Advertisement

Cytotechnology

, Volume 71, Issue 6, pp 1079–1093 | Cite as

The transient expression of CHIKV VLP in large stirred tank bioreactors

  • Peifeng ChenEmail author
  • Jacob Demirji
  • Vera B. Ivleva
  • Joe Horwitz
  • Richard Schwartz
  • Frank Arnold
Original Article

Abstract

Transient gene expression (TGE) bioprocesses have been difficult to scale up in large stirred tank bioreactors with volumes of more than 1.5 L. Low production levels are often observed, but the causes have not been investigated (Gutierrez-Granados et al. in Crit Rev Biotechnol 38:918–940, 2018). Chikungunya Virus-like particle (VLP), expressed by DNA–PEI transient transfection, is a representative case study for these difficulties. Clinical materials were produced in shake flasks, but the process suffered when transferred to large stirred tank bioreactors. The resulting process was not operationally friendly nor cost effective. In this study, a systematic approach was used to investigate the root causes of the poor scale up performance. The transfection conditions were first screened in ambr® 15 high throughput mini bioreactors then examined in 3 L stirred-tank systems. The studies found that production level was negatively correlated with inoculum cell growth status (P < 0.05). The pH range, DNA and PEI levels, order of the reagent addition, and gas-sparging systems were also studied and found to affect process performance. Further hydromechanical characterizations (Re, energy dissipation rates, and P/V, etc.) of shake flasks, ambr® 15, and 3-L stirred tank systems were performed. Overall, the study discovered that the shear stress (caused by a microsparger) and PEI toxicity together were the root causes of scale-up failure. Once the microsparger was replaced by a macrosparger, the scale-up was successful.

Keywords

Chikungunya virus Virus-like particle vaccine Transient gene expression DNA and PEI pH Shear stress Scale-up challenge ambr® 15 bioreactor Large stirred tank bioreactor 

Notes

Acknowledgments

We thank Robin Luedtke and Diane Wycuff (VPP/VRC/NIH) for reviewing the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no competing financial interest.

