Application Note

May 05, 2023
Keywords or phrases:CellGenix® GMP T Cell Medium, generation of CAR-Tcells, expansion and cultivation of T cells

Evaluating the Performance of CellGenix®
GMP TCM and CellGenix® Cytokines
in CAR-T Processes
Benedikt Steinle, Evelien Nijssen, Sarah Carqueville*, Ursula Schultz, Charlotte Tardivet
Sartorius CellGenix GmbH, Am Flughafen 16, 79108 Freiburg* Correspondence
Email: sarah.carqueville@sartorius.com

Abstract
Cell and gene therapies, including chimeric antigen receptor (CAR) T cell therapy, have become a promising avenue for treating
various diseases. However, successfully manufacturing these therapies requires manufacturers to overcome operational
challenges associated with new and emerging modalities.
This study explores the use of CellGenix® GMP T cell culture medium (TCM) and cytokines for generating functional CAR-Tcells with an early differentiated memory T cell phenotype. The study examines the expansion, functionality, and phenotypeof CAR-T cells cultured in CellGenix® GMP TCM and other commercially available T cell media. The findings shed light on thepotential of CellGenix® GMP TCM to optimize CAR-T cell manufacturing.	For further information, visit
www.sartorius.com

Introduction
Cell and gene therapies continue to be the fastest-growing
area of therapeutics, with several new therapies available topatients and hundreds more in development. One of themost prominent examples of cell therapy is the chimericantigen receptor (CAR) T cell therapy, which relies on T cells
that harbor a specific CAR to target and kill tumor cells more
effectively.
Developing such innovative therapies requires biopharmaceutical companies to solve many operational challenges,
particularly during manufacturing, scaling up, and scalingout. Sourcing GMP-grade raw materials that can be used inclinical manufacturing can be very challenging and should
be considered early in the development of these therapies.
This study focuses on generating functional CAR-T cellswith an early differentiated memory T cell phenotype using
CellGenix® GMP TCM and CellGenix® cytokines. CellGenix®
GMP TCM is a ready-to-use xeno-free medium that does not
require the addition of glutamine or human serum. It can beused for various T cell applications when supplemented withCellGenix® GMP IL-7, CellGenix® GMP IL-15, and CellGenix®
GMP IL-2, or for the generation of Tregs with CellGenix®
GMP IL-2 and, CellGenix® GMP TGF-β1.
In this study, we investigated the functionality of CellGenix®
GMP TCM for the generation of CD19 CAR-T cells. Thisapplication note provides an overview of a CAR production
process, including CAR-T cell generation and functional
characterization, such as CD19 CAR expression, killing
capacity, and T cell phenotype. We used a 96-well scaledown model to compare CellGenix® GMP TCM with othercommercially available T cell media.

Materials and Methods
CAR T Cell Generation and Expansion
CD3+ T cells were purified from healthy donor peripheral
blood mononuclear cells (PBMCs) isolated from leukocyte
reduction chambers using a negative selection kit, aliquoted,and cryopreserved. After thawing, cells were activated with
Dynabead Human T-Activator CD3 | CD28 (Thermo Fisher)
at a bead-to-cell ratio of 1:1 in CellGenix® GMP TCM orcompetitor media in the presence of CellGenix® IL-7 andCellGenix® IL-15 (10 ng/mL). Control (CTL) media was preparedas published previously (50% advanced RPMI 1640 (Gibco),
45% Click’s (Irvine Scientific), 2 mmol/L GlutaMAXTM (Gibco),and 5% Human AB Serum).1The cells were seeded in triplicates in 100 µL medium into
96-well plates. Twenty-four hours after seeding, cells weretransduced by adding 100 µl medium containing lenti-CD19
CAR (scFv-41BB-CD3 ζ) viral particles (Creative Biolabs) toachieve a final MOI of 3. Cells were passaged on days 3, 6,
and 8. T cell count and viability were determined by flow
cytometry using the Attune NXT (Thermo Fisher).

In Vitro Cytotoxicity Assay
NALM-6 cells were stained with CellTrace™ Violet (Invitrogen)and co-cultivated with transduced T cells at different effectorto-target (E : T) ratios for 18 hours in CellGenix® GMP TCM.
Wells containing mock transfected T cells or only labeled
NALM-6 cells served as controls. CAR-mediated cytotoxicity
was determined by analyzing the residual alive CellTrace™
Violet target cells at each E : T ratio. Absolute cell counts weredetermined on an Attune NTX (Thermo Fisher).

