Next Generation Cell-Free Synthesis of Functional Glycoproteins

基于细胞的表达系统仍然是合成生物制品的主要方法，尽管从时间和资源的角度来看，这种方法都有很大的局限性。假设使用无细胞蛋白质合成(CFPS)平台可以避免这些低效。基于中国仓鼠卵巢(CHO)细胞的这类系统的最新发展提出了利用CFPS在工业上可行地用于重组蛋白质合成的前景。这将在一个没有传统表达系统所依赖的生物产量和无关的、浪费的细胞代谢之间妥协的系统中实现，但仍然遵守适当的治疗安全要求。然而，CFPS系统仍然存在与蛋白质产量低和质量不足相关的特定限制-特别是在翻译后修饰(PTM)方面。这个项目将着眼于解决前一个问题，利用工具来指导改进的能源产生，使用优化的表达结构和补充两种辅助蛋白质：tGADD34和K3L来提高产量。关于后一个问题，N-连接糖基化--一个影响生物疗法的效率、疗效和半衰期的非模板化和复杂的过程--仍然是一个需要解决的重大挑战。据推测，用于CFPS反应和/或使用固定化糖基转移酶的提取物中的微生物浓缩可以辅助介导糖基化。首先，这项工作将寻求在这一PTM过程中实现高精度和同质性。在均质性方面，仅使用微体浓缩获得的PTM的质量将与通过固定化糖基转移酶系统(人工高尔基反应器(AGR))获得的质量进行比较，并在IgG1的可结晶片段(MFC)的单体片段上进行实验。因此，组合的CHO CFPS-AGR平台有可能大规模生产定制的生物治疗药物，满足个性化药物的需求。到目前为止，产生活性CHO提取物的工作裂解方案已经得到改进，进一步改变了反应混合物。此外，还纯化了tGADD34和K3L，并将其添加到CFPS反应中以促进表达。

Cell-based expression systems remain the predominant method by which biologics are synthesised,despite significant limitations associated with such an approach from both a time and resource perspective.Bypassing these inefficiencies can hypothetically be achieved using Cell-Free Protein Synthesis (CFPS) platforms.Recent developments in such systems based on Chinese Hamster Ovary (CHO) cells raise the prospect that theycould feasibly be used for recombinant protein synthesis in industry using CFPS. This would be achieved in asystem devoid of the compromise between biologic yield and unrelated, wasteful, cellular metabolism thattraditional expression systems rely on, yet still upholding the appropriate safety requirements of therapeutics.However, CFPS systems still bear specific limitations relating to low protein yield and insufficient quality- specifically with regards to post-translational modifications (PTMs). This project will look at addressing theformer issue by utilising tools to direct improved energy source generation, the use of an optimised expressionconstruct and supplementation of two accessory proteins: tGADD34 and K3L to improve yield. With regards tothe latter issue, N-linked glycosylation - a non-templated and complex process affecting efficiency, efficacy andhalf-life of biotherapeutics - remains a significant challenge to address. It is hypothesised that microsomeenrichment in the extract used for CFPS reactions and/or the use of immobilised glycosyltransferases can assistin mediating glycosylation. Primarily, this work will seek to achieve high accuracy and homogeneity in this PTMprocess. The quality, in terms of homogeneity, of PTM achieved solely using microsome enrichment will becompared to that through a system of immobilised glycosyltransferases (an 'Artificial Golgi Reactor' (AGR)), withexperiments trialled on the monomeric fragment of the crystallisable fragment (mFc) of IgG1.Combined CHO CFPS-AGR platforms therefore have the potential for large scale manufacturing ofbespoke biotherapeutics, addressing the necessities of personalised medicine.To date, a working lysate protocol to generate active CHO extract has been improved, further to amodified reaction mix. Furthermore, tGADD34 and K3L have been purified and supplemented into the CFPSreactions to boost expression.