Enabling on-demand biologics manufacturing with ALiCE

关于项目无细胞蛋白质合成（CFPS）具有彻底改变生物制造的潜力。传统的基于细胞的蛋白质表达工作流程需要细胞系转化以表达感兴趣的基因，然后将所得菌株培养至可以提取足够产物的有用体积。这些步骤中的每一个本身都是一个工程挑战，必须针对每一种蛋白质重复和重新优化。CFPS完全绕过了这一点，在几个小时内从细胞的分离翻译机器中产生蛋白质。一种在许多参数上表现良好的无细胞系统被称为ALiCE（几乎活细胞无表达），它包含来自烟草根细胞培养物的浓缩蛋白质合成机器，并由LenioBio商业化。它已显示出提供>3 mg/mL的参考蛋白质产率和从微升到升的容易的缩放潜力。这种真核系统具有产生一系列困难蛋白质的潜力，包括膜蛋白和病毒样颗粒蛋白。尽管取得了这些早期的里程碑式的成功，但在ALiCE系统中还有更多的东西需要理解和设计，以进一步实现按需生物制剂的制造。在这个项目中，我们将调查ALiCE的使用和生产，重点是确定系统的关键质量属性（CQA），这将为最佳实践制造提供信息。通过对ALiCE组分（如核糖体、微粒体和/或线粒体）和蛋白质生产反应的分析和表征，将有助于理解这些CQA，并最终将这些知识转化为商业生产过程。该研究计划的基础将是系统应用和蛋白质治疗剂/候选疫苗规模化生产的机会。这将使该项目的最佳实践开发能够用于现实世界的应用，规模化的蛋白质生产与随后的功能表征相匹配。目的1）建立ALiCE系统的关键质量属性（CQA），并设计可用于过程中和批放行质量控制的分析方法，重点是整个ALiCE裂解物，翻译机器和原生微粒体。2）发展对ALiCE反应的机械理解，以实现工程干预的建模。3）将ALiCE应用于研究方法：候选人将发展生物信息学，微生物学，分子生物学，无细胞蛋白质合成，蛋白质纯化，生物化学/生物物理方法，显微镜，分析和生物加工方面的技能。与EPSRC的战略和研究领域保持一致：该项目与战略优先事项“工程和技术前沿”和“转变健康和医疗保健”以及研究领域“制造技术”保持一致，因为它调查了具有高效率/可靠性和适当精度/灵活性的制造过程，产品和系统。

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.