Bioprocess and Biosystems Engineering

, Volume 41, Issue 5, pp 633–640 | Cite as

Enhanced production of anti-PD1 antibody in CHO cells through transient co-transfection with anti-apoptotic genes Bcl-x L and Mcl-1

  • Xinyu Zhang
  • Lei Han
  • Huifang Zong
  • Kai Ding
  • Yuan Yuan
  • Jingyi Bai
  • Yuexian Zhou
  • Baohong Zhang
  • Jianwei Zhu
Research Paper


Apoptosis has a negative impact on the cell survival state during cell cultivation. To optimize mammalian cell culture for production of biopharmaceuticals, one of the important approaches is to extend cell life through over-expression of anti-apoptotic genes. Here, we reported a cost-effective process to enhance cell survival and production of an antibody through transient co-transfection with anti-apoptotic genes Bcl-x L or Mcl-1 in Chinese hamster ovary (CHO) cells with polyethylenimine (PEI). Under the optimal conditions, it showed reduced levels of apoptosis and improved cell viability after co-transfected with Bcl-x L or Mcl-1. The overall production yield of the antibody anti-PD1 increased approximately 82% in CHO cells co-transfected with Bcl-x L , and 34% in CHO cells co-transfected with Mcl-1. This work provides an effective way to increase viability of host cells through delaying apoptosis onset, thus, raise production yield of biopharmaceuticals without the process of generating stable cell lines and subsequent screening.


Anti-apoptosis Bcl-xL Chinese hamster ovary cell Mcl-1 Transient gene expression 



This project was supported by Natural Science Foundation of Shanghai [Grant No. 15ZR1423800].

Compliance with ethical standards

Conflict of interest

All the authors reviewed and agreed to submit this manuscript. The authors declare that they have no conflict of interest.

Research involving human participants and/or animals

The study does not contain experiments using animals and human studies.

Supplementary material

449_2018_1898_MOESM1_ESM.tif (2.1 mb)
Supplement Fig. 1 Optimization of transfection efficiency in 3D plot showing the transfection efficiencies in CHO cells at different DNA: PEI ratios, DNA concentrations and cell concentrations. Samples were analyzed 48 h after transfection for GFP expression by flow cytometry (TIF 2139 KB)


