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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