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

生物制药行业依靠筛选和适应程序来鉴定稳定的、高产的、能够在确定的培养基中生长以产生重组蛋白（rP）的转染物。当需要筛选和评估潜在的rP药物靶点和/或当更快速地生成重组材料将允许更多分子更快地进入首次人体研究时，该程序在药物开发管道的早期并不理想。因此，瞬时基因表达为此类研究提供了快速生成rP的手段。瞬时表达技术用于生产生物治疗相关的rp，主要是利用悬浮适应的人胚胎肾细胞（HEK293细胞）和磷酸钙沉淀，尽管最近中国仓鼠卵巢（CHO）细胞也被使用。事实上，许多公司正在研究瞬时表达技术，利用非病毒DNA传递系统和生物反应器培养的细胞，在几天内产生数百毫克的核糖核酸。最近的报告还表明，通过系统的方法，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，以及在培养基中添加丙戊酸，有可能提高瞬态过程中的滴度。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可以提高产量，而重链和轻链转录本的基因优化也可以提高CHO细胞瞬时表达期间的mAb生产水平。Lonza Biologics项目拥有CHO悬浮液适应宿主细胞系（CHOK1SV），与其他CHO悬浮液适应细胞系相比，该细胞系能够达到高细胞浓度并保持长时间的活力。龙沙还拥有自己专有的矢量表达系统GS系统。在这个项目中，学生将开发一种新的基于CHOK1SV和GS载体（DNA）的瞬时表达系统，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基础上，这些工作表明，操纵瞬态系统可以提高产量。测试分子将包括模型mAb（基因优化和非优化）、tPA和rhEPO。最初，学生将研究载体修饰后的传递系统和DNA到细胞核的内在化（例如，通过使用核定位序列和分割元素）。学生还将确定环境条件（如温度、pH值）、培养基组成和饲喂对产品产量的影响。该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，学生将专注于细胞周期工程（如p21）和伴侣和折叠酶，我们已经在实验室的瞬态研究中显示，它们增加了模型蛋白的高水平瞬态表达（如Torsin A，翻译起始因子）。最后，在CHO菌株中表达牛痘E3L蛋白对单抗的瞬时表达有积极影响。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，p58参与了预防或调节这种激酶的活性。因此，学生将研究调节PKR活性对瞬时产量的影响。结果：1；开发新的载体，CHOK1SV衍生的宿主和处理（媒体/饲料）技术，用于表达100毫克rps2。了解从CHOK1SV细胞瞬时表达rPs的限制

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