We demonstrate the versatility of our Sefia™ expansion system, which facilitates manufacture of CAR T cells by providing:
- Highly flexible application software (Universal app)
- A single-use disposable kit available in two versions—silicone membrane and fluorinated ethylene propylene (FEP) membrane.
Starting with 1 × 108 isolated T cells (frozen apheresis from healthy donors), the system can generate a CAR T cell patient dose using numerous commercially available serum-free media, in this study denoted media A to E.
Introduction
Autologous chimeric antigen receptor (CAR) T cell therapies have been adopted since 2011 to treat patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL) and help them fight cancer. Recently, multiple new products have received FDA approval, allowing their use in many clinical trials (1–4).
Over the past few years, advancements in engineering strategies, as well as the development of new media and cytokines that allow improved and selective expansion of the T memory stem cell subpopulations have gained increasing interest. Thus, it has become critical that newly developed T cell manufacturing equipment is indeed media agnostic, allowing for T cell activation, transduction, and expansion, regardless of the medium used.
The cell therapy manufacturing market is increasingly shifting towards the use of serum-free media (SFM), instead of traditional serum-containing media. This trend is driven by several factors, including the desire for greater consistency, reproducibility, and control in production processes. Serum-free media offer a defined composition that reduces the variability associated with animal-derived components, which is critical for regulatory compliance and product quality (5).
Materials and methods
We installed the Sefia™ expansion kits into the Sefia™ expansion system as directed by the Universal application. Isolated T-cells were seeded at 3.14 × 105 live cells/cm2, equivalent to 1 × 108 total live T cells in culture vessel 1 (CV1) and activated using T cell TransAct stimulation agent (10 microliter per one million cells) for 24 ± 4 h. We evaluated two different configurations of the disposable single-use kit, according to the cell growth surface material, silicone, and FEP membrane.
We supplemented all media tested with 350 IU/mL IL2 from Cytiva and 1× of HyClone™ penicillin-streptomycin 100×, except medium D, where we used 12.5 ng/mL interleukin (IL)7 and IL15. The total volume for the activation incubation step at the end of day 0 was 150 mL.
On day 1, T-cells were transduced via CAR-lentiviral vector (LVV) and left to incubate for 16 ± 4 h. On day 2, fed-batch cell culture was used up to 450 mL. Cell counts were taken starting on day 5. At this point, workflow conditions and criteria were optimized according to the media and membrane used.
With media A, B, C and E, once CV1 reached ≥ 1 × 106 viable cells/mL or > 17.5 mM lactate concentration, we split the culture (50% in CV1, 50% in CV2), and the media were added up to 600 mL in both vessels. Continuous perfusion was initiated at a 0.5× daily rate for FEP membrane or 1× for the silicone membrane once CV1 and/or CV2 reached ≥ 1 × 106 viable cells/mL or > 17.5 mM lactate concentration.
For medium D, cells were activated using T cell TransAct stimulation agent (20 microliter per one million cells) for 24 ± 4 h, and the LVV was incubated for 48 ± 4 h. Post transduction, a wash step was performed with medium D using the high-speed perfusion feature of the Universal application software (25 mL/min for 60 min) to remove the excess of activator. Post-wash, medium D was added up to a final volume of 450 mL.
We counted cells starting on day 5 and the medium was added up to 600 mL on day 9. Cells were counted on days 5, 9, and 12. Metrics including viable cell density (VCD), total viable cells (TVC), and fold expansion were quantitated. We performed single-vessel expansion (no culture split) with no perfusion strategy.
In all conditions, once the cell culture reached ≥ 2 × 109 total viable cells, we initiated the harvest procedure.
At harvest, T-cell memory phenotype (CD3, CD45RO, CD62L expression), T-cell activation (CD25 expression), and transduction efficiency (CD19+ CAR expression) were assessed by flow cytometry, using the CytoFLEX S flow cytometer (Beckman Coulter), Table 1. Data analysis was carried out with FlowJo software.
Cell counts, viability, and aggregate size were measured using the NucleoCounter (NC)-200 (Chemometec) (Table 1). In contrast, we measured metabolites including pH, glucose, lactate, osmolarity, partial CO2, and partial O2 using the Vi-Cell MetaFLEX bioanalyte analyzer (Beckman Coulter).
