Development of a CHOK1SV transient expression system for rapid generation of recombinant proteins

开发用于快速生成重组蛋白的 CHOK1SV 瞬时表达系统

• 批准号: BB/I015884/1
• 负责人: Christopher Smales
• 金额: $ 11.71万
• 依托单位: University of Kent
• 依托单位国家: 英国
• 项目类别: Training Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI015884%2F1
• 关键词: Development; CHOK1SV; transient; expression; system

The biopharmaceutical industry relies upon screening and adaptation procedures to identify stable, productive transfectants able to grow in defined media to produce a recombinant protein (rP). This procedure is not ideal early in the drug development pipeline when it is necessary to screen and evaluate potential rP drug targets and/or when the more rapid generation of recombinant material would allow more molecules into first-in-human studies faster. Transient gene expression therefore provides a means for the generation of rapid amounts of rP for such studies. Transient expression technology for the production of biotherapeutic relevant rPs has mostly utilised suspension adapted human embryonic kidney cells (HEK293 cells) and calcium phosphate precipitation although recently Chinese hamster ovary (CHO) cells have been used. Indeed, a number of companies are investigating transient expression technology to produce hundreds of mgs of rPs in a matter of days using non-viral DNA delivery systems and cells cultured in bioreactors. Recent reports also demonstrate the potential to improve titres in transient processes using a systematic approach including optimisation of the vector and transfection conditions, use of cell cycle regulators (p18, p21) and fibroblast growth factor, and addition of valproic acid to the media. The optimisation of the ratio of IgG heavy and light chain genes and the use of fed-batch cultures when generating IgG material using CHO transient monoclonal antibody (mAb) expression have been reported to improve yields whilst gene optimisation of heavy and light chain transcripts also increases mAb production levels during transient expression in CHO cells. The project Lonza Biologics has a CHO suspension adapted host cell line (CHOK1SV) which is able to achieve high cell concentrations and maintain viability for long periods of time compared to other suspension adapted CHO lines. Lonza also has its own proprietary vector expression system, the GS System. In this project the student will develop a novel CHOK1SV and GS vector based (DNA) transient expression system for the rapid production of 100's of mg's of rPs. This will build upon preliminary work from both the academic and industrial labs that show manipulation of transient systems can lead to increased yields. The test molecules will include a model mAb (gene optimised and non-optimised), tPA and rhEPO. Initially the student will investigate delivery systems and internalisation of the DNA to the nucleus after modification of the vector (e.g. via use of nuclear localisation sequences and partitioning elements). The student will also determine the influence of environmental conditions (e.g. temperature, pH) and media composition and feeding on product yield. The student will also investigate host cell engineering strategies to improve yield and speed of the transient expression process. In particular the student will focus upon cell cycle engineering (e.g. p21) and chaperones and foldases that we have shown in the laboratory in transient studies increases the high-level transient expression of model proteins (e.g. Torsin A, translation initiation factors). Finally, expression of the Vaccinia E3L protein in CHO strains has a positive effect upon transient expression of mAb. E3L acts to block the activation of cellular genes that respond to viral infection, in particular PKR. Work in the lab at Kent has shown that PKR activity can also be modulated by manipulation of p58 involved in preventing or modulating the activity of this kinase. The student will therefore investigate the effect of modulating PKR activity on transient yields. The outcomes: 1. Development of novel vector, CHOK1SV derived host and processing (media/feeds) technology for the expression of 100's mg of rPs 2. An understanding of the limitations upon transient expression of rPs from CHOK1SV cells

生物制药行业依靠筛选和适应程序来识别稳定、高效的转染子，这些转染子能够在确定的培养基中生长以产生重组蛋白（rP）。在药物开发流程的早期，当需要筛选和评估潜在的 rP 药物靶点和/或当更快地生成重组材料将允许更多的分子更快地进入人体首次研究时，该程序并不理想。因此，瞬时基因表达为此类研究提供了快速产生 rP 的方法。用于生产生物治疗相关 rP 的瞬时表达技术主要利用悬浮适应的人胚肾细胞（HEK293 细胞）和磷酸钙沉淀，尽管最近已使用中国仓鼠卵巢（CHO）细胞。事实上，许多公司正在研究瞬时表达技术，利用非病毒 DNA 传递系统和在生物反应器中培养的细胞，在几天内生产数百毫克的 rP。最近的报告还证明了使用系统方法提高瞬时过程滴度的潜力，包括优化载体和转染条件、使用细胞周期调节剂（p18、p21）和成纤维细胞生长因子以及向培养基中添加丙戊酸。据报道，在使用 CHO 瞬时单克隆抗体 (mAb) 表达生成 IgG 材料时，优化 IgG 重链和轻链基因的比例以及使用补料分批培养物可提高产量，同时重链和轻链转录物的基因优化也可提高 CHO 细胞瞬时表达期间的 mAb 产量水平。 Lonza Biologics 项目拥有一种 CHO 悬浮适应宿主细胞系 (CHOK1SV)，与其他悬浮适应 CHO 系相比，它能够实现高细胞浓度并长时间保持活力。 Lonza 还拥有自己专有的载体表达系统 GS 系统。在该项目中，学生将开发一种新型 CHOK1SV 和 GS 载体（DNA）瞬时表达系统，用于快速生产 100 毫克的 rP。这将建立在学术和工业实验室的初步工作的基础上，这些工作表明瞬态系统的操纵可以提高产量。测试分子将包括单克隆抗体模型（基因优化和非优化）、tPA 和 rhEPO。最初，学生将研究载体修饰后的递送系统和 DNA 内化到细胞核的情况（例如，通过使用核定位序列和分配元件）。学生还将确定环境条件（例如温度、pH 值）、培养基成分和饲喂对产品产量的影响。学生还将研究宿主细胞工程策略，以提高瞬时表达过程的产量和速度。特别是，学生将重点关注细胞周期工程（例如 p21）以及我们在实验室的瞬时研究中显示的分子伴侣和折叠酶，它们会增加模型蛋白（例如 Torsin A、翻译起始因子）的高水平瞬时表达。最后，CHO 菌株中痘苗病毒 E3L 蛋白的表达对 mAb 的瞬时表达具有积极作用。 E3L 的作用是阻断响应病毒感染的细胞基因的激活，特别是 PKR。肯特大学实验室的工作表明，PKR 活性也可以通过操纵 p58 来调节，p58 参与预防或调节该激酶的活性。因此，学生将研究调节 PKR 活性对瞬时产量的影响。结果： 1. 开发新型载体、CHOK1SV 衍生宿主和表达 100 毫克 rP 的加工（培养基/补料）技术 2. 了解 CHOK1SV 细胞瞬时表达 rP 的局限性