1

Somatic Cell Therapy Symposium, Bethesda MDSeptember 28, 2007

Validation of the BacT/Alert as an Alternative Sterility Test for Dendreon’s Autologous Cell Therapy Product

Timothy Wood QC Scientist

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Overview of Manufacturing and Sterility Testing

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Challenges of 21 CFR 610.12 (or USP Method)

• Product can render microbiological media turbid

• Insufficient time for an accurate “go” “no go” pre-harvest read

• Limited shelf life: < 24 hours

• Sub-culture transfer if necessary would occur post-infusion

• No “bulk” product. The Culture Pool (in-process) sterility has been tested to the CFR “bulk”

• Repeat testing rules are not appropriate for our product

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Method Validation Challenges

• Experimental Design– No standard available

• Guidance obtained from the new PDA TR # 33 (Parenteral Drug Assoc.)• Guidance from draft USP <1223> Validation of Alternative Micro Methods• Input from industry colleagues experience

• Equivalence challenges– Fresh samples from donors for seeded studies are limited and can be

logistically challenging– Non-seeded side by side testing not possible (limited dose)– How to demonstrate when the standard (21 CFR 610.12) does not provide

needed assurance?

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Key reasons for selection of BacT/Alert as alternative method• Non-destructive (ability to directly subculture positives for ID)• Faster detection• Sample volume (representative)• No sample filtration needed• Minimal preparation and manipulation (reduces false positives) • Continuous monitoring • Similarities to CFR standard (aerobic, anaerobic, sample to media ratio)

• FDA approved for blood cultures and platelet sterility testing• Testing throughput

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BacT/Alert Validation Plan Outline (Phase I & III Products)

• Installation Qualification (IQ)

• Feasibility (Proof of Concept) Studies- System suitability (detection of USP panel organisms)

- Comparison study with CFR method using fresh healthy donor product samples

• Operational Qualification (OQ)- Verification of BacT/Alert system operations

- Temperature mapping of incubation chambers

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Validation Plan Outline (continued)

• Method Validation Study Design- 6 ATCC strains (USP <71> panel)

Staphylococcus aureus ATCC 6538Bacillus subtilis ATCC6633Pseudomonas aeruginosa ATCC9027Clostridium sporogenes ATCC 11437Candida albicans ATCC 10231Aspergillus niger ATCC 16404

- 6 environmental / product isolatesBacillus dipsosauriStaphylococcus epidermidisBrevibacillus breviMicrococcus luteusRhodococcus sp.Bacillus sp.

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Validation Plan Outline (continued)

• Method Validation Study

- Test article in triplicates vs. non-product control bottles without product (B/F - bacteriostasis/fungistasis)

- BacT/Alert Incubation: 35°C for up to 14 days

- Acceptance criteria:-Specificity (ATCC strains & isolates detected in products)-Detection Time (< 7 days)-LOD (Sensitivity <10 CFU/bottle) -Repeatability/Ruggedness (multiple product lots,

culture bottles, operators)

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Feasibility: Suitability of BacT/Alert Culture bottles

Growth Promotion < 100 CFU Time to Positive BT/A bottles vs CFR/USP Method (typical values)

024487296

B subti li

s 6633

S aureu

s 6538

P aerugin

osa 90

27

C sporo

genes

11437

C albic

ans 1

0231

A nige

r 16404

ATCC strain

Hrs BacT/Alert

USP Method

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Feasibility: BacT/Alert vs CFR (Culture Pool)

Positive Detection Times (3 replicates)

0102030405060

B. s

ubtili

s 46

CFU

B. s

ubtili

s 6 C

FU

B. s

ubtili

s 2 C

FU

S. a

ureu

s 65

CFU

S. a

ureu

s 8 C

FU

S. a

ureu

s 1 C

FU

Hrs

BTA Method

CFR (FTM media)

