July 17-18 2017 CASSS “Manufacturing, Quality, and Regulatory Considerations for Cell & Gene Therapies”.

Measurement Assurance For Regenerative Medicine Advanced Therapies

Anne Plant

National Institute of Standards and TechnologyGaithersburg MD

anne.plant@nist.gov

Snapshot of the Industry

• GSK inks money-back guarantee on $665K Strimvelis, blazing a trail for gene-therapy pricing

• Kite Pharma Initiates Rolling Submission of U.S. Biologics License Application (BLA) for KTE-C19, its Investigational anti-CD19 CAR-T Therapy

• Novartis Confirms Plans to File BLA for CAR T-Cell Therapy During Early 2017

• FDA approves Maci (autologous cultured chondrocytes on porcine collagen membrane), first autologous cellularized scaffold for the repair of cartilage defects of the knee

• First CRISPR gene-editing cells (knock out PD-1) tested in a person for the first time Sichuan University, China; First CRISPR clinical trial gets green light from US panel

• Bayer and Versant Ventures Join Forces to Launch Stem Cell Therapy Company BlueRock Therapeutics with USD 225 Million Series A Financing

• For CAR-Ts deal alone, $2 billion in disclosed upfront payments and $4 billion in additional milestone, royalty and other payments between 2012 and mid 2016.

Regenerative Medicine/Advanced Therapy Industry

Cell-Based Therapies

Gene Therapy

Biologics and Small Molecules

Tissue Engineering: Synthetic Materials, Biomaterials and Scaffolds

Stem Cells for Drug Discovery, Toxicity Testing and Disease Modeling

Biobankinghttp://alliancerm.org/page/industry-snapshot

What are the critical characteristics for clinical effectiveness and safety?

Fundamental ChallengeFor Regenerative Medicine Products

Ideal if MOA is established, but rarely is MOA complete

Quality Attributes• Identity• Quantity• Purity/impurity• Sterility• Viability • Biological activity

Challenge to commercialization of Cellular Therapy

products: Measurements

I. Characterization of product

Assays to measure quality attributes

• Scale Up

• Change in personnel, process, location

• Storage

• New analytical and culture equipment

• Improved assays

• Changes in raw materials

II. Control of the manufacturing process

Assays that assure consistency of product during

"If you can not measure it, you can not improve it.“Lord Kelvin

Easy to imagine for measurement of lead in water

Harder to envision Reference Materials for measuring biological

characteristics

Reference materials can provide traceability

Achieving Comparability and Measurement AssuranceThrough Reference Materials

Product 2Product 1

Reference

Measurements: Fundamental ChallengeFor Regenerative Medicine Products

The Biology is Complex

• What in vitro metrics are predictive of in vivo response?

• Gaps in fundamental understanding.

• Lots of variables (clinical and analytical)

• Variation in starting materials (patients)

• Dynamic nature

• No ground truth

• What is the measurand?

• Is your assay measuring what you intend?

• Are there assay variables (personnel, reagents, unknown factors) that are influencing the assay result?

• Is the measurement robust?

• How much dispersion is due to biological variability?

Can only determine if you know how much of the dispersion is due to variability in the assay.

• Is an assay biologically meaningful? Can’t assess unless you know the reliability of your assay.

Measurement Assurance for Cell Therapies is not always straightforward

Assuring Comparability through Measurement Assurance

Qualifying and Validating Measurements

• Precision: Reproducibility / Repeatability

• Accuracy

• Robustness / sensitivity / specificity

• Dynamic range / response function / LOD

Pre-clinical

RM Product

Relatively easy Hard due to

biological

complexity and

variability

Measurement Assurance = Confidence in Measurements for Decision Making

Different Manufacturers’calibration beads

NIST SRM 1934/ Calibrated fluorimeterFluoresceinNile RedAllophycocyanin (APC)Coumarin 30

Light obscuration flow instrumentFor accurate bead concentration

Equivalent Reference

Fluorophore

(ERF) Number

Assuring comparability in instrumentation:

traceability to a reference material

Flow Cytometry Quantitation Consortium81 Federal Register 136 (15 July 2016), pp. 46054-46055

ERF Value Assignment to Cytometer Calibration Microbeads Submitted by

Consortium Members

Lili Wang

Wang et al Cytometry 2012

NIBSC CD4+ Cell Counting Standard (WHO/BS/10.2153)

• CD4 expression as the reference control

• reduces the variability of CD20 expression measurements

• enables quantitative measure of CD20 expression independent of cytometer platforms used.

