Dela F, Hey-Mogensen M (2012) Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects. J Physiol 590(14):3349–3360. https://doi.org/10.1113/jphysiol.2012.230185
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
Ley D, Pereira S, Pedersen LE, Arnsdorf J, Hefzi H, Davy AM, Ha TK, Wulff T, Kildegaard HF, Andersen MR (2019) Reprogramming AA catabolism in CHO cells with CRISPR/Cas9 genome editing improves cell growth and reduces byproduct secretion. Metab Eng 56:120–129. https://doi.org/10.1016/j.ymben.2019.09.005
Long C, Zeng X, Xie J, Liang Y, Tao J, Tao Q, Liu M, Cui J, Huang Z, Zeng B (2019) High-level production of Monascus pigments in Monascus ruber CICC41233 through ATP-citrate lyase overexpression. Biochem Eng J 146:160–169. https://doi.org/10.1016/j.bej.2019.03.007
Martinelle K, Häggström L (1993) Mechanisms of ammonia and ammonium ion toxicity in animal cells: transport across cell membranes. J Biotechnol 30(3):339–350. https://doi.org/10.1016/0168-1656(93)90148-G
Martínez VS, Dietmair S, Quek L-E, Hodson MP, Gray P, Nielsen LK (2013) Flux balance analysis of CHO cells before and after a metabolic switch from lactate production to consumption. Biotechnol Bioeng 110(2):660–666. https://doi.org/10.1002/bit.24728
Mattick JSA, Kamisoglu K, Ierapetritou MG, Androulakis IP, Berthiaume F (2013) Branched-chain amino acid supplementation: impact on signaling and relevance to critical illness. Willey Interdiscip Rev Syst Biol Med 5(4):449–460. https://doi.org/10.1002/wsbm.1219
McAtee Pereira AG, Walther JL, Hollenbach M, Young JD (2018) 13C Flux analysis reveals that rebalancing medium amino acid composition can reduce ammonia production while preserving central carbon metabolism of CHO cell cultures. Biotechnol J 13(10):1700518. https://doi.org/10.1002/biot.201700518
Morimoto K, Tsuda E, Said AA, Uchida E, Hatakeyama S, Ueda M, Hayakawa T (1996) Biological and physicochemical characterization of recombinant human erythropoietins fractionated by Mono Q column chromatography and their modification with sialyltransferase. Glycoconj J 13(6):1013–1020. https://doi.org/10.1007/BF01053197
Motamedian E, Naeimpoor F (2018) LAMOS: A linear algorithm to identify the origin of multiple optimal flux distributions in metabolic networks. Comput Chem Eng 117:372–377. https://doi.org/10.1016/j.compchemeng.2018.06.014
Narkewicz MR, Sauls SD, Tjoa SS, Teng C, Fennessey PV (1996) Evidence for intracellular partitioning of serine and glycine metabolism in Chinese hamster ovary cells. Biochem J 313(3):991–996. https://doi.org/10.1042/bj3130991
Neinast MD, Jang C, Hui S, Murashige DS, Chu Q, Morscher RJ, Li X, Zhan L, White E, Anthony TG, Rabinowitz JD, Arany Z (2019) Quantitative analysis of the whole-body metabolic fate of branched-chain amino acids. Cell Metab 29(2):417-429.e4. https://doi.org/10.1016/j.cmet.2018.10.013
Park H-S, Kim I-H, Kim I-Y, Kim K-H, Kim H-J (2000) Expression of carbamoyl phosphate synthetase I and ornithine transcarbamoylase genes in Chinese hamster ovary dhfr-cells decreases accumulation of ammonium ion in culture media. J Biotechnol 81(2):129–140. https://doi.org/10.1016/S0168-1656(00)00282-0
Pereira S, Kildegaard HF, Andersen MR (2018) Impact of CHO metabolism on cell growth and protein production: an overview of toxic and inhibiting metabolites and nutrients. Biotechnol J 13(3):1700499. https://doi.org/10.1002/biot.201700499
Reimonn TM, Park S-Y, Agarabi CD, Brorson KA, Yoon S (2016) Effect of amino acid supplementation on titer and glycosylation distribution in hybridoma cell cultures—Systems biology-based interpretation using genome-scale metabolic flux balance model and multivariate data analysis. Biotechnol Prog 32(5):1163–1173. https://doi.org/10.1002/btpr.2335
Savizi ISP, Motamedian E, Lewis NE, Jimenez del Val I, Shojaosadati SA (2021) An integrated modular framework for modeling the effect of ammonium on the sialylation process of monoclonal antibodies produced by CHO cells. Biotechnol J n/a(n/a):2100019. https://doi.org/10.1002/biot.202100019
Savizi ISP, Soudi T, Shojaosadati SA (2019) Systems biology approach in the formulation of chemically defined media for recombinant protein overproduction. Appl Microbiol Biotechnol 103(20):8315–8326. https://doi.org/10.1007/s00253-019-10048-1
