surmounting the developmental challenges of miltenyi's...
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8th Technical Meeting of the Advanced Therapy Medicinal Product (ATMP) Manufacturing Community Surmounting the developmental challenges of Miltenyi's Prodigy Cell Separation System - an integrated, closed-system cell processing device Anne Richter, Research & Development 6th February 2014
Cellular Therapy @ Miltenyi Biotec
• Hematology / Graft engineering (blood & marrow transplantation)
• Adoptive T cell immunotherapy
• Regulatory T cell therapy
• NK cell therapy
• Dendritic cell therapy
• Regenerative medicine (heart, liver, bone, nervous system, …)
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 2
Cell Therapy Manufacture
Workflow
Donor / patient
Blood Bone marrow
Leukapheresis
Cell separation
Cell expansion or differentiation
Quality Control Flow Cytometry
Cryopreservation
Administration to patient
Sample Dissociation Tissue
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 3
Cell Therapy Manufacture
Components
Donor / patient
Blood Bone marrow
Leukapheresis
Cell separation
Cell expansion or differentiation
Quality Control Flow Cytometry
Cryopreservation
CliniMACS
Reagents MACS GMP Media
Cell culture bags
MACS GMP Antigens
MACS GMP Cytokines
Cell differentiation bags
Administration to patient
CryoMACS
Freezing Bags CliniMACS Instruments
Sample Dissociation Tissue
GentleMACS
MACSQuant
MACS GMP Antibodies
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 4
Challenges
• Cellular products undergo various degrees of manufacturing complexity
• Manufacturing complexities can make the ‘transfer of protocols’ difficult
• Manual steps increase variability and increase risk of mistakes
• Cell therapies are developed in many disease areas
• Various regulatory requirements in different european countries
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 5
Designed to …
=
Not approved by FDA! Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 6
+ + + =
+ +
… replace or integrate
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 7
Cellular Therapy @ Miltenyi Biotec
• Hematology / Graft engineering
(blood & marrow transplantation)
• Adoptive T cell immunotherapy - restore immunity - control established infection after HSC/SOT
• Regulatory T cell therapy
• NK cell therapy
• Dendritic cell therapy
• Regenerative medicine (heart, liver, bone, nervous system, …)
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 8
isolation
Antigen-specific T cells for immunotherapy Enrichment of IFN-gamma secreting cells
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 9
CliniMACS Cytokine Capture System (CCS)
Pro − Fast (12 hours) − Not HLA-restricted − CD4+ and CD8+ T cells − Multi-antigen-specific T cells − CE-marked
Con − In vitro stimulation necessary
Clinical data of adoptive transfer AdV-specific T cells Feuchtinger et al. (2006) Br. J. Haematol. Qasim et al. (2013) Br. J. Haematol. CMV-specific T cells Peggs et al. (2011) Clin. Infect. Dis. Brestrich et al. (2009) Am. J. Transplant. EBV-specific T cells Moosmann et al. (2010) Blood Icheva et al. (2012) J. Clin. Oncol.
CliniMACSPlus Cytokine Capture System (IFN-gamma) System
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 10
Challenges for the user
• Laborious protocol with several washing and labeling steps • Heating/Cooling steps: 37°C -> 4°C -> 37°C -> 4°C • 4 hours of antigen stimulation is inconvenient for the user (whole process is around 12 hours) • Customer-specific modifications / lab-to-lab variations • Final formulation of target cells required (therapeutic grade infusion solution) • Quality control of starting material and final product
Leuka- pheresis
Donor
virus- specific T cells
Administration to patient
Sample preparation
Cell selection
Cell stimulation
Cell labeling
CliniMACS Prodigy – main components
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 11
Magnet Unit
Liquid Sensor
Pinch valves
Peristaltic pump
Bag hangers
CentriCult Unit
Bag Compartment
Touch screen
Gas Mix Unit Tube Sealer Bar code reader
CliniMACS Prodigy
Integrated cell processing from cell source to final cell product
• Sample preparation
• Cell washing & density gradient separation
• Cell culture (activation, maturation)
• Cell labeling
• MACS cell separation
• Final product formulation
Automated manufacturing
Closed system
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 12
CliniMACS Prodigy
General software features • Fixed processes
• User programs: flexible programming suite (FPS)
• Tubing set installation guidance
• Tubing set Integrity testing
• Failure handling
• Alarm functionality
• Storage of process protocols (download as PDFs)
• Remote service access for MB TechSupport&Service
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 13