References

  1. Akahata W et al (2010) A VLP vaccine for epidemic chikungunya virus protects non-human primates against infection. Nat Med 16:334–338.  https://doi.org/10.1038/nm.2105 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Ansorge S, Lanthier S, Transfiguracion J, Durocher Y, Henry O, Kamen A (2009) Development of a scalable process for high-yield lentiviral vector production by transient transfection of HEK293 suspension cultures. J Gene Med 11:868–876.  https://doi.org/10.1002/jgm.1370 CrossRefPubMedGoogle Scholar
  3. Backliwal G, Hildinger M, Chenuet S, Wulhfard S, De Jesus M, Wurm FM (2008a) Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions. Nucleic Acids Res 36:e96.  https://doi.org/10.1093/nar/gkn423 CrossRefPubMedPubMedCentralGoogle Scholar
  4. Backliwal G, Hildinger M, Hasija V, Wurm FM (2008b) High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI. Biotechnol Bioeng 99:721–727.  https://doi.org/10.1002/bit.21596 CrossRefPubMedGoogle Scholar
  5. Bollin F, Dechavanne V, Chevalet L (2011) Design of experiment in CHO and HEK transient transfection condition optimization. Protein Expr Purif 78:61–68.  https://doi.org/10.1016/j.pep.2011.02.008 CrossRefPubMedGoogle Scholar
  6. Bos AB et al (2014) Development of a semi-automated high throughput transient transfection system. J Biotechnol 180:10–16.  https://doi.org/10.1016/j.jbiotec.2014.03.027 CrossRefPubMedGoogle Scholar
  7. Boulton-Stone JM, Blake JR (1993) Gas bubbles bursting at a free surface. J Fluid Mech 254:437–466.  https://doi.org/10.1017/S0022112093002216 CrossRefGoogle Scholar
  8. Boussif O, Lezoualc’h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, Behr JP (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci USA 92:7297–7301CrossRefGoogle Scholar
  9. Breunig M, Lungwitz U, Liebl R, Goepferich A (2007) Breaking up the correlation between efficacy and toxicity for nonviral gene delivery. Proc Natl Acad Sci USA 104:14454–14459.  https://doi.org/10.1073/pnas.0703882104 CrossRefPubMedGoogle Scholar
  10. Brunner S, Sauer T, Carotta S, Cotten M, Saltik M, Wagner E (2000) Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus. Gene Ther 7:401–407.  https://doi.org/10.1038/sj.gt.3301102 CrossRefPubMedGoogle Scholar
  11. Carpentier E, Paris S, Kamen AA, Durocher Y (2007) Limiting factors governing protein expression following polyethylenimine-mediated gene transfer in HEK293-EBNA1 cells. J Biotechnol 128:268–280.  https://doi.org/10.1016/j.jbiotec.2006.10.014 CrossRefPubMedGoogle Scholar
  12. Cervera L, Gutiérrez-Granados S, Martínez M, Blanco J, Gòdia F, Segura MM (2013) Generation of HIV-1 Gag VLPs by transient transfection of HEK 293 suspension cell cultures using an optimized animal-derived component free medium. J Biotechnol 166:152–165.  https://doi.org/10.1016/j.jbiotec.2013.05.001 CrossRefPubMedGoogle Scholar
  13. Chang LJ et al (2014) Safety and tolerability of chikungunya virus-like particle vaccine in healthy adults: a phase 1 dose-escalation trial. Lancet 384:2046–2052.  https://doi.org/10.1016/s0140-6736(14)61185-5 CrossRefPubMedGoogle Scholar
  14. de los Milagros Bassani Molinas M, Beer C, Hesse F, Wirth M, Wagner R (2014) Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring. Cytotechnology 66:493–514.  https://doi.org/10.1007/s10616-013-9601-3 CrossRefPubMedGoogle Scholar
  15. Delouvroy F, Siriez G, Tran A-V, Mukankurayija L, Kochanowski N, Malphettes L (2015) ambr™ Mini-bioreactor as a high-throughput tool for culture process development to accelerate transfer to stainless steel manufacturing scale: comparability study from process performance to product quality attributes. BMC Proc 9:P78.  https://doi.org/10.1186/1753-6561-9-s9-p78 CrossRefPubMedCentralGoogle Scholar
