Real-Time AT-PCR for Quantitative Detection of Bovine Parainfluenza Virus Type 3 during the Manufacture of Biologics

생물의약품 제조공정에서 Bovine Parainfluenza Virus Type 3 정량 검출을 위한 Real-Time RT-PCR

  • Lee, Dong-Hyuck (Department of Biological Sciences, College of Life Science and Nanotechnology, Hannam University) ;
  • Kim, Chan-Kyong (Department of Biological Sciences, College of Life Science and Nanotechnology, Hannam University) ;
  • Kim, Tae-Eun (Department of Biological Sciences, College of Life Science and Nanotechnology, Hannam University) ;
  • Kim, In-Seop (Department of Biological Sciences, College of Life Science and Nanotechnology, Hannam University)
  • 이동혁 (한남대학교 생명.나노과학대학 생명과학과) ;
  • 김찬경 (한남대학교 생명.나노과학대학 생명과학과) ;
  • 김태은 (한남대학교 생명.나노과학대학 생명과학과) ;
  • 김인섭 (한남대학교 생명.나노과학대학 생명과학과)
  • Published : 2008.08.29

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 parainfluenza virus type 3 (BPIV3) is one of the common bovine pathogens and has widely been known as a contaminant of biologics. In order to establish the validation system for the BPIV3 safety of biologics, a real-time RT-PCR method was developed for quantitative detection of BPIV3 contamination in raw materials, manufacturing processes, and final products. Specific primers for amplification of BPIV3 RNA was selected, and BPIV3 RNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be 2.8 $TCID_{50}/mL$. The real-time RT-PCR method was validated to be reproducible and very specific to BPIV3. The established real-time RT-PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with BPIV3. BPIV3 RNA could be quantified in CHO cell as well as culture supernatant. Also the real-time RT-PCR assay could detect 7.8 $TCID_{50}/mL$ of BPIV3 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 BPIV3 contamination during the manufacture of biologics.

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