Skip to main content
SpringerLink
Log in

Upstream and Downstream Processes for Viral Nanoplexes as Vaccines

  • Protocol
  • First Online:
Vaccine Delivery Technology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2183))

Abstract

The increasing medical interest in viral nanoplexes, such as viruses or virus-like particles used for vaccines, gene therapy products, or oncolytic agents, raises the need for fast and efficient production processes. In general, these processes comprise upstream and downstream processing. For the upstream process, efficiency is mainly characterized by robustly achieving high titer yields, while reducing process times and costs with regard to the cell culture medium, the host cell selection, and the applied process conditions. The downstream part, on the other hand, should effectively remove process-related contaminants, such as host cells/cell debris as well as host cell DNA and proteins, while maintaining product stability and reducing product losses. This chapter outlines a combination of process steps to successfully produce virus particles in the controlled environment of a stirred tank bioreactor, combined with a platform-based purification approach using filtration-based clarification and steric exclusion chromatography. Additionally, suggestions for off-line analytics in terms of virus characterization and quantification as well as for contaminant estimation are provided.

Keven Lothert and Gregor Dekevic shared first authorship.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Nachbagauer R, Krammer F (2017) Universal influenza virus vaccines and therapeutic antibodies. Clin Microbiol Infect 23(4):222–228. https://doi.org/10.1016/j.cmi.2017.02.009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Patel A, Dunlop J, Owsianka A et al (2015) Current and future prophylactic vaccines for hepatitis C virus. Vaccines Dev Ther 31. https://doi.org/10.2147/VDT.S48437

  3. Wolff MW, Reichl U (2011) Downstream processing of cell culture-derived virus particles. Expert Rev Vaccines 10(10):1451–1475. https://doi.org/10.1586/erv.11.111

    Article  Google Scholar 

  4. Santiago-Ortiz JL, Schaffer DV (2016) Adeno-associated virus (AAV) vectors in cancer gene therapy. J Control Release 240:287–301. https://doi.org/10.1016/j.jconrel.2016.01.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Nestola P, Peixoto C, Silva RRJS et al (2015) Improved virus purification processes for vaccines and gene therapy. Biotechnol Bioeng 112(5):843–857. https://doi.org/10.1002/bit.25545

    Article  CAS  PubMed  Google Scholar 

  6. Kaufman HL, Kohlhapp FJ, Zloza A (2015) Oncolytic viruses: a new class of immunotherapy drugs. Nat Rev Drug Discov 14(9):642–662. https://doi.org/10.1038/nrd4663

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Lawler SE, Speranza M-C, Cho C-F et al (2017) Oncolytic viruses in cancer treatment: a review. JAMA Oncol 3(6):841–849. https://doi.org/10.1001/jamaoncol.2016.2064

    Article  PubMed  Google Scholar 

  8. Weiss K, Gerstenberger J, Salzig D et al (2015) Oncolytic measles viruses produced at different scales under serum-free conditions. Eng Life Sci 15(4):425–436. https://doi.org/10.1002/elsc.201400165

    Article  CAS  Google Scholar 

  9. Grein TA, Loewe D, Dieken H et al (2018) High titer oncolytic measles virus production process by integration of dielectric spectroscopy as online monitoring system. Biotechnol Bioeng 115(5):1186–1194. https://doi.org/10.1002/bit.26538

    Article  CAS  PubMed  Google Scholar 

  10. Genzel Y, Rödig J, Rapp E et al (2014) Vaccine production: upstream processing with adherent or suspension cell lines. Methods Mol Biol 1104:371–393. https://doi.org/10.1007/978-1-62703-733-4_23

    Article  CAS  PubMed  Google Scholar 

  11. Vázquez-Ramírez D, Jordan I, Sandig V et al (2019) High titer MVA and influenza A virus production using a hybrid fed-batch/perfusion strategy with an ATF system. Appl Microbiol Biotechnol 103(7):3025–3035. https://doi.org/10.1007/s00253-019-09694-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Jiang W, Hua R, Wei M et al (2015) An optimized method for high-titer lentivirus preparations without ultracentrifugation. Sci Rep 5(5):13875. https://doi.org/10.1038/srep13875

