Pre-Conference Workshop on

PDA: A Global
Bacterial Endotoxin Testing, 17 Feb 2014

Recombinant Factor C
Association
- EndoLISA ®

Karolina Heed, Hyglos GmbH

Recombinant Factor C - EndoLISA®

Introduction
Test principle
Specificity
Specifications
Advantages
Correlation to other methods
Applications
Validation studies
Kit components and equipment
Technical resources

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 Recombinant Factor C - EndoLISA®
INTRODUCTION

Hyglos GmbH -
a company dedicated to innovation in endotoxin detection
Est. 2009 in Bernried at Lake Starnberg (Munich South)
R&D, Production, QC, Logistics, M&S Units
DIN EN ISO 9001:2008 and DIN EN ISO 13485:2003+AC:2007
Proprietary Phage Recombinant Protein Technology, 8 patent families
Development, fermentation and purification of recombinant proteins and enzymes
Test kit development in close collaboration with MicroCoat GmbH (GLP/ISO)
Focus areas: Endotoxin Detection
Endotoxin Removal
Endotoxin-free Reagents and Accessories
Food and Clinical Bacteria Testing
Decolonization of bacteria (e.g. MRSA)
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 Recombinant Factor C - EndoLISA®
INTRODUCTION

Pyrogen/Endotoxin detection milestones:

1925 Florence Seibert develops the rabbit pyrogen test
1964 Frederik Bang and Jack Levin develop the LAL test
1974 Kobayashi obtains lysate from Tachypleus (TAL reagent)
1979 Mahalanabis obtains lysate from Carcinoscorpius rotundicauda
1981 Iwanaga describes the alternative activation pathway through β-1,3-D-glucans
1995 Whole-blood model (University of Konstanz)
2003 PyroGene™ Recombinant Factor C (rFC) Endotoxin Detection Assay introduced
by Cambrex  rFC derived from Carcinoscorpius rotundicauda (Ding et al.)
2011 EndoLISA® using a LPS-specific phage protein for capture and rFC for detection
introduced by Hyglos  rFC derived from Tachypleus tridentatus (Iwanaga, Muta et al.)

Source: M. Rieth, 2012

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 Recombinant Factor C - EndoLISA®
INTRODUCTION

The EndoLISA development project was

funded by the German Federal Ministry of
Economics and Energy (ZIM - Innovation
Program):

Munich, Germany, 1 September, 2011:

“Hyglos today has the pleasure to present EndoLISA ®,
the world’s first commercially available ELISA-based
endotoxin detection method. EndoLISA® is the result of
several years of research and test development aimed
at establishing a better performing alternative to LAL.“

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Recombinant Factor C - EndoLISA®
TEST PRINCIPLE

EndoLISA® relies on
two principal reactions:

1. LPS Binding
2. Endotoxin Detection

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 Recombinant Factor C - EndoLISA®
TEST PRINCIPLE

1. LPS Binding:
- Microtiter wells pre-coated with LPS-binding Phage protein (recombinant)
- Specific quantitative binding of LPS

Phages infecting an E. coli

Prof. Wanner/Hyglos
©

Rec. Phage Protein

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 Recombinant Factor C - EndoLISA®
TEST PRINCIPLE

2. Endotoxin detection:

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Recombinant Factor C - EndoLISA®
TEST PRINCIPLE

What happens inside of the

EndoLISA® microplate well?

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 Recombinant Factor C - EndoLISA®
TEST PRINCIPLE
Step 1

LPS binding 90 min.

