Dr. Holger Grallert, 04/18/2012, Biotech Forum, analytica 2012
A Novel Method
for Endotoxin Detection
Endotoxin Detection
Background
Endotoxin
-
Endotoxins are breakdown products of Gram-negative bacteria
Lipopolysaccharide of the outer cell membrane
Heterogeneous substance class
Ubiquitary occurance
Highly toxic
Triggers severe physiological reactions (fever, septic shock)
 Testing for endotoxin is mandatory
for the confirmation of safe
manufacturing and release of
pharmaceutical products as well as
highly important in medical- and life
science research.
3
Methods of Endotoxin testing
 Since 1940s:
Rabbit Pyrogen Test
 Since 1970s:
Limulus Amoebocyte Lysate (LAL) Test
“Almost all products interfere to a certain extent with the LAL test”
(written
information
of aLonza)
LAL manufacturer)
(written
information
A summarizing study by Guilfoyle & Munson:
• 587 products were tested
• 78% of them were interfering with LAL
4
Test principle of LAL Test
 homogeneous one step assay
Advantages:
Endotoxin
b-1,3-Glucan
Factor C
- high sensitivity of 0,005 EU/ml (depending on
test format)
Factor C*
Factor B
- time to result
 15 - 90 minutes
(depending on test format and desired
sensitivity)
Factor B*
Factor G*
Factor G
Disadvantages:
Proclotting
enzyme
Read out
Clotting
enzyme
Coagulogen
Coagulin (gel clot)
turbidometric
Chromogenic
substrate
Color development
- Direct contact of sample matrix with detection
enzymes  interference of matrix components
with enzyme reaction
- sample dilution necessary to diminish
interference  decrease in sensitivity
- side reaction by b-1,3-glucan
5
EndoLISA®
EndoLISA®
is the first, commercially available solid-phase based method for
endotoxin detection
Heterogeneous assay:
- which uses a highly stable, LPS specific bacteriophage derived
protein for capturing Endotoxin on microtiter plate
- which uses recombinant Factor C coupled with a fluorogenic
substrate for the detection of endotoxin
7
Test principle of EndoLISA®
 heterogenous 3 steps assay
Step 1
binding
90 minutes
37°C
shaking
Step 2
wash
Advantages:
- no contact of sample matrix with enzyme of
the detection reaction  enzyme reaction
runs always at optimal conditions
- highly stable endotoxin binding protein
allows endotoxin capturing out of complex
matrices  less dilution necessary
3 times
- no side reaction by b-1,3-glucan
Disadvantages:
Step 3
- Time to result  3h 20 min
detection
90 minutes
37°C
fluorescence
8
What are the differences between EndoLISA® and the
commonly used LAL Test ?
LAL Test:
- uses the enzymes of the blood clotting
cascade of the horse shoe crab for the
detection of endotoxin
EndoLISA®:
- uses the first enzyme of the blood clotting
cascade of the horse shoe crab for the
detection of endotoxin
- use of the Hyglos Phage Ligand Technology
for specific Endotoxin capturing
- derived from animal source
- use of recombinant proteins
- homogeneous assay
- heterogeneous assay
9
EndoLISA®
Performance
EndoLISA® Sensitivity
EndoLISA standard curve
LLOQ:
0.05 EU/ml
Dynamic range:
up to 500 EU/ml
Relative Fluorescence Units
rfu 90min-rfu 0min
100000
100000
Lowest limit of
quantification
10000
10000
Standardkurv
1000
1000
100
0,001
0.001
0,01
0.01
0,1
0.1
1
1
EU/ml
10
10
100
100
1000
1000
LPS (EU/ml)
11
Correlation between EndoLISA® and LAL Tests
Comparison study with different LPS samples
LPS
typ
Preparation method
E. coli O111:B4
wt
Phenol extraction
E. coli O26:B6
wt
Phenol extraction
E. coli O128:B12
wt
Phenol extraction
E. coli K235
--
Phenol extraction
E. coli EH100
Ra mutant (rough strain)
Phenol/Chloroform/Petrolether
E. coli F583
Rd mutant (rough strain)
Phenol/Chloroform/Petrolether
Salmonella minnesota
wt
Phenol extraction
Salmonella minnesota
Re mutant
Phenol/Chloroform/Petrolether
Salmonella enteritidis
wt
Phenol extraction
Salmonella abortus equi
wt
Phenol extraction
Salmonella thyphimorium
wt
Phenol extraction
Klebsiella pneumoniae
wt
Phenol extraction
Serratia marcescens
wt
Phenol extraction
Pseudomonas aeroginosa
wt
Phenol extraction
E.coli J5
Rc mutant (rough strain)
Phenol/Chloroform/Petrolether