Table 1. 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 and measured using the NC-200. |
|
Activation marker expression |
Cell activation was measured at day 4/day 5 using flow cytometry via CD25 labeling. |
|
Transduction efficiency at harvest |
Transduction efficiency was measured at harvest using flow cytometry via CD19 labeling. |
Results
Medium A
Using medium A, we expanded over 2 × 109 total viable cells (TVC) in 8 to 9 d on the silicone membrane and 8 to 10 d on the FEP membrane (Fig 1A). Post-activation, the CD25 expression was 88.3% ± 4.5% (silicone) and 89.2% ± 5.0% (FEP), Table 2.
At harvest, transduction efficiency was 52.8% ± 4.3% (silicone) and 43.4% ± 9.8% (FEP), with viability of 96.3% ± 0.2% (silicone) and 96.5% ± 1.1% (FEP, Table 2). The success rate for this medium was 100% on both silicone and FEP membranes (n = 4 healthy donors).
Table 2. Medium A: cell activation, viability at harvest, transduction efficiency, and TFE
|
FEP |
Silicone |
|
|
Activation (CD25+ cells [%]) |
89.2 ± 5.0 |
88.3 ± 4.5 |
|
Viability at harvest (%) |
96.5 ± 1.1 |
96.3 ± 0.2 |
|
Transduction efficiency (CD19+ cells [%]) |
43.4 ± 9.8 |
52.8 ± 4.3 |
|
TFE |
25.0 ± 0.3 |
26.4 ± 3.4 |
Medium B
Using medium B, we expanded over 2 × 109 TVC in 6 to 7 d on the silicone membrane and 7 to 8 d on the FEP membrane (Fig 2). Post-activation, we found that CD25 expression was 90.4% ± 7.6% (silicone), and 93.3% ± 2.2% (FEP), Table 3.
At harvest, transduction efficiency was 28.6% ± 8.1% (silicone) and 24.8% ± 8.8% (FEP), with a viability of 97.2% ± 1.2% (silicone) and 96.1% ± 2.1% (FEP), Table 3. The success rate for this medium was 100% on both silicone and FEP membranes (n = 3 healthy donors).
Table 3. Medium B: T cell activation, viability at harvest, transduction efficiency, and TFE
|
FEP |
Silicone |
|
|
Activation (CD25+ cells [%]) |
93.3 ± 2.2 |
90.4 ± 7.6 |
|
Viability at harvest (%) |
96.1 ± 2.1 |
97.2 ± 1.2 |
|
Transduction efficiency (CD19+ cells [%]) |
24.8 ± 8.8 |
28.6 ± 8.1 |
|
TFE |
25.1 ± 0.8 |
23.4 ± 1.4 |
Medium C
Using medium C, we expanded over 2 × 109 TVC in 7 to 8 d on the silicone membrane and 8 to 10 d on the FEP membrane (Fig 3). Post activation, CD25 expression was 96.6% ± 2.4% (silicone), and 96.6% ± 2.5% (FEP), Table 4.
At harvest, transduction efficiency was 32.1% ± 4.2% (silicone) and 28.0% ± 14.8% (FEP), with a viability of 87.4% ± 3.5% (silicone) and 82.9% ± 3.6% (FEP), Table 4. The success rate for this medium was 44% on the silicone membrane (n = 4 healthy donors) and 100% on the FEP membrane (n = 4 healthy donors).
Table 4. Medium C: T cell activation, viability at harvest, transduction efficiency, and TFE
|
FEP |
Silicone |
|
|
Activation (CD25+ cells [%]) |
96.6 ± 2.5 |
96.6 ± 2.4 |
|
Viability at harvest (%) |
82.9 ± 3.6 |
87.4 ± 3.5 |
|
Transduction efficiency (CD19+ cells [%]) |
28.0 ± 14.8 |
32.1 ± 4.2 |
|
TFE |
22.0 ± 1.3 |
30.7 ± 6.5 |
Medium D
Using medium D, we expanded over 2 × 109 TVC in 12 d on the FEP membrane (Fig 4). Post-activation, CD25 expression was 97.6% ± 2.3%, Table 5.