Phage recombinant protein 37°C
shaking
Matrix component

LPS
Step 2
- Matrix removal
- Wash 3 times

Step 3
Endotoxin 90 min.
Detection 37°C
Fluorescence

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 Recombinant Factor C - EndoLISA®
SPECIFICITY

Specificity and Binding Site Analysis of the Recombinant Phage

Protein (Grallert et al.):

LPS source Type EndoLISA® result

E. coli O11:B4 WT +
E. coli EH100 Ra mutant +
E. coli J5 Rc mutant +
E. coli F583 Rd mutant +
Salmonella minnesota Re mutant +
Lipid A (Salmonella) Synthetic -
Lipid A (E. coli) Synthetic -

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 Recombinant Factor C - EndoLISA®
SPECIFICITY

Since Factor C has a LPS binding region that exhibits exceptionally high affinity for
Lipid A, it is deduced that the binding site of the phage protein in EndoLISA lays in
the conserved Kdo region of the LPS molecule (Grallert et al.):

Smooth strains Rough strains

Putative binding
region of the
EndoLISA phage
protein

Lipopolysaccharide (mod. Alexander and Rietschel, 2001)

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 Recombinant Factor C - EndoLISA®
SPECIFICATIONS

EndoZyme® EndoLISA®
Specificity: LPS (single) LPS (double)

Solid phase: No, homogeneous assay Yes, LPS-specific phage protein

Detection reagent: rFC rFC

Reaction with β-Glucan: No No

Measurement range: 0 to 50 EU/ml 0 to 500 EU/ml

Sensitivity: 0.005 EU/ml 0.05 EU/ml

Salt/Detergent tolerance: As LAL, dilution needed High, undiluted e.g. 6M Urea, 2% Tween 20

Wash step: No Yes

Incubation step: No Yes

Time-to-result: 1.5h 3h

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 Recombinant Factor C - EndoLISA®
SPECIFICATIONS

Standard curve of the EndoLISA test; concentrations of the LPS

standard are plotted against the relative fluorescence signal:

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 Recombinant Factor C - EndoLISA®
ADVANTAGES

Highly stable endotoxin binding phage protein:

 Endotoxin capturing out of complex matrices possible

No contact of sample matrix with the detection enzyme (rFC):

 Enzyme reaction running at optimal conditions

Removal of potentially interfering substances:

 Minimized need for dilution

Double LPS-specificity:
 Side reaction by β-1,3-glucan excluded

Heterogeneous test format:

 High potential for developing demasking protocol

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 Recombinant Factor C - EndoLISA®
EndoLISA® vs. LAL - Maximum concentrations with valid spike ADVANTAGES
recovery in different matrices and agents (Grallert et al.):
Substance Solvent/ Diluent EndoLISA LAL Improvement
Factor

Buffer/pH Acetate (pH 4.0) 100 mM NaCl 50 mM 12.5 mM 4

Acetate (pH 5.0) 100 mM NaCl 100 mM1) 12.5 mM >8
MES (pH 6.0) 100 mM NaCl 100 mM1) 5 mM >20
Potassium phosphate (pH 7.2) 100 mM NaCl 100 mM1) 50 mM >2
Imidazole (pH 7.4) Water 500 mM 40 mM >12.5
HEPES (pH 7.5) 100 mM NaCl 100 mM1) 100 mM1) 1
Sodium borate (pH 9.0) 100 mM NaCl 100 mM1) 50 mM >2

NaCl Water 1M 0.5 M 2

Salt KCl Water 1M 0.25 M 4

Chaotropic agent Urea Water 6M 0.5 M 12

Guanidinium chloride Water 1M 0.05 M 20

Methanol - 20 %1) 5% >4

Organic solvent Ethanol - 30 % 0.5 % 60
2-Propanol - 20 % 0.2 % 100
DMSO - 10 % 2% 5

SDS Water 0.05 % 0.001 % 50

Detergent CTAB Water 0.004 % 0.0001 % 40
Zwittergent 3-14 Water 0.02 % 0.005 % 4
Tween 20 Water 2% 0.1 % 20
Triton X-100 Water 0.02 % 0.005 % 4