Immunophenotyping of CAR-T Cells
Staining with CD19 CAR detection reagent (Miltenyi) andflow cytometric measurements were used to determine thepercentage of CD19 CAR-expressing T cells. To assess theT cell memory phenotype, the expression of CD4, CD8,
CD45RA, and CD27 were analyzed by flow cytometry.
The memory T cell populations were defined as previously
published.22Results
High T Cell Expansion in CellGenix®
GMP TCM

Generation of High CD19 CAR-T Cell
Yield in CellGenix® GMP TCM

The generation of autologous CAR-T cells is a multi-stepprocess performed ex vivo. In the case of autologous CAR-T
cell therapies, patient cells are collected by leukapheresis,
after which T cells are isolated. Those T cells are activatedand then transduced predominantly with lentiviral vectors
to introduce a gene for a CAR. To reach high numbers of
CAR-T cells, they are further expanded before they areinjected back into the patient. These cells should then beable to recognize and eliminate specific cancer cells, e.g.,
CD19-positive cancer cells.

Besides the total yield of T cells at the end of cultivation, it iscrucial that the majority of cells carry the CAR transgene.
The expression of the inserted CD19 CAR gene was analyzed
by flow cytometry. In both CellGenix® GMP TCM and CTL
medium with 5% human serum, 88% of the T cells at the endof the cultivation period expressed the CD19 CAR gene on
their surface. In contrast, competitor A reached only 64%
CD19 CAR- T cells, and competitor B 68% (Figure 2A).
By combining the cell number, viability, and proportion ofCD19 CAR-expressing cells, the final yield of CD19 CAR-Tcells per mL of harvested cells can be calculated (Figure 2B).
Based on this calculation, a CD19 CAR-T cell yield of
1.61 × 108 per mL could be reached through cultivation inCellGenix® GMP TCM.

In this study, we mimicked the CAR-T process to show thesuitability of CellGenix® GMP TCM and the CellGenix®
GMP cytokines IL-7 and IL-15 for this application. The firstparameter we looked at was the expansion of CAR-T cells in
the CellGenix® GMP TCM compared to other commerciallyavailable T cell media. Within 10 days of ex vivo cultivation,the T cells expanded 390-fold in CellGenix® GMP TCM(Figure 1A). The T cell expansion in CellGenix® GMP TCM isslightly lower compared to CTL media with 5% serum.
However, it is still much higher than cultivation in competitor
media A and B. The viability of all expanded CAR-T cells wasabove 90%, with the exception of competitor B (Figure 1B).
Additionally, cells expanded in CellGenix® GMP TCM showed
less donor variance than in competitor media.

Figure 1: CellGenix ® GMP TCM Supports Greater T Cell Expansion Than Other Serum-Free Competitor Media While
Maintaining a High Level of Viability.
AB

10080400Viability [%]Fold Expansion

500300200Donor
261262263987

1000CellGenix®
GMP TCM

Competitor A Competitor B CTL + 5% HS

6040Donor
261262263987

200CellGenix®
GMP TCM

Competitor A Competitor B CTL + 5% HS

Note. CD3+ T cells purified from healthy human donors (n=4) were cultured in triplicates for 10 days with CellGenix® GMP TCM or competitor media.
T cell expansion (A) and viability (B) were determined by flow cytometry in technical triplicates.

3B80

CD19-CAR+ T Cells [%]A 100

CD19-CAR+ T Cells [mL]

Figure 2: Ex Vivo Expansion of Lentivirus-Transduced T Cells Results in a High Proportion of CD19 CAR-T Cells
in CellGenix ® GMP TCM

6040Donor
261
262
263
987

200CellGenix®
GMP TCM

Competitor A Competitor B CTL + 5% HS

2.0e+08

1.5e+08

1.0e+08
Donor
261
262
263
987

5.0e+070CellGenix®
GMP TCM

Competitor A Competitor B CTL + 5% HS

Note. Cultivated T cells were transduced on day 1 with lenti-CD19 CAR viral particles and cultivated for 10 days. Expression of CD19 CAR was analyzed
on day 10 (A). Based on the number of viable cells and CD19 CAR expression, the yield of CD19 CAR-T cell per mL was calculated (B).

Functionality of Anti-CD19 CAR-T Cells
Following cognate antigen recognition, CAR-T cells shouldbe able to kill the antigen-bearing tumor cell. To mimic thisand to verify the functionality of the generated CD19 CAR-Tcells, we used the NALM-6 cell line, which expresses theCD19 antigen.
The CellGenix® GMP TCM- expanded CD19 CAR-T cells
eliminated 68% of the NALM-6 cells when co-cultivatedin a 1:2 effector to-target ratio (Figure 3). Compared toCellGenix® GMP TCM, the CD19 CAR-T cells generated incompetitor A medium showed a lower killing capacity at all
ratios, and a higher donor-dependent variance. Thus, expansion
with CellGenix® GMP TCM may contribute to the reductionof failed production runs.
The CD19 CAR-T cells cultivated in the human serum
containing CTL medium showed comparable specific killingas the CellGenix® GMP TCM CD19 CAR-T cells. Nevertheless,using human serum in clinical production carries uncontrollable risks such as potential viral contaminations, highlot-to-lot variations, and supply shortages.

