Surmounting the developmental challenges of Miltenyi's...

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


Cell Therapy Manufacture

Components

Donor / patient

Blood Bone marrow

Leukapheresis

Cell separation

Cell expansion or differentiation


Cryopreservation

CliniMACS

Reagents MACS GMP Media

Cell culture bags

MACS GMP Antigens

MACS GMP Cytokines

Cell differentiation bags


CryoMACS

Freezing Bags CliniMACS Instruments

Sample Dissociation Tissue

GentleMACS

MACSQuant

MACS GMP Antibodies


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


Designed to …

=

Not approved by FDA! Februar 6, 2014 Anne Richter // R&D Reagent Department // AMC Page 6

+ + + =

+ +

… replace or integrate


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, …)


isolation

Antigen-specific T cells for immunotherapy Enrichment of IFN-gamma secreting cells


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


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


Sample preparation

Cell selection

Cell stimulation

Cell labeling

CliniMACS Prodigy – main components


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


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


Process for enrichment of IFN-γ secreting T cells using the CliniMACS Prodigy system


Tubing set, buffer, media Preparation

Manual steps

Automated steps

0.5 h

Tubing set installation




Manual steps

Automated steps

0.5 h

Tubing set installation

0.5 h Priming of TS



0.5 h


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



0.5 h

1.5 h


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





4 h

0.5 h

1.5 h

Tubing set, buffer, media



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



4 h

2 h 10 min

0.5 h

1.5 h

50 min

1 h 30 min


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



CentriCult Unit

Cooling down or warming up liquids in a short time period (4-38°C)



4 h

2 h 10 min

0.5 h

1.5 h

50 min

1 h 30 min

1 h 10 min


5 min

Preparation

Priming of TS

Preparation

Sample preparation

Antigen incubation

Delay



Labeling Enrichment

Reagent/Wash

Magnetic Separation

Take QC sample

Manual steps

Automated steps

0.5 h

0.5 h



• Suitable for various CliniMACS column sizes

• Cell enrichment and depletion

• Multi-step separations possible



4 h

2 h 10 min

0.5 h

1.5 h

50 min

1 h 30 min

1 h 10 min


Enriched IFN-γ+ cell fraction in <10 mL injectable solution

5 min

Preparation

Priming of TS

Preparation

Sample preparation

Antigen incubation

Delay



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


(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


Purity of enriched IFN-γ+ T cells

CliniMACS CD4+ IFN-γ+

Prodigy CD4+ 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


Recovery of IFN-γ+ T cells


Prodigy CD4+ IFN-γ+ CliniMACS 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


Generation of virus-specific T cells for adoptive therapy

Workflow


Sample preparation

Cell selection

Cell stimulation

Administration to patient Donor

CCS Reagent

MACS GMP PepTivator


Cell labeling

MACS GMP Media

Buffer

Leuka- pheresis

Generation of virus-specific T cells for adoptive therapy

Workflow


Sample preparation

Cell selection

Cell stimulation

Leuka- pheresis

Administration to patient Donor

secreted IFN-γ

CD4 CD8

99.7% 97.3%


CCS Reagent

MACS GMP PepTivator

w/o Adv BZLF1 EBNA1 LMP2A pp65

CD

4 C

D8

I FN-g



Cell labeling

MACS GMP Media

Buffer


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)


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


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


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


nene

n=8

Enrichment of IFN-g secreting T cells using CCS -> 3 log depletion of unwanted cells

Achievements and Benefits


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


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!


Date post:	13-Aug-2020
Category:	Documents
Upload:	others
View:	1 times
Download:	0 times

Surmounting the developmental challenges of Miltenyi's...

Documents