At harvest, transduction efficiency and viability were 42.0% ± 5.6% and 90.5% ± 4.3%, respectively, Table 5. The success rate for this medium was 100% on FEP membrane (n = 3 healthy donors).
Table 5. Medium D: T cell activation, viability at harvest, transduction efficiency, and TFE
|
FEP |
Silicone |
|
|
Activation (CD25+ cells [%]) |
97.6 ± 2.3 |
n/a |
|
Viability at harvest (%) |
90.5 ± 4.3 |
n/a |
|
Transduction efficiency (CD19+ cells [%]) |
42.0 ± 5.6 |
n/a |
|
TFE |
36.2 ± 11.4 |
n/a |
Medium E
Using medium E, we expanded over 2 × 109 TVC in 6 to 8 d on the FEP membrane (Fig 5). Post-activation, CD25 expression was 95.3% ± 1.9%, Table 6.
At harvest, transduction efficiency and viability were 19.7% ± 3.0% and 94.8% ± 1.2%, respectively, Table 6. The success rate for this medium was 100% on the FEP membrane (n = 3 healthy donors).
Table 6. Medium E: T cell activation, viability at harvest, transduction efficiency, and TFE
|
FEP |
Silicone |
|
|
Activation (CD25+ cells [%]) |
95.3 ± 1.9 |
n/a |
|
Viability at harvest (%) |
94.8 ± 1.2 |
n/a |
|
Transduction efficiency (CD19+ cells [%]) |
19.7 ± 3.0 |
n/a |
|
TFE |
27.2 ± 3.2 |
n/a |
In all media conditions tested, whether on the silicone or on the FEP membrane, the T cells at harvest had a central memory phenotype as shown in Figures 1 to 5.
Fig 1. T cell viability, expansion, and memory phenotype at harvest for medium A. Each run represents a separate donor. The same donor may not have been used for different media and/or different membranes.
Fig 2. T cell viability, expansion, and memory phenotype at harvest for medium B. Each run represents a separate donor. The same donor may not have been used for different media and/or different membranes.
Fig 3. T cell viability, expansion, and memory phenotype at harvest for medium C. Each run represents a separate donor. The same donor may not have been used for different media and/or different membranes.
Fig 4. T cell viability, expansion, and memory phenotype at harvest for medium D. Each run represents a separate donor. The same donor may not have been used for different media and/or different membranes.
Fig 5. T cell viability, expansion, and memory phenotype at harvest for medium E. Each run represents a separate donor. The same donor may not have been used for different media and/or different membranes.
Conclusions
The Sefia™ expansion system offers an application software (Universal app) with high flexibility to execute multiple workflows, by enabling the selection of desired user parameters, according to diverse workflows and culture media selections.
Here, multiple commercially available serum-free T cell media have been tested. All media types were supplemented with IL2 or IL7/15 (in case of medium D), and T cells were activated using TransAct stimulation agent. Cell activation, transduction efficiency, viability, and proliferation data demonstrate the high versatility of the Sefia™ expansion system.
The data reported in this application note suggest that, when starting with a low cell seeding number (1 × 108 TVC), the selection of the Sefia™ expansion kit (silicone vs FEP membrane) may have a significant impact on cell activation, viability, and proliferation, according to the media used. In general, the FEP membrane seems to promote a faster and more robust T cell activation (i.e., medium C data), while the silicone membrane may induce better proliferation (i.e., medium A and B data), allowing you to achieve higher cell numbers at the end of the process, due to better gas permeability of the silicone membrane compared to FEP.
In these studies, T cells from various donors were used for all tests conducted on silicone or FEP, as well as with different media. The variation in performance observed across different media may be attributed to donor variability.
We observed that T cell culture in media D was sensitive to daily sampling (starting from day 5). A significant decrease in cell viability was observed over time, possibly due to frequent mixing. For this reason, we decided to minimize the number of times we sampled when using these media.
In summary, based on above results, we are showing that Sefia™ expansion system can effectively support the activation, transduction, and expansion of autologous CAR T cells, using various serum-free media. Its high flexibility allows you to easily modify and select workflow parameters, as well as choose your preferred single-use kit (silicone or FEP).
References
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