Scientific/User

Devices and Manufacturing Equipment
Used for Production of Immune Cell
Therapy and Cell Expansion:
Scientific/User Issues
Bruce Levine, Ph.D.
Department of Pathology and Laboratory Medicine
University of Pennsylvania
Activated T Cell Therapy for Infection or Malignancy
Cell Processing
1. Obtain cells from patient and
enrich or isolate particular cells
of interest
6. Quality Control
Reinfuse
cells
5. Remove
beads and
Bead
2. Stimulate cells
to grow
wash cells
3. Insert potentially
therapeutic genes
into cells
4. Large scale cell
expansion
Activated T Cell Processing Flow
Day 0
Leukocyte
Apheresis
Day ~10
Remove Beads
(Baxter Maxsep)
Harvester
(Baxter Fenwal)
Gambro Elutra depletion
of monocytes
Day 3 to 5
Seed WAVE
Bioreactor
QC Testing
QA Review
Stimulation with antiCD3/anti-CD28 Dynal
Beads, seed Baxter gas
permeable culture bag
Final Activated T Cell
Product
Patients enrolling in
clinical trials or their
donors undergo
leukapheresis at the
Hospital Blood Bank.
Particular
populations of
cells can be
enriched or
isolated in the
laboratory.
Gambro Elutra
Increasing rate of Buffer Flow
Gambro Elutra for Monocyte Isolation
Gambro Elutra
• Procedure modified for optimal lymphocyte
collection based on company experience with
settings for optimal monocyte collection
• Company provided:
– multi-day technical training
– Feedback on data collected during validation
runs
– Letter of cross-reference to DMF
Activated T Cell Processing Flow
Day 0
Leukocyte
Apheresis
Day ~10
Remove Beads
(Baxter Maxsep)
Harvester
(Baxter Fenwal)
Gambro Elutra depletion
of monocytes
Day 3 to 5
Seed WAVE
Bioreactor
QC Testing
QA Review
Stimulation with antiCD3/anti-CD28 Dynal
Beads, seed Baxter gas
permeable culture bag
Final Activated T Cell
Product
T Cell Activation with Antibody Coated Beads
JJ Immunol
Immunol 1997;
1997; 159:
159: 5921
5921
Science
Science 1997;
1997; 276:
276: 273
273
Immunol
Immunol.. Rev.
Rev. 1997;
1997; 160:
160: 43
43
Mol.
Mol. Ther
Ther.. 2004;
2004; 9;
9; 902
902
T o ta l C e lls
Polyclonal Replicative Potential of
Adult CD4 T Cells In Vitro
1E+20
1E+19
1E+18
1E+17
1E+16
1E+15
1E+14
1E+13
1E+12
1E+11
1E+10
1E+09
1E+08
1E+07
1E+06
IL-2 Added Day 49
No IL-2 Day 50
Exponential growth (days) 60-100
Mean log10 growth (fold) 9-12
Mean Pop Doubling
30-40
0
10
20
30
40
50
60
70
80
90
100
Day
Nc020795.xls
Activated T Cell Processing Flow
Day 0
Leukocyte
Apheresis
Day ~10
Remove Beads
(Baxter Maxsep)
Harvester
(Baxter Fenwal)
Gambro Elutra depletion
of monocytes
Day 3 to 5
Seed WAVE
Bioreactor
QC Testing
QA Review
Stimulation with
cell-based aAPC’s,
seed Baxter gas
permeable culture bag
Final Activated T Cell
Product
Off-the-Shelf Dendritic Cell Substitute:
reagent for the in vitro expansion of T lymphocytes
Polyclonal T Cell Stimulation with LV-aAPC
CD8 CD8
CD8
10,000
RADS
Polyclonal
stimulation
CD6
es
l
u
y: lec
r
a
ibr mo
l
LV stim ines
•co tok
•cy
CD8
28
4
CD64
LV-a
APC
(KT6
4)
CD8 Polyclonal
CD8
Stimulation
CD28
TCR
3
CD3
CD8
Next Generation: Cell-Based Artificial APC
LV-aAPC
T Cell
2 microns
Suhoski et al. Molecular Therapy 15:981-8, 2007
Baxter Lifecell Flasks- gas permeable bags
Activated T Cell Processing Flow
Day 0
Leukocyte
Apheresis
Day ~10
Remove Beads
(Baxter Maxsep)
Harvester
(Baxter Fenwal)
Gambro Elutra depletion
of monocytes
Day 3 to 5
Seed WAVE
Bioreactor
QC Testing
QA Review
Stimulation with antiCD3/anti-CD28 Dynal
