Virus Separation Using Membranes

  • Tanja A. Grein
  • Ronald Michalsky
  • Peter Czermak
Part of the Methods in Molecular Biology book series (MIMB, volume 1104)


Industrial manufacturing of cell culture-derived viruses or virus-like particles for gene therapy or vaccine production are complex multistep processes. In addition to the bioreactor, such processes require a multitude of downstream unit operations for product separation, concentration, or purification. Similarly, before a biopharmaceutical product can enter the market, removal or inactivation of potential viral contamination has to be demonstrated. Given the complexity of biological solutions and the high standards on composition and purity of biopharmaceuticals, downstream processing is the bottleneck in many biotechnological production trains. Membrane-based filtration can be an economically attractive and efficient technology for virus separation. Viral clearance, for instance, of up to seven orders of magnitude has been reported for state of the art polymeric membranes under best conditions.

This chapter summarizes the fundamentals of virus ultrafiltration, diafiltration, or purification with adsorptive membranes. In lieu of an impractical universally applicable protocol for virus filtration, application of these principles is demonstrated with two examples. The chapter provides detailed methods for production, concentration, purification, and removal of a rod-shaped baculovirus (Autographa californica M nucleopolyhedrovirus, about 40 × 300 nm in size, a potential vector for gene therapy, and an industrially important protein expression system) or a spherical parvovirus (minute virus of mice, 22–26 nm in size, a model virus for virus clearance validation studies).

Key words

Ultrafiltration Diafiltration Membrane chromatography Adsorptive membrane Baculovirus Parvovirus Gene therapy Vaccine Viral vector 


