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

Abstract

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.

Keywords

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

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