Chelator EDTA (pH 8.0) Water 0.4 mM 0.4 mM 1

Citrate (pH 7.5) Water 10 mM 10 mM 1

Protease inhibitor Benzamidine Water 100 mM1) 0.1 mM >1000

PMSF 2-Propanol/Water 5 mM < 0.05 mM >100

Antibiotics Rifampicin Methanol/Water 3.5 mg/ml 0.04 mg/ml 100

Chloramphenicol Ethanol/Water 3.5 mg/ml 0.1 mg/ml 35

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 Recombinant Factor C - EndoLISA®
ADVANTAGES

Removal efficiency of interfering substances - Testing of matrix

interference:
Benzamidine DMSO
1000,0 spike re cov e ry in DM SO

1000,0
% spike recovery

100,0
100,0

% spike recovery
EndoLISA
LAL EndoLISA
10,0
LAL
10,0

1,0

1,0 0,1
0,01 0,10 1,00 10,00 100,00 0,1 1 10 100

benzamidine (mM) DMSO (% )

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 Recombinant Factor C - EndoLISA®
CORRELATION TO OTHER METHODS
Comparison study with different LPS samples:

LPS Type Preparation method Serial dilutions of LPS samples

were prepared in water.
E. coli O111:B4 wt Phenol extraction
E. coli O26:B6 wt Phenol extraction Samples were measured with
E. coli O128:B12 wt Phenol extraction EndoLISA, LAL and rFC assay of
E. coli K235 -- Phenol extraction two different manufacturers.
E. coli EH100 Ra mutant (rough strain) Phenol/Chloroform/Petrolether
Log values of determined EU/ml
E. coli F583 Rd mutant (rough strain) Phenol/Chloroform/Petrolether
were plotted against each other.
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

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 Recombinant Factor C - EndoLISA®
CORRELATION TO OTHER METHODS

EndoLISA® and LAL (Corr.: 91.8%):

EndoLISA® and PyroGene rFC (Corr.: 91.4%):

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 Recombinant Factor C - EndoLISA®
APPLICATIONS
Selecting the ideal endotoxin test for your samples

Increasing complexity of Matrix/Sample

Water Buffer / Complex matrix Masked LPS

EndoZyme® EndoLISA® EndoLISA® SP

Introduced 2013 Introduced 2011 Planned introduction 2014

Quality control, research, health and safety Sample preparation for

Pharmacopoeia de-masking of endotoxin

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 Recombinant Factor C - EndoLISA®
APPLICATIONS

Selection samples processed by EndoLISA® users:

Albumin from human Plasma [25%] Monoclonal antibody (10 mg/ml)

Bacteriophage [Endotoxin-free water with 0.9% NaCl, pH 7] Monoclonal antibody (49 mg/ml)

Bovine Serum Albumin [40%] Mouse plasma

Brucella abortus antigen Ocriplasmin [5mM citric acid pH 3.1, 6% Mannitol]

Buffer (48.5% MeOH, 44% EtOH, 7.5% H2O) Protein (120 mg/ml) [Propylene glycol, boric acid]

Cholesteryl hemisuccinate Protein (0.1 mg/ml) [DPBS, Glycerol, pH 7.2]

Clay samples diluted in endotoxin-free water (100 mg/ml) Protein [50 mM NaHPO4, 200 mM NaCl, pH 8]

Factor VIII from human Plasma Protein, recombinant full-length human S100A12

Fucosylated protein (15 mg/ml) [PBS, pH 7.0] Protein, recombinant (1 mg/ml) [20 mM Tris-Cl, 0.2 µM CaCl 2
Environmental water samples (rivers, lakes, tap-water,
Protein, recombinant [350 mM arginine phosphate, pH 7.5]
fountain water, table water, etc) filtered by a 0.45 μm membrane-filter
Hay dust suspensions Protein, recombinant [30 mM citrate]

Herbal dry extract powder (20 mg/ml) [Ethanol 50% (v/v)] Proteoliposomes [PBS, detergents]

Laminin (0.1 mg/ml) [PBS, 10% Glycerol, 0.02 % NaN 3] Recombinant Mammary Serum Amyloid A3 Protein

