Real-Time RT-PCR for Validation of Reovirus Type 3 Safety During the Manufacture of Mammalian Cell Culture-Derived Biopharmaceuticals

세포배양 유래 생물의약품 생산 공정에서 Reovirus Type 3 안전성 검증을 위한 Real-Time RT-PCR

  • Lee, Dong-Hyuck (Department of Biological Sciences, Hannam University) ;
  • Jeong, Hyo-Sun (Tissue Engineering Division, Research and Development Dept., Hans Daedeok R&D Center, Hans Biomed Corp.) ;
  • Kim, Tae-Eun (Department of Biological Sciences, Hannam University) ;
  • Oh, Seon-Hwan (Department of Biological Sciences, Hannam University) ;
  • Lee, Jung-Suk (Tissue Engineering Division, Research and Development Dept., Hans Daedeok R&D Center, Hans Biomed Corp.) ;
  • Kim, In-Seop (Department of Biological Sciences, Hannam University)
  • 이동혁 (한남대학교 생명.나노과학대학 생명과학과) ;
  • 정효선 (한스바이오메드(주) 한스대덕연구소) ;
  • 김태은 (한남대학교 생명.나노과학대학 생명과학과) ;
  • 오선환 (한남대학교 생명.나노과학대학 생명과학과) ;
  • 이정숙 (한스바이오메드(주) 한스대덕연구소) ;
  • 김인섭 (한남대학교 생명.나노과학대학 생명과학과)
  • Published : 2008.09.30

Abstract

Validation of viral safety is essential in ensuring the safety of mammalian cell culture-derived biopharmaceuticals, because numerous adventitious viruses have been contaminated during the manufacture of the products. Mammalian cells are highly susceptible to Reovirus type 3 (Reo-3), and there are several reports of Reo-3 contamination during the manufacture of biopharmaceuticals. In order to establish the validation system for the Reo-3 safety, a real-time RT-PCR method was developed for quantitative detection of Reo-3 in cell lines, raw materials, manufacturing processes, and final products as well as Reo-3 clearance validation. Specific primers for amplification of Reo-3 RNA was selected, and Reo-3 RNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be $3.2{\times}10^0\;TCID_{50}/ml$. The real-time RT-PCR method was proven to be reproducible and very specific to Reo-3. The established real-time RT-PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with Reo-3. Reo-3 RNA could be quantified in CHO cell as well as culture supernatant. When the real-time RT-PCR assay was applied to the validation of virus removal during a virus filtration process, the result was similar to that of virus infectivity assay. Therefore, it was concluded that this rapid, specific, sensitive, and robust assay could replace infectivity assay for detection and clearance validation of Reo-3.

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