Beads, seed Baxter gas
permeable culture bag
Final Activated T Cell
Product
WAVE Biotech Bioreactor
WAVE Feed and Harvest via Perfusion
Small (0.1 to 5 liter)
Wave Bioreactor
SYSTEM 2/10
Wave 2/10 perfusion w/pinch valves
WAVE Parameters
• Day of seeding after transduction and
vector wash
• Cell concentration
• Rock rate, rock angle
• Media perfusion rate
• Media formulation (ARV, foaming, IL-2,
supplements
• Nutrient usage, metabolite production
Small Scale Flask vs WAVE
Population Doubling Level
10
8
Small Scale
WAVE
PDL
6
4
2
0
-2
0
1
2
3
4
5
Days of Culture
6
7
8
9
Lifecell Static vs WAVE
Population Doubling Level
5.00
4.00
PDL
3.00
2.00
C1-Wave
C2-Lifecell
1.00
0.00
-1.00
0
1
2
3
4
5
6
-2.00
Day of Culture
7
8
9
10
WAVE Bioreactor
• Company provided:
– some technical training
– Protocols, manuals, references available on
website
– Letter of cross-reference to DMF
• References directly applicable to CD3/CD28
stimulated T cells
– Hami LS, Green C, Leshinsky N, Markham E, Miller K, Craig S: GMP
production and testing of Xcellerated T Cells for the treatment of patients
with CLL. Cytotherapy (2004); 6(6):554-562.
– Hami L, et.al. Comparison of a Static Process and a Bioreactor-based
Process for the GMP Manufacture of Autologous Xcellerated T Cells for
Clinical Trials. BioProcessing (2003); 2(6):1-10.
Activated T Cell Processing Flow
Day 0
Leukocyte
Apheresis
Day ~10
Remove Beads
(Baxter Maxsep)
Harvester
(Baxter Fenwal)
Gambro Elutra depletion
of monocytes
Day 3 to 5
Seed WAVE
Bioreactor
QC Testing
QA Review
Stimulation with antiCD3/anti-CD28 Dynal
Beads, seed Baxter gas
permeable culture bag
Final Activated T Cell
Product
Baxter MaxSep
The magnetic beads used to grow the T cells must be
removed prior to reinfusing the cells to the patient. The
cell culture is passed Baxter MaxSep magnets where the
beads are retained and the cells flow through.
Activated T Cell Processing Flow
Day 0
Leukocyte
Apheresis
Day ~10
Remove Beads
(Baxter Maxsep)
Harvester
(Baxter Fenwal)
Gambro Elutra depletion
of monocytes
Day 3 to 5
Seed WAVE
Bioreactor
QC Testing
QA Review
Stimulation with antiCD3/anti-CD28 Dynal
Beads, seed Baxter gas
permeable culture bag
Final Activated T Cell
Product
Fenwal (former subsidiary of Baxter) Harvester
Built on apheresis machine CS3000 frame. Concentrates and washes cells at up to 700mls per minute
continuous flow, we run at 300-400 mls per minute to increase cell yield. cells per minute depends on starting
concentration. If static bag @ 1E+06/ml = 3-7E+09 cells/minute, not including washes. If WAVE @ 10E+06/ml
= 3-7E+09 cells/minute, not including washes. closed system disposable set.
Cells concentrated in belt
chamber, end volume ~250mls
Fenwal Harvester Reservoir set,
Manifold set, and Harvester set
The volume of the cell
culture can be as much
as 10 liters (2.5 gallons)
or more depending on
cell dose. The cells are
washed out of culture
media, concentrated
and resuspended into
250mls (8 oz) infusible
solution.
Fenwal Harvester
• Company initially provided:
– some technical training
– Repair/PM service
– Manual
• Current status
– No longer manufactured (~10 years)
– No technical, scientific, regulatory support
– Kits no longer manufactured (mid-2008?)
– Absence of comparable devices
Autotransfusion devices as an alternative?
• Different requirements for
351 vs 361?
• Following validation,
submit data in IND
annual report or
ammendment?