Advertisement

Insight into the roles of tyrosine on rCHO cell performance in fed-batch cultures

  • Hongping Tang
  • Xintao Zhang
  • Weijian Zhang
  • Li Fan
  • Haibin Wang
  • Wen-Song TanEmail author
  • Liang ZhaoEmail author
Biotechnological products and process engineering
  • 25 Downloads

Abstract

Tyrosine (Tyr), as one of the least soluble amino acids, is essential to monoclonal antibody (mAb) production in recombinant Chinese hamster ovary (rCHO) cell cultures since its roles on maintaining the specific productivity (qmAb) and avoiding Tyr sequence variants. To understand the effects of Tyr on cell performance and its underlying mechanisms, rCHO cell–producing mAbs were cultivated at various cumulative Tyr addition concentrations (0.6 to 5.5 mM) in fed-batch processes. Low Tyr concentrations gave a much lower peak viable cell density (VCD) during the growth phase and also induced rapid cell death and pH decrease during the production phase, resulting in a low efficient fed-batch process. Autophagy was initiated following the inhibition of mTOR under the Tyr starvation condition. Excessive autophagy subsequently induced autophagic cell death, which was found as the major type of cell death in this study. Additionally, the results obtained here demonstrate that the decrease in culture pH under the Tyr starvation condition was associated with the autophagy and such pH drop might be attributed to the lysosome acidification and cell lysis.

Keywords

Chinese hamster ovary cells Tyrosine Cell death Autophagy Culture pH 

Notes

Funding information

This work was supported by the Fundamental Research Funds for the Central Universities (No. 22221818014).

Compliance with ethical standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2019_9921_MOESM1_ESM.pdf (294 kb)
ESM 1 (PDF 293 kb)