    Article  PubMed  PubMed Central  Google Scholar 

  13. Tapia F, Vogel T, Genzel Y et al (2014) Production of high-titer human influenza A virus with adherent and suspension MDCK cells cultured in a single-use hollow fiber bioreactor. Vaccine 32(8):1003–1011. https://doi.org/10.1016/j.vaccine.2013.11.044

    Article  CAS  PubMed  Google Scholar 

  14. Grein TA, Loewe D, Dieken H et al (2019) Aeration and shear stress are critical process parameters for the production of oncolytic measles virus. Front Bioeng Biotechnol 7:78. https://doi.org/10.3389/fbioe.2019.00078

    Article  PubMed  PubMed Central  Google Scholar 

  15. Firquet S, Beaujard S, Lobert P-E et al (2015) Survival of enveloped and non-enveloped viruses on inanimate surfaces. Microbes Environ 30(2):140–144. https://doi.org/10.1264/jsme2.ME14145

    Article  PubMed  PubMed Central  Google Scholar 

  16. Black FL (1959) Growth and stability of measles virus. Virology 7:184–192. https://doi.org/10.1016/0042-6822(59)90186-2

    Article  CAS  PubMed  Google Scholar 

  17. Kohn A, Yassky D (1962) Growth of measles virus in kb cells. Virology 17:157–163. https://doi.org/10.1016/0042-6822(62)90092-2

    Article  CAS  PubMed  Google Scholar 

  18. Weiss K, Salzig D, Mühlebach MD et al (2012) Key parameters of measles virus production for oncolytic virotherapy. Am J Biochem Biotechnol 8(2):81–98. https://doi.org/10.3844/ajbbsp.2012.81.98

    Article  CAS  Google Scholar 

  19. Trabelsi K, Majoul S, Rourou S et al (2012) Development of a measles vaccine production process in MRC-5 cells grown on Cytodex1 microcarriers and in a stirred bioreactor. Appl Microbiol Biotechnol 93:1031–1040. https://doi.org/10.1007/s00253-011-3574-y

    Article  CAS  PubMed  Google Scholar 

  20. Weiss K, Salzig D, Röder Y et al (2013) Influence of process conditions on measles virus stability. Am J Biochem Biotechnol 9(3):243–254. https://doi.org/10.3844/ajbbsp.2013.243.254

    Article  Google Scholar 

  21. Loewe D, Häussler J, Grein TA et al (2019) Forced degradation studies to identify critical process parameters for the purification of infectious measles virus. Viruses 11(8). https://doi.org/10.3390/v11080725

  22. Rapp F, Butel JS, Wallis C (1965) Protection of measles virus by sulfate ions against thermal inactivation. J Bacteriol 90:132–135

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Chisti Y (2000) Animal-cell damage in sparged bioreactors. Trends Biotechnol 18:420–432. https://doi.org/10.1016/S0167-7799(00)01474-8

    Article  CAS  PubMed  Google Scholar 

  24. Crouch CF, Fowler HW, Spier RE (1985) The adhesion of animal cells to surfaces: the measurement of critical surface shear stress permitting attachment or causing detachment. J Chem Tech Biotechnol 35:273–281

    Article  Google Scholar 

  25. Cherry RS, Papoutsakis ET (1989) Growth and death rates of bovine embryonic kidney cells in turbulent microcarrier bioreactors. Bioprocess Eng 4:81–89. https://doi.org/10.1007/BF00373735

    Article  Google Scholar 

  26. Baczko K, Lazzarini RA (1979) Efficient propagation of measles virus in suspension cultures. J Virol 31:854–855

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Sviben D, Forčić D, Kurtović T et al (2016) Stability, biophysical properties and effect of ultracentrifugation and diafiltration on measles virus and mumps virus. Arch Virol 161(6):1455–1467. https://doi.org/10.1007/s00705-016-2801-3

    Article  CAS  PubMed  Google Scholar 

  28. Wolff MW, Reichl U (2008) Downstream processing: from egg to cell culture-derived influenza virus particles. Chem Eng Technol 31(6):846–857. https://doi.org/10.1002/ceat.200800118

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Weigel T, Soliman R, Wolff MW et al (2019) Hydrophobic-interaction chromatography for purification of influenza A and B virus. J Chromatogr B Analyt Technol Biomed Life Sci 1117:103–117. https://doi.org/10.1016/j.jchromb.2019.03.037