  1. 1.
    Reis R, Zydney AL (2001) Membrane separations in biotechnology. Curr Opin Biotechnol 12:208–211CrossRefGoogle Scholar
  2. 2.
    GlaxoSmithKline-Biologicals SA (2007) Cervarix® product information, human papillomavirus vaccine types 16 and 18 (recombinant, as04 adjuvanted). Document approved by the Therapeutic Goods AdministrationGoogle Scholar
  3. 3.
    Justice C et al (2011) Process control in cell culture technology using dielectric spectroscopy. Biotechnol Adv 29(4):391–401CrossRefGoogle Scholar
  4. 4.
    Betting DJ et al (2009) Enhanced immune stimulation by a therapeutic lymphoma tumor antigen vaccine produced in insect cells involves mannose receptor targeting to antigen presenting cells. Vaccine 27(2):250–259. doi: 10.1016/j.vaccine.2008.10.055 CrossRefGoogle Scholar
  5. 5.
    Lacroix J et al (2010) Parvovirus H1 selectively induces cytotoxic effects on human neuroblastoma cells. Int J Cancer 127(5):1230–1239CrossRefGoogle Scholar
  6. 6.
    Weiss K et al (2012) Parameters for optimizing production of measles virus with regard to oncolytic virotherapy. Am J Biochem Biotechnol 8(2): 81–98CrossRefGoogle Scholar
  7. 7.
    Marques BF, Roush DJ, Göklen KE (2009) Virus filtration of high-concentration monoclonal antibody solutions. Biotechnol Prog 25(2):483–491CrossRefGoogle Scholar
  8. 8.
    Wickramasinghe SR et al (2010) Understanding virus filtration membrane performance. J Membr Sci 365(1–2):160–169CrossRefGoogle Scholar
  9. 9.
    Bolton G et al (2005) Normal-flow virus filtration: detection and assessment of the endpoint in bioprocessing. Biotechnol Appl Biochem 42(2):133–142. doi: org/10.1042/BA20050056 CrossRefGoogle Scholar
  10. 10.
    Gottschalk U (2009) Process scale purification of antibodies. Wiley, Hoboken, NJCrossRefGoogle Scholar
  11. 11.
    Nehring D et al (2004) Experimental and modeling study of a membrane filtration process using ceramic membranes to increase retroviral pseudotype vector titer. J Membr Sci 237(1–2):25–38CrossRefGoogle Scholar
  12. 12.
    Grzenia DL et al (2006) Purification of densonucleosis virus by tangential flow ultrafiltration. Biotechnol Prog 22(5):1346–1353. doi: 10.1021/bp060077c CrossRefGoogle Scholar
  13. 13.
    Reis RV, Zydney AL (1999) Protein ultrafiltration. In: Flickinger MC, Drew SW (eds) Enzyclopedia of bioprocess technology: fermentation, biocatalysis and bioseparation. Wiley, New York, pp 2197–2213Google Scholar
  14. 14.
    Vicente T et al (2009) Purification of recombinant baculoviruses for gene therapy using membrane processes. Gene Ther 16(6): 766–775CrossRefGoogle Scholar
  15. 15.
    Liew MWO, Chuan YP, Middelberg APJ (2012) Reactive diafiltration for assembly and formulation of virus-like particles. Biochem Eng J 68:120–128CrossRefGoogle Scholar
  16. 16.
    Barsoum J (1999) Concentration of recombinant baculovirus by cation-exchange chromatography. Biotechniques 26(5):834–836, 838, 840Google Scholar
  17. 17.
    Grein TA 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. doi: 10.1016/j.jviromet.2012. 03.031 CrossRefGoogle Scholar
  18. 18.
    Wu C, Soh KY, Wang S (2007) Ion-exchange membrane chromatography method for rapid and efficient purification of recombinant baculovirus and baculovirus gp64 protein. Hum Gene Ther 18(7):665–672CrossRefGoogle Scholar
  19. 19.
    Charcosset C (2012) Membrane processes in biotechnologies and pharmaceutics, 1st edn. Elsevier, Amsterdam, 336Google Scholar
  20. 20.
    Kim JS, Akeprathumchai S, Wickramasinghe SR (2001) Flocculation to enhance microfiltration. J Membr Sci 182:161–172CrossRefGoogle Scholar
  21. 21.
    Wickramasinghe SR et al (2004) Improved permete flux by flocculation of biological feeds: comparision between experiment and theory. J Membr Sci 242:57–71CrossRefGoogle Scholar
  22. 22.
    Belfort G, Davis RH, Zydney AL (1994) The behavior of suspensions and macromolecular solutions in crossflow microfiltration. J Membr Sci 96:1–58CrossRefGoogle Scholar
  23. 23.
    Ghosh R, Wang L (2006) Purification of humanized monoclonal antibody by hydrophobic interaction membrane chromatography. J Chromatogr A 1107(1–2):104–109CrossRefGoogle Scholar
  24. 24.
    Hensgen MI et al (2009) Purification of minute virus of mice using high performance tangential flow filtration. Desalination 250(3):1121–1124CrossRefGoogle Scholar
  25. 25.
    Jornitz MW, Soelkner PG, Meltzer TH (2002) Sterile filtration: a review of the past and present technologies. PDA J Pharm Sci Technol 56(4):192–195Google Scholar
  26. 26.
    Grzenia DL, Carlson JO, Wickramasinghe SR (2008) Tangential flow filtration for virus purification. J Membr Sci 321(2):373–380CrossRefGoogle Scholar
  27. 27.
    Fikar M, Kovacs Z, Czermak P (2010) Dynamic optimization of batch diafiltration processes. J Membr Sci 355:168–174CrossRefGoogle Scholar
  28. 28.
    Zydney A, Kuriyel R (2000) Protein ultrafiltration. In: Desai M (ed) Methods in biotechnology downstream protein processing, vol 9. Humana Press, Totowa, NJ, pp 35–46CrossRefGoogle Scholar
  29. 29.
    Foley G (1999) Minimisation of process time in ultrafiltration and continuous diafiltration: the effect of incomplete macrosolute rejection. J Membr Sci 163(2):349–355CrossRefGoogle Scholar
