近日，國際著名雜志Molecular Biotechnology 在線刊登了上海巴斯德研究所周保羅研究組的最新研究成果“High Yield of Human Monoclonal Antibody Produced by Stably Transfected Drosophila Schneider 2 Cells in Perfusion Culture Using Wave Bioreactor,，”這是首次關(guān)于用波浪生物反應(yīng)器灌注培養(yǎng)穩(wěn)轉(zhuǎn)了人單克隆抗體基因的果蠅S2細(xì)胞來產(chǎn)生人單克隆抗體的報道。
治療性人單克隆抗體已經(jīng)增長為數(shù)十億美元的產(chǎn)業(yè),，因而建立一個穩(wěn)定的培養(yǎng)條件以滿足人單克隆抗體的大規(guī)模表達(dá)是非常重要的,。波浪生物反應(yīng)器于二十世紀(jì)九十年代后期被用于哺乳動物及昆蟲細(xì)胞的蛋白表達(dá)，但是用波浪生物反應(yīng)器來灌注培養(yǎng)穩(wěn)轉(zhuǎn)了人單克隆抗體基因的果蠅穩(wěn)轉(zhuǎn)細(xì)胞表達(dá)人單克隆抗體蛋白尚屬首次,。
在該研究中上海巴斯德所的研究助理王璐嵐和博士生胡紅星在周保羅教授的指導(dǎo)下克隆了抗高致病性禽流感病毒的血凝蛋白的人單克隆抗體基因,，并構(gòu)建到果蠅誘導(dǎo)型表達(dá)載體里面，穩(wěn)轉(zhuǎn)了果蠅S2細(xì)胞系,，通過有限稀釋法拿到高表達(dá)人單克隆抗體的單細(xì)胞克隆,。對穩(wěn)轉(zhuǎn)了人單克隆抗體的單克隆細(xì)胞株分別用來在灌注和非灌注的生物反應(yīng)器中進(jìn)行比較培養(yǎng)，發(fā)現(xiàn)灌注培養(yǎng)方法在獲得細(xì)胞數(shù)量與表達(dá)抗體蛋白產(chǎn)量上明顯優(yōu)于后者,，且這兩種方法生產(chǎn)的抗體都是有中和活性的,。這些結(jié)果表明用波浪生物反應(yīng)器進(jìn)行灌注培養(yǎng)S2穩(wěn)轉(zhuǎn)細(xì)胞系進(jìn)行大規(guī)模蛋白表達(dá)是非常好的方法。
該研究是與GE公司研發(fā)部Fast Trak中心的楊建軍博士合作完成的,，得到了國家自然科學(xué)基金及李嘉誠基金會的資助,。（生物谷Bioon.com）
doi:10.1007/s12033-011-9484-5
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High Yield of Human Monoclonal Antibody Produced by Stably Transfected Drosophila Schneider 2 Cells in Perfusion Culture Using Wave Bioreactor
Lulan Wang, Hongxing Hu, Jianjun Yang, Feng Wang, Christian Kaisermayer and Paul Zhou
Since it was first introduced in late 1990s Wave bioreactor has been used for protein production by mammalian and insect cell lines. However, using Wave bioreactor to produce human monoclonal antibody by stable Drosophila Schneider 2 (S2) cell transfectants has not been reported before. In this study, S2 cells were co-transfected with an inducible vector expressing human monoclonal antibody heavy and light chains, respectively, specific for hemagglutinin (HA) of H5N1 influenza virus. Stable S2 transfectant clone was selected by limiting dilution assay. Stable S2 transfectant clone that produce the highest amount of human monoclonal antibody was inoculated into two 2-l disposable cellbags, where cell growth and antibody production were compared between batch and perfusion cultures using Wave bioreactor. Here, we report that maximum viable cell density reached 1.06 × 107 cells/ml in batch culture; whereas 1.04 × 108 cells/ml was achieved in perfusion culture. The maximum volumetric antibody productivity in batch culture was 52 mg/l/day; while perfusion culture yielded 1,437 mg/l/day. As a result, the total antibody production was 201 mg in batch culture and 8,212 mg in perfusion culture. The antibody produced by both cultures displays full neutralizing activity. Thus, our results provide strong support for using Wave bioreactor in perfusion culture for a large-scale production of human monoclonal antibody by stable S2 cell transfectants.