Membrane protein [Buffer containing detergents and sarcosyl] Recombinant Serum Amyloid A

Metalloprotease [50 mM Hepes, 1M NaCl, 2 mM CaCl 2, pH 8.2] Rifampicin (5 mg/ml)

Metal working fluids Surface-retained organic matter

Mitogen-activated protein 1 (MAP-1) [0.9 % NaCl, 1 mM CaCl2] Vaccines [Detergents, Glucans, organic solvents, proteolytic
enzymes, high salt concentrations]

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 Recombinant Factor C - EndoLISA®
APPLICATIONS

Measuring protein samples - EndoLISA® and LAL in comparison:

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
Customer protein 1 Unknown < 0.25 EU/ml* < 0.125 EU/ml*
Customer protein 2 PBS buffer 192.3 EU/mg 188.3 EU/mg

Customer protein 3 350 mM Supernatant: Supernatant:

Argininphosphate 0.512 EU/mg 0.227 EU/mg
Buffer, pH 7.5
Suspension: Suspension:
1.24 EU/mg invalid spike
Customer protein 4 1,2-Propandiol and 24.47 EU/mg Invalid spike
boric acid

Comparable results to LAL

Superior performance in suspension or in „extreme“ buffers

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 Recombinant Factor C - EndoLISA®
APPLICATIONS
Endotoxin: 5 EU/mL CSE
Detection: EndoLISA®, Gel-Clot LAL
Sample: 15 mg/ml Rifampicin in 100% Methanol

Antibiotics - Rifampicin: EndoLISA® LAL

Required
Dilution 1:12 dilution Dilution 1:600
in 30 % MeOH in H2O

Detection limit: Detection limit:

0.04 EU/mg 2.00 EU/mg
Molecular formula: C43H58N4O12
Molecular weight: 823.0
water solubility: 1.4 mg/mL(at 25 °C)
QC using Gel-Clot LAL could result in
patient exposure > 1000 EU

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 Recombinant Factor C - EndoLISA®
VALIDATION STUDIES

Validated parameters:

Sensitivity (LOD, LLOQ)

Precision (intra-assay, inter assay)
Accuracy (intra-assay, inter assay, recovery)
Consistence (Lot-to-lot, operator to operator, day to day)
Measurement Hook Effect

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Recombinant Factor C - EndoLISA®
VALIDATION STUDIES

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 Recombinant Factor C - EndoLISA®
KIT COMPONENTS AND EQUIPMENT

Included: EndoLISA® Plates (16 well-strips)

Water (endotoxin-free)
Additional material needed:
Binding Buffer
Fluorescence Microplate Reader
Endotoxin Standard (1000 EU; E. coli O55,:B5) Incubator 37°C

Wash Buffer Plate shaker, 0-800 rpm

Vortexer, 0-1500 rpm
Enzyme (Recombinant Factor C)
Pipettes and tips (endotoxin-free)
Substrate Glass Test Tubes (endotoxin-free)
Reagent reservoir (endotoxin-free)
Assay Buffer
Cover Foils

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Recombinant Factor C - EndoLISA®
EQUIPMENT
Fluorescence Microplate Reader:

Microplate fluorescence readers from different suppliers may be used for reading of
EndoLISA® and EndoZyme® results.

The EndoLISA® and EndoZyme® assays have been developed and validated on the
FLx800™ Fluorescence Microplate Reader reader supplied by BioTek Instruments
GmbH. Using an instrument from another supplier, the parameters stated in the
package insert should be used and validated .

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 Recombinant Factor C - EndoLISA®
TECHNICAL RESOURCES

www.endolisa.com
Manuals, Video User Guide (YouTube), Application Notes, Certificates of Analysis,
Validation Certificate, MSDS, Software Templates, FAQ, Reference Customers and
Publications
Complete technical support contact information

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Recombinant Factor C - EndoLISA®

Thank you!
[email protected]

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