Dr. Holger Grallert, 04/18/2012, Biotech Forum, analytica 2012

A Novel Method for Endotoxin Detection

Endotoxin Detection

Background

3

- 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.

Endotoxin

4

Methods of Endotoxin testing

(written information of a LAL manufacturer)

5

Test principle of LAL Test

homogeneous one step assay

Advantages:

- time to result 15 - 90 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 -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 GFactor G*

-1,3-Glucan

Read out

EndoLISA®

7

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

8

Test principle of EndoLISA®

heterogenous 3 steps assay

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 -1,3-glucan

Disadvantages:

-Time to result 3h 20 min

binding

wash

detection

Step 1

Step 2

Step 3

90 minutes37°Cshaking

3 times

90 minutes37°Cfluorescence

9

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

- 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

EndoLISA®

Performance

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EndoLISA standard curve

Standardkurve EU/ml 90min

EU/ml

rfu

90

min

-rfu

0m

in

0,001 0,01 0,1 1 10 100 1000100

1000

10000

100000

0.001 0.01 0.1 1 10 100 1000

100000

1000

LPS (EU/ml)

Rel

ativ

e F

luo

resc

ence

Un

its Lowest limit of

quantification

10000

EndoLISA® Sensitivity

LLOQ: 0.05 EU/ml

Dynamic range:up to 500 EU/ml

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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

Correlation between EndoLISA® and LAL Tests

Comparison study with different LPS samples

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Comparison to chromogenic kinetic LAL assays

92% and 89 % correlation to LAL assays from two different manufacturers

Results:Corre lation of EndoLISA to LAL (Endochrom e-K; Charles

River)

y = 1,0658x - 0,2216R2 = 0,9183

-3,00

-2,00

-1,00

0,00

1,00

2,00

-3,00 -2,00 -1,00 0,00 1,00 2,00

LAL (EU/m l)

En

do

LIS

A (

EU

/ml)

Corre lation of EndoLISA to LAL (Kinetic-QCL; Lonza)

y = 0,9368x - 0,063R2 = 0,8893

-3,00

-2,00

-1,00

0,00

1,00

2,00

-3,00 -2,00 -1,00 0,00 1,00 2,00

LAL (EU/m l)E

nd

oL

ISA

(E

U/m

l)

Manufacturer 1 Manufacturer 2

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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

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Testing of matrix interference

Example: Arginine solution (pH 8.0)

EndoLISA®: no test interference at 400 mM arginine

LAL Test: test interference above 50 mM arginine

0,1

1,0

10,0

100,0

1000,0

10 100 1000

Arginine (mM)

% s

pik

e re

cove

ry

EndoLISA

LAL

200%

50%

range of valid spike recovery

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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 NaCl100 mM NaCl100 mM NaCl100 mM NaCl

Water100 mM NaCl100 mM NaCl

50 mM100 mM1)

100 mM1)

100 mM1)

500 mM1)

100 mM1)

100 mM1)

12.5 mM12.5 mM

5 mM50 mM40 mM

100 mM1)

50 mM

4>8

>20>2

>12.51

>2

Salt NaClKCl

WaterWater

1M1M

0.5 M0.25 M

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Chaotropic agent

UreaGuanidinium chloride

WaterWater

6M1M

0.5 M0.05 M

1220

Organic solvent

MethanolEthanol2-Propanol DMSO

--------

20 %1)

30 %20 %10 %

5 %0.5 %0.2 %

2%

>460

1005

Detergent SDSCTABZwittergent 3-14Tween 20Triton X-100

WaterWaterWaterWaterWater

0.05 %0.004 %0.02 %

2 %0.02 %

0.001 %0.0001 %0.005 %

0.1 %0.005 %

50404

204

Chelator EDTA (pH 8.0)Citrate (pH 7.5)

WaterWater

0.4 mM10 mM

0.4 mM10 mM

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Protease inhibitor

BenzamidinePMSF

Water2-Propanol

100 mM1)

5 mM0.1 mM

< 0.05 mM>1000>100

Antibiotic RifampicinChloramphenicol

MethanolEthanol

3.5 mg/ml3.5 mg/ml

0.04 mg/ml0.1 mg/ml

10035

1) Highest concentration tested

EndoLISA®

vs. LAL TestSpike recovery in different matrices and agents

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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 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

suspension: 1,24 EU/mg

supernatant: 0,227 EU/mg

suspension: invalid spike

Custumer protein 4 1,2-Propandiol and boric acid

24.47 EU/mg invalid spike

- Comparable results to LAL- Superior performance in suspension or in „extreme“ buffers

* lowest detection level

Procedure:

- serial dilutions of sample in water were analyzed in EndoLISA and LAL

- validity of results were confirmed by sample spiking

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

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19

Outlook

• Validation for entrance in EP/USP

• Extension of protocol for blood/plasma samples

• Launch of homogeneous rFC assay

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