Development and Implementation of Metrology Standards for Mammalian Cell-Free Systems

The development of cell-free transcription-translation systems, coupled with synthetic biology methodologies, has has led to innovations in metabolic engineering, biosensor development, and high-throughput protein synthesis (Reyes, Kuruma, & Tsuda, 2017; Sullivan et al., 2016; Wen et al., 2017). Mammalian cell-free systems are particularly interesting since they can facilitate the production of high molecular weight, complex protein structures (Brödel, Sonnabend, & Kubick, 2014). This includes proteins requiring post-translational modifications, including mammalian proteins for pharmaceutical applications such as antibodies (Martin et al., 2017; Stech et al., 2017). Cell-free systems are open reactions which enables accessible sampling and experimentation thus making it easier to better understand the dynamics of the system - including bottlenecks to protein synthesis, inhibitory by-product formation and resource depletion (Caschera & Noireaux, 2015). Understanding these characteristics may lead to insights that help improve cell-free reaction activity, in vitro protein production yields, extend reaction run-times and minimise batch-to-batch variabilities. To this end, we are developing and systematically characterising mammalian cell-free systems that are based upon HeLa, HEK293 and CHO cell extracts. We are developing standardised protocols for preparing mammalian cell extracts and utilising LC-MS, coupled with flux analyses, to rationally improve reaction energy mix components. Initially this involved investigation into the optimum growth phase and cell density for harvesting cells, and the method of lysis. Furthermore, by understanding how the cellular lysate environment changes overtime and between batches we can highlight key parameters and determine their optimum range. Measurement standards developed for these parameters will enable fingerprinting of future lysates. Importantly, these metrology standards will enable absolute quantification of components allowing for cross-study comparisons whilst enabling us to rationally improve the methods for generating cell-free extracts and energy mix components. Ultimately, we intend to develop improved, automated, cell-free protocols and metrology toolkits for mammalian cell-free synthetic biology applications.