3/3 3/32/3

1/3

3/3

3/33/3

3/31/3

1/3

0/3 0/3

CFR: 10 mL sample into 100mL TSB (20-25°C) and 100 mL FTM (30-35°C)BacT/Alert: 5 mL sample into 40 mL aerobic and anaerobic bottles (35°C)

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Feasibility: BacT/Alert vs CFR (Final Product)

Positive Detection Times (2 replicates)

020406080

100

S. a

ure u

s 69

CFU

S. a

ure u

s 7 C

FU

S. a

ure u

s 1 C

FU

Hrs

BTA MethodCFR (FTM)CFR (TSB)

2/2 2/2

2/2

2/21/2

1/2

2/2 0/2

2/2

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Feasibility Study Conclusions

• Cell product is compatible with the BacT/Alert method• Suitability (growth promotion) of culture bottles confirmed• Overall BacT/Alert performed better than CFR

– CFR false negative reads (visual): 4– BacT/Alert false negatives: 0– Detection time range BacT/Alert: 12.1 - 18.4 hrs

CFR: 24 hrs – not detected (14 days)

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Method Validation: B/F Study of ATCC Panel <100 CFU Final product vs non-product control bottles (35°C)

13.9

15.8

16.7 25

.1

51

39.9

12.9 20

.3

18.5

18.4 27

20.3

44

NE

G (1

4 da

ys)

12.3

0102030405060

B subtili

s 43 C

FU

S aureus 5

3 CFU (A

ST)

S aureus 5

3 CFU (N

ST)

P aerug inos

a 52 CFU

C albican

s 23 C

FU

C sporogen

es 16 C

FU (NST)

A n iger 2

5 CFU

Neg Contr

ols 0 C

FU

Avg

Hrs

to P

ositi

ve (n

=3)

Cell Product

Pos Control

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Method Validation: Environmental & Product Isolates Final product vs non-product control bottles (35°C)

11.1 23

.9

25.9

28.1

46.6

24.9 31

.2

11.3 24 26

.1

91.7

47.6

73.7

33.5

NEG

(14

days

)

020

4060

80100

Bac il lus dipso

sauri 24 C

FU

Microco

ccus l

uteus 73 CFU

Sta ph epidermidis

37 CFU (A

ST)

Staph epidermidis

37 CFU (N

ST)

Rhodococcus sp

p 103 CFU

Brev ibac il lus brevis

68 CFU

Bac il lus sp

p 7 CFU

Neg Cont ro

ls 0 C

FU

Hrs

to P

ositi

ve (n

=3)

Cell Product

Pos Control

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Method Validation: Sensitivity of Detection (LOD)

Target < 10 CFU/bottle

20.0

20.9 28

.3

29.0

22.7

51.7

12.4

30.3

31.4

21.5

60

53.7

14.7

NEG

010203040506070

B. subt i

lis 6 CF

U

S. aur eu

s 4 CFU

(AST)

S. au

r eus 4

CFU (N

ST)

P. aeru

ginosa 5

CFU

C. alb ican

s 8 CFU

C. sporoge

nes 1 CFU (N

ST)

A. n ige

r 10 C

FU

Bacillu

s dip so sau

r i 5 C

FU

Micr oco

ccus l

ut eus 5

CFU

Staph epid

ermidi

s 5 CFU

(AST)

St aph ep

idermidi

s 5 C

FU (NST

)

Rhodococ

cus sp. 3

CFU

Breviba cillu

s brevis

4 CFU

Bacillus s

p . 2 CFU

ATCC Strains and Isolates

Avg

Hrs

to P

ositi

ve D

etec

tion

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Method Validation Initial Conclusions

• B/F studies concluded no effect of the product on detection• All unseeded product controls were determined negative• Range of average detection time (<100 CFU in product)

– 9.4 hrs Bacillus dipsosauri (isolate)– 51 hrs Clostridium sporogenes (ATCC)

• Sensitivity (LOD)– Range 1-10 CFU/culture bottle

• Repeatability/Ruggedness– Reproducible results between multiple products, culture bottle

lots, and operators

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Further studies were generated following correspondence with FDA on validation progress

