Enabling on-demand biologics manufacturing with ALiCE

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About the ProjectCell-free protein synthesis (CFPS) has the potential to revolutionise biomanufacturing. Traditional, cell-based protein expression workflows require cell line transformation to express a gene of interest, followed by culture of the resulting strain to useful volumes where sufficient product can be extracted. Each of these steps is an engineering challenge in and of itself and must be repeated and reoptimized for every protein. CFPS circumvents this entirely, producing protein in a matter of hours from the isolated translational machinery of cells. One cell-free system that performs well across many parameters is called ALiCE (Almost Living Cell Free Expression), it contains the concentrated protein synthesis machinery derived from tobacco root cell cultures and is commercialised by LenioBio. It has been shown to provide reference protein yields of >3 mg/mL and facile scaling potential from microliters through to liters. This eukaryotic system has the potential to produce a range of difficult proteins, including membrane proteins and virus-like particle proteins. Despite these early landmark successes, there is more to understand and engineer within the ALiCE system to further enable on-demand biologics manufacturing. Aims In this project we will investigate the use and production of ALiCE, with an emphasis on identifying Critical Quality Attributes (CQAs) of the system that will inform best practice manufacturing. Analysis and characterisation of ALiCE components (e.g. ribosomes, microsomes and/or mitochondria) and the protein production reaction will be employed to understand these CQAs and ultimately translate these learnings into a commercial manufacturing process.Underpinning this program of research, will be the opportunity for system application and the scaled production of protein therapeutics/vaccine candidates. This would allow the best practice developments of the project to be leveraged for a real-world application, with scaled protein production matched by subsequent functional characterisation. Objectives1) Establish Critical Quality Attributes (CQAs) for the ALiCE system and devise analytical procedures that could be implemented for in-process and batch release quality control, with focus on whole ALiCE lysate, translation machinery and native microsomes.2) Develop a mechanistic understanding of the ALiCE reaction to enable modelling of engineering interventions.3) Apply ALiCE for on-demand production of therapeutics or vaccine candidates combined with physiochemical and functional protein characterisation.Research methods:The candidate will develop skills in bioinformatics, microbiology, molecular biology, cell-free protein synthesis, protein purification, biochemical/biophysical methods, microscopy, analytics, and bioprocessing. Alignment to EPSRC's strategies and research areas:This project is aligned with the Strategic Priorities 'Frontiers in Engineering and Technology' and 'Transforming Health and Healthcare,' as well as the Research Area 'Manufacturing Technologies', because it investigates manufacturing processes, products, and systems that function with high efficiency/reliability and appropriate precision/flexibility.