Discussion

Here we described the development and testing of the GPEx® Lightning technology. Combining GPEx®, GS selection, and targeted recombination technologies leverages the advantages of both semi-targeted integration and site-specific recombination for cell line development. This technology consistently results in stable cell pools containing approximately 50+ copies of recombined Boat gene construct per cell on average. Clones isolated from these pools can contain 150 to 200+ copies of Boat expression construct. This represents a total of up to 1.4 million base pairs of DNA stably inserted into the CHO genome at multiple chromosome sites from a single transfection.
Generic fed-batch production of the resulting pools and clones showed that Boat gene copy number correlated with final titer. Titers from pool populations typically reach 3-7 grams per liter in non-optimized conditions and up to 12 g/L with minimal optimization. Clones typically reach 4-12 g/L and up to 15 g/L. Both pools and clones reach high cell density with prolonged cell viability. Recent efforts focusing on process intensification (i.e. higher inoculation density, feed rates, and temperature) have achieved similar titers in shorter production runs (not shown). Additionally, these pools and clones show very high genetic and production stability over greater than 40 generations. The N-glycan distribution across 40+ generations also remains remarkably consistent, especially the amount of high-mannose and total fucosylated species. Clones show N-glycan profiles consistent with the pools from which they were isolated mitigating the risk of clones having divergent protein quality.
This technology also allows for shortened development timelines. Once the Boat gene construct is cloned and purified, transfection and selection typically take less than 25 days to produce pool research cell banks. These banks can then be scaled for large scale production. When combined with the Berkeley Lights Beacon instrument for cloning, clones can be produced, expanded, banked, and characterized in an additional 12 weeks.
Many pharmaceutical/biotech companies and CDMOs have moved away from random integration approaches to cell line development in favor of semi-targeted or targeted approaches to improve process consistency, increase yield, and reduce timelines (Rajendra, 2017; Rajendran, 2021; Feary, 2020; Ng, 2021). To our knowledge this is the first example of a technology that combines the two latter approaches, allowing for a very high number of transgenes to be inserted at consistent locations in the genome. The speed of development, high and stable expression with consistent glycosylation, and ability to scale to bioreactors make GPEx Lightning pool-derived material suitable for early phase clinical studies. The use of pool-derived material should also allow for cGMP production of diagnostic assay components, molecules that are part of medical devices, or process reagents needed to produce cell/gene therapy products to be produced not only in a more cost-effective manner but also with greatly reduced timelines. As we have seen with the COVID pandemic, reducing timelines and costs through approaches like this can have significant impact for drug development, patients, and society.