  16. Derouazi M, Girard P, Van Tilborgh F, Iglesias K, Muller N, Bertschinger M, Wurm FM (2004) Serum-free large-scale transient transfection of CHO cells. Biotechnol Bioeng 87:537–545.  https://doi.org/10.1002/bit.20161 CrossRefPubMedGoogle Scholar
  17. 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:E9CrossRefGoogle Scholar
  18. Fliedl L, Kaisermayer C (2011) Transient gene expression in HEK293 and vero cells immobilised on microcarriers. J Biotechnol 153:15–21.  https://doi.org/10.1016/j.jbiotec.2011.02.007 CrossRefPubMedGoogle Scholar
  19. Florea B, Meaney C, Junginger HE, Borchard G (2002) Transfection efficiency and toxicity of polyethylenimine in differentiated Calu-3 and nondifferentiated COS-1 cell cultures. AAPS PharmSci 4:4.  https://doi.org/10.1208/ps040312 CrossRefGoogle Scholar
  20. Garcia-Briones MA, Chalmers JJ (1994) Flow parameters associated with hydrodynamic cell injury. Biotechnol Bioeng 44:1089–1098.  https://doi.org/10.1002/bit.260440910 CrossRefPubMedGoogle Scholar
  21. Gebhart CL, Kabanov AV (2001) Evaluation of polyplexes as gene transfer agents. J Control Release 73:401–416CrossRefGoogle Scholar
  22. Grieger JC, Soltys SM, Samulski RJ (2016) Production of recombinant adeno-associated virus vectors using suspension HEK293 cells and continuous harvest of vector from the culture media for GMP FIX and FLT1. Clin Vector Mol Ther 24:287–297.  https://doi.org/10.1038/mt.2015.187 CrossRefGoogle Scholar
  23. Grosjean F, Batard P, Jordan M, Wurm FM (2002) S-phase synchronized CHO cells show elevated transfection efficiency and expression using CaPi. Cytotechnology 38:57–62.  https://doi.org/10.1023/A:1021197830091 CrossRefPubMedPubMedCentralGoogle Scholar
  24. Grosse S, Thevenot G, Monsigny M, Fajac I (2006) Which mechanism for nuclear import of plasmid DNA complexed with polyethylenimine derivatives? J Gene Med 8:845–851.  https://doi.org/10.1002/jgm.915 CrossRefPubMedGoogle Scholar
  25. Gutierrez-Granados S, Cervera L, Kamen AA, Godia F (2018) Advancements in mammalian cell transient gene expression (TGE) technology for accelerated production of biologics. Crit Rev Biotechnol 38:918–940.  https://doi.org/10.1080/07388551.2017.1419459 CrossRefPubMedGoogle Scholar
  26. Han X, Fang Q, Yao F, Wang X, Wang J, Yang S, Shen BQ (2009) The heterogeneous nature of polyethylenimine-DNA complex formation affects transient gene expression. Cytotechnology 60:63.  https://doi.org/10.1007/s10616-009-9215-y CrossRefPubMedPubMedCentralGoogle Scholar
  27. Hu W, Berdugo C, Chalmers JJ (2011) The potential of hydrodynamic damage to animal cells of industrial relevance: current understanding. Cytotechnology 63:445–460.  https://doi.org/10.1007/s10616-011-9368-3 CrossRefPubMedPubMedCentralGoogle Scholar
  28. Janakiraman V, Kwiatkowski C, Kshirsagar R, Ryll T, Huang YM (2015) Application of high-throughput mini-bioreactor system for systematic scale-down modeling, process characterization, and control strategy development. Biotechnol Prog 31:1623–1632.  https://doi.org/10.1002/btpr.2162 CrossRefPubMedGoogle Scholar
  29. Kafil V, Omidi Y (2011) Cytotoxic impacts of linear and branched polyethylenimine nanostructures in A431. Cells Bioimpacts 1:23–30.  https://doi.org/10.5681/bi.2011.004 CrossRefPubMedGoogle Scholar
  30. Kim TK, Eberwine JH (2010) Mammalian cell transfection: the present and the future. Anal Bioanal Chem 397:3173–3178.  https://doi.org/10.1007/s00216-010-3821-6 CrossRefPubMedPubMedCentralGoogle Scholar
