Bovine Herpesvirus Type 1 정량 검출을 위한 Real-Time PCR

이동혁,정효선,이정희,김태은,이정숙,김인섭,Lee, Dong-Hyuck,Jeong, Hyo-Sun,Lee, Jung-Hee,Kim, Tae-Eun,Lee, Jung-Suk,Kim, In-Seop 한국미생물학회 2008 미생물학회지 Vol.44 No.1

소의 혈액, 세포, 조직, 기관 등은 생물의약품과 조직공학제제, 세포치료제의 원료로 널리 사용되고 있다. 소유래 물질을 원료로 사용한 제제의 경우 소유래 원료 물질에 다양한 바이러스가 오염된 사례가 있기 때문에 바이러스 안전성 검증이 필수적이다. Bovine herpesvirus type 1 (BHV-1)은 소에게 가장 흔하게 감염되는 바이러스중의 하나이다. 소유래 물질을 원료로 하는 생물의약품, 조직공학제제, 세포치료제 등에서 BHV-1안전성을 확보하기 위해, 원료물질, 제조공정, 완제품에서 BHV-1을 정량적으로 검출하고, 제조공정에서 BHV-1 제거 검증을 위한 시험법으로 활용이 가능한 BHV-1 real-time PCR 시험법을 확립하였다. BHV-1에 특이적인 primer를 선별하였으며, 형광염료 SYBR Green I을 사용하여 BHV-1 DNA 정량 검출 시험법을 최적화하였다. 세포배양법에 의한 감염역가와 비교한 결과 real-time PCR 민감도는 $2\;TCID_{50}/ml$이었다. 확립된 시험법의 신뢰성(reliability)을 보증하기 위해 시험법 검증을 실시한 결과 특이성(specificity)과 재현성(reproducibility)이 우수함을 확인하였다. 확립된 real-time PCR을 생물의약품 제조공정 검증에 적용할 수 있는지 확인하기 위하여 인위적으로 BHV-1을 오염시킨 Chinese hamster ovary (CHO) 세포주와 소유래 콜라겐에서 BHV-1 검출 시험을 실시하였다. BHV-1을 감염시킨 CHO 세포에서 세포변병효과를 관찰할 수 없었지만, 세포와 세포배양 상청액에서 BHV-1을 정량적으로 검출할 수 있었다. 소유래 콜라겐에서도 $10\;TCID_{50}/ml$까지 정량적으로 검출할 수 있었다. 위와 같은 결과에서 확립된 BHV-1 real-time PCR 시험법은 생물의약품 안전성 보증을 위한 세포주 검증, 생물의약품생산공정 검증, 바이러스 제거 공정 검증 등에서 감염역가 시험법과 같은 생물학적 시험법을 대신할 수 있는 신속하고, 특이성과 민감성이 우수한 시험법임을 확인하였다. Bovine blood, cell, tissue, and organ are used as raw materials for manufacturing biopharmaceuticals, tissue engineered products, and cell therapy. Manufacturing processes for the biologicals using bovine materials have the risk of viral contamination. Therefore viral validation is, essential in ensuring the safety of the products. Bovine herpesvirus type 1 (BHV-1) is the most common bovine pathogen found in bovine blood, cell, tissue, and organ. In order to establish the validation system for the BHV-1 safety of the products, a real-time PCR method was developed for quantitative detection of BHV-1 in raw materials, manufacturing processes, and final products as well as BHV-1 clearance validation. Specific primers for amplification of BHV-1 DNA was selected, and BHV-1 DNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be $2\;TCID_{50}/ml$. The real-time PCR method was validated to be reproducible and very specific to BHV-1. The established real-time PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with BHV-1. BHV-1 DNA could be quantified in CHO cell as well as culture supernatant. Also the real-time PCR assay could detect $10\;TCID_{50}/ml$ of BHV-1 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 BHV-1 contamination during the manufacture of biologics.

