Optimization of the Production Process and Characterization of the Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1), a SARS Vaccine Candidate.
Optimization of the Production Process and Characterization of the Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1), a SARS Vaccine Candidate.
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DOI：
10.1016/j.xphs.2017.04.037
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发表时间：
2017-08
影响因子：
3.8
通讯作者：
Hotez PJ
中科院分区：
文献类型：
--
作者：
Chen WH;Chag SM;Poongavanam MV;Biter AB;Ewere EA;Rezende W;Seid CA;Hudspeth EM;Pollet J;McAtee CP;Strych U;Bottazzi ME;Hotez PJ
从2002年到2003年，严重急性呼吸系统综合征（SARS）在全球范围内蔓延到五大洲，造成8000例呼吸道感染和800例死亡。为了减轻未来爆发的影响以及为生物防御做准备，正在开发生产重组蛋白疫苗候选物的方法。在此之前，我们报道了一种有前途的重组SARS疫苗候选物RBD 219-N1的5 L规模表达和纯化，RBD 219-N1是SARS冠状病毒的218个氨基酸残基的受体结合结构域（RBD）在酵母-巴斯德毕赤酵母X-33中表达。当用氢氧化铝作为佐剂时，该蛋白质引起高中和抗体滴度和高RBD特异性抗体滴度。然而，RBD 219-N1的产率（每升发酵上清液60 mg RBD 219-N1; 60 mg/L FS）仍需要改进以达到我们的>100 mg/L FS的目标。本研究优化了10 L规模的生产工艺，使FS的产量提高了6 ~ 7倍，达到400 mg/L，纯化回收率> 50%。一组表征测试表明，该工艺具有重现性，纯化的无标签RBD 219-N1蛋白具有高纯度和明确的结构，因此是当前药品生产质量管理规范和未来1期临床试验下生产的合适候选物。
From 2002 to 2003, a global pandemic of severe acute respiratory syndrome (SARS) spread to 5 continents and caused 8000 respiratory infections and 800 deaths. To ameliorate the effects of future outbreaks as well as to prepare for biodefense, a process for the production of a recombinant protein vaccine candidate is under development. Previously, we reported the 5 L scale expression and purification of a promising recombinant SARS vaccine candidate, RBD219-N1, the 218–amino acid residue receptor-binding domain (RBD) of SARS coronavirus expressed in yeast–Pichia pastoris X-33. When adjuvanted with aluminum hydroxide, this protein elicited high neutralizing antibody titers and high RBD-specific antibody titers. However, the yield of RBD219-N1 (60 mg RBD219-N1 per liter of fermentation supernatant; 60 mg/L FS) still required improvement to reach our target of >100 mg/L FS. In this study, we optimized the 10 L scale production process and increased the fermentation yield 6- to 7-fold to 400 mg/L FS with purification recovery >50%. A panel of characterization tests indicated that the process is reproducible and that the purified, tag-free RBD219-N1 protein has high purity and a well-defined structure and is therefore a suitable candidate for production under current Good Manufacturing Practice and future phase-1 clinical trials.
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影响因子：
4.8
作者：
Cao Z;Liu L;Du L;Zhang C;Jiang S;Li T;He Y
通讯作者：
He Y
影响因子：
4.8
作者：
Curti E;Seid CA;Hudspeth E;Center L;Rezende W;Pollet J;Kwityn C;Hammond M;Matsunami RK;Engler DA;Hotez PJ;Elena Bottazzi M
通讯作者：
Elena Bottazzi M
DOI：
10.1016/j.bbrc.2009.05.003
发表时间：
2009-07-10
影响因子：
3.1
作者：
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通讯作者：
Jiang, Shibo
影响因子：
4.8
作者：
Curti, Elena;Kwityn, Clifford;Bottazzi, Maria Elena
通讯作者：
Bottazzi, Maria Elena
影响因子：
2.2
作者：
Du, Lanying;Zhao, Guangyu;Jiang, Shibo
通讯作者：
Jiang, Shibo