Advances in purification of SARS-CoV-2 spike ectodomain protein using high-throughput screening and non-affinity methods.
Advances in purification of SARS-CoV-2 spike ectodomain protein using high-throughput screening and non-affinity methods.
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DOI：
10.1038/s41598-022-07485-w
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发表时间：
2022-03-15
影响因子：
4.6
通讯作者：
Gowetski DB
中科院分区：
文献类型：
--
作者：
Cibelli N;Arias G;Figur M;Khayat SS;Leach K;Loukinov I;Shadrick W;Chuenchor W;Tsybovsky Y;Vaccine Production Program Analytical Development;Gulla K;Gowetski DB
大流行病毒SARS-CoV-2的尖峰(S)糖蛋白是疫苗设计和治疗开发的关键靶点。对于稳定的、可溶的、天然的S蛋白胞外结构域，需要一个高产、可扩展、符合cGMP的下游流程，以满足正在进行的研究和开发的广泛材料需求。截至2021年6月，S蛋白已独家使用亲和层析和尺寸排除层析等难以规模化、低产率的方法进行纯化。在这里，我们提出了第一种已知的非亲和纯化方法，以在哺乳动物细胞系CHO-DG44中表达两个S结构物S_DF2P和HexaPro。Tecan Freedom EVO200自动化生物处理工作站上的高通量树脂筛选使离子交换树脂被确定为可行的纯化步骤。对色谱单元操作以及用于病毒清除、低pH处理和20纳米过滤的行业标准方法进行了可行性评估。将改进的工艺应用于从CHO-DG44稳定的池收获中纯化HexaPro，获得了迄今报道的最高纯度的S蛋白。我们的结果表明，商用层析树脂适合于制备SARS-CoV-2刺突蛋白的cGMP。我们预计，我们的结果将为全球生物制药生产实验室提供蓝图，并为工艺强化提供起点。
The spike (S) glycoprotein of the pandemic virus, SARS-CoV-2, is a critically important target of vaccine design and therapeutic development. A high-yield, scalable, cGMP-compliant downstream process for the stabilized, soluble, native-like S protein ectodomain is necessary to meet the extensive material requirements for ongoing research and development. As of June 2021, S proteins have exclusively been purified using difficult-to-scale, low-yield methodologies such as affinity and size-exclusion chromatography. Herein we present the first known non-affinity purification method for two S constructs, S_dF_2P and HexaPro, expressed in the mammalian cell line, CHO-DG44. A high-throughput resin screen on the Tecan Freedom EVO200 automated bioprocess workstation led to identification of ion exchange resins as viable purification steps. The chromatographic unit operations along with industry-standard methodologies for viral clearances, low pH treatment and 20 nm filtration, were assessed for feasibility. The developed process was applied to purify HexaPro from a CHO-DG44 stable pool harvest and yielded the highest yet reported amount of pure S protein. Our results demonstrate that commercially available chromatography resins are suitable for cGMP manufacturing of SARS-CoV-2 Spike protein constructs. We anticipate our results will provide a blueprint for worldwide biopharmaceutical production laboratories, as well as a starting point for process intensification.
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影响因子：
56.9
作者：
Hsieh, Ching-Lin;Goldsmith, Jory A.;McLellan, Jason S.
通讯作者：
McLellan, Jason S.
影响因子：
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Amanat F;Stadlbauer D;Strohmeier S;Nguyen THO;Chromikova V;McMahon M;Jiang K;Arunkumar GA;Jurczyszak D;Polanco J;Bermudez-Gonzalez M;Kleiner G;Aydillo T;Miorin L;Fierer DS;Lugo LA;Kojic EM;Stoever J;Liu STH;Cunningham-Rundles C;Felgner PL;Moran T;García-Sastre A;Caplivski D;Cheng AC;Kedzierska K;Vapalahti O;Hepojoki JM;Simon V;Krammer F
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Krammer F
影响因子：
3
作者：
Mastronarde, DN
通讯作者：
Mastronarde, DN
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作者：
Scheres, Sjors H. W.
通讯作者：
Scheres, Sjors H. W.
影响因子：
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作者：
Kim, Eun;Erdos, Geza;Gambotto, Andrea
通讯作者：
Gambotto, Andrea