    Article  CAS  PubMed  Google Scholar 

  30. Wolff MW, Siewert C, Lehmann S et al (2010) Capturing of cell culture-derived modified Vaccinia Ankara virus by ion exchange and pseudo-affinity membrane adsorbers. Biotechnol Bioeng 105(4):761–769. https://doi.org/10.1002/bit.22595

    Article  CAS  PubMed  Google Scholar 

  31. Wolff MW, Siewert C, Hansen SP et al (2010) Purification of cell culture-derived modified vaccinia ankara virus by pseudo-affinity membrane adsorbers and hydrophobic interaction chromatography. Biotechnol Bioeng 107(2):312–320. https://doi.org/10.1002/bit.22797

    Article  CAS  PubMed  Google Scholar 

  32. Orr V, Zhong L, Moo-Young M et al (2013) Recent advances in bioprocessing application of membrane chromatography. Biotechnol Adv 31(4):450–465. https://doi.org/10.1016/j.biotechadv.2013.01.007

    Article  CAS  PubMed  Google Scholar 

  33. Grein TA, Michalsky R, Vega López M et al (2012) Purification of a recombinant baculovirus of Autographa californica M nucleopolyhedrovirus by ion exchange membrane chromatography. J Virol Methods 183(2):117–124. https://doi.org/10.1016/j.jviromet.2012.03.031

    Article  CAS  PubMed  Google Scholar 

  34. Peixoto C, Ferreira TB, Sousa MFQ et al (2008) Towards purification of adenoviral vectors based on membrane technology. Biotechnol Prog 24(6):1290–1296. https://doi.org/10.1002/btpr.25

    Article  CAS  PubMed  Google Scholar 

  35. Nestola P, Peixoto C, Villain L et al (2015) Rational development of two flowthrough purification strategies for adenovirus type 5 and retro virus-like particles. J Chromatogr A 1426:91–101. https://doi.org/10.1016/j.chroma.2015.11.037

    Article  CAS  PubMed  Google Scholar 

  36. Opitz L, Salaklang J, Büttner H et al (2007) Lectin-affinity chromatography for downstream processing of MDCK cell culture derived human influenza A viruses. Vaccine 25(5):939–947. https://doi.org/10.1016/j.vaccine.2006.08.043

    Article  CAS  PubMed  Google Scholar 

  37. Weigel T, Solomaier T, Wehmeyer S et al (2016) A membrane-based purification process for cell culture-derived influenza A virus. J Biotechnol 220:12–20. https://doi.org/10.1016/j.jbiotec.2015.12.022

    Article  CAS  PubMed  Google Scholar 

  38. Fortuna AR, van Teeffelen S, Ley A et al (2019) Use of sulfated cellulose membrane adsorbers for chromatographic purification of cell cultured-derived influenza A and B viruses. Sep Purif Technol 226:350–358. https://doi.org/10.1016/j.seppur.2019.05.101

    Article  CAS  Google Scholar 

  39. Opitz L, Lehmann S, Reichl U et al (2009) Sulfated membrane adsorbers for economic pseudo-affinity capture of influenza virus particles. Biotechnol Bioeng 103(6):1144–1154. https://doi.org/10.1002/bit.22345

    Article  CAS  PubMed  Google Scholar 

  40. Carvalho SB, Fortuna AR, Wolff MW et al (2018) Purification of influenza virus-like particles using sulfated cellulose membrane adsorbers. J Chem Technol Biotechnol 93(7):1988–1996. https://doi.org/10.1002/jctb.5474

    Article  CAS  Google Scholar 

  41. Hoffmann D, Leber J, Loewe D et al (2019) Purification of new biologicals using membrane-based processes. In: Basile A, Charcosset C (eds) Current trends and future developments on (bio-) membranes. Elsevier, Amsterdam, pp 123–150

    Chapter  Google Scholar 

  42. Transfiguracion J, Jorio H, Meghrous J et al (2007) High yield purification of functional baculovirus vectors by size exclusion chromatography. J Virol Methods 142(1–2):21–28. https://doi.org/10.1016/j.jviromet.2007.01.002

  43. Kalbfuss B, Wolff M, Morenweiser R et al (2007) Purification of cell culture-derived human influenza A virus by size-exclusion and anion-exchange chromatography. Biotechnol Bioeng 96(5):932–944. https://doi.org/10.1002/bit.21109

