(ler) assessment

A3 ‐ Endotoxin Testing
Presented by: John Dubczak
A COMPARATIVE
IN-VITRO AND IN-VIVO
LOW ENDOTOXIN RECOVERY
(LER) ASSESSMENT
WHAT IS LOW ENDOTOXIN RECOVERY
(LER)?
• An Experimental Result When RSE/CSE Spikes
Are Not Recovered from Undiluted Finished
Products and/or In-Process Samples
• Identified by Genentech/Roche During a Container
Validation Study
• First Described by Joseph Chen & Anders Vinther
at the 2013 Annual PDA Meeting
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A3 ‐ Endotoxin Testing
Presented by: John Dubczak
WHAT LER IS NOT
• An LAL Method Validation Issue
– Method Validations Document Testing at the Non‐Inhibitory Concentration
• A Public Health Issue !!
3
1977 PYROGEN TEST RESULTS
FROM BAXTER TRAVENOLS’
PLANTS IN THE UNITED STATES
Total Number of Rabbit Tests Performed
28,410
Total Number of LAL Tests Performed
143,196
Total Number of Pyrogenic Tests Tested By Both Procedures
37
Total Number of LAL Pyrogen Test Failures
37
Total Number of USP Rabbit Test Failures
4
Mascoli and Weary (1979). Application and advantages of the Limulus amebocyte lysate (LAL) pyrogen test for parenteral injectable products. Pages 381‐402, Biomedical Applications of the Horseshoe Crab (Limulidae), Alan R. Liss, Inc.
2
A3 ‐ Endotoxin Testing
Presented by: John Dubczak
Mascoli and Weary (1979). Application and advantages of the Limulus amebocyte lysate (LAL) pyrogen test for parenteral injectable products. Pages 381‐402, Biomedical Applications of the Horseshoe Crab (Limulidae), Alan R. Liss, Inc.
CURRENT LER ISSUES
• ARE ISSUES WITH PURIFIED
ENDOTOXIN STANDARDS
• RSE/CSE Are Not Stable In Solutions Containing Tween/Chelating Agent Combinations:
– Tween/Citrate
– Tween/Phosphate
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A3 ‐ Endotoxin Testing
Presented by: John Dubczak
PURIFIED
ENDOTOXIN STANDARDS
• Prepared From Both Smooth and Rough Strains:
– Smooth Strains via Phenol/Water
– Rough Strains via Phenol/Chloroform/Petroleum
Ether
• Composition of All Is Approximately 90% LPS
• In Aqueous Solutions, These Structures Exist as:
–
–
–
–
Micelles
Filamentous Ribbons
Hexagonal Lattices
Spherical Discs and Vesicles
THE ULTRASTRUCTURAL MOPHOLOGY OF ENDOTOXIN AND LIPOPOLYSACCHARIDES
K.A. Brogden & M. Phillips
Electron Microscopy Reviews
Vlol.1, pp261-277, 1988
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A3 ‐ Endotoxin Testing
Presented by: John Dubczak
LOSING ENDOTOXIN ACTIVITY
• Purified Endotoxin Is Not As Stable As
Naturally Occurring Endotoxin And Can Be
The Cause Of PPC Failure.
• The Materials Used To Prepare The Control
Standard And Sample Can Also Be A Source
Of Inhibition.
STERILE WATER for IRRIGATION
(SWI)
RSE INTERFERENCE
50 EU/ml
Released SWI
Rejected SWI
489.9
1003.4
5.0
“
707.1
2584.7
0.5
“
1033.7
>3371.9
0.05 “
1534.8
>3504.2
0.005 “
2484.3
>3581.8
Neg Control
>3080.9
>3523.3
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A3 ‐ Endotoxin Testing
Presented by: John Dubczak
NATURAL ENDOTOXINS
• Chemically They Contain:
– LPS
– Outer Membrane Surface
Proteins
– Lipoproteins
– Phospholipids
• Exist As:
– Spherical Vesicles
– Tubular Blebs
THE ULTRASTRUCTURAL MOPHOLOGY OF ENDOTOXIN AND LIPOPOLYSACCHARIDES
K.A. Brogden & M. Phillips
Electron Microscopy Reviews
Vlol.1, pp261-277, 1988
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A3 ‐ Endotoxin Testing
Presented by: John Dubczak
PREPARATION OF A NATIVE ENDOTOXIN
• Source of Gram Negative Organism Should Be:
– Authenticated
– Preserved
– Available for Distribution
• Cultivation Methods Should Be Easy and Reproducible
• Endotoxin From This Organism Should Be Derived from Nutritive Limiting Conditions
ENDOSAFES’ NATIVE ENDOTOXIN
PREPARATIONS
• Utilized:
– E. cloacae ATCC 7256
– E. coli 055:B5 ATCC 12014
– S. marcescens ATCC 14756
– P. aeruginosa ATCC 9027
– R. pickettii ATCC 29791
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A3 ‐ Endotoxin Testing
Presented by: John Dubczak
ENDOSAFES’ NATIVE ENDOTOXIN
PREPARATION
– Depyrogenated Nutrient Broth:
• < 0.005 EU/ml with PPC Recoveries
– Adapted / Stressed Organisms to Grow in 1% Nutrient
Broth/99% Sterile Water for Irrigation:
• Each Successive Sub-Culture Was Verified for Purity
• An Isolate From Each Successive Sub-Culture Was
Identified via MALDI-TOF / Acugenix Axcess Data
Base
– All Cultures Were Incubated at 25 Degrees C
– Sterilized via Double 0.2 µm Filtration
15
8
A3 ‐ Endotoxin Testing
Presented by: John Dubczak
LAL METHOD COMPARISON
Chromogenic
Sample Endotoxin Time Zero
LRW Control RSE
9.47
LRW Control E. cloacae 7.00
Tween Citrate RSE
0.56
Tween Citrate E. cloacae 4.36
24 Hrs 48 Hrs 120 Hrs
9.21
9.57
10.36
7.18
6.99
11.71
<0.05 <0.05 <0.05
4.64
4.62
7.04
Turbidimetric
Sample Endotoxin Time Zero
LRW Control RSE
9.77
LRW Control E. cloacae 6.56
Tween Citrate RSE
0.11
Tween Citrate E. cloacae 8.73
24 Hrs 48 Hrs 120 Hrs
8.16
9.12
10.70
5.44
6.22
10.14
<0.05 <0.05 <0.05
7.13
7.92
12.10
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A3 ‐ Endotoxin Testing
Presented by: John Dubczak
WHOLE BLOOD
MONOCYTE ACTIVATION TEST
• Can be Used to Estimate the In-Vivo Activity of
Bacterial Endotoxins
• Involves the Use of Diluted Heparinized Whole Blood
WHOLE BLOOD MONOCYTE
ACTIVATION TEST (MAT)
METHODOLOGY
• Overnight Stimulation Of Blood With Sample and
Endotoxin Standards:
– IL-1ß Generated When Endotoxins Are
Present
•
Following Overnight Incubation All Are Transferred
Onto Anti-body Coated Micro Titer Plate With
Conjugate Antibody:
– IL-1ß Binds To The Plate
– Conjugate Antibody Bind to IL-1ß
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A3 ‐ Endotoxin Testing
Presented by: John Dubczak
WHOLE BLOOD MONOCYTE
ACTIVATION TEST (MAT)
METHODOLOGY
• Remove Non-binding Plasma Proteins By Washing
• Add TMB Substrate
– Color Change Is Proportional To The Amount Of IL1ß Conjugate Enzyme That Is Bound To The Plate
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THRESHOLD TEST SUITABILITY
AND
INTERPRETATION
• 0.5 EU/ml Pyrogen Control:
– Must Exhibit OD That Is > 1.6 Times The Negative
Control OD
• Positive Product Control Must Be > 0.5 EU/ml Std
• Samples Are Deemed Pyrogenic If The OD Readings
Are > 0.5EU/ml Standards.
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A3 ‐ Endotoxin Testing
Presented by: John Dubczak
COMPARATIVE IN-VITRO 48 HOUR
LER TESTS
50 EU/ml INOCULATION
Sample Endotoxin
NaCl control RSE
NaCl control E. cloacae
Tween Citrate RSE
Tween Citrate E. cloacae
Cust Protein RSE
Cust Protein E. cloacae
LAL
38.2661
47.0597
0.8894
51.8562
<0.5000
9.0792
MAT
+
+
+
-
IN-VITRO METHOD COMPARISONS
• Examined:
– LAL
– MAT
– Ligand Based Assay
• Utilized:
– RSE
– Enterobacter cloacae ATCC 7256 Endotoxin
– E. coli 055:B5 ATCC 120114 Endotoxin
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A3 ‐ Endotoxin Testing
Presented by: John Dubczak
LIGAND BASED ENDOTOXIN
DETECTION METHOD
• Add Standards And Samples / Spikes to Microplate
(Ligand Bound To Plate)
• Add Binding Buffer
• Shake and Incubate for 16 Hours
– 90 Minutes To 18 Hours Recommended
• Discarded Solution in The Microplate and Performed
3X Wash of The Wells.
LIGAND BASED ENDOTOXIN
DETECTION METHOD
• Introduced rFC Assay Reagent (Assay Buffer, Enzyme,
Substrate) to Each Well of the Microplate
• Initial Time Zero Read On Fluorescence Reader
(Excitation 380/20, Emission 440/40)
• Incubated for 90 Minutes (Longer Incubation Increases
Sensitivity)
• Obtained a Final Read.