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

이동혁,정효선,김태은,오선환,이정숙,김인섭,Lee, Dong-Hyuck,Jeong, Hyo-Sun,Kim, Tae-Eun,Oh, Seon-Hwan,Lee, Jung-Suk,Kim, In-Seop 한국미생물학회 2008 미생물학회지 Vol.44 No.3

세포배양 유래 생물의 약품 생산 공정에서 다양한 바이러스가 오염된 사례가 있기 때문에 바이러스 안전성 검증이 필수적이다. Reovirus type 3 (Reo-3)는 동물 세포주와 동물 세포 배양 공정에 오염되는 대표적인 바이러스이다. 세포배양 유래 생물의약품의 Reo-3 안전성을 확보하기 위해, 세포주, 원료물질, 제조공정, 완제품에서 Reo-3를 정략적으로 검출하고, 제조공정에서 Reo-3 제거 검증을 위한 시험법으로 활용이 가능한 real-time RT-PCR 시험법을 확립하였다. Reo-3에 특이적인 primer를 선별하였으며, 형광염료 SYBR Green I을 사용하여 Reo-3 RNA 정략 검출 시험법을 최적화하였다. 세포배양법에 의한 감염역가와 비교한 결과 real-time RT-PCR 민감도는 $3.2{\times}10^0\;TCID_{50}/ml$이었다. 확립된 시험법의 신뢰성(reliability)을 보증하기 위해 시험법 검증을 실시한 결과 특이성(specificity)과 재현성(reproducibility)이 우수함을 확인하였다. 확립된 real-time RT-PCR을 생물의약품 제조공정 검증에 적용할 수 있는지 확인하기 위하여 인위적으로 Reo-3를 오염시킨 CHO 세포에서 Reo-3 검출 시험을 실시한 결과 Reo-3를 감염시킨 CHO 세포와 세포배양 상청액에서 Reo-3를 정략적으로 검출할 수 있었다. 또한 바이러스필터 공정에서 Reo-3제거 효과를 감염역가 시험법과 비교 검증한 결과 더 빠른 시간에 동일한 결과를 얻을 수 있었다. 위와 같은 결과에서 확립된 Reo-3 real-time RT-PCR 시험법은 생물의약품 안전성 보증을 위한 세포주 검증, 생물의 약품 생산 공정 검증, 바이러스 제거 공정 검증 등에서 감염 역가 시험법을 대신할 수 있는 신속하고, 특이성과 민감성이 우수한 시험법임을 확인하였다. 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.

경량합성벽체의 전단성능 및 압축내력 평가

이동혁,이상섭,배규웅,문태섭,Lee, Dong Hyuck,Lee, Sang Sup,Bae, Kyu Woong,Moon, Tae Sup 한국강구조학회 2005 한국강구조학회 논문집 Vol.17 No.1

본 논문은 스틸스터드로 구성된 골조에 경량기포모르터를 타설 양생하여 제작한 경량합성벽체의 반복가력 전단실험에 의한 에너지 소산능력과 압축내력 실험을 통한 압축성능의 평가에 대한 연구이다. 경량기포모르터의 유무, 경량기포모르터의 비중(0.6, 0.8, 1.0, 1.2), 마감재(경량기포모르터, OSB, 석고보드) 및 브레이스의 유무, 벽체의 단위(1단위-$900mm{\times}2,400mm$, 2단위-$1,800mm{\times}2,400mm$)를 변수로 하여 실시하였던 경량합성벽체의 전단내력 평가 실험 가운데 반복 실험 결과를 동일 조건의 단조 실험과 비교하였다. 또한 기존 스틸하우스벽체와 경량합성벽체의 압축내력도 실험을 통해 조사하였다. 반복가력 실험결과, 단조가력 실험체의 거동과 약간의 차이를 보이고 있으며 이는 벽체에 채운 경량기포모르터의 비중에 따른 차이라고 생각된다. 압축내력 실험결과, 기존형 실험체에 비하여 경량기포모르터를 타설한 실험체의 최대내력은 2~2.5배, 초기강성은 2~3배 정도 증가하였다. This paper presents the test results and evaluations for the energy dissipation capacity and compressive performance of light-weight hybrid panels. A total of 26 full-scale specimens of light-weight hybrid panels were tested. The parameters include the presence of light-weight foamed mortar, the specific gravity of light-weight foamed mortar (0.6, 0.8, 1.0, 1.2), the finishing materials (light-weight foamed mortar, OSB [Oriented Strand Board], gypsum board), the shape of bracing (x, ~), and the size of panels (1P-900 mm 2,400 mm, 2P-1,800 mm 2,400 mm). The results of the cyclic tests are somewhat different from those of monotonic tests, due to the different specific gravity of light-weight foamed mortar. It was found from the compressive tests that the ultimate strength and initial stiffness are increased by means of light-weight foamed mortar (2~2.5 times in ultimate strength and 2~3 times in initial stiffness).

