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剂的主要方法，尽管从时间和资源的角度来看，这种方法存在显著的局限性。假设使用无细胞蛋白质合成（CFPS）平台可以实现这些低效率。细胞提高了前景，他们可以切实用于重组蛋白的合成在工业上使用CFPS。这将在一个系统中实现，该系统不需要在生物产量和传统表达系统所依赖的不相关的、浪费的细胞代谢之间进行妥协，但仍然保持治疗的适当安全性要求。然而，CFPS系统仍然具有与低蛋白产量和质量不足相关的特定限制-特别是关于翻译后修饰（PTM）。该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，使用优化的表达构建体和补充两种辅助蛋白：tGADD 34和K3 L以提高产量。关于后一个问题，N-连接的糖基化--一个影响生物治疗药物的效率、功效和半衰期的非模板化和复杂过程--仍然是一个需要解决的重大挑战。假设用于CFPS反应的提取物中的微粒体富集和/或固定化糖基转移酶的使用可以帮助介导糖基化。首先，这项工作将寻求在此PTM过程中实现高精度和均匀性。就均一性而言，单独使用微粒体富集获得的PTM的质量将优于通过固定化糖基转移酶系统获得的PTM的质量（“人工高尔基体反应器”（AGR）），在IgG 1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平台具有大规模生产定制生物治疗剂的潜力，迄今为止，已经改进了产生活性CHO提取物的工作裂解物方案，进一步改进了反应混合物。此外，tGADD 34和K3 L已被纯化并补充到CFPS反应中以加强表达。

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.