Next Generation Cell-Free Synthesis of Functional Glycoproteins

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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.