    Article  CAS  PubMed  Google Scholar 

  44. Lee J, Gan HT, Latiff SMA et al (2012) Principles and applications of steric exclusion chromatography. J Chromatogr A 1270:162–170. https://doi.org/10.1016/j.chroma.2012.10.062

    Article  CAS  PubMed  Google Scholar 

  45. Ingham KC (1978) Precipitation of proteins with polyethylene glycol: characterization of albumin. Arch Biochem Biophys 186(1):106–1013

    Article  CAS  PubMed  Google Scholar 

  46. Atha DH, Ingham KC (1981) Mechanism of precipitation of proteins by polyethylene glycols. J Biol Chem 256(23):12108–12117

    CAS  PubMed  Google Scholar 

  47. Polson A (1977) A theory for the displacement of proteins and viruses with polyethylene glycol. Prep Biochem 7:129–154

    CAS  PubMed  Google Scholar 

  48. Arakawa T, Timasheff SN (1985) Mechanism of poly(ethylene glycol) interaction with proteins. Biochemistry 24:6756–6762

    Article  CAS  PubMed  Google Scholar 

  49. Timasheff SN, Arakawa T (1988) Mechanism of protein precipitation and stabilization by co-solvents. J Cryst Growth 90:39–46

    Article  CAS  Google Scholar 

  50. Sim S-L, He T, Tscheliessnig A et al (2012) Branched polyethylene glycol for protein precipitation. Biotechnol Bioeng 109(3):736–746. https://doi.org/10.1002/bit.24343

    Article  CAS  PubMed  Google Scholar 

  51. Wang Y, Sarkar M, Smith AE et al (2012) Macromolecular crowding and protein stability. J Am Chem Soc 134(40):16614–16618. https://doi.org/10.1021/ja305300m

    Article  CAS  PubMed  Google Scholar 

  52. Lewis GD, Metcalf TG (1988) Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus, from oyster, water, and sediment samples. Appl Environ Microbiol 54(8):1983–1988

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Ludwig A-K, de Miroschedji K, Doeppner TR et al (2018) Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales. J Extracell Vesicles 7(1):1528109. https://doi.org/10.1080/20013078.2018.1528109

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Gagnon P, Toh P, Lee J (2014) High productivity purification of immunoglobulin G monoclonal antibodies on starch-coated magnetic nanoparticles by steric exclusion of polyethylene glycol. J Chromatogr A 1324:171–180. https://doi.org/10.1016/j.chroma.2013.11.039

    Article  CAS  PubMed  Google Scholar 

  55. Tao S-P, Zheng J, Sun Y (2015) Grafting zwitterionic polymer onto cryogel surface enhances protein retention in steric exclusion chromatography on cryogel monolith. J Chromatogr A 1389:104–111. https://doi.org/10.1016/j.chroma.2015.02.051

    Article  CAS  PubMed  Google Scholar 

  56. Wang C, Bai S, Tao S-P et al (2014) Evaluation of steric exclusion chromatography on cryogel column for the separation of serum proteins. J Chromatogr A 1333:54–59. https://doi.org/10.1016/j.chroma.2014.01.059

    Article  CAS  PubMed  Google Scholar 

  57. Marichal-Gallardo P, Pieler MM, Wolff MW et al (2017) Steric exclusion chromatography for purification of cell culture-derived influenza A virus using regenerated cellulose membranes and polyethylene glycol. J Chromatogr A 1483:110–119. https://doi.org/10.1016/j.chroma.2016.12.076

    Article  CAS  PubMed  Google Scholar 

  58. Lothert K, Sprick G, Beyer F et al (2019) Membrane-based steric exclusion chromatography for the purification of a recombinant baculovirus and its application for cell therapy. J Virol Methods 275:113756. https://doi.org/10.1016/j.jviromet.2019.113756

    Article  CAS  PubMed  Google Scholar 

  59. (1998) WHO Expert Committee on biological standardization. World Health Organ Tech Rep Ser 878: i–vi, 1–101

    Google Scholar 

  60. Sheng-Fowler L, Lewis AM, Peden K (2009) Issues associated with residual cell-substrate DNA in viral vaccines. Biologicals 37(3):190–195. https://doi.org/10.1016/j.biologicals.2009.02.015