  1. 1.
    Zhu J (2012) Mammalian cell protein expression for biopharmaceutical production. Biotechnol Adv 30:1158–1170CrossRefGoogle Scholar
  2. 2.
    Birch JR, Onakunle Y (2005) Biopharmaceutical proteins: opportunities and challenges. In: Smales CM, James DC (eds) Methods in molecular biology. Therapeutic proteins: methods and protocols, vol 308. Humana Press, Inc., Totowa, NJ, pp 1–16Google Scholar
  3. 3.
    Trill JJ, Shatzman AR, Ganguly S (1995) Production of monoclonal antibodies in COS and CHO cells. Curr Opin Biotechnol 6:553–560CrossRefGoogle Scholar
  4. 4.
    Hacker DL, De Jesus M, Wurm FM (2009) 25 years of recombinant proteins from reactor-grown cells—where do we go from here? Biotechnol Adv 27:1023–1027CrossRefGoogle Scholar
  5. 5.
    DrugBank (2017) About DrugBank. Accessed 14 Nov 2017
  6. 6.
    Zhu J (2013) Update on production of recombinant therapeutic protein: transient gene expression. Smithers Rapra, ShropshireGoogle Scholar
  7. 7.
    Ding K, Han L, Zong H, Chen J, Zhang B, Zhu J (2017) Production process reproducibility and product quality consistency of transient gene expression in HEK293 cells with anti-PD1 antibody as the model protein. Appl Microbiol Biotechnol 101:1889–1898CrossRefGoogle Scholar
  8. 8.
    Daramola O, Stevenson J, Dean G, Hatton D, Pettman G, Holmes W, Field R (2014) A high-yielding CHO transient wystem: coexpression of genes encoding EBNA-1 and GS enhances transient protein expression. Biotechnol Prog 30:132–141CrossRefGoogle Scholar
  9. 9.
    Cain K, Peters S, Hailu H, Sweeney B, Stephens P, Heads J, Sarkar K, Ventom A, Page C, Dickson A (2013) A CHO cell line engineered to express XBP1 and ERO1-Lalpha has increased levels of transient protein expression. Biotechnol Prog 29:697–706CrossRefGoogle Scholar
  10. 10.
    Rajendra Y, Hougland MD, Alam R, Morehead TA, Barnard GC (2015) A high cell density transient transfection system for therapeutic protein expression based on a CHO GS-knockout cell line:process development and product quality assessment. Biotechnol Bioeng 112:977–986CrossRefGoogle Scholar
  11. 11.
    Steger K, Brady J, Wang WL, Duskin M, Donato K, Peshwa M (2015) CHO-s antibody titers > 1 gram/liter using flow electroporation-mediated transient gene expression followed by rapid migration to high-yield stable cell lines. J Biomol Screen 20:545–551CrossRefGoogle Scholar
  12. 12.
    Jain NK, Barkowski-Clark S, Altman R, Johnson K, Sun F, Zmuda J, Liu CY, Kita A, Schulz R, Neill A, Ballinger R, Patel R, Liu J, Mpanda A, Huta B, Chiou H, Voegtli W, Panavas T (2017) A high density CHO-S transient transfection system: comparison of ExpiCHO and Expi293. Protein Expr Purif 134:38–46CrossRefGoogle Scholar
  13. 13.
    Arden N, Betenbaugh MJ (2004) Life and death in mammalian cell culture:strategies for apoptosis inhibition. Trends Biotechnol 22:174–180CrossRefGoogle Scholar
  14. 14.
    Goswami J, Sinskey AJ, Steller H, Stephanopoulos GN, Wang DIC (1999) Apoptosis in batch cultures of Chinese Hamster Ovary cells. Biotechnol Bioeng 62:632–640CrossRefGoogle Scholar
  15. 15.
    Al-Rubeai M, Emery AN (1990) Mechanisms and kinetics of monoclonal antibody synthesis and secretion in synchronous and asynchronous hybridoma cell cultures. J Biotechnol 16:67–85CrossRefGoogle Scholar
  16. 16.
    Singh RP, Al-Rubeai M, Gregory CD, Emery AN (1994) Cell death in bioreactors: a role for apoptosis. Biotechnol Bioeng 44:720–726CrossRefGoogle Scholar
  17. 17.
    Kim JY, Kim YG, Lee GM (2012) CHO cells in biotechnology for production of recombinant proteins: current state and further potential. Appl Microbiol Biotechnol 93:917–930CrossRefGoogle Scholar
  18. 18.
    Youle RJ, Strasser A (2008) The Bcl-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9:47–59CrossRefGoogle Scholar
  19. 19.
    Mastrangelo AJ, Betenbaugh MJ (1998) Overcoming apoptosis: new methods for improving protein-expression systems. Trends Biotechnol 16:88–95CrossRefGoogle Scholar
  20. 20.
    Yang J, Liu XS, Bhalla K, Kim CN, Ibrado AM, Cai JY, Peng TI, Jones DP, Wang XD (1997) Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275:1129–1132CrossRefGoogle Scholar
  21. 21.
    Chiang GG, Sisk WP (2005) Bcl-xL mediates increased production of humanized monoclonal antibodies in Chinese hamster ovary cells. Biotechnol Bioeng 91:779–792CrossRefGoogle Scholar
  22. 22.
    Tey BT, Singh RP, Piredda L, Piacentini M, Al-Rubeai M (2000) Influence of Bcl-2 on cell death during the cultivation of a Chinese hamster ovary cell line expressing a chimeric antibody. Biotechnol Bioeng 68:31–43CrossRefGoogle Scholar
  23. 23.
    Meents H, Enenkel B, Eppenberger HM, Werner RG, Fussenegger M (2002) Impact of coexpression and coamplification of sICAM and antiapoptosis determinants Bcl-2/Bcl-xL on productivity, cell survival, and mitochondria number in CHO-DG44 grown in suspension and serum-free media. Biotechnol Bioeng 80:706–716CrossRefGoogle Scholar
  24. 24.
    Mastrangelo AJ, Hardwick JM, Bex F, Betenbaugh MJ (2000) Part I. Bcl-2 and Bcl-xL limit apoptosis upon infection with alphavirus vectors. Biotechnol Bioeng 67:544–554CrossRefGoogle Scholar