Assuring comparability in instrumentation:

traceability to numbers of cell surface receptors

Diffe

rence

s in a

bsolu

te

absorb

ance

Differences in

response

functions

Differences in

measurement

uncertainties

Assuring comparability: Interlaboratory studies,

Design of Experiment for Robust Protocols

DoE

John Elliott

Result: robust protocol and comparable results

We observe a large difference between NC-200 and Hemocytometer counts, which counting method is more

appropriate?

Dilution

Fraction

NC-200

(CV)

Hem I

(CV)

Hem II

(CV)

0.2 0.05 0.09 0.11

0.5 0.07 0.26 0.23

0.8 0.07 0.16 0.10

Precision

Assuring comparability: Cell Counting

Evaluating the performance of a cell counting method for relative

accuracy and precision: Experimental design and statistical analysis

Sumona Sarkar

Assuring comparability in cell counting

through statistical models

Experts are not perfect at classifying.

Scale bar = ~ 0.5mm

Testing Assumptions

Michael Halter

Expert 2 Scores Expert 1 Scores

1 2 3 4 5

1 7 5 0 0 02 2 31 19 11 43 1 21 52 49 114 0 4 35 105 435 0 0 2 25 22

479 colonies scored by two experts

Are Assumptions about Biomarkers Correct?

H9 hESC expressing GFP inserted at OCT4 site

• What will happen to this colony in time?

• What did it look like at an earlier stage?

• How does it compare to other colonies?

• How reproducible are different preparations of cells?

Quantifying large numbers of colonies over long times

How do individual colonies respond in time?

oQuantify diversity of gene expression

oQuantify differences in preparations

oQuantify dynamic changes over time

oQuantify effects of media etc.

o Large statistical sampling: 680 colonies/ 5days

o Methods for handling, viewing and analyzing

~1TB of data

Bhadriraju et al Stem Cell Research 2016

The more rare the event (ie,

nonexpressors), the larger the sampling

that is required.

Power analysis of

Fisher’s exact test with

increasing sample size

The number of colonies sampled effects the sensitivity for detecting differences

in Oct-4 expression between the relative proportions of “homogeneous”,

“heterogeneous”, and “non-expressing” colonies in the preparations.

Measurement Assurance is Necessary for Confident Decisions

Challenge: Significant effort will be required.

Risk: Data that are not reliable, and results and

conclusions that are not correct.

Solution: May require a community effort.

"If you can not measure it, you can not improve it.“

Lord Kelvin

NIST Workshop on Measurement Challenges for CAR-T Biomanufacturing January, 2016; IBBR

Genome Editing Standards WorkshopNIST Hosted, ASGCT partnered May 2016

NIST Workshops on RMAT Measurements

Strategies to achieve measurement

assurance for cell therapy products May 11-12, 2015; NIST

• NIST-FDA Cell Counting Workshop: Sharing practices in cell

counting measurements April 10, 2017; NIST Gaithersburg, MD

• NIST-FDA Flow Cytometry Workshop Oct 2017, NIST Gaithersburg, MD

Standards for Regenerative Medicine and

Advanced Therapies

Sec. 3036 of 21st Century Cure Act

Requires HHS/FDA in consulation with NIST and stakeholders to “facilitate an effort to coordinate and prioritize the development of standards…”

Key language: • Introduced a specific Regenerative

Medicine/Advanced Therapy (RMAT) product designation

• Language intended to optimize the FDA’s approval pathways RMAT without reducing safety and efficacy

Dec 16, 2016: U.S. Secretary of Commerce Penny PritzkerAnnounces Biopharmaceutical Manufacturing Institute Joining Manufacturing USA Network

NIST Laboratory Programs

• Industry engagement • USG coordination • Convene workshops

• ISO/TC 276 leadership• ASTM F04 leadership• Expert contributions to

SDOs

• Innovative measurement solutions

• Industry collaborations

NIST Efforts

Thank You