Real-Time PCR for Quantitative Detection of Bovine Parvovirus during Manufacture of Biologics

생물의약품 제조공정에서 Bovine Parvovirus 정량 검출을 위한 Real-Time PCR

  • Lee, Dong-Hyuck (Department of Biological Sciences, Hannam University) ;
  • Lee, Jung-Hee (Department of Biological Sciences, Hannam University) ;
  • Kim, Chan-Kyong (Department of Biological Sciences, Hannam University) ;
  • Kim, Tae-Eun (Department of Biological Sciences, Hannam University) ;
  • Bae, Jung-Eun (Department of Biological Sciences, Hannam University) ;
  • Kim, In-Seop (Department of Biological Sciences, Hannam University)
  • 이동혁 (한남대학교 생명.나노과학대학 생명과학과) ;
  • 이정희 (한남대학교 생명.나노과학대학 생명과학과) ;
  • 김찬경 (한남대학교 생명.나노과학대학 생명과학과) ;
  • 김태은 (한남대학교 생명.나노과학대학 생명과학과) ;
  • 배정은 (한남대학교 생명.나노과학대학 생명과학과) ;
  • 김인섭 (한남대학교 생명.나노과학대학 생명과학과)
  • Published : 2008.09.28


Bovine blood, cell, tissue, and organ are used as raw materials for manufacturing biologics such as biopharmaceuticals, tissue-engineered products, and cell therapy. Manufacturing processes for the biologics have the risk of viral contamination. Therefore viral validation is essential in ensuring the safety of the products. Bovine parvovirus (BPV) is one of the common bovine pathogens and has widely been known as a possible contaminant of biologics. In order to establish the validation system for the BPV safety of biologics, a real-time PCR method was developed for quantitative detection of BPV contamination in raw materials, manufacturing processes, and final products. Specific primers for amplification of BPV DNA were selected, and BPV DNA was quantified by use of SYBR Green 1. The sensitivity of the assay was calculated to be $1.3{\times}10^{-1}\;TCID_{50}/mL$. The real-time PCR method was validated to be reproducible and very specific to BPV. The established real-time PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with BPV. BPV DNA could be quantified in CHO cell as well as culture supernatant. Also the real-time PCR assay could detect $1.3{\times}10^0\;TCID_{50}/mL$ of BPV artificially contaminated in bovine collagen. The overall results indicated that this rapid, specific, sensitive, and robust assay can be reliably used for quantitative detection of BPV contamination during manufacture of biologics.


Bovine parvovirus;Chinese hamster ovary (CHO) cell;collagen;real-time PCR;virus validation


