The Sefia™ cell therapy manufacturing platform allows the commercialization of innovative CAR T therapy production as a modular, functionally closed, end-to-end cell therapy manufacturing platform. Here, we describe how the platform was used step-by-step for magnetic isolation, cell activation, transduction, expansion, harvest, and formulation of CAR T cells from fresh and frozen apheresis samples from healthy donors.
Introduction
Since the first immunotherapy treatments in patients with chronic lymphocytic leukemia (CLL) in 2011 (1) and acute lymphoblastic leukemia (ALL) in 2012 (2), chimeric antigen receptor (CAR) T cell therapies have gained increasing interest, with the approval and licensing of multiple new products and the initiation of numerous clinical trials. Over the past few years, the development of new engineering modalities with bispecific antigens (dual CAR T cells) and novel gene-editing tools (CRISPR) have broadened the scope of clinical applications to treat solid tumors and other diseases, showing improved safety as well as enhanced antitumoral potency and cytokine activity (3,4).
Despite ongoing advancements in engineering strategies, the fundamental steps of CAR T cell therapy manufacturing—T cell isolation, activation, transduction, and expansion—remain consistent. As investigators and physicians seek to extend the reach of these therapies to new patient populations, a significant challenge lies in the manual labor required to produce these therapies at commercial scale, hindering widespread treatment availability.
The Sefia™ cell therapy manufacturing platform is our end-to-end solution for the commercialization of these breakthrough therapies. To meet these evolving needs, we designed a flexible, modular, and digitally integrated technology, that consists of two functionally closed systems:
- The Sefia Select™ system to magnetically isolate T cells from fresh or frozen apheresis, and to harvest and formulate the final product.
- The Sefia™ expansion system, which automates the workflow steps of activation, transduction, and expansion of CAR T cells in a single instrument with a dedicated application software (Universal application), and a single-use kit (Sefia™ expansion kit). The Universal application software offers high flexibility to execute multiple types of workflows by selecting the desired user parameters, according to diverse reagents and input materials.
In this study, we present the results obtained from the processing of fresh and frozen apheresis samples from healthy donors to produce CAR T cells using our Sefia™ cell therapy manufacturing platform.
Materials and methods
Magnetic labeling and isolation of target T cells with the Sefia Select™ system
Frozen (Charles River, n = 3) and fresh (CytoCare, n = 3) leukopaks from six healthy donors were used in this process (Fig 1). VIA Thaw™ dry thawer was used to cautiously thaw the frozen leukopaks. Subsequently, leukopaks were connected to the Sefia Select™ system using a single-use CT-400.1 kit for magnetic labeling of the target T cells and their isolation from other immune cells. Initially, we executed a cell-washing procedure to eliminate over 80% of the platelets. Following this wash, immune cells underwent incubation with CD4/CD8 nanomagnetic beads under controlled temperature and mixing conditions. Next, a post-incubation wash was carried out to remove excess nanobeads, aiming for a minimum 3-log reduction. The subsequent step involved magnetically isolating the bead-bound target cells. Upon completion of isolation, we resuspended the cells in the expansion medium for direct transfer to the Sefia™ expansion system.
Fig 1. The experimental process used in this study.
Target T cell activation, transduction, and expansion with the Sefia™ expansion system
The single-use kit was installed into the Sefia™ expansion system as directed by the Universal application software (Fig 2, Table 1). Isolated T cells were seeded at 3.14 × 105 live cells/cm2, which is equivalent to 100 × 106 total live T cells in the Sefia™ expansion system culture vessel (CV) 1 and activated using TransAct stimulation reagent (10 µL/1 × 106 cells) for 24 ± 4 h (Fig 1 and 3). Studies implemented newly developed serum-independent, xeno-free Akron ImmunoCell growth medium (Akron ICGM), supplemented with 350 IU/mL of Xuri™ Interleukin-2 (IL2) growth factor (Cytiva) and 1× of HyClone™ penicillin-streptomycin 100× solution (Cytiva). The total volume for the activation step at the end of day 0 was 150 mL in the Sefia™ expansion system.
Fig 2. Setup of the Sefia expansion system single-use kit.
