Case Study: Poseida Therapeutics

Alerion^TM Microbubble Cell Separation System Delivers Improved Cell Health and Viability for More Efficacious Cell Therapies

Poseida Therapeutics, a clinical-stage allogeneic-focused therapeutic developer leveraging proprietary technologies to create life-saving cell therapies, selected the Akadeum platform, consisting of the new Alerion^TM Microbubble Cell Separation System, the innovative BioRise^TM consumable, and the Human T Cell Leukopak Isolation Kit, to improve workflows for its cancer and genetic disease solutions.

A critical step in developing these therapies is isolating T cells from leukapheresis material. This requires an isolation process that ensures cell health and viability, and must be highly consistent and efficient for CAR-T cell manufacturing and for effective therapeutics.

Poseida Therapeutics chose the Alerion^TM Microbubble Cell Separation System due to:

• Superior cell health in negative selection compared to positive selection
• Superior scalability, providing a solution avoiding particles directly bound to T cells
• Simple, easy-to-follow workflow
• Superior yield and purity
• Significant flexibility
• Instrument ease of use and walk away capability

Objective

The study sought to assess Akadeum’s Alerion^TM Microbubble Cell Separation System’s effectiveness in isolating T cells for CAR-T manufacturing at an increased processing scale. The goal was to improve cell health, cell viability, impurity clearance, T cell recovery, scalability, process consistency, and processing time compared to their optimized magnetic cell separation method.

Kit Used

The Alerion^TM Microbubble Cell Separation System was paired with Akadeum’s Human T Cell Leukopak Isolation Kit and BioRise^TM consumable.

Experimental Design

Poseida Therapeutics conducted two major types of experiments: split-run and large-scale. These experiments aimed to compare the performance of the Alerion^TM system directly with Poseida’s optimized magnetic cell separation method.

Split-Run Procedure

1. Sample preparation: Leukopak samples were obtained from three different healthy donors and processed using Poseida’s benchmark wash procedure. Each leukopak was divided into two equal fractions.
2. Labeling and isolation: One fraction from each of the three donors was processed using the Alerion^TM Microbubble Cell Separation System for negative selection. The other fraction from each of the three donors was labeled with magnetic microbeads and processed through the optimized  CD4/CD8 positive selection system.
3. Post-isolation processing: Isolated cells from both fractions were seeded in culture media and allowed to rest overnight before further processing.

Large-Scale Procedure

1. Sample preparation: Leukopak samples were obtained from three different donors and processed using Akadeum’s manual washing procedure to achieve the desired cell concentration.
2. Labeling and isolation: Each leukopak was processed in its entirety in a single run using the Alerion^TM Microbubble Cell Separation System and one BioRise^TM consumable for labeling and isolation.
3. Post-isolation processing: Isolated cells were seeded into Wilson Wolf G-Rex flasks to rest overnight before electroporation and downstream processing.

Figure 1: Schematic of experimental design. Courtesy of Poseida Therapeutics, Inc. 

Performance Metrics

The study evaluated several key performance metrics to assess the effectiveness of the Alerion^TM system compared to their optimized method. These metrics were:

• Post-isolation T cell purity and recovery: Measurement of overall purity and recovery levels post-isolation
• Cell growth and expansion: Comparison of fold growth data from isolated T cells
• Detailed impurity clearance: Percentage of monocytes, B cells, NK cells, and NKT cells removed by Alerion^TM and optimized magnetic cell separation methods
• Scalability: Evaluation of purity performance at larger cell loads compared to results from small-scale split run isolation procedures
• Processing time: Total processing time from leukopak handling to seeding in culture media

Key Findings

1. Gentler, Shorter Separations: More Healthy Cells for Therapies

The system also had superior leukopak-to-overnight-rest T cell recovery rates, with up to 40% more cells to engineer for CAR-T therapies A significant improvement was consistent across all split-run experiments; microbubbles offer gentler, shorter separations, which are important drivers of cell health in cell therapy manufacturing.

