腸病毒71型(Enterovirus 71, EV71)在腸病毒屬中致病力最高，容易在孩童引發嚴重的神經系統併發症。台灣自1998年爆發疫情以來，至今約有2000位孩童感染重症，將近250人死亡，對於兒童的生命極具威脅性。然而，目前尚未有有效治療或預防腸病毒71型的方法。本實驗室先前的研究證實了以桿狀病毒/昆蟲細胞表現系統生產的類病毒顆粒(virus-like particles, VLP)能在動物體內誘發良好的免疫反應，對於曾在台灣引發大流行的不同基因型腸病毒71型皆具有保護力，可見其具有做為腸病毒71型疫苗的潛力。本研究接續先前的成果，希望開發使用新型拋棄式生物反應器來進行腸病毒71行類病毒顆粒大量生產的完整程序。拋棄式生物反應器因具有不需清洗與操作簡易等優點，近年來漸漸成為市場主流。我們與國內賽宇細胞科技公司合作，使用其開發的潮汐式填充床反應器來達成EV71類病毒顆粒的大量生產。在進行製程放大時需考量成本的問題，因此將病毒劑量固定在0.1 MOI作探討。我們先利用250 ml搖瓶小量測試培養基與感染時機和EV71 類病毒顆粒產量的關係，得到在實驗室等級生產的最佳值。在初步的反應器量產測試中，我們使用同為賽宇細胞科技公司開發的BelloCell®反應器，了解昆蟲細胞(High Five™)於潮汐式填充床反應器中的生長情形，找出感染時機並且調整反應器參數以找出最適當的病毒感染策略。在得到BelloCell® 500反應器的生產條件後，我們放大製程到大型TideCell®反應器中，提升總培養體積至10 L，進行更大量的EV71類病毒顆粒生產，期許能使EV71類病毒顆粒的生產程序更符合經濟效益，以加速EV71 類病毒顆粒疫苗工業化的應用。

Enterovirus 71 (EV71) causes significant morbidity among children, threatening children's health with severe neurological complications. Since the first outbreak in Taiwan in 1998, approximately 2,000 children have been affected with severe disease caused by EV71, resulting in 250 deaths. Up to now, there are still neither effective anti-EV71 therapies nor preventing vaccines available. Our group has confirmed in animal models that EV71 virus-like particles (VLP) produced by baculovirus/insect cell system can elicit effective immune responses, suggesting that EV71 VLP is a potential vaccine candidate. This study thus aims to develop a convenient process for mass production by using the novel oscillating bioreactor. Taking the cost of mass production into consideration, the cells were infected with the recombinant baculovirus at an MOI (multiplicity of infection) of 0.1 and the production parameters were optimized in small shaker flasks. The production process was further transferred to BelloCell®, a 500 ml disposable packed-bed reactor developed by Cesco Bioengineering for high-density cell culture for the VLP production. After determining the process parameters in BelloCell® system, we further utilized the larger TideCell® to make the whole process much more efficient.

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