    Article  CAS  PubMed  Google Scholar 

  61. Akpinar F, Yin J (2015) Characterization of vesicular stomatitis virus populations by tunable resistive pulse sensing. J Virol Methods 218:71–76. https://doi.org/10.1016/j.jviromet.2015.02.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Yang L, Yamamoto T (2016) Quantification of virus particles using nanopore-based resistive-pulse sensing techniques. Front Microbiol 7:1500. https://doi.org/10.3389/fmicb.2016.01500

    Article  PubMed  PubMed Central  Google Scholar 

  63. Murphy RM (1997) Static and dynamic light scattering of biological macromolecules: what can we learn? Curr Opin Biotechnol 8(1):25–30. https://doi.org/10.1016/S0958-1669(97)80153-X

    Article  CAS  PubMed  Google Scholar 

  64. Citkowicz A, Petry H, Harkins RN et al (2008) Characterization of virus-like particle assembly for DNA delivery using asymmetrical flow field-flow fractionation and light scattering. Anal Biochem 376(2):163–172. https://doi.org/10.1016/j.ab.2008.02.011

    Article  CAS  PubMed  Google Scholar 

  65. Driskell JD, Jones CA, Tompkins SM et al (2011) One-step assay for detecting influenza virus using dynamic light scattering and gold nanoparticles. Analyst 136(15):3083–3090. https://doi.org/10.1039/C1AN15303J

    Article  CAS  PubMed  Google Scholar 

  66. Vestad B, Llorente A, Neurauter A et al (2017) Size and concentration analyses of extracellular vesicles by nanoparticle tracking analysis: a variation study. J Extracell Vesicles 6(1):1344087. https://doi.org/10.1080/20013078.2017.1344087

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Soo CY, Song Y, Zheng Y et al (2012) Nanoparticle tracking analysis monitors microvesicle and exosome secretion from immune cells. Immunology 136(2):192–197. https://doi.org/10.1111/j.1365-2567.2012.03569.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Kramberger P, Ciringer M, Štrancar A et al (2012) Evaluation of nanoparticle tracking analysis for total virus particle determination. Virol J 9:265. https://doi.org/10.1186/1743-422X-9-265

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Harris A, Cardone G, Winkler DC et al (2006) Influenza virus pleiomorphy characterized by cryoelectron tomography. Proc Natl Acad Sci U S A 103(50):19123–19127. https://doi.org/10.1073/pnas.0607614103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Ruigrok RW, Andree PJ, van Hooft Huysduynen RA et al (1984) Characterization of three highly purified influenza virus strains by electron microscopy. J Gen Virol 65(Pt 4):799–802. https://doi.org/10.1099/0022-1317-65-4-799

    Article  CAS  PubMed  Google Scholar 

  71. Pease LF, Lipin DI, Tsai D-H et al (2009) Quantitative characterization of virus-like particles by asymmetrical flow field flow fractionation, electrospray differential mobility analysis, and transmission electron microscopy. Biotechnol Bioeng 102(3):845–855. https://doi.org/10.1002/bit.22085

    Article  CAS  PubMed  Google Scholar 

  72. Li Y, Wang Q, Bergmann SM et al (2019) Preparation of monoclonal antibodies against KHV and establishment of an antigen sandwich ELISA for KHV detection. Microb Pathog 128:36–40. https://doi.org/10.1016/j.micpath.2018.12.034

    Article  CAS  PubMed  Google Scholar 

  73. Pfefferkorn M, Böhm S, Schott T et al (2018) Quantification of large and middle proteins of hepatitis B virus surface antigen (HBsAg) as a novel tool for the identification of inactive HBV carriers. Gut 67(11):2045–2053. https://doi.org/10.1136/gutjnl-2017-313811

    Article  CAS  PubMed  Google Scholar 

  74. Rajendran M, Sun W, Comella P et al (2018) An immuno-assay to quantify influenza virus hemagglutinin with correctly folded stalk domains in vaccine preparations. PLoS One 13(4):e0194830. https://doi.org/10.1371/journal.pone.0194830

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Jensen KS, Adams R, Bennett RS et al (2018) Development of a novel real-time polymerase chain reaction assay for the quantitative detection of Nipah virus replicative viral RNA. PLoS One 13(6):e0199534. https://doi.org/10.1371/journal.pone.0199534