Table 1. User-defined parameters applied in the Sefia™ expansion system in these studies via the Universal application*
| Parameters | Abridged setpoints | |
| Activation | Pre-seeding reagent • Reagent 1: Enabled • Transfer type: Transfer by mass (150 g) • Reagent incubation: Enabled (0.5 h ± 0.1 h) • Vessel draining: Enabled (100 g/min) |
|
| Initial cellular product, manual mixing before transfer: Enabled (20 s) • Seeding type: Seeding by target mass (transfer all, rinse bag [50 g]) |
||
| Post-seeding reagents • Reagent 2: Enabled • Transfer type: Transfer all (100 g/min) |
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| Incubation • Target mass before incubation: 150 g • Incubation time: 24 h ± 4 h |
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| Sampling • Mixing time: 240 s • Circulation mass flow rate: 100 g/min • Circulation time: 180 s |
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| Transduction | Culture vessel to use • Vessel 1: Enabled |
|
| Post-seeding reagents • Reagent 1: Enabled • Transfer type: Fill by syringe (100 g/min, 60 s) |
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| Incubation • Target mass before incubation: 150 g • Incubation time: 16 h ± 4 h |
||
| Sampling • Mixing time: 240 s • Circulation mass flow rate: 100 g/min • Circulation time: 180 s |
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| Expansion | Pre-expansion dilution • Dilution target: Fixed target mass: 450 g |
|
| Cell culture • Culture split: Enabled (mixing 180 s, 50% split) • Dilution after split: Enabled (600 g target mass) • Double-vessel perfusion—perfusion cycle time per vessel: 2 h |
||
| Feeding period 1 • Fed-batch: Enabled (240 h) |
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| Feeding period 2 • Perfusion: Enabled (240 min settling time) • Feeding period time: 240 h • Sampling frequency: by operator |
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| Total expansion time • Minimum time: 0 h • Maximum time: 480 h |
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| Sampling • Mixing time: 240 s • Circulation mass flow rate: 100 g/min • Circulation time: 180 s |
||
| Harvest | • Mass flow rate: 100 g/min • Vessel rinsing with media: Disabled • Harvest time: By operator |
|
| General alarms and setpoints | • CO2 - CO2: 5% - Enable CO2 deviation alarms: Enabled (default) • Temperature: - Enable system warm-up: Enabled (default) - Culture vessel temperature: 37°C - Enable temperature alarms: Enabled (default) • Mixing time: rinse mixing time: 30 s, pre-incubation mixing time: 15 s • Transferred mass accuracy alarm: default |
|
| Troubleshooting | • Enable next button on screen: Disabled • Enable automatic drawer locking: Enabled • Enable kit test: Enabled • Vessel tare values: Default |
|
* All transfers in the parameter group are set in grams, as the instrument measures mass. However, for simplification, we use mL throughout the rest of the main text when referring to volumes
Fig 3. Workflow steps used in the Sefia™ expansion system.
On day 1, T cells were transduced via CAR-lentiviral vector (LVV) and left to incubate for 16 ± 4 h.
On day 2, cell culture was fed-batch up to 450 mL in CV1. Cell counts were taken starting on day 5 onwards. Once CV1 reached ≥ 1.0 × 106 viable cells/mL or > 17.5 mM lactate concentration, the culture was split (50% in CV1, 50% in CV2) and topped up to 600 mL in both vessels. Continuous perfusion, at a 0.5 daily rate was initiated once CV1 and/or CV2 reached ≥ 106 viable cells/mL or > 17.5 mM lactate concentration. Cells were harvested from the Sefia™ expansion system, once ≥ 2.0 × 109 total viable cells were counted (CV1 + CV2). Cell recovery, purity, viability, and phenotype were quantitated on days 0, 5, and at harvest. Cell counts, viability, and aggregate size were measured using the NucleoCounter (NC)-200 (Chemometec) with n = 3 cell counts per sample. Metabolites including pH, glucose, lactate, osmolarity, partial carbon dioxide, and oxygen were measured using the Vi-Cell MetaFLEX bioanalyte analyzer (Beckman Coulter). Purity (CD3), phenotype (central memory: CD45RO+, CD62L+, effector memory: CD45RO+, CD62L-, effector: CD45RO-, CD62L-, naïve: CD45RO-, CD62L+, CD4, CD8), activation (CD25), and transduction efficiency (CD19) were measured using the CytoFLEX S flow cytometer (Beckman Coulter) and further analyzed using FlowJo software.
Post-harvest formulation
We conducted post-harvest formulation with the Sefia Select™ system. Following harvest, the collection bag volume was reduced to 75 mL through buffer exchange (DPBS + 1% human serum albumin) using the S-Wash application with the CT-200.1 single-use kit. Subsequently, the ReadySelect application with CT-350.1 single-use kit was used for the product's final formulation, including the cryopreparation (cryosolution with 5% DMSO) and the splitting in three cryobags. This crucial step ensures ± 2 mL volume precision in each cryobag, with less than 3 mL of air per bag and a cell number deviation of less than 15% between the bags.