The Alerion^TM Microbubble Cell Separation System consistently yielded a higher purity of T cells compared to their optimized magnetic cell separation method. The Alerion^TM system outperformed the optimized method in all split-run experiments and matched the testers’ previous experience with manual microbubble kits.

Figure 2: T cell purity and recovery rates. (Left) The overall percentage of T cells recovered post-isolation for both Alerion^TM and benchmark methods. (Right) Normalized purity data of T cells using the Alerion^TM system compared to the benchmark magnetic cell separation method. Courtesy of Poseida Therapeutics, Inc. 

2. Strong Cell Growth: Essential for CAR-T Manufacturing Success

Fold growth data from all runs showed strong expansion of Alerion^TM-isolated cells, which is essential for successful CAR-T cell manufacturing.

Figure 3: T cell fold growth. Fold growth of T cells post-isolation for both Alerion^TM and optimized (benchmark) magnetic cell separation methods. Courtesy of Poseida Therapeutics, Inc. 

3. Superior Clearance of Monocytes and NK Cells Improves Transduction Efficiency

Akadeum’s Alerion^TM Microbubble Cell Separation System achieved greater clearance of monocytes and NK cells compared to the optimized magnetic cell separation method. Alerion^TM’s monocyte clearance approached 100% in all side-by-side runs, a critical clearance factor as residual monocytes contribute to poor transduction efficiency. Clearance of monocytes and NK cells while preserving the desired T Cell population was significantly better with Alerion^TM, enabling better transduction efficiency downstream,  with greater consistency observed among runs.

Figure 4: Cell clearance. Comparison of the clearance rates of monocytes, B cells, NK cells, and NKT cells between the Alerion^TM and optimized (benchmark) methods. Courtesy of Poseida Therapeutics, Inc.

4. Successful Large Scale Cell Separation: Enabling Negative Selection

The Alerion^TM Microbubble Cell Separation System performed well in large-scale isolation procedures, demonstrating that it can handle higher cell loads in a single processing run without compromising purity in recovered cells. This is a benefit to cell therapy workflows where large-scale negative selection was desired but previously not possible. The negative selection isolation workflow enables movement away from positive selection-based magnetic particles that directly bind to T cells. When normalized, the purity levels from full leukopak runs were within or close to the range of smaller-scale split runs.

Figure 5: Purity in large-scale runs. Purity levels from large-scale isolation procedures using the Alerion^TM system, with purity levels of smaller-scale split-runs indicated by the dotted lines. Courtesy of Poseida Therapeutics, Inc.

5. Efficient Processing Times

Akadeum’s Alerion^TM Microbubble Cell Separation System provided comparable or better processing times than the optimized magnetic separation method. The ease of operation, processing of greater cell volumes, more cell product at the end of the process, and the ability to process multiple donors simultaneously were additional benefits noted during the evaluation.

Figure 6: Processing time comparison. Total processing times for T cell isolation using Alerion^TM and the optimized (benchmark) method. Courtesy of Poseida Therapeutics, Inc.

Discussion

The Alerion^TM Microbubble Cell Separation System showed significantly improved T cell isolation compared to the traditional fully optimized magnetic cell separation method. Crucial for CAR-T therapy manufacturing, shorter processing times and enhanced purity and recovery rates translate to more available, healthier T cells for genetic modification and expansion, improving treatment efficacy. Superior monocyte and NK cell clearance improves transduction efficiency, enhancing final product quality.

The system’s scalability and efficient processing times suit both small research settings and large-scale manufacturing environments, enabling a method that does not result in particles directly bound to T cells at the end of the process. The ease of operation allows streamlined workflows, more donors processed and reduced labor costs.

“We found the system easy to operate and were even able to process multiple donors on the Alerion^TM simultaneously during our runs, allowing us to increase our experimental throughput, which was an additional benefit.” – Julian Kopelove, Senior Process Development Associate, Poseida Therapeutics

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