  1. Adamson, S. R. 1999. Experiences of virus, retrovirus and retrovirus-like particles in chinese hamster ovary (CHO) and hybridoma cells used for production of protein therapeutics. Dev. Biol. Stand. 93: 89-96
  2. Barnes, M. A., R. E. Wright, A. B. Bodine, and C. F. Alberty. 1982. Frequency of bluetongue and bovine parvovirus infection in cattle in South Carolina dairy herds. Am. J. Vet. Res. 43: 1078-1080
  3. Celis, P. and G. Silvester. 2004. European regulatory guidance on virus safety of recombinant proteins, monoclonal antibodies and plasma derived medicinal products. Dev. Biol. Stand. 118: 3-10
  4. Code of federal regulation 9 (9CFR), animal and animal products. 1996. part 113.53. Requirement for ingredients of animal origin used for production of biologics
  5. Jennings, A. 1999. Detecting viruses in sera: methods used and their merits. Dev. Biol. Stand. 99: 51-59
  6. Ryu, S.-R., J.-H. Shin, S.-Y. Baek, J.-O. Kim, K.-I. Min, B.- S. Min, B.-G. Kim, D.-K. Kim, M.-K. Park, M.-J. Ahn, K.- S. Chae, H.-S. Jeong, S.-H. Lee, and S.-N. Park. 2003. Evaluation of limit of detection and range of quantitation for RT-PCR, real-time RT-PCR and RT-PCR-ELISA detection of bovine viral diarrhea virus contamination in biologics derived from cell cultures. J. Bacteriol. Virol. 33: 161-168
  7. Storz, J. and R. C. Bates. 1973. Parvovirus infection in calves. J. Am. Vet. Med. Assoc. 163: 884-886
  8. Belak, S. 2007. Molecular diagnosis of viral diseases, present trends and future aspects: A view from the OIE collaborating centre for the application of polymerase chain reaction methods for diagnosis of viral diseases in veterinary medicine. Vaccine 25: 5444-5452
  9. Cho, H. M., D. H. Lee, H. M. Kim, and I. S. Kim. 2008. Real-time RT-PCR for quantitative detection of bovine viral diarrhoea virus during manufacture of biologics. Kor. J. Microbiol. Biotechnol. 36: 34-42
  10. Garnick, R. L. 1998. Raw materials as a source of contamination in large-scale cell culture. Dev. Biol. Stand. 93: 21-29
  11. Merten, O.-W. 2002. Virus contamination of cell cultures-a biotechnological view. Cytotechnol. 39: 91-116
  12. Parkman, P. D. 1996. Safety of biopharmaceuticals: a current perspective. Dev. Biol. Stand. 88: 5-7
  13. Belak, S. and P. Thoren. 2001. Molecular diagnosis of animal diseases: some experiences over the past decade. Expert Rev. Mol. Diagn. 1: 434-443
  14. Lee, D. H., H. S. Jeong, J. H. Lee, T. E. Kim, J. Lee, and I. S. Kim. 2008. Real-time PCR for quantitative detection of bovine herpesvirus type 1. Kor. J. Microbiol. 44: 14-21
  15. Erickson, G. A., S. R. Bolin, and J. G. Landgraf. 1991. Viral contamination of fetal bovine serum used for tissue culture: risks and concerns. Dev. Biol. Stand. 75: 173-175
  16. Kil, T. G., W. J. Kim, D. H. Lee, Y. Kang, H. M. Sung, S. H. Yoo, S.-N. Park, and I. S. Kim. 2005. Quantitative real-time PCR of porcine parvovirus as a model virus for cleaning validation of chromatography during manufacture of plasma derivatives. Kor. J. Microbiol. 41: 216-224
  17. Gajiwala, K. and A. L. Gajiwala. 2004. Evaluation of lyophilised, gamma-irradiated amnion as a biological dressing. Cell Tissue Bank. 5: 73-80
  18. Yarlagadda, P. K., M. Chandrasekharan, and J. Y. Shyan. 2005. Recent advances and current developments in tissue scaffolding. Biomed. Mater. Eng. 15: 159-177
  19. Jeong, H.-S., J,-H. Shin, Y.-N. Park, J.-Y. Choi, Y.-L. Kim, B.-G. Kim, S.-R. Ryu, S.-Y. Baek, S.-H. Lee, and S.-N. Park. 2003. Development of real-time RT-PCR for evaluation of JEV clearance during purification of HPV type 16 L1 virus-like particles. Biologicals 31: 223-229
  20. Faraj, K. A., T. H. van Kuppevelt, and W. F. Daamen. 2007. Construction of collagen scaffolds that mimic the three-dimensional architecture of specific tissues. Tissue Eng. 13: 2387-2394
  21. Choi, Y.-M., J.-K. Kim, J.-I. Park, and S.-W. Jeong. 2006. Evaluation of bovine amniotic membrane for the treatment of superficial canine corneal ulcer. J. Vet. Clinics 23: 334-336
  22. Lee, D. H., H. M. Cho, H. M. Kim, J. Lee, and I. S. Kim. 2008. Real-time PCR for validation of minute virus of mice safety during the manufacture of mammalian cell culture-derived biopharmaceuticals. Kor. J. Microbiol. Biotechnol. 36: 12-20
  23. Chen, K. C., B. C. Shull, E. A. Moses, M. Lederman, E. R. Stout, and R. C. Bates. 1986. Complete nucleotide sequence and genome organization of bovine parvovirus. J. Virol. 60: 1085-1097
  24. Rudnick, A. 2006. Advances in tissue engineering and use of type 1 bovine collagen particles in wound bed preparation. J. Wound Care 15: 402-404
  25. International Conference on Harmonisation. 1998. Guidance on viral safety evaluation of biotechnology products derived from cell lines of human or animal origin; availability. Federal Resister 63: 51074-51084
  26. Sandals, W. C. D., R. C. Povey, and A. H. Meek. 1995. Prevalence of bovine parvovirus infection in Ontario dairy cattle. Can. J. Vet. Res. 59: 81-86
  27. Storz, J., S. Young, E. J. Carroll, R. C. Bates, R. A. Bowen, and D. A. Keney. 1978. Parvovirus infection of the bovine fetus: Distribution of infection, antibody response, and agerelated susceptibility. Am. J. Vet. Res. 39: 1099-1102
  28. Watzinger, F., K. Ebner, and T. Lion. 2006. Detection and monitoring of virus infections by real-time PCR. Mol. Aspects Med. 27: 254-298
  29. Horaud, F. 1991. Introductory remark: viral safety of biologicals. Dev. Biol. Stand. 75: 3-7
  30. Schneider, G. 2003. Bioimplants-characteristics and use. Laryngorhinootologie 82: 839-852
  31. Darling, A. 2002. Validation of biopharmaceutical purification process for virus clearance evaluation. Mol. Biotechnol. 21: 57-83