- serial dilutions of LPS samples were prepared in water
- samples were measured with EndoLISA and LAL Test of two different manufacturers
- log values of determined EU/ml were plotted against each other
12
Comparison to chromogenic kinetic LAL assays
Results:
Correlation of EndoLISA to LAL (Endochrom e-K; Charles
River)
Correlation of EndoLISA to LAL (Kinetic-QCL; Lonza)
Manufacturer 1
Manufacturer 2
2,00
y = 0,9368x - 0,063
R2 = 0,8893
2,00
y = 1,0658x - 0,2216
R2 = 0,9183
1,00
EndoLISA (EU/ml)
EndoLISA (EU/ml)
1,00
0,00
-1,00
-1,00
-2,00
-2,00
-3,00
-3,00
0,00
-2,00
-1,00
0,00
LAL (EU/m l)
1,00
2,00
-3,00
-3,00
-2,00
-1,00
0,00
1,00
2,00
LAL (EU/m l)
 92% and 89 % correlation to LAL assays from two
different manufacturers
13
Testing of matrix interference
Procedure:
• serial dilutions of samples were made in water
• samples were spiked with 5 EU/ml of standard LPS
• EU/ml content of sample were determined in EndoLISA and LAL Test
• percentage of spike recovery relating to nominal concentration was calculated
Criterion of validity:
spike recovery is in the range of 50% - 200% of the nominal spiked concentration
14
Testing of matrix interference
Example: Arginine solution (pH 8.0)
1000,0
200%
range of valid
spike recovery
% spike recovery
100,0
50%
EndoLISA
10,0
LAL
1,0
0,1
10
100
1000
Arginine (mM)
EndoLISA®: no test interference at 400 mM arginine
LAL Test: test interference above 50 mM arginine
15
EndoLISA®
vs. LAL Test
Substance
Solvent
EndoLISA
LAL assay
Improvement
Factor
100 mM NaCl
100 mM NaCl
100 mM NaCl
100 mM NaCl
Water
100 mM NaCl
100 mM NaCl
50 mM
100 mM1)
100 mM1)
100 mM1)
500 mM1)
100 mM1)
100 mM1)
12.5 mM
12.5 mM
5 mM
50 mM
40 mM
100 mM1)
50 mM
4
>8
>20
>2
>12.5
1
>2
Buffer/pH
Acetate (pH 4.0)
Acetate (pH 5.0)
MES (pH 6.0)
Potassium phosphate (pH 7.2)
Imidazole (pH 7.4)
HEPES (pH 7.5)
Sodium borate (pH 9.0)
Salt
NaCl
KCl
Water
Water
1M
1M
0.5 M
0.25 M
2
4
Chaotropic
agent
Urea
Guanidinium chloride
Water
Water
6M
1M
0.5 M
0.05 M
12
20
Organic
solvent
Methanol
Ethanol
2-Propanol
DMSO
-----
20 %1)
30 %
20 %
10 %
5%
0.5 %
0.2 %
2%
>4
60
100
5
Detergent
SDS
CTAB
Zwittergent 3-14
Tween 20
Triton X-100
Water
Water
Water
Water
Water
0.05 %
0.004 %
0.02 %
2%
0.02 %
0.001 %
0.0001 %
0.005 %
0.1 %
0.005 %
50
40
4
20
4
Chelator
EDTA (pH 8.0)
Citrate (pH 7.5)
Water
Water
0.4 mM
10 mM
0.4 mM
10 mM
1
1
Protease
inhibitor
Benzamidine
PMSF
Water
2-Propanol
100 mM1)
5 mM
0.1 mM
< 0.05 mM
>1000
>100
Antibiotic
Rifampicin
Chloramphenicol
Methanol
Ethanol
3.5 mg/ml
3.5 mg/ml
0.04 mg/ml
0.1 mg/ml
100
35
Spike recovery in
different matrices
and agents
1) Highest concentration tested
16
EndoLISA®
Real life samples
Procedure:
- serial dilutions of sample
in water were analyzed in
EndoLISA and LAL
- validity of results were
confirmed by sample
spiking
Comparison of protein samples to LAL
Sample
Solvent
EndoLISA®
LAL
BSA fraction V, very low endotoxin
10 mM Tris pH 8.0
0,05 EU/mg
0,1 EU/mg
HSA fraction V
10 mM Tris pH 8.0
0,9 EU/mg
0,9 EU/mg
Ovalbumin
Water
0,3 EU/mg
0,42 EU/mg
Custumer protein 1
Unknown
< 0,25 EU/ml*
< 0,125 EU/ml*
Custumer protein 2
PBS buffer
192.3 EU/mg
188.3 EU/mg
Custumer protein 3
350 mM
Argininphosphate
Buffer, pH 7.5
supernatant:
0,512 EU/mg
supernatant:
0,227 EU/mg
suspension:
1,24 EU/mg
suspension:
invalid spike
1,2-Propandiol and
boric acid
24.47 EU/mg
invalid spike
Custumer protein 4
* lowest detection level
- Comparable results to LAL
- Superior performance in suspension or in „extreme“
buffers
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Summary
• Sensitivity of 0.05 EU/ml
• Dynamic range up to 500 EU/ml
• Good correlation to LAL
• Superior performance in complex matrix formulations
• Minimal dilution necessary
• No use of animal source
18
Outlook
• Validation for entrance in EP/USP
• Extension of protocol for blood/plasma samples
• Launch of homogeneous rFC assay
19
Thank you for your attention!
For more information about EndoLISA® and Hyglos Endotoxin
Removal products EndoTrap ® visit:
or the Hyglos booth at
the analytica 2012:
Hall A3, booth 262