A platform for context-specific genetic engineering of recombinant protein production by CHO cells.

An increasing number of engineered therapeutic recombinant proteins with unpredictable manufacturability are currently filling industrial cell line development pipelines. These proteins can be "difficult-to-express" (DTE) in that production of a sufficient quantity of correctly processed recombinant product by engineered mammalian cells is difficult to achieve. In these circumstances, identification of appropriate cell engineering strategies to increase yield is difficult as constraints are cell line and product-specific. Here we describe and validate the development of a high-throughput microscale platform for multiparallel testing of multiple functional genetic components at varying stoichiometry followed by assessment of their effect on cell functional performance. The platform was used to compare and identify optimal cell engineering solutions for both transient and stable production of a model DTE IgG1 monoclonal antibody. We simultaneously tested the functional effect of 32 genes encoding discrete ER or secretory pathway components, each at varying levels of expression and utilized in different combinations. We show that optimization of functional gene load and relative stoichiometry is critical and optimal cell engineering solutions for stable and transient production contexts are significantly different. Our analysis indicates that cell engineering workflows should be cell line, protein product and production-process specific; and that next-generation cell engineering technology that enables precise control of the relative expression of multiple functional genetic components is necessary to achieve this.

Whole synthetic pathway engineering of recombinant protein production.

The output from protein biomanufacturing systems is a function of total host cell biomass synthetic capacity and recombinant protein production per unit cell biomass. In this study, we describe how these two properties can be simultaneously optimized via design of a product-specific combination of synthetic DNA parts to maximize flux through the protein synthetic pathway and the use of a host cell chassis with an increased capability to synthesize both cell and product biomass. Using secreted alkaline phosphatase (SEAP) production in Chinese hamster ovary cells as our example, we demonstrate how an optimal composition of input components can be assembled from a minimal toolbox containing rationally designed promoters, untranslated regions, signal peptides, product coding sequences, cell chassis, and genetic effectors. Product titer was increased 10-fold, compared with a standard reference system by (a) identifying genetic components that acted in concert to maximize the rates of SEAP transcription, translation, and translocation, (b) selection of a cell chassis with increased biomass synthetic capacity, and (c) engineering the host cell factory's capacity for protein folding and secretion. This whole synthetic pathway engineering process to design optimal expression cassette-chassis combinations should be applicable to diverse recombinant protein and host cell-type contexts.