A summary of the criteria evaluated in this study is found in Table 2.
Table 2. Criteria evaluated
| Criteria to evaluate |
Test execution description | |
| Cell viability and expansion | To determine viability and cell count (VCD, TVC, and fold expansion), samples were collected using a syringe welded onto the single-use kit sampling loop. After mixing, samples were collected and analyzed on days 4 and 5 to harvest daily using the NC-200 cell counter. | |
| Cell activation | Flow cytometry (5.0 × 105 total viable cells) was used to determine the expression of the activation marker, CD25. | |
| Memory phenotype | Memory phenotype was measured via flow cytometry at harvest: Central memory: CD45RO+, CD62L+, effector memory: CD45RO+, CD62L-, effector: CD45RO-, CD62L-, naïve: CD45RO-, CD62L+ | |
| Transduction efficiency | Transduction efficiency was measured at days 4 and 5 and harvest using flow cytometry via CD19 labeling. | |
| Post-harvest recovery | Once cells reached 2 × 109 total viable cells— confirmed via sampling—CV1 and CV2 were mixed and harvested. The harvest bag was weighed and sampled to quantitate total viable cells that were collected. Recovery was quantitated by comparing post-harvest cells to total viable cells present in CV1 and CV2 before harvest. | |
Results
Sefia Select™ magnetic isolation
The Sefia Select™ system using the MagnetSelect application and the CT-400.1 single-use kit, achieved an average T cell recovery of 54.8 ± 8.5% and 66.3 ± 11.5% and a purity of 92.0 ± 1.6% and 88.0 ± 4.7% for frozen and fresh apheresis, respectively (Table 3).
Table 3. MagnetSelect application results
| MagnetSelect application | Frozen apheresis healthy donor (n = 3) | Fresh apheresis healthy donor (n = 3) |
| Initial total white blood cells | 2.5 × 109 ± 2.2 × 108 | 5.7 × 109 ± 4.6 × 109 |
| Initial percentage of CD3 | 64% ± 4.0% | 45.4% ± 11.6% |
| Initial percentage of CD19 | 11.8% ± 5.3% | 17.6% ± 2.9% |
| Average processing time | 3.3 h | 2.5 h |
| Average T cell recovery | 54.8% ± 8.5% | 66.3% ± 11.5% |
| Average T cell purity | 92.0% ± 1.6% | 88.0% ± 4.7% |
| Average cell viability drop | 0.2% | 2.2% |
| Average platelet depletion | N/A | 98.8% ± 1.2% |
| Average RBC depletion | N/A | 93.5% ± 5.2% |
| Final volume accuracy | + 0.01 mL | + 1.8 mL |
Sefia™ expansion system
On day 5, average viability remained high (frozen: 95.2 ± 0.4%; fresh: 96.7 ± 1.4%) in the Sefia™ expansion system starting with frozen or fresh material (Fig 4 and 5).
Fig 4. Cell expansion in frozen donors (n = 3).
Fig 5. Cell expansion in fresh donors (n = 3).
Flow cytometry analysis indicated cell activation on day 5, with 84.2 ± 2.7% (frozen) and 99.3 ± 0.5% (fresh) CD25+ T cells in the Sefia™ expansion system (Table 4). Average transduction efficiency at day 5 was > 47.0% (47.6 ± 3.1% [frozen]; 56.1 ± 8.6% [fresh]), see Figure 6 and Table 4). Once the system reached ≥ 2 billion total live cells (Fig 4 and 5), they were harvested from the Sefia™ expansion system with > 94% average recovery (94.8 ± 2.4% [frozen], 96.0 ± 2.6% [fresh]). Cell counts demonstrated a total fold expansion of 25.5 ± 3.6 (frozen) and 34.9 ± 8.0 (fresh). Viability remained high at harvest for both frozen (96.3 ± 0.3%) and fresh conditions (98.1 ± 0.5%). T cell phenotype indicated a shift towards central memory (> 82.0%) in all conditions (Fig 7). Results indicated transduction efficiency slightly increased to 52.2 ± 5.1% (frozen) and 58.3 ± 10.7% (fresh) at harvest for T cells in the Sefia™ expansion system (Fig 6).