Schmidt C, Seibel R, Wehsling M, Le Mignon M, Wille G, Fischer M, Zimmer A (2020) Keto leucine and keto isoleucine are bioavailable precursors of their respective amino acids in cell culture media. J Biotechnol 321:1–12. https://doi.org/10.1016/j.jbiotec.2020.06.013
Schneider M, Marison IW, von Stockar U (1996) The importance of ammonia in mammalian cell culture. J Biotechnol 46(3):161–185. https://doi.org/10.1016/0168-1656(95)00196-4
Sellick CA, Croxford AS, Maqsood AR, Stephens GM, Westerhoff HV, Goodacre R, Dickson AJ (2015) Metabolite profiling of CHO cells: molecular reflections of bioprocessing effectiveness. Biotechnol J 10(9):1434–1445. https://doi.org/10.1002/biot.201400664
Seth G, Hossler P, Yee JC, Hu W-S (2006) Engineering cells for cell culture bioprocessing – physiological fundamentals. In: Hu W-S (ed) Cell Culture Engineering. Springer Berlin Heidelberg, Berlin, Heidelberg, pp 119–164
Sheikholeslami Z, Jolicoeur M, Henry O (2014) Elucidating the effects of postinduction glutamine feeding on the growth and productivity of CHO cells. Biotechnol Prog 30(3):535–546. https://doi.org/10.1002/btpr.1907
Synoground BF, McGraw CE, Elliott KS, Leuze C, Roth JR, Harcum SW, Sandoval NR (2021) Transient ammonia stress on Chinese hamster ovary (CHO) cells yield alterations to alanine metabolism and IgG glycosylation profiles. Biotechnol J n/a(n/a):2100098. https://doi.org/10.1002/biot.202100098
Templeton N, Dean J, Reddy P, Young JD (2013) Peak antibody production is associated with increased oxidative metabolism in an industrially relevant fed-batch CHO cell culture. Biotechnol Bioeng 110(7):2013–2024. https://doi.org/10.1002/bit.24858
Wahrheit J, Nicolae A, Heinzle E (2014) Dynamics of growth and metabolism controlled by glutamine availability in Chinese hamster ovary cells. Appl Microbiol Biotechnol 98(4):1771–1783. https://doi.org/10.1007/s00253-013-5452-2
Walker V, Mills GA (1995) Quantitative methods for amino acid analysis in biological fluids. Ann Clin Biochem 32(1):28–57. https://doi.org/10.1177/000456329503200103
Walsh G (2010) Biopharmaceutical benchmarks 2010. Nat Biotechnol 28(9):917–924. https://doi.org/10.1038/nbt0910-917
Xing Z, Kenty B, Koyrakh I, Borys M, Pan S-H, Li ZJ (2011) Optimizing amino acid composition of CHO cell culture media for a fusion protein production. Process Biochem 46(7):1423–1429. https://doi.org/10.1016/j.procbio.2011.03.014
Xu P, Dai X-P, Graf E, Martel R, Russell R (2014) Effects of glutamine and asparagine on recombinant antibody production using CHO-GS cell lines. Biotechnol Prog 30(6):1457–1468. https://doi.org/10.1002/btpr.1957
Yang M, Butler M (2000a) Effect of ammonia on the glycosylation of human recombinant erythropoietin in culture. Biotechnol Prog 16(5):751–759. https://doi.org/10.1021/bp000090b
Yang M, Butler M (2000b) 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)68:4%3c370::AID-BIT2%3e3.0.CO;2-K
Yoon SK, Choi SL, Song JY, Lee GM (2005) Effect of culture pH on erythropoietin production by Chinese hamster ovary cells grown in suspension at 32.5 and 37.0°C. Biotechnol Bioeng 89(3):345–356. https://doi.org/10.1002/bit.20353
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 X, Jiang R, Lin H, Xu S (2020) Feeding tricarboxylic acid cycle intermediates improves lactate consumption and antibody production in Chinese hamster ovary cell cultures. Biotechnol Prog 36(4):e2975. https://doi.org/10.1002/btpr.2975
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This study was funded by the Tarbiat Modares University (Grant NO: IG-39702).
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ISPS designed and carried out the experiments, analyzed the data, interpreted the results, and wrote the manuscript. NM provided cells and material and reviewed the manuscript. EM and NEL gave advice and reviewed the manuscript. SAS supervised the project, reviewed the manuscript, and provided the funding.
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Savizi, I.S.P., Maghsoudi, N., Motamedian, E. et al. Valine feeding reduces ammonia production through rearrangement of metabolic fluxes in central carbon metabolism of CHO cells. Appl Microbiol Biotechnol 106, 1113–1126 (2022). https://doi.org/10.1007/s00253-021-11755-4
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DOI: https://doi.org/10.1007/s00253-021-11755-4