Applying synthetic biology to upscale and de-risk biologics production by CHO cell transient transfection.

点击翻译按钮获取中文摘要

Mammalian cell transient transfection typically involves subjecting cells to procedures that result in a burst of transgene expression that increases in intensity over 48 hours then rapidly fades. Transient expression of heterologous proteins in mammalian cells is a powerful way to rapidly generate protein reagents. However, it has historically suffered from poor yields and reproducibility compared to methods where the recombinant gene is stably integrated into the genome and high expressing clones isolated. To date approaches to improve these metrics for transient transfection include: i) the use of design of experiments (DoE) to quickly measure the influence of multiple parameters on protein expression levels ii) addition of small molecules such as N, N-Dimethyl acetamide (DMA) to enhance gene expression and iii) co-transfection with additional transgenes encoding proteins that enhance protein folding. This project proposes to capture proteomic data on the up- and down- regulation of genes that results from the biological shock caused by transient transfection reagents. These data will provide a valuable knowledge base for the design of synthetic gene networks (SGNs) that are switched on, or indirectly triggered, by the transfection procedure. These SGNs would then be inserted into the CHO cell genome using precise CRISPR techniques to direct expression of proteins that enhance transfection performance, without contributing negatively to metabolic burdens placed on the cell.Little work has been done to characterise the metabolic status and requirements of cells during this burst of high-level transgene expression. A further element of the project will involve capturing metabolic data during transient transfection and refining metabolic models. Outputs will inform media formulation and supplementation with the goal of shortening the expression burst period to the first 24 hours whilst preserving or increasing total yield.All elements of the project will be validated across a suite of macromolecular therapeutic proteins ranging from highly standard platform entities such as monoclonal antibodies (MAbs), to difficult-to-express proteins such as highly glycosylated enzymes for replacement therapies. Different scales of cultivation will be tested in order to establish accurate scale mimics and to demonstrate the robustness and scalability of novel methods arising from the project.