  31. Kioukia N, Nienow AW, Al-Rubeai M, Emery AN (1996) Influence of agitation and sparging on the growth rate and infection of insect cells in bioreactors and a comparison with hybridoma culture. Biotechnol Prog 12:779–785.  https://doi.org/10.1021/bp9600703 CrossRefGoogle Scholar
  32. Kroll C, Rathert P (2018) Stable expression of epigenome editors via viral delivery and genomic integration. Methods Mol Biol 1767:215–225.  https://doi.org/10.1007/978-1-4939-7774-1_11 CrossRefPubMedGoogle Scholar
  33. Ma N, Koelling KW, Chalmers JJ (2002) Fabrication and use of a transient contractional flow device to quantify the sensitivity of mammalian and insect cells to hydrodynamic forces. Biotechnol Bioeng 80:428–437.  https://doi.org/10.1002/bit.10387 CrossRefPubMedGoogle Scholar
  34. Ma N, Chalmers JJ, Aunins JG, Zhou W, Xie L (2004) Quantitative studies of cell-bubble interactions and cell damage at different pluronic F-68 and cell concentrations. Biotechnol Prog 20:1183–1191.  https://doi.org/10.1021/bp0342405 CrossRefPubMedGoogle Scholar
  35. Mennesson E, Erbacher P, Kuzak M, Kieda C, Midoux P, Pichon C (2006) DNA/cationic polymer complex attachment on a human vascular endothelial cell monolayer exposed to a steady laminar flow. J Control Release 114:389–397.  https://doi.org/10.1016/j.jconrel.2006.06.006 CrossRefPubMedGoogle Scholar
  36. Muller N, Derouazi M, Van Tilborgh F, Wulhfard S, Hacker DL, Jordan M, Wurm FM (2007) Scalable transient gene expression in Chinese hamster ovary cells in instrumented and non-instrumented cultivation systems. Biotechnol Lett 29:703–711.  https://doi.org/10.1007/s10529-006-9298-x CrossRefPubMedGoogle Scholar
  37. Nienow AW (2006) Reactor engineering in large scale animal cell culture. Cytotechnology 50:9–33.  https://doi.org/10.1007/s10616-006-9005-8 CrossRefPubMedPubMedCentralGoogle Scholar
  38. Park JY, Lim BP, Lee K, Kim YG, Jo EC (2006) Scalable production of adeno-associated virus type 2 vectors via suspension transfection. Biotechnol Bioeng 94:416–430.  https://doi.org/10.1002/bit.20776 CrossRefPubMedGoogle Scholar
  39. Peter CP, Suzuki Y, Buchs J (2006) Hydromechanical stress in shake flasks: correlation for the maximum local energy dissipation rate. Biotechnol Bioeng 93:1164–1176.  https://doi.org/10.1002/bit.20827 CrossRefPubMedGoogle Scholar
  40. 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 84:332–342.  https://doi.org/10.1002/bit.10774 CrossRefPubMedGoogle Scholar
  41. 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 112:977–986.  https://doi.org/10.1002/bit.25514 CrossRefPubMedGoogle Scholar
  42. Rawat J, Gadgil M (2016) Shear stress increases cytotoxicity and reduces transfection efficiency of liposomal gene delivery to CHO-S cells. Cytotechnology 68:2529–2538.  https://doi.org/10.1007/s10616-016-9974-1 CrossRefPubMedPubMedCentralGoogle Scholar
  43. Raymond C, Tom R, Perret S, Moussouami P, L’Abbe D, St-Laurent G, Durocher Y (2011) A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications. Methods 55:44–51.  https://doi.org/10.1016/j.ymeth.2011.04.002 CrossRefPubMedGoogle Scholar
  44. Recillas-Targa F (2006) Multiple strategies for gene transfer, expression, knockdown, and chromatin influence in mammalian cell lines and transgenic animals. Mol Biotechnol 34:337–354.  https://doi.org/10.1385/mb:34:3:337 CrossRefPubMedGoogle Scholar
  45. Rosser MP et al (2005) Transient transfection of CHO-K1-S using serum-free medium in suspension: a rapid mammalian protein expression system. Protein Expr Purif 40:237–243.  https://doi.org/10.1016/j.pep.2004.07.015 CrossRefPubMedGoogle Scholar
  46. Schlaeger EJ, Christensen K (1999) Transient gene expression in mammalian cells grown in serum-free suspension culture. Cytotechnology 30:71–83.  https://doi.org/10.1023/a:1008000327766 CrossRefPubMedPubMedCentralGoogle Scholar
