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

Endotoxin Detection Background 2

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 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

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 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

EndoLISA® 6

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

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

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

EndoLISA® Performance 10

Relative Fluorescence Units 0.001 0.01 0.1 1 10 100 1000 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

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

Comparison to chromogenic kinetic LAL assays Results: Manufacturer 1 Manufacturer 2  92% and 89 % correlation to LAL assays from two different manufacturers 13 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 14

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

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 % 0.0001 % 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

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: 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 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

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

Outlook Validation for entrance in EP/USP Extension of protocol for blood/plasma samples Launch of homogeneous rFC assay 19 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 20