A Design of Experiments Approach for Enhancing Room Temperature Stability of a Lyophilised and Paper-Based Bacterial Cell-Free System

Centralised cell-based biomanufacturing severely limits applicability in low-resource and extreme environments, where a largely untreated human population is present. Cell-free protein synthesis (CFPS) can surpass many of these limitations, due to its flexibility and low maintenance. After initial optimisation for high-level expression, we conceptualised CFPS platforms composed of lyophilised pellets and cellulose stacks for ease of storage and distribution. The latter platform consisted of lyophilised components on cellulose discs, which were layered and rehydrated to kickstart protein synthesis. Such paper-encompassed reactions were capable of robust expression, where the system can be modulated by simply changing the DNA layer. Using an initial screening design followed by a minimalistic design of experiments approach, we were able to improve the shelf life of lyophilised CFPS at room temperature from <1 week to 100% preservation at month 1. We anticipate that our strategy will enable quicker and more efficient stability optimisation for sustainable applications in all environments.