References

  1. Aggarwal SR (2014) What's fueling the biotech engine—2012 to 2013. Nat Biotechnol 32(1):32–39Google Scholar
  2. 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 Google Scholar
  3. Carrera AC (2004) TOR signaling in mammals. J Cell Sci 117(Pt 20:4615–4616.  https://doi.org/10.1242/jcs.01311 Google Scholar
  4. Castilho L, Maroes A, Augusto E, Butler M (2008) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy. Taylor & Francis Group, New YorkGoogle Scholar
  5. Chaabane W, User SD, El-Gazzah M, Jaksik R, Sajjadi E, Rzeszowska-Wolny J, Los MJ (2013) Autophagy, apoptosis, mitoptosis and necrosis: interdependence between those pathways and effects on cancer. Arch Immunol Ther Exp 61(1):43–58.  https://doi.org/10.1007/s00005-012-0205-y Google Scholar
  6. Choi AMK, Ryter SW, Levine B (2013) Autophagy in human health and disease. New Engl J Med 368(19):1845–1846.  https://doi.org/10.1056/NEJMra1205406 Google Scholar
  7. Feeney L, Carvalhal V, Yu XC, Chan B, Michels DA, Wang YJ, Shen A, Ressl J, Dusel B, Laird MW (2013) Eliminating tyrosine sequence variants in CHO cell lines producing recombinant monoclonal antibodies. Biotechnol Bioeng 110(4):1087–1097.  https://doi.org/10.1002/bit.24759 Google Scholar
  8. Figueroa B, Ailor E, Osborne D, Hardwick JM, Reff M, Betenbaugh MJ (2007) Enhanced cell culture performance using inducible anti-apoptotic genes E1B-19K and Aven in the production of a monoclonal antibody with chinese hamster ovary cells. Biotechnol Bioeng 97(4):877–892.  https://doi.org/10.1002/bit.21222 Google Scholar
  9. Fu T, Zhang C, Jing Y, Jiang C, Li Z, Wang S, Ma K, Zhang D, Hou S, Dai J, Kou G, Wang H (2016) Regulation of cell growth and apoptosis through lactate dehydrogenase C over-expression in Chinese hamster ovary cells. Appl Microbiol Biotechnol 100(11):5007–5016.  https://doi.org/10.1007/s00253-016-7348-4 Google Scholar
  10. Galluzzi L, Morselli E, Vicencio JM, Kepp O, Joza N, Tajeddine N, Kroemer G (2008) Life, death and burial: multifaceted impact of autophagy. Biochem Soc T 36:786–790.  https://doi.org/10.1042/Bst0360786 Google Scholar
  11. Gozuacik D, Kimchi A (2007) Autophagy and cell death. Curr Top Dev Biol 78:217–245.  https://doi.org/10.1016/S0070-2153(06)78006-1 Google Scholar
  12. Guo Y, Pei X (2019) Tetrandrine-induced autophagy in MDA-MB-231 triple-negative breast cancer cell through the inhibition of PI3K/AKT/mTOR signaling. Evid Based Complement Alternat Med 2019:7517431.  https://doi.org/10.1155/2019/7517431 Google Scholar
  13. Hansen HA, Emborg C (1994) Extra- and intracellular amino acid concentrations in continuous Chinese hamster ovary cell culture. Appl Microbiol Biotechnol 41(5):560–564Google Scholar
  14. Hwang SO, Lee GM (2008) Nutrient deprivation induces autophagy as well as apoptosis in Chinese hamster ovary cell culture. Biotechnol Bioeng 99(3):678–685.  https://doi.org/10.1002/bit.21589 Google Scholar
  15. Jayapal KR, Wlaschin KF, Hu WS, Yap MGS (2007) Recombinant protein therapeutics from CHO cells - 20 years and counting. Chem Eng Prog 103(10):40–47Google Scholar
  16. Jeon MK, Yu DY, Lee GM (2011) Combinatorial engineering of ldh-a and bcl-2 for reducing lactate production and improving cell growth in dihydrofolate reductase-deficient Chinese hamster ovary cells. Appl Microbiol Biotechnol 92(4):779–790.  https://doi.org/10.1007/s00253-011-3475-0 Google Scholar
  17. Kang S, Mullen J, Miranda LP, Deshpande R (2012) Utilization of tyrosine- and histidine-containing dipeptides to enhance productivity and culture viability. Biotechnol Bioeng 109(9):2286–2294.  https://doi.org/10.1002/bit.24507 Google Scholar
  18. Kawai A, Uchiyama H, Takano S, Nakamura N, Ohkuma S (2014) Autophagosome-lysosome fusion depends on the pH in acidic compartments in CHO cells. Autophagy 3(2):154–157.  https://doi.org/10.4161/auto.3634 Google Scholar
  19. Kim NS, Lee GM (2002) Response of recombinant Chinese hamster ovary cells to hyperosmotic pressure: effect of Bcl-2 overexpression. J Biotechnol 95(3):237–248Google Scholar
