Cellular Therapies for Repair Cellular Therapies for Repair and Regeneration of Joint and Regeneration of Joint

Surfaces—Product Surfaces—Product Characterization and Characterization and

TestingTesting

Malcolm Moos Jr., M.D., Ph.D.Malcolm Moos Jr., M.D., Ph.D.

Medical OfficerMedical Officer

CBER/OCTGT/DCGTCBER/OCTGT/DCGT

moos@cber.fda.govmoos@cber.fda.gov

SourceSourceProcesProces

ss

ProducProduct t

TestingTesting

““Device”Device” ““Biologic”Biologic”

• Well-characterizedWell-characterized

• Precisely defined materialsPrecisely defined materials

• Precisely defined structurePrecisely defined structure

• Tests that measure critical Tests that measure critical product parameters reliablyproduct parameters reliably

• Data defining these Data defining these parametersparameters

• Poorly characterizedPoorly characterized

• Heterogeneous Heterogeneous

• Complex molecular Complex molecular entitiesentities

• Inadequate analytical Inadequate analytical proceduresprocedures

• Insufficient scientific Insufficient scientific basis correlating basis correlating measurable properties measurable properties with biological activitywith biological activity

ReleaseReleaseTestingTesting

ProcessProcesscontrolcontrol

““Device”Device” ““Biologic”Biologic”

• Well-characterizedWell-characterized

• Precisely defined materialsPrecisely defined materials

• Precisely defined structurePrecisely defined structure

• Tests that measure critical Tests that measure critical product parameters reliablyproduct parameters reliably

• Data defining these Data defining these parametersparameters

• Poorly characterizedPoorly characterized

• Heterogeneous Heterogeneous

• Complex molecular Complex molecular entitiesentities

• Inadequate analytical Inadequate analytical proceduresprocedures

• Insufficient scientific Insufficient scientific basis correlating basis correlating measurable properties measurable properties with biological activitywith biological activity

ReleaseReleaseTestingTesting

ProcessProcesscontrolcontrol

SourceSourceProcesProces

ss

ReleasRelease e

TestingTesting

Strategic Strategic considerationsconsiderations

““Measure twice, cut once”:Measure twice, cut once”: Up-front investment in each area Up-front investment in each area

may prevent costly waste of time may prevent costly waste of time and resources on sub-optimal and resources on sub-optimal process or variable/ineffective process or variable/ineffective productproduct

Better characterization makes Better characterization makes many things easiermany things easier

Source ControlSource Control

Anatomic siteAnatomic sitePathologic involvementPathologic involvementCellular Cellular

homogeneity/inhomogeneityhomogeneity/inhomogeneity

Freshly Isolated ChondrocytesFreshly Isolated Chondrocytes

In vitroIn vitro culture is not required culture is not required

HACDC in HACDC in regenerating muscleregenerating muscle

Product TestingProduct Testing

Is guided by detailed Is guided by detailed understanding of the product understanding of the product and the manufacturing and the manufacturing processprocess

Should ensure consistency of Should ensure consistency of process and product process and product

Should predict Should predict in vivoin vivo activityactivity

Product TestingProduct Testing

——An example of An example of new alternativesnew alternatives

Terminal markers may not predict biological functionTerminal markers may not predict biological functionCharacterization of functional chondroprogenitors Characterization of functional chondroprogenitors Qualification by meaningful biological responsesQualification by meaningful biological responses

0

20

40

60

80

100

0 20 40 60 80 100

Islet cell transplantation in Islet cell transplantation in diabetic micediabetic mice

Live/dead Staining (%)Live/dead Staining (%)

0 20 40 80 10060

OC

R/D

NA

(%

)O

CR

/DN

A (

%)

20

40

80

100

60

0

Theoreticalcorrelation

OOxygen xygen CConsumption onsumption RRate and dye ate and dye exclusion are exclusion are uncorrelateduncorrelated

(K. Papas, University of Minnesota Department of Surgery, in preparation)

Normalized Oxygen Consumption Rate, OCR/DNA (nmol/min•mgDNA)

Oxy

gen

Con

sum

ptio

n R

ate,

OC

R (

nmol

/min

)

Fractional Viability (%)