• Address slow-growing/low CO2 producing bacteria• Include another mold

• Other microorganisms selected:– Penicillium chrysogenum (low temperature mold, slow-growing)– Propionibacterium acnes (slow-growing)– Pseudomonas fluorescens (associated with contaminated blood products)

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Other Microorganisms Final product vs non-product control bottles (35°C)

Final Product Sample

18.4 20.3

8391

19.3 21.6

90101

020406080

100120

P fluor es

cens 49838 >200 C

FU

P fluor es

cens 49838 26

CFU

P acnes 1182

7 90 CFU

P acnes 1

1827 10 CFU

P chrys

ogen um 10106 3

7 CFU

P chrys

ogenum 10

106 4 CF

U

Neg Con

tr ols

0 CFU

Avg

Hrs

to P

ositi

ve (n

=3)

Cell Product Pos Control

NE

G (1

4 da

ys)

NE

G (1

4 da

ys)

NE

G (1

4 da

ys)

Penicillium chyrsogenum: No growth or detection at 35°C

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CO2 production and detection of P. chrysogenum at lower incubation temperatures (standard algorithm)

Penicillium chrysogenum incubation at 32°C CFU count = 29

Penicillium chrysogenum incubation at 28°C CFU count = 10

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Method Validation Study Final Conclusions

• Wide range of organisms detected in products (<100 CFU)• Products are compatible (0 false positives) • Low-level sensitivity: 1-10 CFU/bottle• Longest seeded detection time noted:

– Propionibacterium acnes 3 CFU @ 129 hours (5.4 days)

• Lower temperature increased sensitivity for Penicillium chrysogenum

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Proposed BacT/Alert Sterility Method

• Feb 2006: BacT/Alert 7-day sterility method approved by FDA as an alternate sterility test for autologous Phase III product

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Additional Data Requested by FDA

• Equivalence of BacT/Alert vs. CFR/USP– Show comparison of product B/F results using panel of ATCC organisms– Manufacturer’s literature (aerobic & anaerobic bottles vs. TSB & FTM media)

• Comparability of detection algorithms (BacT/Alert Classic vs 3D Models)

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Equivalence Data:B/F Comparison between CFR and BacT/Alert (Culture Pool)

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Equivalence Data: Manufacturer’s literature

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BacT/Alert Detection Comparability (with standard algorithm)Classic Model (Site 1-2002) vs 3D Model (Site 2-2006)

45.044.039.933.632.540.7aerobic (iAST)A. niger 16404

20.519.75120.222.130.9anaerobic (iNST)

C. sporogenes11437

29.427.425.124.624.625.2aerobic (iAST)C. albicans 10231

17.918.816.717.819.117.7aerobic (iAST)

P. aeruginosa9027

15.816.615.815.516.315.6anaerobic (iNST)S. aureus 6538

14.516.113.914.215.814.4aerobic (iAST)S. aureus 6538

12.112.812.312.612.812.6aerobic (iAST)B. subtilis 6633

BT/A 3D Incubator 2

Site 2(2006)

BT/A 3D Incubator 1

Site 2(2006)

BT/A Classic Model 120

Site 1(2002)

BT/A 3D Incubator 2

Site 2(2006)

BT/A 3D Incubator 1

Site 2(2006)

BT/A Classic Model 120

Site 1(2002)

Final ProductCulture pool

BT/ABottle type

Microorganism & ATCC Number (<100 CFU)

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Moving Forward

• Established BacT/Alert validation requirements for new installations / new processing facilities – Instrument IQ/OQ/PQ– Performed local B/F study– Included local isolates (to be licensed facility)

• Replace Gram stain release assay with rapid method– Real-time results ( ≤ 4 hrs)– Sensitivity ≤ 103 cfu– Some possible technologies

• PCR (Total viable bacteria / yeast & mold)• Fluorescent cytometry• PGD Test (Verax Biomedical)• BacSTAT (GenPrime)