  47. Segura MM, Garnier A, Durocher Y, Coelho H, Kamen A (2007) Production of lentiviral vectors by large-scale transient transfection of suspension cultures and affinity chromatography purification. Biotechnol Bioeng 98:789–799.  https://doi.org/10.1002/bit.21467 CrossRefPubMedGoogle Scholar
  48. Shaddeau AW, Schneck NA, Li Y, Ivleva VB, Arnold FJ, Cooper JW, Lei QP (2019) Development of a new tandem ion exchange and size exclusion chromatography method to monitor vaccine particle titer in cell culture media. Anal Chem 91:6430–6434.  https://doi.org/10.1021/acs.analchem.9b00095 CrossRefPubMedGoogle Scholar
  49. Shin HS, Kim HJ, Sim SJ, Jeon NL (2009) Shear stress effect on transfection of neurons cultured in microfluidic devices. J Nanosci Nanotechnol 9:7330–7335.  https://doi.org/10.1166/jnn.2009.1769 CrossRefPubMedGoogle Scholar
  50. Simon F, Javelle E, Oliver M, Leparc-Goffart I, Marimoutou C (2011) Chikungunya virus infection current infectious disease reports 13:218–228.  https://doi.org/10.1007/s11908-011-0180-1 CrossRefPubMedGoogle Scholar
  51. Smalley C, Erasmus JH, Chesson CB, Beasley DWC (2016) Status of research and development of vaccines for chikungunya. Vaccine 34:2976–2981.  https://doi.org/10.1016/j.vaccine.2016.03.076 CrossRefPubMedGoogle Scholar
  52. Sun X, Hia HC, Goh PE, Yap MGS (2008) High-density transient gene expression in suspension-adapted 293 EBNA1 cells. Biotechnol Bioeng 99:108–116.  https://doi.org/10.1002/bit.21537 CrossRefPubMedGoogle Scholar
  53. Tait AS, Brown CJ, Galbraith DJ, Hines MJ, Hoare M, Birch JR, James DC (2004) Transient production of recombinant proteins by Chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents. Biotechnol Bioeng 88:707–721.  https://doi.org/10.1002/bit.20265 CrossRefPubMedGoogle Scholar
  54. Tuvesson O, Uhe C, Rozkov A, Lüllau E (2008) Development of a generic transient transfection process at 100 L scale. Cytotechnology 56:123–136.  https://doi.org/10.1007/s10616-008-9135-2 CrossRefPubMedPubMedCentralGoogle Scholar
  55. van Gaal EV, van Eijk R, Oosting RS, Kok RJ, Hennink WE, Crommelin DJ, Mastrobattista E (2011) How to screen non-viral gene delivery systems in vitro? J Control Release 154:218–232.  https://doi.org/10.1016/j.jconrel.2011.05.001 CrossRefPubMedGoogle Scholar
  56. Varley J, Birch J (1999) Reactor design for large scale suspension animal cell culture. Cytotechnology 29:177–205.  https://doi.org/10.1023/a:1008008021481 CrossRefPubMedPubMedCentralGoogle Scholar
  57. Velez-Suberbie ML, Betts JPJ, Walker KL, Robinson C, Zoro B, Keshavarz-Moore E (2018) High throughput automated microbial bioreactor system used for clone selection and rapid scale-down process optimization. Biotechnol Prog 34:58–68.  https://doi.org/10.1002/btpr.2534 CrossRefPubMedGoogle Scholar
  58. Venereo-Sanchez A et al (2016) Hemagglutinin and neuraminidase containing virus-like particles produced in HEK-293 suspension culture: an effective influenza vaccine candidate. Vaccine 34:3371–3380.  https://doi.org/10.1016/j.vaccine.2016.04.089 CrossRefPubMedGoogle Scholar
  59. Won Y-Y, Sharma R, Konieczny SF (2009) Missing pieces in understanding the intracellular trafficking of polycation/DNA complexes. J Control Release 139:88–93.  https://doi.org/10.1016/j.jconrel.2009.06.031 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Vaccine Production Program, Vaccine Research CenterNational Institute of Allergy and Infectious Diseases, National Institutes of HealthGaithersburgUSA
  2. 2.Amicus TherapeuticsCranburyUSA
  3. 3.CRISPR TherapeuticsCambridgeUSA
  4. 4.Tunnell ConsultingKing of PrussiaUSA

Personalised recommendations