형질전환 벼 현탁세포 배양에서 hCTLA4Ig 생산을 위한 disposable bioreactor의 적용

Title
형질전환 벼 현탁세포 배양에서 hCTLA4Ig 생산을 위한 disposable bioreactor의 적용
Authors
양용석
Keywords
형질전환벼현탁세포배양에서hctla4ig생산을위한disposablebioreactor의적용
Issue Date
2011
Publisher
인하대학교
Abstract
During the last several years, disposable bioreactors have been increasingly incorporated into preclinical, clinical, and production-scale biotechnological facilities where process simplicity, safety and flexibility are top priority. Driven by the demands of the market and the manufacturing industry, numerous studies reporting their advantage in use for mammalian expression system have contributed to new developments. As a result, many types of disposable bioreactors are now available. However, there are few studies regarding their suitability for the production of pharmaceutical proteins in plant cell cultures. Plant cell-based production system has been studied as a potentially cheap, safe, and scalable alternative to mammalian and bacterial production of biopharmaceuticals. In this study, two kinds of disposable bioreactors were successfully applied to transgenic rice cell cultures for the production of recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) and compared with 5-L stirred tank bioreactor system. The first one was wave-mixed bag, the most common disposable bioreactor, modified with a fritted sparger to increase oxygen transfer. The second was an airlift disposable bioreactor which has advantages in simple process and low cost operation. In both systems, a fed-batch culture method was used for the efficient production of hCTLA4Ig by feeding concentrated amino acids. Dissolved oxygen level was regulated at 30%. Agitation and aeration rate during the cultures in wave-mixed disposable bioreactor and airlift disposable bioreactor were determined for the same mixing time. The results from wave-mixed and airlift disposable bioreactor showed similar maximum cell density (12.6 g DCW/L vs. 11.9 g DCW/L), doubling time (5.03 day vs. 4.84 day) and maximum hCTLA4Ig concentration (44.5 mg/L vs. 43.7 mg/L). Relatively higher cell viability was sustained in airlift disposable bioreactor during the culture. However, the time needed to obtain
Description
1. 서론 1 1.1 생물의약품(biopharmaceuticals) 1 1.1.1 생물의약품(biopharmaceuticals) 1 1.1.2 동등생물의약품(biosimilars) 4 1.2 식물세포배양 5 1.2.1 Plant-made pharmaceuticals (PMPs) 5 1.2.2 형질전환된 벼 세포 8 1.2.2.1 RAmy3D promoter 8 1.2.2.2 Human cytotoxic T lymphocyte-associated antigen4-immunoglobulin (hCTLA4Ig) 9 1.3 Bioreactor engineering과 식물세포배양 10 1.3.1 Bioreactor types 10 1.3.2 Bioreactor design의 고려요소 13 1.4 Disposable bioreactor 15 1.4.1 Aeration과 mixing 전략 16 1.4.2 식물세포 배양의 적용 21 2. 연구 목적 25 3. 재료 및 방법 27 3.1 세포주 27 3.2 배양조건 27 3.3 Flask 배양 29 3.4 Bioreactor 배양 29 3.5 분석 25 3.5.1 세포분석 29 3.5.1.1 세포량 측정 34 3.5.1.2 Cell viability 측정 34 3.5.2 단백질 분석 35 3.5.2.1 hCTLA4Ig의 정량분석 35 3.5.2.2 hCTLA4Ig의 정성분석 35 3.5.3 Protease 활성 측정 38 3.3.4 삼투압 측정 38 4. 결과 및 고찰 39 4.1 hCTLA4Ig 생산을 위한 유가식 배양 적용 39 4.1.1 Stirred tank bioreactor 배양 39 4.2 Pneumatically driven bioreactor 배양 전략 44 4.2.1 Airlift bioreactor 배양 44 4.2.2 Stirred tank bioreactor와 비교 49 4.3 Disposable bioreactor 배양 53 4.3.1 Disposable bioreactor design 53 4.3.2 세포생장 및 hCTLA4Ig 생산 확인 55 4.3.3 Disposable bioreactor의 mixing time 변화에 따른 세포생장 및 hCTLA4Ig 생산비교 63 4.3.3.1 Airlift disposable bioreactor 63 4.3.3.2 Wave-mixed disposable bioreactor 69 4.3.4 Disposable bioreactor와 stirred tank bioreactor 비교 및 평가 73 5. 결론 76 6. 참고문헌 78
URI
http://dspace.inha.ac.kr/handle/10505/22839
Appears in Collections:
College of Natural Science(자연과학대학) > Ocean Sciences (해양과학) > Theses(해양과학 석박사 학위논문)
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