Trimeric SARS-CoV-2 Spike Proteins Produced from CHO Cells in Bioreactors Are High-Quality Antigens
Trimeric SARS-CoV-2 Spike Proteins Produced from CHO Cells in Bioreactors Are High-Quality Antigens
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DOI：
10.3390/pr8121539
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发表时间：
2020-12-01
期刊：
影响因子：
3.5
通讯作者：
Wurm, Florian M.
中科院分区：
文献类型：
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作者：
Pino, Paco;Kint, Joeri;Wurm, Florian M.
大流行的人类冠状病毒的刺突蛋白对于它进入人类细胞是必不可少的。事实上，大多数针对严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的中和抗体都是针对病毒表面暴露的Spike蛋白，使其成为疫苗和诊断测试的首选抗原。在当前的大流行背景下，全球对尖峰蛋白的需求迅速增加，每年可能超过数百克至1000克。冠状病毒尖峰是大的高度糖基化的高三聚体复合体，具有内在的不稳定性。糟糕的可制造性现在威胁到这些蛋白质用于疫苗和诊断测试的可用性。在这里，我们概述了可扩展的、符合良好制造规范(GMP)和化学定义的生产两种细胞分泌稳定形式的三聚体刺突蛋白(武汉和D614G变体)的工艺。这些过程是化学定义的，基于克隆悬浮-CHO细胞群体，并通过两步可扩展的下游过程进行蛋白质纯化。用电子显微镜和高效液相分析证实了三聚体的构象。病毒抑制实验显示与敏感细胞结合。检测血清SARS-CoV-2特异性免疫球蛋白分子的敏感性和特异性均高于刺激物片段(刺激物亚基-1、S1和受体结合区，RBD)。这里描述的方法将能够生产足够高质量的三聚体尖峰蛋白，以满足全球对SARS-CoV-2诊断测试和潜在疫苗的需求。
The spike protein of the pandemic human corona virus is essential for its entry into human cells. In fact, most neutralizing antibodies against Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) are directed against the Virus-surface exposed spike protein, making it the antigen of choice for use in vaccines and diagnostic tests. In the current pandemic context, global demand for spike proteins has rapidly increased and could exceed hundreds of grams to kilograms annually. Coronavirus spikes are large heavily glycosylated homo-trimeric complexes, with inherent instability. The poor manufacturability now threatens the availability of these proteins for vaccines and diagnostic tests. Here, we outline scalable, Good Manufacturing Practice (GMP) compliant, and chemically defined processes for the production of two cell-secreted stabilized forms of the trimeric spike proteins (Wuhan and D614G variant). The processes are chemically defined and based on clonal suspension-CHO cell populations and on protein purification via a two-step scalable downstream process. The trimeric conformation was confirmed using electron microscopy and HPLC analysis. Binding to susceptible cells was shown using a virus-inhibition assay. The diagnostic sensitivity and specificity for detection of serum SARS-CoV-2-specific-immunoglobulin molecules was found to exceed that of spike fragments (Spike subunit-1, S1 and Receptor Binding Domain, RBD). The process described here will enable production of sufficient high-quality trimeric spike protein to meet the global demand for SARS-CoV-2 diagnostic tests and potentially vaccines.