  20. Kim YG, Kim JY, Mohan C, Lee GM (2009) Effect of Bcl-xL overexpression on apoptosis and autophagy in recombinant Chinese hamster ovary cells under nutrient-deprived condition. Biotechnol Bioeng 103(4):757–766.  https://doi.org/10.1002/bit.22298 Google Scholar
  21. Kim YJ, Baek E, Lee JS, Lee GM (2013) Autophagy and its implication in Chinese hamster ovary cell culture. Biotechnol Lett 35(11):1753–1763.  https://doi.org/10.1007/s10529-013-1276-5 Google Scholar
  22. Klionsky DJ, Elazar Z, Seglen PO, Rubinsztein DC (2014) Does bafilomycin A1block the fusion of autophagosomes with lysosomes? Autophagy 4(7):849–850.  https://doi.org/10.4161/auto.6845 Google Scholar
  23. Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, Baehrecke EH, Blagosklonny MV, El-Deiry WS, Golstein P, Green DR, Hengartner M, Knight RA, Kumar S, Lipton SA, Malorni W, Nunez G, Peter ME, Tschopp J, Yuan J, Piacentini M, Zhivotovsky B, Melino G, Nomenclature Committee on Cell D (2009) Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ 16(1):3–11.  https://doi.org/10.1038/cdd.2008.150 Google Scholar
  24. Kroemer G, Levine B (2008) Autophagic cell death: the story of a misnomer. Nat Rev Mol Cell Biol 9(12):1004–1010.  https://doi.org/10.1038/nrm2529 Google Scholar
  25. Krysko DV, Vanden Berghe T, D'Herde K, Vandenabeele P (2008) Apoptosis and necrosis: detection, discrimination and phagocytosis. Methods 44(3):205–221.  https://doi.org/10.1016/j.ymeth.2007.12.001 Google Scholar
  26. Lee JS, Lee GM (2012) Monitoring of autophagy in Chinese hamster ovary cells using flow cytometry. Methods 56(3):375–382.  https://doi.org/10.1016/j.ymeth.2011.11.006 Google Scholar
  27. Levine B (2005) Eating oneself and uninvited guests: autophagy-related pathways in cellular defense. Cell 120(2):159–162.  https://doi.org/10.1016/j.cell.2005.01.005 Google Scholar
  28. Levine B, Yuan JY (2005) Autophagy in cell death: an innocent convict? J Clin Invest 115(10):2679–2688.  https://doi.org/10.1172/Jc126390 Google Scholar
  29. Luo H, Yu YY, Chen HM, Wu W, Li Y, Lin H (2019) The combination of NVP-BEZ235 and rapamycin regulates nasopharyngeal carcinoma cell viability and apoptosis via the PI3K/AKT/mTOR pathway. Exp Ther Med 17(1):99–106.  https://doi.org/10.3892/etm.2018.6896 Google Scholar
  30. Martinet W, De Meyer GR, Herman AG, Kockx MM (2005) Amino acid deprivation induces both apoptosis and autophagy in murine C2C12 muscle cells. Biotechnol Lett 27(16):1157–1163.  https://doi.org/10.1007/s10529-005-0007-y Google Scholar
  31. Mauvezin C, Neufeld TP (2015) Bafilomycin A1 disrupts autophagic flux by inhibiting both V-ATPase-dependent acidification and Ca-P60A/SERCA-dependent autophagosome-lysosome fusion. Autophagy 11(8):1437–1438.  https://doi.org/10.1080/15548627.2015.1066957 Google Scholar
  32. Meneses-Acosta A, Mendonca RZ, Merchant H, Covarrubias L, Ramirez OT (2001) Comparative characterization of cell death between Sf9 insect cells and hybridoma cultures. Biotechnol Bioeng 72(4):441–457.  https://doi.org/10.1002/1097-0290(20000220)72:4<441::Aid-Bit1006>3.0.Co;2-3 Google Scholar
  33. Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140(3):313–326.  https://doi.org/10.1016/j.cell.2010.01.028 Google Scholar
  34. Muthing J, Kemminer SE, Conradt HS, Sagi D, Nimtz M, Karst U, Peter-Katalinic J (2003) Effects of buffering conditions and culture pH on production rates and glycosylation of clinical phase I anti-melanoma mouse IgG3 monoclonal antibody R24. Biotechnol Bioeng 83(3):321–334.  https://doi.org/10.1002/bit.10673 Google Scholar
  35. Okada H, Mak TW (2004) Pathways of apoptotic and non-apoptotic death in tumour cells. Nat Rev Cancer 4(8):592–603.  https://doi.org/10.1038/nrc1412 Google Scholar
  36. Pattingre S, Tassa A, Qu XP, Garuti R, Liang XH, Mizushima N, Packer M, Schneider MD, Levine B (2005) Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 122(6):927–939.  https://doi.org/10.1016/j.cell.2005.07.002 Google Scholar
