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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
2
Overview of Manufacturing and Sterility Testing
3
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
4
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?
5
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
6
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
7
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.
8
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)
9
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
10
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)
11
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
12
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)
13
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
14
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
15
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
16
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
17
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)
18
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
19
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
20
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
21
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
22
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)
23
Equivalence Data:B/F Comparison between CFR and BacT/Alert (Culture Pool)
24
Equivalence Data: Manufacturer’s literature
25
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)
26
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)