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A3 ‐ Endotoxin Testing
Presented by: John Dubczak
NaCl DILUENT
Sample Value (EU/mL)
Diluent
LPS
Source
LAL
MAT
Ligand
0 hr
24 hr
0 hr
24 hr
0 hr
24 hr
NaCl
RSE
62.6
70.1
76.0
68.2
80.7
33.1
NaCl
E. cloacae
56.6
73.1
35.1
32.1
0.0
0.0
NaCl
E. coli
51.4
65.2
71.6
96.0
89.4
48.4
LER MATRIX:
TWEEN/CITRATE/SALINE
Sample Value (EU/mL)
Diluent
LPS Source
LAL
MAT
Ligand
0 hr
24 hr
0 hr
24 hr
0 hr
24 hr
Tween/Citrate
RSE
<0.5
<0.5
<25
<25
0
0.0
Tween/Citrate
E. cloacae
31.4
70.9
29.6
31.8
0.0
0.0
Tween/Citrate
E. coli
82.6
49.4
68.9
>100
66.8
45.5
14
A3 ‐ Endotoxin Testing
Presented by: John Dubczak
CUSTOMER PROTEIN IN
TWEEN/CITRATE/SALINE MATRIX
Sample Value (EU/mL)
Diluent
LPS Source
LAL
MAT
Ligand
0 hr
24 hr
0 hr
24 hr
0 hr
24 hr
Customer Protein
RSE
4.5
2.0
<25
28.4
2.7
5.0
Customer Protein
E. cloacae
52.0
72.6
38.4
53.8
6.0
7.5
Customer Protein
E. coli
23.8
23.4
53.9
43.5
23.4
10.3
30
15
A3 ‐ Endotoxin Testing
Presented by: John Dubczak
PARALLEL LAL/RABBIT PYROGEN
TEST PROTOCOL
• Utilized Tween/Citrate/Saline Buffer
• RSE and 5 Native Endotoxins
• Prepared Stock Concentration of Approximately 50
EU/ml
• Injection Dose of 1.0 ml / Kg
• Coordinated LAL and Rabbit Testing
– True Zero Time Testing
– RSE Decay in Tween/Citrate is Rapid
RSE DECAY IN TWEEN/CITRATE/SALINE MATRIX
6.0000
5.0000
4.0000
EU/ml
Trial 2
Trial 1
3.0000
2.0000
1.0000
0.0000
0
15
30
45
60
minutes
16
A3 ‐ Endotoxin Testing
Presented by: John Dubczak
ZERO TIME
(EU/ml)
24 HOURS at 2 - 8° C
(EU/ml)
RSE
58.0
<0.5
E. cloacae
60.7
55.4
E. coli
27.3
20.2
S. marcescens
59.2
63.2
P. aeruginosa
58.0
47.3
R. pickettii
37.2
21.9
33
ZERO TIME RABBIT DATA
34
17
A3 ‐ Endotoxin Testing
Presented by: John Dubczak
24 HOUR RABBIT DATA: 2 – 8°C STORAGE
35
36
18
A3 ‐ Endotoxin Testing
Presented by: John Dubczak
37
38
19
A3 ‐ Endotoxin Testing
Presented by: John Dubczak
ZERO
TIME
(EU/ml)
24 HOURS
at 2 - 8° C
(EU/ml)
48 HOURS
at 37° C
(EU/ml)
RSE
58.0
<0.5
ND
E. cloacae
60.7
55.4
0.8
E. coli
27.3
20.2
<0.5
S. marcescens
59.2
63.2
3.7
P. aeruginosa
58.0
47.3
0.8
R. pickettii
37.2
21.9
<0.5
39
RABBIT RESULTS: 48 HOUR STORAGE AT 37°C
40
20
A3 ‐ Endotoxin Testing
Presented by: John Dubczak
CONCLUSIONS
• Lipopolysaccharide is Unstable in the Tween/Citrate/Saline Matrix
• Native Endotoxins Exhibit Temperature Dependent Stability in the Tween/Citrate/Saline Matrix
• The LAL Assay Correlates with Rabbit Fever Response to Endotoxins
• LAL/MAT/Rabbit Assays (Biological Amplifcation Assays) Measure the Biological Activity of Endotoxins
– Not Absolute Concentrations
41
ACKNOLOWLEDGEMENTS
•
•
•
•
•
•
•
•
Brad Parish
Cynthia Black
Clare Lenich
Dr. Masakazu Tsuchiya
James Bunn
Jill Schultz
Daniel Fernandez
Barbara Edwards
21

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