Non-viral Engineering of T Cells

With the growth of gene-modified cell therapies, advanced technologies are needed to enhance cell viability and allow for the scaled manufacturing of CAR-T cell therapies for increased treatment of cancer and other hematological conditions.

As both viral and non-viral methods evolve, there is a growing demand for electroporation platforms that reduce cell mortality and improve fold expansion and knockout efficiency at a larger scale. These improvements are crucial to improve the effectiveness and scalability of gene-modified cell therapies, ultimately accelerating the delivery of innovative treatments to patients.

However, traditional high-voltage electroporation has its drawbacks, including potential damage to cell membrane, increased cell death, and challenges with scaling up for large-volume cell therapy production.

As Charles River is always looking to identify leading cell therapy manufacturing platforms that improve and automate life-saving therapies for patients, we conducted a study with Kytopen's Flowfect Tx™ and two other top, commercially available electroporation platforms.

The 10-day study was performed using the same two donors with each platform. Cell viability, cell density, knockout efficiency, negative viable cells, and fold expansion were all measured.

Results of CRISPR/Cas9 TRAC gene knock-out experiments

Using CRISPR/Cas9 TRAC gene knock-out experiments in primary T cells to assess the performance and capabilities of the electroporation platforms, cells were transfected on day 2 and sampling points were taken on days 2 (pre-transfection), 4, 7, and 10 for both cell counts and flow cytometry.

Study results revealed that the Flowfect Tx platform delivers greater cell viability and editing efficiency compared to conventional, commercially available electroporation platforms.

A key finding was the considerably higher yields at multiple timepoints of gene-edited, viable cells with the Flowfect Tx platform that have the potential to be used as the final drug substance. These results confirm that Kytopen is prepared to accelerate the development and manufacture of cell therapies to yield the highest quality advanced cellular therapies.

Most significantly, the total number of TRAC- negative viable cells was considerably higher after transfection with the Flowfect Tx platform at day 10, showing the ability of the Flowfect technology to retain cell health post transfection and to deliver larger amounts of gene-edited cells.

"This is a milestone for Kytopen as many cell therapies are developed and manufactured by CDMOs and we are excited to present this data from such a prestigious CDMO as Charles River," said Kevin Gutshall, Chief Commercial Officer at Kytopen, in a recent press release. "We have a GMP-scale manufacturing platform that has already been evaluated for tech transfer and this study further highlights the ease of implementation to develop superior yields of final drug substance."

The Flowfect technology combines mechanical, electrical, and chemical forces, and allows the adjustment of multiple parameters to maximize transfection efficiency, cell health, and cell yield. An additional key differentiator is that the Flowfect Tx platform is not restricted to the batch processing of conventional electroporators that can overheat and harm cells, but is designed with continual flow processing enabling the handling of hundreds of billions of cells in just minutes.

"We were drawn to the Flowfect™ technology because it utilizes the benefits of electroporation, while eliminating the drawbacks in that it utilizes mechanical and chemical forces to lower the overall voltage requirements and allow for a gentler treatment of cells," said Alex Sargent, Director, Process Development, Cell Therapy CDMO Services at Charles River. "Scale up and ease of use are important factors in optimizing cell therapy manufacturing. For this study, we simply plugged in the instrument, added cells and payload, and ran a single protocol without any protocol or reagent changes or optimization. It truly is just plug and play."

Process Development Innovation

Because cell therapy development is a rapidly evolving, complex, and competitive field, our process development team continues to grow its experience and expertise using the most cutting-edge technology while establishing commercial viability and regulatory compliance.

With our tech-agnostic capabilities, we can optimize all stages of cell therapy process development by leveraging a variety of platforms. Our processes include:

• Cell selection and enrichment
• Gene editing and viral transduction
• pDNA and vector production
• Cell culture and expansion
• Harvest, formulation, fill and finish, and cryopreservation
• Moving processes from manual to automated, open to closed

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