  25. 25.
    Charbonneau JR, Furtak T, Lefebvre J, Gauthier ER (2003) Bcl-xL expression interferes with the effects of l-glutamine supplementation on hybridoma cultures. Biotechnol Bioeng 81:279–290CrossRefGoogle Scholar
  26. 26.
    Simpson NH, Singh RP, Perani A, Goldenzon C, Al-Rubeai M (1998) In hybridoma cultures, deprivation of any single amino acid leads to apoptotic death, which is suppressed by the expression of the Bcl-2 gene. Biotechnol Bioeng 59:90–98CrossRefGoogle Scholar
  27. 27.
    Figueroa B Jr, Sauerwald TM, Oyler GA, Hardwick JM, Betenbaugh MJ (2003) A comparison of the properties of a Bcl-xL variant to the wild-type anti-apoptosis inhibitor in mammalian cell cultures. Metab Eng 5:230–245CrossRefGoogle Scholar
  28. 28.
    Figueroa B Jr, Chen S, Oyler GA, Hardwick JM, Betenbaugh MJ (2004) Aven and Bcl-xL enhance protection against apoptosis for mammalian cells exposed to various culture conditions. Biotechnol Bioeng 85:589–600CrossRefGoogle Scholar
  29. 29.
    Jung D, Cote S, Drouin M, Simard C, Lemieux R (2002) Inducible expression of Bcl-xL restricts apoptosis resistance to the antibody secretion phase in hybridoma cultures. Biotechnol Bioeng 79:180–187CrossRefGoogle Scholar
  30. 30.
    Gauthier ER, Piche L, Lemieux G, Lemieux R (1996) Role of Bcl-xL in the control of apoptosis in murine myeloma cells. Cancer Res 56:1451–1456Google Scholar
  31. 31.
    Charbonneau JR, Gauthier ER (2000) Prolongation of murine hybridoma cell survival in stationary batch culture by Bcl-xL expression. Cytotechnology 34:131–139CrossRefGoogle Scholar
  32. 32.
    Majors BS, Betenbaugh MJ, Pederson NE, Chiang GG (2009) Mcl-1 overexpression leads to higher viabilities and increased production of humanized monoclonal antibody in Chinese Hamster Ovary Cells. Biotechnol Prog 25:1161–1168CrossRefGoogle Scholar
  33. 33.
    Zhai D, Jin C, Huang Z, Satterthwait AC, Reed JC (2008) Differential regulation of Bax and Bak by anti-apoptotic Bcl-2 family proteins Bcl-B and Mcl-1. J Biol Chem 283:9580–9586CrossRefGoogle Scholar
  34. 34.
    Kozopas KM, Yang T, Buchan HL, Zhou P, Craig RW (1993) Mcl-1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to Bcl-2. Proc Natl Acad Sci USA 90:3516–3520CrossRefGoogle Scholar
  35. 35.
    O’Donnell JS, Long GV, Scolyer RA, Teng MW, Smyth MJ (2017) Resistance to PD1/PDL1 checkpoint inhibition. Cancer Treat Rev 52:71–81CrossRefGoogle Scholar
  36. 36.
    Pham PL, Perret S, Doan HC, Cass B, St-Laurent G, Kamen A, Durocher Y (2003) Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency. Biotechnol Bioeng 84:332–342CrossRefGoogle Scholar
  37. 37.
    Osmekhina E, Neubauer A, Klinzing K, Myllyharju J, Neubauer P (2010) Sandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylase. Microb Cell Fact 9:48CrossRefGoogle Scholar
  38. 38.
    Liu C, Dalby B, Chen W, Kilzer JM, Chiou HC (2008) Transient transfection factors for high-level recombinant protein production in suspension cultured mammalian cells. Mol Biotechnol 39:141–153CrossRefGoogle Scholar
  39. 39.
    Zustiak MP, Jose L, Xie YQ, Zhu JW, Betenbaugh MJ (2014) Enhanced transient recombinant protein production in CHO cells through the co-transfection of the product gene with Bcl-xL. Biotechnol J 9:1164–1174CrossRefGoogle Scholar
  40. 40.
    Ye J, Kober V, Tellers M, Naji Z, Salmon P, Markusen JF (2009) High-level protein expression in scalable CHO transient transfection. Biotechnol Bioeng 103:542–551CrossRefGoogle Scholar
  41. 41.
    Durocher Y, Perret S, Kamen A (2002) High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res 30(2):e9CrossRefGoogle Scholar
  42. 42.
    Han YK, Ha TK, Lee SJ, Lee JS, Lee GM (2011) Autophagy and apoptosis of recombinant Chinese hamster ovary cells during fed-batch culture: effect of nutrient supplementation. Biotechnol Bioeng 108:2182–2192CrossRefGoogle Scholar
  43. 43.
    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 103:757–766CrossRefGoogle Scholar
  44. 44.
    Majors BS, Betenbaugh MJ, Chiang GG (2007) Links between metabolism and apoptosis in mammalian cells: applications for anti-apoptosis engineering. Metab Eng 9:317–326CrossRefGoogle Scholar
  45. 45.
    Majors BS, Betenbaugh MJ, Pederson NF, Chiang GG (2008) Enhancement of transient gene expression and culture viability using Chinese hamster ovary cells overexpressing Bcl-xL. Biotechnol Bioeng 101:567–578CrossRefGoogle Scholar
  46. 46.
    Sandhu KS, Al-Rubeai M (2009) The effect of Bcl-2, YAMA, and XIAP over-expression on apoptosis and adenovirus production in HEK293 cell line. Biotechnol Bioeng 104:752–765Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Xinyu Zhang
    • 1
  • Lei Han
    • 1
  • Huifang Zong
    • 1
  • Kai Ding
    • 1
  • Yuan Yuan
    • 1
  • Jingyi Bai
    • 1
  • Yuexian Zhou
    • 1
  • Baohong Zhang
    • 1
  • Jianwei Zhu
    • 1
    • 2
  1. 1.Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education; School of PharmacyShanghai Jiao Tong UniversityShanghaiPeople’s Republic of China
  2. 2.Jecho Laboratories, Inc. 7320 Executive WayFrederickUSA

Personalised recommendations