Viable Islet E

quivalents, VIE

QResponse to Rat Islet Transplants in Diabetic Balb/C Mice (Anti-CD4)

2 / 0 / 0 1 / 0 / 0

2 / 0 / 0

2 / 0 / 0

6/ 0 / 0

4/ 0 / 0

3 / 0 / 0

3 / 0 / 0

1 / 0 / 10 / 1 / 2

1 / 0 / 1

2 / 0 / 0

1 / 0 / 0

0 / 1 / 0

1 / 0 / 0

3 / 0 / 0

2 / 0 / 0

3 / 0 / 0

all curemixedall fail

3 / 0 /0

2 / 0 / 0

1 / 0 / 2

1 / 0 / 0

0

4

5

6

0 50 100 150 200 250 300 350 400 450

100800

700

600

500

400

300

200

100

0

0 50402010 6030 8070 90

0 / 1 / 1

0 / 0 / 4

0 / 0 / 6

0 / 0 / 3

0 / 0 / 2

1

2

3

0 / 0 / 30 / 0 / 2

Cure rates and days to cure in mice Cure rates and days to cure in mice receiving 1,000 human IE according to receiving 1,000 human IE according to

ATP/DNAATP/DNA

00

2020

4040

6060

8080

100100

00 1010 2020 3030 4040

Days after transplantDays after transplant

Cu

re r

ate

(%)

Cu

re r

ate

(%)

ATP < 110ATP < 110 pmol/µg DNApmol/µg DNA

ATP ATP >> 110 110 pmol/µg DNApmol/µg DNA

P=0.008P=0.008

(B. Hering, University (B. Hering, University of Minnesota of Minnesota Department of Department of Surgery, with Surgery, with permission) permission)

The nude mouse modelThe nude mouse model

Dell’Accio et al, A&R, 2001Dell’Accio et al, A&R, 2001

The ShmooThe Shmoo

——Al Capp, ca. 1948Al Capp, ca. 1948

Instructions go both Instructions go both waysways

DonorDonor RecipientRecipient

Donor-Host Donor-Host Interactions?Interactions?

Cell productCell product

Articular surfaceArticular surface

Subchondral boneSubchondral boneRemnants of calcified cartilageRemnants of calcified cartilage

CoverCover

Synovial Joint FluidSynovial Joint Fluid

Competence Competence FactorsFactors

gene expression

Biological Response

P

β-actin

ALK-1

BMP-2

Col2a1

FGFR-3

Population doublings FI 2 4 10 12

Safranin O

Molecular profile of the stable chondrocyte

His

tolo

gy s

core

His

tolo

gy s

core

Gene scoreGene score

-5-5 -3-3 -1-1 11 33 55

00

11

22

33

Relation of Gene score to Relation of Gene score to Stable Cartilage FormationStable Cartilage Formation

Stable cartilageStable cartilage

Space—the final Space—the final frontier?frontier?

Thomas and Moos, 2004 (unpublished)

Joint-specific growth factorEnzymeOverlap growth factor activation joint surface specification

Molecular Molecular Boundaries in Tissue Boundaries in Tissue

FormationFormation

400x Thomas and Moos, 2005 (submitted)

Thought QuestionsThought Questions

Acceptance, selection, or instruction of Acceptance, selection, or instruction of starting materialstarting material

Consider competence factors sufficient Consider competence factors sufficient to define chondroprogenitors (rather to define chondroprogenitors (rather than characteristics of terminal than characteristics of terminal differentiation)differentiation)

Distribution of characteristics in 3D may Distribution of characteristics in 3D may be criticalbe critical

Qualification of potential tests against Qualification of potential tests against appropriate biological responses appropriate biological responses in in vivovivo

•Criteria for obtaining starting tissueCriteria for obtaining starting tissue

•Characteristics of functional chondroprogenitor cellsCharacteristics of functional chondroprogenitor cells

•Analytical methods to determine these characteristicsAnalytical methods to determine these characteristics

•Qualification of these tests in preclinical modelsQualification of these tests in preclinical models

•Potency assaysPotency assays

•Special issues for cells contained in natural or artificial matricesSpecial issues for cells contained in natural or artificial matrices

Questions for DiscussionQuestions for Discussion