Cell‐free protein synthesis (CFPS) is an established method for rapid recombinant protein production. Advantages like short synthesis times and an open reaction environment make CFPS a desirable platform for new and difficult‐to‐express products. Most recently, interest has grown in using the technology to make larger amounts of material. This has been driven through a variety of reasons from making site specific antibody drug conjugates, to emergency response, to the safe manufacture of toxic biological products. We therefore need robust methods to determine the appropriate reaction conditions for product expression in CFPS. Here we propose a process development strategy for Escherichia coli lysate‐based CFPS reactions that can be completed in as little as 48 hr. We observed the most dramatic increases in titer were due to the E. coli strain for the cell extract. Therefore, we recommend identifying a high‐producing cell extract for the product of interest as a first step. Next, we manipulated the plasmid concentration, amount of extract, temperature, concentrated reaction mix pH levels, and length of reaction. The influence of these process parameters on titer was evaluated through multivariate data analysis. The process parameters with the highest impact on titer were subsequently included in a design of experiments to determine the conditions that increased titer the most in the design space. This proposed process development strategy resulted in superfolder green fluorescent protein titers of 0.686 g/L, a 38% improvement on the standard operating conditions, and hepatitis B core antigen titers of 0.386 g/L, a 190% improvement.

中文翻译：

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提高基于大肠杆菌的无细胞蛋白质合成反应中效价的合理方法

无细胞蛋白质合成（CFPS）是一种快速生产重组蛋白的成熟方法。短合成时间和开放反应环境等优势使 CFPS 成为新的和难以表达的产品的理想平台。最近，人们对使用该技术制造大量材料的兴趣日益浓厚。这是由多种原因驱动的，从制造位点特异性抗体药物偶联物到应急响应，再到安全生产有毒生物产品。因此，我们需要稳健的方法来确定 CFPS 中产品表达的适当反应条件。在这里，我们提出了大肠杆菌的工艺开发策略基于裂解物的 CFPS 反应可在短短 48 小时内完成。我们观察到最显着的滴度增加是由于大肠杆菌细胞提取物的应变。因此，我们建议第一步为感兴趣的产品鉴定高产量的细胞提取物。接下来，我们控制了质粒浓度、提取量、温度、浓缩反应混合物的 pH 值和反应时间。通过多变量数据分析评估这些工艺参数对滴度的影响。随后将对效价影响最大的工艺参数包括在实验设计中，以确定在设计空间中增加效价最多的条件。该提议的工艺开发策略导致超级折叠绿色荧光蛋白滴度为 0.686 g/L，比标准操作条件提高了 38%，乙肝核心抗原滴度为 0.386 g/L，提高了 190%。