Table 4. Summary of results for Sefia™ cell therapy manufacturing platform
| Frozen (n = 3) | Fresh (n = 3) |
|
| MagnetSelect recovery | 54.8 ± 8.5% | 66.3 ± 11.5% |
| Activation: Viability | 95.2 ± 0.4% | 96.7 ± 1.4% |
| Activation: CD25+ | 84.2 ± 2.7% | 99.3 ± 0.5% |
| D5 transduction efficiency | 47.6 ± 3.1% | 56.1 ± 8.6% |
| Harvest: Recovery | 94.8 ± 2.4% | 96.0 ± 2.6% |
| Harvest: Total fold expansion | 25.5 ± 3.6 | 34.9 ± 8.0 |
| Harvest: Viability | 96.3 ± 0.3% | 98.1 ± 0.5% |
| Harvest: Transduction efficiency | 52.2 ± 5.1% | 58.3 ± 10.7% |
| S-Wash recovery | 92.5 ± 8.9% | 90.9 ± 14.7% |
| ReadySelect recovery | 99.8 ± 10.7% | 106.14% |
Fig 6. Transduction efficiency (%).
Fig 7. T cell memory phenotype at harvest.
S-Wash application
Post-harvest volume reduction was performed in Sefia Select™ system using the S-Wash application and the CT200.1 single-use kit. We achieved a recovery of 92.5 ± 8.9% and 90.9 ± 14.7% and a viability drop of 1.0 ± 0.1% and 1.3 ± 1.1% for frozen and fresh apheresis, respectively (Table 5).
Table 5. S-Wash application results
| S-Wash | Frozen apheresis healthy donor (n = 3) | Fresh apheresis healthy donor (n = 3) |
| Typical processing time | 1.3 h | 1.3 h |
| Washout efficiency | 99.9% | 99.9% |
| Average cell recovery for expanded cells | 92.5% ± 8.9% | 90.9% ± 14.7% |
| Average cell viability loss | 1.0% ± 0.1% | 1.3% ± 1.1% |
| Average standard deviation for final volume | ± 0.3 mL | ± 0.8 mL |
ReadySelect application
In the final formulation step, we used the ReadySelect application and the CT-350.1 single-use kit. We achieved a cell recovery of 99.8 ± 10.7% and 106.1 ± 23.4% for frozen and fresh apheresis, respectively. Post-formulation, the cells are in 5% DMSO solution. Even though we use a 50× dilution factor prior to counting the cells, we believe that the accuracy of cell counts is impacted by the residual presence of DMSO. Finally, we observe only a < 3.0% viability drop among the three cryobags in both frozen and fresh apheresis (Table 6).
Table 6. ReadySelect application results
| ReadySelect application | Frozen apheresis healthy donor (n = 3) | Fresh apheresis healthy donor (n = 3) |
| Average final volume accuracy | ± 3.1% | ± 4.9% |
| Average cell repartition accuracy | 2.8% ± 6.0% | 3.4% ± 7.9% |
| Average cell recovery | 99.8% ± 10.7% |
106.1% ± 23.4% |
| Average cell viability loss | 2.6% | 1.5% |
Conclusion
To further commercialize and extend the reach of CAR T therapies, there is an urgent need to facilitate the large-scale manufacturing of CAR T cells. The Sefia™ cell therapy manufacturing platform promotes the commercialization of innovative CAR T therapy production as a modular, functionally closed, end-to-end cell therapy manufacturing platform consisting of the Sefia Select™ system for T cell magnetic isolation and final formulation, and Sefia™ expansion system for activation, transduction, and expansion.
The Sefia™ expansion system implements a dedicated application software (Universal application) that accommodates many workflows and input products for different users. To provide flexibility, the Universal application offers many user-definable parameters. These parameters allow the procedure to be customized to fit customers’ needs for activation, transduction and expansion steps of the workflow. For instance, you can define transfer method for initial cellular product, activator, viral vector, and other reagents, as well as volumes and time of incubation, feeding strategy (batch, fed-batch, perfusion), perfusion rates, etc. The system is paired with a single-use kit (Sefia™ expansion kit) consisting of two culture vessels, weldable lines for attaching media and reagent bags, and a sampling loop consisting of six tails designed for sterile connection to facilitate viral syringe addition and sampling. Together, our results show:
- The Sefia Select™ system magnetically isolated T cells from fresh and frozen leukopaks with > 54.0% recovery and > 87.0% purity.
- The Sefia™ expansion system activated (> 84.0%), transduced (> 52.0% at harvest), and expanded (> 25-fold) T cells using Akron ImmunoCell growth medium and TransAct as an activator.
- Final product formulation was achieved with > 99.0% recovery using the Sefia Select™ unit.
Our work indicates that these components offer the flexible execution of workflows with user-defined parameters to perform T-cell isolation, activation, transduction, and expansion in frozen and fresh samples.
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