Process for enrichment of IFN-γ secreting T cells using the CliniMACS Prodigy system
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 14
Tubing set, buffer, media Preparation
Manual steps
Automated steps
0.5 h
Tubing set installation
Process for enrichment of IFN-γ secreting T cells using the CliniMACS Prodigy system
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 15
Tubing set, buffer, media Preparation
Manual steps
Automated steps
0.5 h
Tubing set installation
0.5 h Priming of TS
Process for enrichment of IFN-γ secreting T cells using the CliniMACS Prodigy system
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 16
0.5 h
Tubing set, buffer, media Preparation
Priming of TS
Preparation
Delay
Manual steps
Automated steps
0.5 h
0.5 h
Cell sample, reagents, Programming
(time of process end)
Bar code reader GUI
Process for enrichment of IFN-γ secreting T cells using the CliniMACS Prodigy system
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 17
0.5 h
1.5 h
Tubing set, buffer, media Preparation
Priming of TS
Preparation
Sample preparation
Delay
Manual steps
Automated steps
0.5 h
0.5 h
CentriCult Unit Centrifuge unit
Gradient separation Volume reduction Exchange of fluids up to 400 g acceleration Boundary layer detection Temperature control
Cell Washing
Different washing programs Continuous and discontinuous centrifugation
Cell sample, reagents, Programming
(time of process end)
Process for enrichment of IFN-γ secreting T cells using the CliniMACS Prodigy system
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 18
4 h
0.5 h
1.5 h
Tubing set, buffer, media
Cell sample, reagents, Programming
(time of process end)
Preparation
Priming of TS
Preparation
Sample preparation
Antigen incubation
Delay
Manual steps
Automated steps
0.5 h
0.5 h
CentriCult Unit Cell culturing
Heat Exchange Cartridge as important part of the tubing set
Cooling down or warming up liquids in a short time period (4-38°C)
Customer defines time of process end
Process for enrichment of IFN-γ secreting T cells using the CliniMACS Prodigy system
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 19
4 h
2 h 10 min
0.5 h
1.5 h
50 min
1 h 30 min
Tubing set, buffer, media
5 min
Preparation
Priming of TS
Preparation
Sample preparation
Antigen incubation
Delay
Wash/Labeling Catch Matrix
Secretion Phase/Wash
Labeling Enrichment
Reagent/Wash Take QC sample
Manual steps
Automated steps
0.5 h
0.5 h
Centrifuge unit Gradient separation Volume reduction Exchange of fluids up to 400 g acceleration Boundary layer detection Temperature control
Cell Washing
Different washing programs Continuous and discontinuous centrifugation
Cell sample, reagents, Programming
(time of process end)
CentriCult Unit
Cooling down or warming up liquids in a short time period (4-38°C)
Process for enrichment of IFN-γ secreting T cells using the CliniMACS Prodigy system
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 20
4 h
2 h 10 min
0.5 h
1.5 h
50 min
1 h 30 min
1 h 10 min
Tubing set, buffer, media
5 min
Preparation
Priming of TS
Preparation
Sample preparation
Antigen incubation
Delay
Wash/Labeling Catch Matrix
Secretion Phase/Wash
Labeling Enrichment
Reagent/Wash
Magnetic Separation
Take QC sample
Manual steps
Automated steps
0.5 h
0.5 h
Cell sample, reagents, Programming
(time of process end)
• Suitable for various CliniMACS column sizes
• Cell enrichment and depletion
• Multi-step separations possible
Process for enrichment of IFN-γ secreting T cells using the CliniMACS Prodigy system
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 21
4 h
2 h 10 min
0.5 h
1.5 h
50 min
1 h 30 min
1 h 10 min
Tubing set, buffer, media
Enriched IFN-γ+ cell fraction in <10 mL injectable solution
5 min
Preparation
Priming of TS
Preparation
Sample preparation
Antigen incubation
Delay
Wash/Labeling Catch Matrix
Secretion Phase/Wash
Labeling Enrichment
Reagent/Wash
Magnetic Separation
Final Handling
Take QC sample
Manual steps
Automated steps
Seal off target cells, removal of TS
0.5 h
0.5 h
Cell sample, reagents, Programming
(time of process end) Tube Sealer
secreted IFN-γ
Semi-automated process CliniMACS® Plus
CD
4
CD
8
CD
4
CD
8
automated process CliniMACS® Prodigy
Original fraction
Negative fraction
Positive fraction
1.18%
0.33%
97.8%
1.68%
0.27%
99.7%
0.08%
0.39%
88.0%
0.16%
0.04%
97.3%
LP 5, 15/11/12
Enrichment of IFN-γ secreting T cells from leukapheresis stimulated with PepTivator CMV pp65 and EBV EBNA-1
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 22
Purity of enriched IFN-γ+ T cells
CliniMACS CD4+ IFN-γ+
Prodigy CD4+ IFN-γ+
CliniMACS CD8+ IFN-γ+
Prodigy CD8+ IFN-γ+
IFN
-γ+ c
ells
am
ong
T ce
lls (
%)
pp65/ EBNA-1 pp65/ EBNA-1 Hexon PepTivator
0
20
40
60
80
100
LP5 LP7 LP3
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 23
Recovery of IFN-γ+ T cells
CliniMACS CD4+ IFN-γ+
Prodigy CD4+ IFN-γ+ CliniMACS CD8+ IFN-γ+