4High Proportion of Early DifferentiatedCD8+ T Cells in CellGenix® GMP TCM
Another important quality attribute of the final CAR-T cell
product is the phenotype of the T cells. After T cell activation,different memory T cell populations associated with different
therapeutic potentials are formed. Stem cell-like memory
T cells (Tscm , CD45RA+CD27+) and central memory T cells(Tcm , CD45RA-CD27+) are associated with longevity orlymphoid homing, whereas effector memory T cells(Tem , CD45RA- CD27-) and terminal effector memory
CD45RA+ T cells (Temra , CD45RA+CD27-) are associatedwith effector functions and peripheral homing.2
In each cultivation medium tested, the CD8+ T cells had highproportions of T cells with Tscm and Tcm phenotype (Figure 4).
The proportion of Tscm and Tcm in the CD4+ T cell population
is significantly lower. Culture conditions such as pH anddissolved oxygen are known to affect the differentiation stateof T cells. These parameters cannot be controlled in a 96-wellformat. Culturing T cells in a bioreactor offers the possibility
to optimize the culture conditions pH and dissolved oxygen
which can further enhance the proportion of a less differentiatedT cell phenotype.3

Figure 3: Generated CAR-T Cells Specifically Target and Kill CD19 + NALM-6 Cells
CellGenix® GMP TCM

Competitor A

CTL + 5% HS

100% Nalm-6 Cell Lysis

80

60

40Donor
261
262
263
987

200
1:2

1:4

1:8

1:2

1:4

1:8

1:2

1:4

1:8

Ration (Effector : Target)

Note. On day 10, the CAR-T cells were co-cultivated with CTV-labeled NALM-6 cells in 1:2, 1:4, and 1:8 effector to-target ratios. After 18 hours, CTV+ Nalm-6cells were counted. Specific lysis of NALM-6 cells was calculated as the percentage of NALM-6 cells remaining in the co-culture compared to the control culture.

Figure 4: Early Differentiated Tscm and Tcm Phenotype of CAR-T Cells
CellGenix® GMP TCM

Competitor A

Competitor B

CTL + 5% HS

Proportion of Memory T Cells [%]100908070605040MemorySubtype:30Temra

20Tem

10Tcm
Tscm

0CD4CD8CD4CD8CD4CD8CD4CD8Note. The phenotype of the CAR-T cells analyzed by flow cytometry on day 10. The cells were stained with CD4, CD8, CD45RA and CD27 antibodies
and the memory populations were defined as published. ²5Conclusion

References

Successful cell and gene therapy manufacturing depends
on high-quality raw materials (ancillary materials, according
to the United States Pharmacopeia). Results from this study
indicate that CellGenix® GMP TCM supplemented withCellGenix® GMP IL-7 and CellGenix® GMP IL-15 provides
a significant benefit over other commercially available
serum-free T cell media, for the generation of CAR-T cells.

1. Gerdemann, Ulrike et al. “Rapidly generated multivirusspecific cytotoxic T lymphocytes for the prophylaxis andtreatment of viral infections.” Molecular therapy : thejournal of the American Society of Gene Therapy
vol. 20,8 (2012): 1622-32.
2. Mahnke, Y.D., Brodie, T.M., Sallusto, F., Roederer, M. andLugli, E. (2013), The who's who of T-cell differentiation:
Human memory T-cell subsets. Eur. J. Immunol.,
43: 2797-2809.
3. Lamas, R., Ulrey R., Ahuja S., and Sargent A. (2022),
Changes to culture pH and dissolved oxygen enhancechimeric receptor T-cell generation and differentiation.
Biotechnology Progress, 38(5).

The CAR-T cells show robust expansion and maintain theirfunctionality and a favorable phenotype. Previously it has
been shown that CellGenix® GMP TCM, in combination withCellGenix® GMP cytokines IL-7 and IL-15, is also suitable forT cell cultivation in static bioreactor systems as well as instirred systems. This suggests that CellGenix® GMP TCM
with CellGenix® GMP IL-7 and IL-15 gives rise to a morefavorable CAR T-cell therapy product and can be used indifferent manufacturing modalities.

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