Standardization of Flow Cytometry Workflows for Cell

Standardization of Flow Cytometry Workflows for Cell Therapy Applications Novartis Cell and Gene Therapies Unit (CGTU) Sadik H. Kassim, Senior Fellow ISCT-Las Vegas (May 27, 2015)

Disclaimer

The information contained in this document belongs to Novartis and/or its affiliates. Novartis does not make and expressly disclaims: (a) any representation or warranty (express or implied) with respect to the information shown in this presentation; and (b) any liability relating to the accuracy or completeness of the information.

2

Flow Cytometry is a Keystone Analytical Platform for Cell Therapy: cGMP Setting D0 Composition

Product Release: CoA

In Process: Transduction

1. Porter DL, et al. N Engl J Med. 2011;365(8):725-733. 2. Porter DL, et al. J Cancer. 2011;2:331-332. 3. Kalos M, et al. Sci Transl Med. 2011;3:95ra73. 3 | ISCT Flow Cytometry Workshop | Sadik Kassim| May 27, 2015 |

Flow Cytometry is a Keystone Analytical Platform for Cell Therapy: Research and Development Setting Lead CAR selection involves choosing the optimal ScFv/Signaling Domain Configurations

Alfred Garfall et al. Discovery Medicine, 2014

Marc Cartellieri et al. JBB, 2010 4 | ISCT Flow Cytometry Workshop | Sadik Kassim| May 27, 2015 |

-Phenotype -Function

Flow Cytometry Assays: Cell Based Cancer Immunotherapies 1) 2) 3) 4) 5)

Multi-parameter immunophenotyping of cells. Intracellular cytokine staining (ICS). Cytotoxicity assays. Proliferation assays. Cell-free cytokine analysis.

5 | ISCT Flow Cytometry Workshop | Sadik Kassim| May 27, 2015 |

Flow Cytometry Workflows: What Needs to be Standardized?

1. Sample Preparation

4. Data Analysis 2. Instrument Setup 3. Data Acquisition

6

Flow Cytometry Workflow Requirements Regulatory and Quality Standards

Throughput

Drug Discovery

Target Identification

7

Target Validation

Preclinical

Pharmaceutical Formulation

| ISCT Flow Cytometry Workshop | Sadik Kassim| May 27, 2015 |

Clinical

Toxicity Evaluation

Clinical Trials

Automating Flow Cytometry Workflows  Automation can potentially address the analytical challenges on the path to commercialization.

 Automation is not just about increasing throughput. • “Automation” means “walkaway” sample prep, acquisition, and analysis

 Key benefits: • Rigorous management/tracking of multi-patient, multi-assay sample flows • Reproducibility and robustness of notoriously variable cell-based assays • Ease-of-transfer of analytical methods • Applicability across portfolio.

8 | ISCT Flow Cytometry Workshop | Sadik Kassim| May 27, 2015 |

Flow Cytometry Workflow Standardization Solutions: GNF High Throughput Flow Cytometry  Industry-first, fully automated screening system dedicated to processing and reading cells using flow cytometry

Incubators

• 1536- or 384-well plates • 50,000 wells per day, 0.9 manual

• Exceptions under further investigation (small values, etc)

automated 20 | ISCT Flow Cytometry Workshop | Sadik Kassim| May 27, 2015 |

Manual vs Automated sample prep

 All analysis automated (v2)

 Each point shows the

manual

manual and automated prep values for 1 population from 1 sample

 Overall correlation improved • r2 = 0.998

automated 21 | ISCT Flow Cytometry Workshop | Sadik Kassim| May 27, 2015 |

Manual vs Automated sample prep

r2 = 0.999

manual

automated rep 2

r2 = 0.998

automated

automated rep 1

 Replicate samples processed in same plate present the “best case scenario” 22 | ISCT Flow Cytometry Workshop | Sadik Kassim| May 27, 2015 |

Automated Flow Cytometry Analysis Manual Analysis Variability – 3 FlowJo Users UPCC04409-29G1 100

80

60

Mean +/- SD CV

40

4.55

1.76 20

1.79 12.69

5.33

37.37

42.70

0 CAR+

CAR+CD4+

Easy 23

CAR+CD8+

CD45RA+CCR7+

CD45RA+CCR7–

CD45RA–CCR7+

CD45RA–CCR7–

Naive

EMRA

Central Memory

Effector Memory

Messy

Conclusions and Future Directions -Standardization of flow cytometry workflows, in research, development, and manufacturing/QC will be important for commercialization of cell therapies. -Automation provides a potential cost-effective and reproducible solution. -In house data demonstrates that automated and manual flow cytometry workflows produce similar results. -Instrument qualification and assay validation will be critical for the effective deployment of these approaches.

24 | ISCT Flow Cytometry Workshop | Sadik Kassim| May 27, 2015 |

Acknowledgements Arnaud Colantonio

Chelsea Xue

Sean Garrity

Andrew Beavis

Viraj Tyagi

Joe Sieling

Brian Gismonde

Margit Jeschke

Nebojsa Milovic

Marty Giedlin

Mark Dudley

John Scott

Amy Shaw

Jeannine Larrieux

Lana Parent

Erik Rutjens

Olja Pulluqi

Poonam Vaidya

Piotr Pierog Junxia Wang Rod Rietze Joe Chin Ted Manley Brian Duffy Olivier Graf 25

Dan Sipes Dan Rines John Scott Jen Brogdon Boris Engels Fay Dufort