유전자조작, 균주분리 세포배양 유래 생물의약품 생산 공정에서 Minute Virus of Mice 안전성 검증을 위한 Real-Time PCR

이동혁 ( Dong Hyuck Lee ),조항미 ( Hang Mee Cho ),김현미 ( Hyun Mi Kim ),이정숙 ( Jung Suk Lee ),김인섭 ( In Seop Kim ) 한국미생물생명공학회 2008 한국미생물·생명공학회지 Vol.36 No.1

산지유통시설의 물류현황과 개선방안

이동혁(Dong Hyuck Lee) 한국식품유통학회 2000 食品流通硏究 Vol.17 No.1

N/A

농수산물도매시장 평가제도 운영현황과 발전방향

이동혁(Dong Hyuck Lee) 한국식품유통학회 1998 食品流通硏究 Vol.15 No.2

N/A

공영도매시장 시설 정비 방안

이동혁 ( Dong Hyuck Lee ) 한국식품유통학회 2006 食品流通硏究 Vol.23 No.3

The objectives of this research are to make a confrontation plan for the agricultural wholesale markets to fulfill the central role continuously in the agricultural marketing by solving the competitiveness problems of the increases in the physical distribution expenses caused by the small and old facilities. It is needed to expand or to complement of the essential facilities of the agricultural wholesale markets to accomplish the improvement in the market conditions and the mid-term & long-term market development, and it is needed to make the plans for the complementation & the modernization of the facilities such as the distribution facilities (ex. the cold chain facilities and the sorting facilities) and the waste disposal facilities to confront the new marketing environment. It is appropriate to set up and to fulfill the basic direction of the complementation of the facilities of the agricultural wholesale markets by means of the promotion of facilities complementation coping with the condition changes, the complementation by the thorough budget plan and the guaranteeing of the accounting transparency, the introduction of the various complement methods, the fulfillment of the marketing information system & the distribution efficiency, the reinforcement of the safety and hygiene and the anti-pollution measures.

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

이동혁(Dong Hyuck Lee),김찬경(Chan Kyong Kim),김태은(Tae Eun Kim),김인섭(In Seop Kim) 한국생물공학회 2008 KSBB Journal Vol.23 No.4

생물의약품 제조공정에서 Bovine Parvovirus 정량 검출을 위한 Real-Time PCR

이동혁 ( Dong Hyuck Lee ),이정희 ( Jung Hee Lee ),김찬경 ( Chan Kyong Kim ),김태은 ( Tae Eun Kim ),배정은 ( Jung Eun Bae ),김인섭 ( In Seop Kim ) 한국미생물생명공학회 2008 한국미생물·생명공학회지 Vol.36 No.3

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 I. The sensitivity of the assay was calculated to be 1.3 × 10-1 TCID50/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 × 100 TCID50/ 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.

국내 도시공원을 통한 무장애 공원 계획 방향에 관한 연구

이동혁(Lee Dong-Hyuck),강병근(Kang Byoung-Keun),박현수(Park Hyeon-Soo) 대한건축학회 2011 대한건축학회 학술발표대회 논문집 - 계획계/구조계 Vol.31 No.2(계획계)

Takes place daily in the space of modern society, the physical environment in urban areas, as well as the general public to persons with disabilities and the elderly is significant. Disabled and elderly people in urban spaces due to the physical constraints ‘access’ and ‘usability’ significant drop compared to the general public. Therefore, there is no obstacle to the disabled and elderly in order to plan a city or town several architectural modifications regarding the situation on the need to study jeongbina. In this study, along with improving the quality of modern life, more and more the availability of the Riverside Park were shown. For the disabled and elderly urban planning and design of the basic planning direction and to propose standards.