Fast and robust recombinant protein production utilizing episomal stable pools in WAVE bioreactors.

Protein reagents are essential resources for several stages of drug discovery projects from structural biology and assay development through lead optimization. Depending on the aim of the project different amounts of pure protein are required. Small-scale expressions are initially used to determine the reachable levels of production and quality before scaling up protein reagent supply. Commonly, amounts of several hundreds of milligrams to grams are needed for different experiments, including structural investigations and activity evaluations, which require rather large cultivation volumes. This implies that cultivation of large volumes of either transiently transfected cells or stable pools/stable cell lines is needed. Hence, a production process that is scalable, speeds up the development projects, and increases the robustness of protein reagent quality throughout scales. Here we present a protein production pipeline with high scalability. We show that our protocols for protein production in Chinese hamster ovary cells allow for a seamless and efficient scale-up with robust product quality and high performance. The flexible scale of the production process, as shown here, allows for shorter lead times in drug discovery projects where there is a reagent demand for a specific protein or a set of target proteins.

High throughput generation of a resource of the human secretome in mammalian cells.

The proteins secreted by human tissues and blood cells, the secretome, are important both for the basic understanding of human biology and for identification of potential targets for future diagnosis and therapy. Here, a high-throughput mammalian cell factory is presented that was established to create a resource of recombinant full-length proteins covering the majority of those annotated as 'secreted' in humans. The full-length DNA sequences of each of the predicted secreted proteins were generated by gene synthesis, the constructs were transfected into Chinese hamster ovary (CHO) cells and the recombinant proteins were produced, purified and analyzed. Almost 1,300 proteins were successfully generated and proteins predicted to be secreted into the blood were produced with a success rate of 65%, while the success rates for the other categories of secreted proteins were somewhat lower giving an overall one-pass success rate of ca. 58%. The proteins were used to generate targeted proteomics assays and several of the proteins were shown to be active in a phenotypic assay involving pancreatic ß-cell dedifferentiation. Many of the proteins that failed during production in CHO cells could be rescued in human embryonic kidney (HEK 293) cells suggesting that a cell factory of human origin can be an attractive alternative for production in mammalian cells. In conclusion, a high-throughput protein production and purification system has been successfully established to create a unique resource of the human secretome.