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KEYNOTE PRESENTATION: New Parts and Systems for CHO Cell Synthetic Biology
University of Sheffield
How Novel Technologies can be Applied to Advance CHO-K1 Manufacturing Cell Lines
Boehringer Ingelheim Pharma GmbH & Co.KG
Systems Biology, Omics and Big Data Integration in Cell Line Development and Engineering
Synthetic biology based on the “Design-Build-Test-Learn” cycle offers a new paradigm for CHO cell engineering, where it is possible to engineer the host cell factory in a product specific manner via combinatorial “tuning” of discrete cellular synthetic processes. This approach permits “one-size-fits-all” genetic vectorology and mechanistically blind screening of transfected cells to be replaced with tailored design and construction of specifically fit-for-purpose cell factories. This engineering design system relies upon a toolbox of synthetic parts with user-defined functionality that work in synchrony to enable product manufacturability.
CHO cells are the most widely used host for large-scale production of recombinant therapeutic proteins. Using transcriptomic approaches we have identified target genes involved in productivity and product quality. Subsequently a variety of novel parental CHO cell lines were generated applying cell line engineering techniques. These novel knockout CHO cell lines are superior in respect to productivity and/or product quality.
Progress in Host Cell Engineering to Improve Cell Quality and Growth
Genome editing, next-generation sequencing (NGS) and cell line engineering using microRNAs (miRNAs) have emerged as key technologies towards successful biopharmaceutical process development. We have evaluated and integrated these innovative tools to enhance our state-of-the-art cell line development and characterization processes using our novel BI-HEX® CHO-K1GS manufacturing cell line. This talk will present an overview on our recent achievements in employing these technologies to provide solutions for future bioprocessing challenges.
The utility of CHO cell factories derives from exploitation of their acquired genetic/functional variation, which enable industry to identify cell lineages with desirable manufacturing properties. Here we discuss novel technologies engineered to provide a desired level of process control while at the same time enabling optimal leverage of the cell factory using ChemStress Fingerprinting.
Yield is still an area that requires significant improvement for many promising recombinant proteins and antibodies. Novel vector technology enables rapid generation of stable, CHO cell lines able to provide at least 10-fold more product per cell.
CRISPR and Genome Editing Technologies
CHO cells are widely used in the industry as a host for the production of complex pharmaceutical proteins. Thus, accelerated genome engineering of CHO cell factories to improve product yield and quality is of great interest. In this talk, our recent efforts in accelerating genome engineering of CHO cell factories will be presented. Topics will include targeted integration and multiplexing of gene knockouts.
The continued growth of biotherapeutics has created a critical need for technologies that facilitate scalable biomanufacturing and precise genome modification. In this presentation, we present recent data for a high-performance cell engineering technology that enables improved transient CHO productivity and streamlined stable cell line generation as well as custom CRISPR-mediated gene editing for construction of high-yield CHO cell lines.
At CFB we have established a high throughput genome engineering pipeline using CRISPR-Cas9. The pipeline is being used to generate a panel of engineered CHO cells with improved properties for the production of recombinant therapeutic proteins. We now have a collection of cell lines generating predictable and tailormade glycoprofiles, better product quality and improved growth phenotypes.
Cell Line and Host Cell Engineering Technologies
A whole genome mouse siRNA screening was applied to suspension CHO cells producing and secreting GFP. With this screen, two genes were identified, that, upon knockdown, boost the productivity in CHO cells. The effect of knockdown of these genes varied in different production clones producing either an antibody or an Erythropoietin-Fc fusion protein, indicating cell- and/or subclone specific limitations. However, in each cell line, at least one of the two knockdowns enhanced specific productivity.
Despite their low costs, rapid deployment and flexibility, the current Single Use facilities are limited by their output, typically 500 kg/year for a 6 x 2000 L facility at 3 g/L Fed Batch titer. This also limits their usefulness for commercial scale manufacturing of multiple midsize portfolio products. Upstream process intensification can solve this limitation. With consistent effective titers of 10 g/L and beyond, annual outputs of 1500 kg/year can be realized from Single Use facilities so they become an even more attractive option for commercial manufacturing of multiple products.
To this point however the development and scale-up of intensified upstream has been cumbersome and time consuming. In this presentation a platform of upstream process intensification tools and technologies are shown, that can greatly speed-up and improve process development, scale-up and commercial scale process control.
Examples will be shown including an effective titer boost from 3 to 10 g/L in 12 days of culture, using commercially available platform tools, including cell line, media, process development tools and commercial scale manufacturing tools.