Building and commercialisation of a new mammalian cell factory platform

目前，用于制造分泌治疗蛋白的行业标准细胞工厂平台是基于CHO细胞的变体，其中变体是天然的或工程化的营养缺陷型，用于合成对支持细胞生长至关重要的氨基酸或核苷酸的关键中间体。目前最先进的重组蛋白生产使用遗传载体，其使用适当的CHO细胞变体共表达所需的重组基因加上选择标记。只有那些摄取共表达载体的细胞可以在缺乏添加的谷氨酰胺（GS）或核苷（DHFR）的培养基上选择。这两种系统都有可能与小分子量抑制剂-甲硫氨酸磺胺（MSX）的GS和氨甲蝶呤（MTX）的DHFR的扩增。在选择过程中包括抑制剂使得能够分离具有更高拷贝的GS或DHFR的细胞，所述GS或DHFR与共表达的重组基因遗传连锁，导致重组基因的更高表达。因此，每个细胞的产量可以大幅增加，并导致适合商业用途的生产率。在Dickson实验室中使用CHO细胞进行的代谢组学研究已经确定了一种代谢靶标，其以类似于（但不同于）GS的方式提供了一种强大的共表达遗传载体选择系统，可进行敲除（再次类似于Lonza开发的GS Xceed 1系统）。几种小分子量化学物质是代谢靶点的有效抑制剂（类似于MSX），为选择扩增的基因拷贝提供了可能性。确定的代谢目标是细胞生长的基础，是在一个关键的代谢途径，不像GS，不能绕过其他营养素，如天冬酰胺。它对于潜在的细胞宿主应用也是细胞不可知的。我们假设这个平台在载体开发和宿主细胞系工程方面为合成干预提供了巨大的潜力，提供了一种替代和改进的代谢选择系统来生产治疗性蛋白质。该代谢靶点和相关基因构成了新的知识产权。一旦举例说明，其目的是该技术将获得专利，并且该专利，所产生的材料，该博士生将负责平台的技术开发，新型细胞系，细胞培养基和其他合成生物学构建体的设计和应用组件的生成，制备敲除细胞系和细胞培养组分如培养基。一旦开发完成，这些关键组件将使用相关的工业分子对平台进行概念验证评估。蛋白质的生产对于治疗性和非治疗性蛋白质都至关重要。全球治疗性蛋白质的年销售额超过2000亿英镑，生物制造业是英国的经济领导者。在这个博士学位中开发的平台将作为已经为Lonza，Fuji和Horizon等公司获得商业红利的技术的潜在竞争对手。目前，由于缺乏经营自由和进入成本高，制造成本昂贵，限制了具有创新产品的新公司的出现，无法获得所需的技术。该项目的一个主要目标是开发一个创新的、行业领先的、开源的表达平台，具有非常灵活的许可条款，从而推动行业的发展。收入将通过销售平台组件、技术支持和伙伴关系产生。不受严格许可费限制的表达平台的可用性将对中小企业和学术创新者具有极大的吸引力。这个表达系统已经完成了Pathway Bioburma和Dickson实验室的一名理学硕士学生之间进行的概念项目的初步证明。

Currently industry standard cell factory platforms for manufacture of secreted therapeutic proteins are based around variants of CHO cells, where variants are either natural or engineered auxotroph's for key intermediates in the synthesis of amino acids or nucleotides, essential to support cell growth. Current state-of-the-art recombinant protein production uses genetic vectors which co-express a desired recombinant gene plus a selection marker, using an appropriate CHO cell variant. Only those cells which take up the co-expression vector can be selected on medium that lacks added glutamine (GS) or nucleosides (DHFR). Both systems have the potential for amplification with small molecular weight inhibitors - methionine sulphoxamine (MSX) for GS and methotrexate (MTX) for DHFR. Inclusion of inhibitors in the selection process enables the isolation of cells with higher copies of GS or DHFR which, being genetically linked to the co-expressed recombinant gene, leads to higher expression of the recombinant gene. Consequently, production per cell can be extensively increased and results in productivity suitable for commercial use. Metabolomics studies with CHO cells in the Dickson laboratory have identified a metabolic target that, in a manner analogous to (but distinct from) that of GS, offers a powerful co-expression genetic vector selection system, amenable to knockdown (again analogous to the GS Xceed1 system, developed by Lonza). Several small molecular weight chemicals are potent inhibitors of the metabolic target (analogous to MSX), offering the potential to selection of amplified gene copy. The identified metabolic target is fundamental to cell growth, is in a critical metabolic pathway and, unlike GS, cannot be bypassed by other nutrients such as asparagine. It is also cell-agnostic for potential cell host application. We hypothesise that this platform provides huge potential for synthetic interventions in terms of vector development and host cell line engineering, offering an alternative and improved metabolic selection system to produce therapeutic proteins. This metabolic target and associated gene constitute novel intellectual property. Once exemplified it is intended that the technology would be patented, and that this patent, the materials generated (such as cell lines, vectors and media) would support future commercialisation The PhD student will be responsible for the technical development of the platform, generation of novel cell lines, cell culture medium and other components with design and application of synthetic biology constructs, preparation of knockout cell lines and cell culture components such as medium. Once developed these key components will enable a proof-of-concept evaluation of the platform using a relevant industrial molecule. Production of proteins is of key importance for both therapeutic and nontherapeutic proteins. With global annual sales of therapeutic proteins of >£200Bn, the biomanufacturing sector is an economic leader for the UK. The platform developed in this PhD would be offered as a potential competitor to the technology already reaping commercial dividends for companies such as Lonza, Fuji and Horizon. Currently the lack of freedom to operate and the high cost of entry makes manufacturing expensive and limits the emergence of new companies with innovative products, lacking access to the required technology. A key aim of this project is to develop an innovative, industry-leading, open-source expression platform with significantly flexible license terms, thereby driving significant industry uptake. Revenues would be generated through sales of platform components, technical support and partnerships. The availability of expression platforms un-encumbered by stringent licensing fees would have great attraction to SME's and academic innovators. This expression system has completed an initial proof of concept project undertaken between Pathway Biopharma and an MSc student in the Dickson lab.