  37. Petibone DM, Majeed W, Casciano DA (2017) Autophagy function and its relationship to pathology, clinical applications, drug metabolism and toxicity. J Appl Toxicol 37(1):23–37.  https://doi.org/10.1002/jat.3393 Google Scholar
  38. Salazar A, Keusgen M, von Hagen J (2016) Amino acids in the cultivation of mammalian cells. Amino Acids 48(5):1161–1171.  https://doi.org/10.1007/s00726-016-2181-8 Google Scholar
  39. Sharma A, Sharma R, Singh SP, Khinchi M (2017) A Brief Review on Apoptosis. Asian J Pharm Res Dev:1–10Google Scholar
  40. Tang H, Miao S, Zhang X, Fan L, Liu X, Tan WS, Zhao L (2018) Insights into the generation of monoclonal antibody acidic charge variants during Chinese hamster ovary cell cultures. Appl Microbiol Biotechnol 102(3):1203–1214.  https://doi.org/10.1007/s00253-017-8650-5 Google Scholar
  41. Trummer E, Fauland K, Seidinger S, Schriebl K, Lattenmayer C, Kunert R, Vorauer-Uhl K, Weik R, Borth N, Katinger H, Muller D (2006) Process parameter shifting: Part I. Effect of DOT, pH, and temperature on the performance of Epo-Fc expressing CHO cells cultivated in controlled batch bioreactors. Biotechnol Bioeng 94(6):1033–1044.  https://doi.org/10.1002/bit.21013 Google Scholar
  42. von Hagen J, Hecklau C, Seibel R, Pering S, Schnellbaecher A, Wehsling M, Eichhorn T (2017) Simplification of Fed-Batch Processes with a Single-Feed Strategy. BioProcess InternationalGoogle Scholar
  43. Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22(11):1393–1398.  https://doi.org/10.1038/nbt1026 Google Scholar
  44. Xu P, Dai XP, 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 Google Scholar
  45. Yang JD, Lu C, Stasny B, Henley J, Guinto W, Gonzalez C, Gleason J, Fung M, Collopy B, Benjamino M, Gangi J, Hanson M, Ille E (2007) Fed-batch bioreactor process scale-up from 3-L to 2,500-L scale for monoclonal antibody production from cell culture. Biotechnol Bioeng 98(1):141–154.  https://doi.org/10.1002/bit.21413 Google Scholar
  46. Yoon YH, Cho KS, Hwang JJ, Lee SJ, Choi JA, Koh JY (2010) Induction of lysosomal dilatation, arrested autophagy, and cell death by chloroquine in cultured ARPE-19 cells. Invest Ophthalmol Vis Sci 51(11):6030–6037.  https://doi.org/10.1167/iovs.10-5278 Google Scholar
  47. Yu L, Lenardo MJ, Baehrecke EH (2004) Autophagy and caspases: a new cell death program. Cell Cycle 3(9):1122–1124Google Scholar
  48. Yu M, Hu Z, Pacis E, Vijayasankaran N, Shen A, Li F (2011) Understanding the intracellular effect of enhanced nutrient feeding toward high titer antibody production process. Biotechnol Bioeng 108(5):1078–1088.  https://doi.org/10.1002/bit.23031 Google Scholar
  49. Zanghi JA, Schmelzer AE, Mendoza TP, Knop RH, Miller WM (1999) Bicarbonate concentration and osmolality are key determinants in the inhibition of CHO cell polysialylation under elevated pCO2 or pH. Biotechnol Bioeng 65(2):182–191Google Scholar
  50. Zhang X, Tang H, Sun Y-T, Liu X, Tan W-S, Fan L (2015) Elucidating the effects of arginine and lysine on a monoclonal antibody C-terminal lysine variation in CHO cell cultures. Appl Microbiol Biotechnol 99(16):6643–6652.  https://doi.org/10.1007/s00253-015-6617-y
  51. Zhu MM, Goyal A, Rank DL, Gupta SK, Boom TV, Lee SS (2005) Effects of elevated pCO2 and osmolality on growth of CHO cells and production of antibody-Fusion Protein B1: a case study. Biotechnol Prog 21(1):70–77Google Scholar
  52. Zustiak MP, Pollack JK, Marten MR, Betenbaugh MJ (2008) Feast or famine: autophagy control and engineering in eukaryotic cell culture. Curr Opin Biotechnol 19(5):518–526.  https://doi.org/10.1016/j.copbio.2008.07.007

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.The State Key Laboratory of Bioreactor EngineeringEast China University of Science and TechnologyShanghaiChina
  2. 2.Zhejiang Hisun Pharmaceutical Co., Ltd.ZhejiangChina

Personalised recommendations