  30. 30.
    Paulen R et al (2011) Minimizing the process time for ultrafiltration/diafiltration under gel polarization conditions. J Membr Sci 380(1–2):148–154CrossRefGoogle Scholar
  31. 31.
    Foley G (2006) Water usage in variable volume diafiltration: comparison with ultrafiltration and constant volume diafiltration. Desalination 196(1–3):160–163CrossRefGoogle Scholar
  32. 32.
    Klein E (2000) Affinity membranes: a 10-year review. J Membr Sci 179(1–2):1–27CrossRefGoogle Scholar
  33. 33.
    Yang H et al (2002) Purification of a large protein using ion-exchange membranes. Ind Eng Chem Res 41(6):1597–1602CrossRefGoogle Scholar
  34. 34.
    EMEA (2001) Guideline on plasma derived medicinal products. The European Agency of the Evaluation of Medicinal Products. CPMP/BWP/269/95, rev. 3Google Scholar
  35. 35.
    FDA (1997) U.F.a.D.A., Points to consider in the manufacture and testing of monoclonal antibody products for human use. Center for Biologics Evaluation and ReseachGoogle Scholar
  36. 36.
    ICH, Q2(R1) (1996) Validation of analytical procedures: text and methodology. International conference on harmonisationGoogle Scholar
  37. 37.
    WHO (1998) Forty-seventh report technical report series, No 878. WHO Expert Committee on Biological StandardizationGoogle Scholar
  38. 38.
    WHO (2004) Guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products. WHO technical report series no. 924Google Scholar
  39. 39.
    EMEA (2012) Guideline on process validation. EMA/CHMP/CVMP/QWP/70278/2012-Rev1Google Scholar
  40. 40.
    Schmidt S, Kauling J (2006) UV-Inaktivierung von Viren und Bakterien mit einem innovativen Wendelrohrreaktor im Labor- und Prozessmaßstab. Chem Ing Tech 78(11):1739–1745CrossRefGoogle Scholar
  41. 41.
    Committee, f.P.M.P., Note for guidance on plasma derived products. CPMP/BWP/269/95Google Scholar
  42. 42.
    ICH-Q5A(R1), Q5(R1) (1999) Viral safety evaluation of biotechnology products derived from cell lines of human or animal origin. International conference on harmonisationGoogle Scholar
  43. 43.
  44. 44.
    Hongo-Hirasaki T et al (2006) Removal of small viruses (parvovirus) from IgG solution by virus removal filter Planova®20N. J Membr Sci 278(1–2):3–9CrossRefGoogle Scholar
  45. 45.
    Guideline on virus safety evaluation of biotechnological investigational medicinal products (2008) EMEA/CHMP/BWP/398498/2005 Retrieved 07 Nov 2013
  46. 46.
    Michalsky R et al (2008) Effects of temperature and shear force on infectivity of the baculovirus Autographa californica M nucleopolyhedrovirus. J Virol Methods 153(2):90–96CrossRefGoogle Scholar
  47. 47.
    O’Reilly DR, Miller LK, Luckow VA (1994) Baculovirus expression vectors: a laboratory manual. Oxford University Press, New YorkGoogle Scholar
  48. 48.
    Roldão A et al (2009) Error assessment in recombinant Baculovirus titration: evaluation of different methods. J Virol Methods 159(1):69–80CrossRefGoogle Scholar
  49. 49.
    Tsujikawa M et al (2011) Caution in evaluation of removal of virus by filtration: misinterpretation due to detection of viral genome fragments by PCR. J Virol Methods 178(1–2):39–43CrossRefGoogle Scholar
  50. 50.
    Guo H et al (2010) Low-pressure membrane integrity tests for drinking water treatment: a review. Water Res 44(1):41–57CrossRefGoogle Scholar
  51. 51.
    DiLeo AJ, Philips MW (1994) Integrity test for membranes. US Patent 5282380Google Scholar
  52. 52.
    Hanson LE, Howell CR (2004) Elicitors of plant defense responses from biocontrol strains of trichoderma viren. Phytopathology 94(2):171–176CrossRefGoogle Scholar
  53. 53.
    Czermak P, Nehring D, Wickramasinghe R (2007) Membrane filtration in animal cell culture. In: Pörtner (ed) Animal cell biotechnology: methods and protocols. Humana Press, Totoba, pp 397–420CrossRefGoogle Scholar
  54. 54.
    Clarke TE, Clem RJ (2002) Lack of involvement of haemocytes in the establishment and spread of infection in Spodoptera frugiperda larvae infected with the baculovirus Autographa californica M nucleopolyhedrovirus by intrahaemocoelic injection. J Gen Virol 83(7):1565–1572Google Scholar
  55. 55.
    Reed LJ, Muench H (1938) A simple method of estimating fifty percent endpoints. Am J Epidemiol 27(3):493–497Google Scholar
  56. 56.
    O’Reilly DR, Miller LK, Luckow VA (1993) Baculovirus expression vectors: a laboratory manual. Oxford University Press, New YorkGoogle Scholar
  57. 57.
    Aucoin MG, Mena JA, Kamen AA (2010) Bioprocessing of baculovirus vectors: a review. Curr Gene Ther 10(3):174–186CrossRefGoogle Scholar
  58. 58.
    Stanbridge LJ, Dussupt V, Maitland NJ (2003) Baculoviruses as vectors for gene therapy against human prostate cancer. J Biomed Biotechnol 2003(2):79–91CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Tanja A. Grein
    • 1
  • Ronald Michalsky
    • 1
  • Peter Czermak
    • 1
    • 2
    • 3
  1. 1.Institute of Bioprocess Engineering and Pharmaceutical TechnologyUniversity of Applied Sciences MittelhessenGiessenGermany
  2. 2.Department of Chemical EngineeringKansas State UniversityManhattanUSA
  3. 3.Faculty of Biology and ChemistryJustus-Liebig-University of GiessenGiessenGermany

Personalised recommendations