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Abachin E, Convers S, Falque S et al (2018) Comparison of reverse-transcriptase qPCR and droplet digital PCR for the quantification of dengue virus nucleic acid. Biologicals 52:49–54. https://doi.org/10.1016/j.biologicals.2018.01.001

    Article  CAS  PubMed  Google Scholar 

  77. Rush BS, Coughlin ML, Sanyal G (2018) In vitro infectivity of oncolytic Newcastle Disease Virus: correlation between plaque and fluorescent focus assays. J Virol Methods 251:69–74. https://doi.org/10.1016/j.jviromet.2017.09.029

    Article  CAS  PubMed  Google Scholar 

  78. Tobita K, Sugiura A, Enomoto C et al (1975) Plaque assay and primary isolation of influenza a viruses in an established line of canine kidney cells (MDCK) in the presence of trypsin. Med Microbiol Immunol 162(1):9–14. https://doi.org/10.1007/BF02123572

    Article  CAS  PubMed  Google Scholar 

  79. Tatsumoto N, Miyauchi T, Arditi M et al (2018) Quantification of infectious sendai virus using plaque assay. Bio Protoc 8(21). https://doi.org/10.21769/BioProtoc.3068

  80. Karlsson EA, Meliopoulos VA, Tran V et al (2018) Measuring influenza virus infection using bioluminescent reporter viruses for in vivo imaging and in vitro replication assays. Methods Mol Biol 1836:431–459. https://doi.org/10.1007/978-1-4939-8678-1_21

    Article  CAS  PubMed  Google Scholar 

  81. Lambeth CR, White LJ, Johnston RE et al (2005) Flow cytometry-based assay for titrating dengue virus. J Clin Microbiol 43(7):3267–3272. https://doi.org/10.1128/JCM.43.7.3267-3272.2005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Vlasak J, van Hoang M, Christanti S et al (2016) Use of flow cytometry for characterization of human cytomegalovirus vaccine particles. Vaccine 34(20):2321–2328. https://doi.org/10.1016/j.vaccine.2016.03.067

    Article  CAS  PubMed  Google Scholar 

  83. Konz JO, Lee AL, Lewis JA et al (2005) Development of a purification process for adenovirus: controlling virus aggregation to improve the clearance of host cell DNA. Biotechnol Prog 21(2):466–472. https://doi.org/10.1021/bp049644r

    Article  CAS  PubMed  Google Scholar 

  84. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1–2):248–254. https://doi.org/10.1016/0003-2697(76)90527-3

    Article  CAS  PubMed  Google Scholar 

  85. Smith PK, Krohn RI, Hermanson GT et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150(1):76–85. https://doi.org/10.1016/0003-2697(85)90442-7

    Article  CAS  PubMed  Google Scholar 

  86. Weber C, Gokorsch S, Czermak P (2007) Expansion and chondrogenic differentiation of human mesenchymal stem cells. Int J Artif Organs 30:611–618. https://doi.org/10.1177/039139880703000709

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Bioprocess Engineering and Pharmaceutical Technology (IBPT), Technische Hochschule Mittelhessen (THM) - University of Applied Sciences, Giessen, Germany

    Keven Lothert, Gregor Dekevic, Daniel Loewe, Denise Salzig, Peter Czermak & Michael W. Wolff

  2. Faculty of Biology and Chemistry, Justus-Liebig-University Giessen, Giessen, Germany

    Peter Czermak

  3. Division Bioresources, Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Giessen, Germany

    Peter Czermak

Authors
  1. Keven Lothert
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gregor Dekevic
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Daniel Loewe
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Denise Salzig
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peter Czermak
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michael W. Wolff
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michael W. Wolff .

Editor information

Editors and Affiliations

  1. Department of Chemical and Biological Engineering, University at Buffalo, The State University of New York, Buffalo, NY, USA

    Blaine A. Pfeifer

  2. Department of Chemical and Biological Engineering, University at Buffalo, The State University of New York, Buffalo, NY, USA

    Andrew Hill

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Lothert, K., Dekevic, G., Loewe, D., Salzig, D., Czermak, P., Wolff, M.W. (2021). Upstream and Downstream Processes for Viral Nanoplexes as Vaccines. In: Pfeifer, B.A., Hill, A. (eds) Vaccine Delivery Technology. Methods in Molecular Biology, vol 2183. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0795-4_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0795-4_12

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0794-7

  • Online ISBN: 978-1-0716-0795-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation