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首页> 外文会议>Cell culture engineering XV >AN INTEGRATED CELL LINE DEVELOPMENT PLATFORM FOR GENERATION OF HIGH YIELDING CHO STABLE CELL LINES EXPRESSING A STABILIZED TRIMERIC PRE-FUSION RSV F RECOMBINANT VIRAL GLYCOPROTEIN
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AN INTEGRATED CELL LINE DEVELOPMENT PLATFORM FOR GENERATION OF HIGH YIELDING CHO STABLE CELL LINES EXPRESSING A STABILIZED TRIMERIC PRE-FUSION RSV F RECOMBINANT VIRAL GLYCOPROTEIN

机译：用于生成表达稳定的三聚体预融合RSV F重组病毒糖蛋白的高产量CHO稳定细胞系的综合细胞系发育平台

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Accelerating timelines to deliver stable cell lines with high productivity is challenging, especially for biologics portfolios which include complex recombinant vaccine constructs. The VRC's current integrated approach to shorten CHO cell line development timeline utilizes host cells pre-adapted to production medium, optimization of the expression vectors, improved pre and post-transfection methodology, automated ClonePix™ technology and top clone selection and ranking in micro-scale ambr bioreactor technology. This platform was developed with monoclonal antibodies as model proteins. While this integrated platform enabled generation of high yielding clones expressing monoclonal antibodies, it was initially much less successful with a difficult to express recombinant stabilized trimeric RSV F protein. Initial stable CHO cell lines produced the RSV F protein at very low levels that were not suitable for GMP manufacturing and the protein did not have quality attributes similar to the initial transient expression-based research protein. The RSV F trimer requires enzymatic cleavage of the protein for proper folding and trimer formation. However, the need for stable co-expression of a furin-like molecule in CHO cells was initially unknown. This case study summarizes the successful development of stable CHO cell lines expressing the stabililzed RSV F trimer and a cell line development protocol for difficult to express trimeric viral glycoproteins for clinical manufacturing. Implications for cell line development of next generation trimeric HIV envelope vaccines will be discussed.
机译：加快时间表以高生产率交付稳定的细胞系具有挑战性，特别是对于包括复杂重组疫苗构建体的生物制剂产品组合而言。 VRC当前缩短CHO细胞系开发时间表的整合方法利用了预先适应生产培养基的宿主细胞，表达载体的优化，改进的转染前后方法，自动化的ClonePix™技术以及顶级克隆的选择和微观规模ambr生物反应器技术。该平台是用单克隆抗体作为模型蛋白开发的。尽管此集成平台能够生成表达单克隆抗体的高产克隆，但最初却很难成功表达重组稳定的三聚体RSV F蛋白。最初的稳定CHO细胞系产生的RSV F蛋白水平非常低，不适合GMP的生产，并且该蛋白的品质属性与基于瞬时表达的初始研究蛋白相似。 RSV F三聚体需要蛋白质的酶促裂解才能正确折叠和形成三聚体。然而，最初未知在CHO细胞中稳定表达弗林蛋白酶样分子的需要。该案例研究总结了表达稳定的RSV F三聚体的稳定CHO细胞系的成功开发，以及难以表达用于临床生产的三聚体病毒糖蛋白的细胞系开发方案。将讨论下一代三聚体HIV包膜疫苗对细胞系发展的影响。

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