Prodigy CD8+ IFN-γ+
corr
. Rec
over
y (%
)
cell
coun
t 0
20
40
60
80
100
LP5 LP7 LP310000
100000
1000000
LP5 LP7 LP3
pp65/ EBNA-1 pp65/ EBNA-1 Hexon pp65/ EBNA-1 pp65/ EBNA-1 Hexon
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 24
Generation of virus-specific T cells for adoptive therapy
Workflow
virus- specific T cells
Sample preparation
Cell selection
Cell stimulation
Administration to patient Donor
CCS Reagent
MACS GMP PepTivator
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 25
Cell labeling
MACS GMP Media
Buffer
Leuka- pheresis
Generation of virus-specific T cells for adoptive therapy
Workflow
virus- specific T cells
Sample preparation
Cell selection
Cell stimulation
Leuka- pheresis
Administration to patient Donor
secreted IFN-γ
CD4 CD8
99.7% 97.3%
Quality Control Flow Cytometry
CCS Reagent
MACS GMP PepTivator
w/o Adv BZLF1 EBNA1 LMP2A pp65
CD
4 C
D8
I FN-g
Quality Control Flow Cytometry
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 26
Cell labeling
MACS GMP Media
Buffer
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 27
Characterization of T cell responses
Rapid and multiple-sample analysis of antigen-specific T cells by ICS
Cell culture (Stimulation)
Cell analysis (Flow cytometry)
MACSQuant Express Mode for automated cell processing
and analysis of ag-specific T cells
PepTivator – HT (pre-coated 8x12 strip well
format)
Rapid Cytokine Inspector
Cell analysis (Fixation/Permeabilization/Staining)
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 28
Characterization of T cell responses
Rapid and multiple-sample analysis of antigen-specific T cells by ICS
Cell culture (Stimulation)
Cell analysis (Flow cytometry)
MACSQuant Express Mode for automated cell processing
and analysis of ag-specific T cells
PepTivator – HT (pre-coated 8x12 strip well
format)
Rapid Cytokine Inspector
Cell analysis (Fixation/Permeabilization/Staining)
Applications: • Process/Quality control - screening of donor cells for viral antigen reactive T cells - (multi) virus-specific T cell therapy: determination of final cell product composition • Immunomonitoring - immune reconstitution after stem cell transplantation or adoptive T cell therapy
Screening of PBMC (LP7) for virus-specific T cells using PepTivator – HT and Rapid Cytokine Inspector
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 29
w/o AdV Hexon BZLF1 EBNA1 LMP2A pp65
CD
4-A
PC
CD
8-FI
TC
Anti-IFN-γ-PE
Gated CD3+ T cells
Immunophenotyping: Identification of leukocyte subsets
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 30
FSC-
H
SSC
SSC
FSC-A CD45-VioBlue FSC CD19-PE-Vio770
CD4-PerCP CD14-FITC
CD3-
APC
CD8-
APC-
Vio7
70
SSC
CD56/CD16-PE CD56/CD16-PE
SSC
CD3-
APC
Cell type Markers Color Cells/µL % live cells Leukocytes CD45+ 5.51×103 100 T cells CD45+, CD3+ 1.21×103 22 CD4+ T cells CD45+, CD3+, CD4+ 8.08×102 15 CD8+ T cells CD45+, CD3+, CD8+ 3.33×102 6 B cells CD45+, CD19+ 1.32×102 2.4 Monocytes CD45+, CD14+ 4.19×102 7.6 NK cells SSClow, CD45+, CD14–, CD16+, CD56+ 3.56×102 6.5 NKT cells SSClow, CD45+, CD14–, CD56+, CD16+, CD3+ 0.46×102 0.83 Eosinophils SSChigh, CD45+, CD14–, CD16– 1.98×102 3.6 Neutrophils SSChigh, CD45+,CD14–, CD16+ 3.23×103 59
Cellular Composition of the CCS Final Cell Product
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 31
nene
n=8
Enrichment of IFN-g secreting T cells using CCS -> 3 log depletion of unwanted cells
Achievements and Benefits
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 32
CliniMACS Prodigy CCS process Performance comparable to
CliniMACSPlus semi-automated procedure
Significant reduction of hands-on time (from 12 to 3-4 hours!)
Automation of eliminate laborious steps and minimize mistakes
Final formulation of the target cells in a small volume (<10 mL)
CE mark for instrument and TS; CE mark for reagent expected classified as ATMP Tools and protocols for QC
Safety • Product safety – limit manual steps and
number of connection and reconnection
steps
• Operator safety – minimize potential
exposure of operator to agents
Costs • Reduced hands-on time
• Reduced clean room requirements
• Manufacturing of cells in OR
Reproducibility/ease of use • Fully-automated and controlled process
Outlook - Roll-out plan CE-applications 2014
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 33
Q1/2014 Q2/2014 Q3/2014 Q4/2014 Q1/2015
BM-133 Enrichment
CCS-IFN Enrichment (CE-mark TS 500 Nov 4th 2013)
LP-TCRab-19 Depletion
LP-3-19 Depletion
LP-34 Enrichment CE-Mark TS 310
Medical CE Research Use Development
LP-14 Enrichment for MoDCs
LP-304 Enrichment for PDCs
CE-Mark TS 510
Viral Transduction process: CAR-T cells
CD133
CCS
TCRa/b/CD19
CD14 -> MoDC
PDC
Gene-modified T cells
CD34
CD3/CD19
Thank You for Your Attention!
Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 34
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