CHO synthetic promoters improve expression and product quality of biotherapeutic proteins.

When expressing complex biotherapeutic proteins, traditional expression plasmids and methods may not always yield sufficient levels of high-quality product. High-strength viral promoters commonly used for recombinant protein (rProtein) production in mammalian cells allow for maximal expression, but provide limited scope to alter their transcription dynamics. However, synthetic promoters designed to provide tunable transcriptional activity offer a plasmid engineering approach to more precisely regulate product quality, yield or to reduce product related contaminants.

Control of Multigene Expression Stoichiometry in Mammalian Cells Using Synthetic Promoters.

To successfully engineer mammalian cells for a desired purpose, multiple recombinant genes are required to be coexpressed at a specific and optimal ratio. In this study, we hypothesized that synthetic promoters varying in transcriptional activity could be used to create single multigene expression vectors coexpressing recombinant genes at a predictable relative stoichiometry. A library of 27 multigene constructs was created comprising three discrete fluorescent reporter gene transcriptional units in fixed series, each under the control of either a relatively low, medium, or high transcriptional strength synthetic promoter in every possible combination.

A novel hydrogen peroxide evolved CHO host can improve the expression of difficult to express bispecific antibodies.

The manufacture of bispecific antibodies by Chinese hamster ovary (CHO) cells is often hindered by lower product yields compared to monoclonal antibodies. Recently, reactive oxygen species have been shown to negatively impact antibody production. By contrast, strategies to boost cellular antioxidant capacity appear to be beneficial for recombinant protein expression.

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.

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.

In silico design of context-responsive mammalian promoters with user-defined functionality.

Comprehensive de novo-design of complex mammalian promoters is restricted by unpredictable combinatorial interactions between constituent transcription factor regulatory elements (TFREs). In this study, we show that modular binding sites that do not function cooperatively can be identified by analyzing host cell transcription factor expression profiles, and subsequently testing cognate TFRE activities in varying homotypic and heterotypic promoter architectures. TFREs that displayed position-insensitive, additive function within a specific expression context could be rationally combined together in silico to create promoters with highly predictable activities.