1. for Endotoxin Detection
Dr. Holger Grallert, 04/18/2012, Biotech Forum, analytica 2012
A Novel Method
for Endotoxin Detection 1

2. Endotoxin Detection
Background 2

3. 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

4. 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 Lonza)
A summarizing study by Guilfoyle & Munson:
587 products were tested
78% of them were interfering with LAL
(written information of a LAL manufacturer) 4 4

5. Test principle of LAL Test  homogeneous one step assay
Advantages:
time to result  minutes (depending on test format and desired sensitivity)
high sensitivity of 0,005 EU/ml (depending on test format)
Disadvantages:
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
Factor C
Factor C*
Endotoxin
Factor B
Factor B*
Proclotting enzyme
Clotting enzyme
Coagulogen
Chromogenic substrate
Coagulin (gel clot)
turbidometric
Color development
Factor G
Factor G*
b-1,3-Glucan
Read out 5 5

6. EndoLISA® 6

7. EndoLISA® Heterogeneous assay:
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 7

8. Test principle of EndoLISA®  heterogenous 3 steps assay
binding
wash
detection
Step 1
Step 2
Step 3
90 minutes
37°C
shaking
3 times
fluorescence
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
no side reaction by b-1,3-glucan
Disadvantages:
Time to result  3h 20 min 8 8

9. What are the differences between EndoLISA® and the commonly used LAL Test ?
uses the enzymes of the blood clotting cascade of the horse shoe crab for the detection of endotoxin
derived from animal source
homogeneous assay
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
use of recombinant proteins
heterogeneous assay 9 9

10. EndoLISA®
Performance 10

11. Relative Fluorescence Units
100000
1000
LPS (EU/ml)
Relative Fluorescence Units
Lowest limit of quantification
10000
EndoLISA® Sensitivity
LLOQ:
0.05 EU/ml
Dynamic range:
up to 500 EU/ml 11 11

12. 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
E. coli O128:B12
E. coli K235 --
E. coli EH100
Ra mutant (rough strain)
Phenol/Chloroform/Petrolether
E. coli F583
Rd mutant (rough strain)
Salmonella minnesota
Re mutant
Salmonella enteritidis
Salmonella abortus equi
Salmonella thyphimorium
Klebsiella pneumoniae
Serratia marcescens
Pseudomonas aeroginosa
E.coli J5
Rc mutant (rough strain)
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 12

13. Comparison to chromogenic kinetic LAL assays
Results:
Manufacturer 1
Manufacturer 2
 92% and 89 % correlation to LAL assays from two different manufacturers 13 13

14. 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 14

15. Testing of matrix interference
Example: Arginine solution (pH 8.0)
200%
50%
range of valid spike recovery
EndoLISA®: no test interference at 400 mM arginine
LAL Test: test interference above 50 mM arginine 15 15

16. EndoLISA® vs. LAL Test Spike recovery in different matrices and agents
Substance
Solvent
EndoLISA
LAL assay
Improvement
Factor
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)
100 mM NaCl
Water
50 mM
100 mM1)
500 mM1)
12.5 mM
5 mM
40 mM 4
>8
>20
>2
>12.5 1
Salt
NaCl
KCl 1M
0.5 M
0.25 M 2
Chaotropic agent
Urea
Guanidinium chloride 6M
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
0.05 %
0.004 %
0.02 %
2 %
0.001 % %
0.005 %
0.1 % 50 40
Chelator
EDTA (pH 8.0)
Citrate (pH 7.5)
0.4 mM
10 mM
Protease inhibitor
Benzamidine
PMSF
0.1 mM
< 0.05 mM
>1000
>100
Antibiotic
Rifampicin
Chloramphenicol
3.5 mg/ml
0.04 mg/ml
0.1 mg/ml 35
1) Highest concentration tested 16 16

17. suspension: invalid spike
EndoLISA®
Real life samples
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
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: ,512 EU/mg
suspension: ,24 EU/mg
supernatant: ,227 EU/mg
suspension: invalid spike
Custumer protein 4
1,2-Propandiol and boric acid
24.47 EU/mg
invalid spike
Procedure:
serial dilutions of sample in water were analyzed in EndoLISA and LAL
validity of results were confirmed by sample spiking
* lowest detection level
Comparable results to LAL
Superior performance in suspension or in „extreme“ buffers 17 17

18. 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 18

19. Outlook Validation for entrance in EP/USP
Extension of protocol for blood/plasma samples
Launch of homogeneous rFC assay 19 19

20. 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 20