Resolving LAL Test Interference Problems For Accurate Detection of Endotoxin in Snake Antivenom.pdf

Resolving LAL Test Interference Problems For Accurate Detection of Endotoxin in Snake Antivenom.pdf

1. Resolving LAL Test Interference Problems for Accurate
Detection of Endotoxin in Snake Antivenom
Dr. Norhan Saif Sherabah, PhD, TQMD
Building1 IPC Manager, VACSERA
Mansoura University, Faculty of Pharmacy
First Pharmaschool International Conference
of Pharmaceutical Sciences
Februrary,15-16,2016

2. 1.Preliminary Screening /Interference Assay is performed on
one batch of product to determine a compatible dilution,
2.Inhibition Enhancement / Validation Study where three
batches of product were tested at this compatible dilution.
GM of endotoxin standard series in both sample dilution
and LRW can be achieved with the same efficiency.
Sorting out the interfering factors before validating the gel
clot LAL test to avoid false positive results.
Aim of Oral Presentation:
2
Approach:

3. 3
Define Polyvalent Snake Antivenom
Quality, safety and efficacy problems
What is endotoxin ?
Why Bacterial Endotoxin Testing?
Techniques of Endotoxin Detection in Snake Antivenom
Resolving LAL interference
FDA Gel Clot LAL Guideline-Key Points
Performing Gel Clot Test
Topics of Presentation
References:
Sorting out of interference in detection of endotoxins in antitoxic sera preparations
3rd FUE International Conference of Pharmaceutical Sciences, February 9-11, 2015

4. A biological preparation of either whole antibodies(IgG)
or antibody fragments [F(ab’)2] against the venom's
active molecule which can then be harvested from the
animal's blood and used to treat envenomation against
snake venoms, like :
Cerastes cerastes,
Naja haje (cobra),
Naja nigricollis
Polyvalent Snake Antivenom
4
10R Polyvalent Snake Antivenom Vial

5. Why are antisera essential?
No alternative successful therapy
High degree of mortality and
morbidity in the absence of
treatment.
The diseases in which they are
used represent a heavy toll of human
suffering.
Largely affects children and
farmers
in rural communities.
5
Snake bite victim in Ecuador
Photo: D.A. Warrell

6. Immunization of
horses (or sheep)
Bleeding and
separation of plasma
Selection and
collection of venom
Quality control for
endotoxin
Final product
Fractionation and
purification of IgG or (Fab')
2
Production of Animal Antivenom
6

7. Poor
Physicochemical
Characteristics of
Antisera
Protein
Aggregates
Bacterial
Lipopolysaccharides
Poor
Stability of
Proteins
High
Protein
Load
Residual
and non-IgG
Proteins and
Fragments
7
Quality, safety and efficacy problems

8. Pyrogen is a substance that produces a fever when injected
into blood or cerebrospinal fluid.
Endotoxinis the natural form of lipopolysaccharide occurring in
the outer layer of the bi-layered gram negative bacterial cell
wall.
All endotoxins are pyrogens but not all pyrogens are endotoxin.
Endotoxinis a potent pyrogen.
What is endotoxin ?
8
Section through membrane of gram-negative bacteria (Caroff et al., 2003)

9. 9
Structure and Chemical Nature of Endotoxin
1. Schematic Representation of the Chemical Structure of LPS
2. Structures of Endotoxin Aggregates in Aqueous Solutions

10. 10
Endotoxin
Tumor Necrosis
Factor
Interleukins
(1,6,8)
Clinical Associations of Endotoxin:
Urticaria, itching, fever, vomiting,
headache, bronchospasm,
hypotension, angioedema.
Macrophages
Cutaneous reaction
Photo:InstitutoButantan

11. Sources of Endotoxin Contamination
Airborne
bacteria
Personnel
Behavior
Raw Materials
as contaminated
plasma
Waterborne
Bacteria
Inadequate Cleaning
& Disinfection
Contaminated
Surfaces &
equipments
High Levels of
Relative
Humidity
Poor
implementation of
GMP
11

12. 12
Why Bacterial Endotoxin Testing?
Heavy demand for Large Volume
Parenteral therapy;
Need for an official USP compendia
test to ensure the absence of
endotoxins in intravenous
preparations;
Monitoring of high purity water as a
prime or processing agent for all
biologicals, drugs and medical
devices.

13. Techniques of Endotoxin Detection in Polyvalent
Snake Antivenom
The first USP official rabbit pyrogen test was in 1942.
limited due to labour intensity and cost.
1970 -Bang and Levin prepared the first Lysate and the rate of
reaction was dependent on the concentration of endotoxin present.
LAL is Limulus Amebocyte Lysate and is extracted from the
amebocytes of the Limulus crab (Horseshoe crab).
In 1987,FDA established guidelines for LAL Test of pharmaceutical
and medical devices.
The LAL test has evolved over time
to replace the rabbit pyrogenicity test
13

14. Disinfection of cardiac
membrane
Making of Limulus Amebocyte Lysate Reagent
Donation Bleeding of Crabs
Centrifugation to
separate the serum
Cell lysing
Lysate is allowed to
age at 2-8 degrees C
Filling, Lyophilization , Crimping
14

15. Principal of LAL: Endotoxin Initiated Cascade Enzymatic Rx
Endotoxin + Mg++
Factor CActivated Factor C
Factor BActivated Factor B
Proclotting EnzymeClotting Enzyme
CoagulagenCoagulin gel
(1-3)-ß-D-Glucan
Factor GActivated Factor G
15
The Reaction Pathways of Endotoxins and ß-Glucan with LAL Reagents.

16. Resolving LAL Interferences
Inhibition
Enhancement
Interference:asignificantdifferencebetweentheend
pointsofPWCandPPCusingstandardendotoxin.
1.Inhibition:amaterialinterfereswiththeabilityoftheLAL
reagenttoreactwithendotoxin,causingunderestimationof
theamountofendotoxinpresent.
2.Enhancement:interferencethatincreasesthesensitivityof
theassay,resultinginoverestimationoftheendotoxin
concentrationofthesample.
Enhancementislessdangerousthaninhibition.
16
ThefirststepincreatingaLALforapharmaceuticalentityis
identificationofacompatibleconcentrationforroutinetesting

17. 1. Calculation of Endotoxin Limit (EL) =K/M
17
Quantities of endotoxins are expressed in defined Endotoxin
Unit (EU).
1 EU=1IU
K = Minimum Pyrogenic Dose .
M = Maximum Dose of the drug substance per kg per hour.
Table: Endotoxin Limits of Various Parenteral Products
Type of Product Route of AdministrationK per Kg
All parenteral preparations Intrathecal 0.2
Radiopharmaceuticals Intravenous 2.0
All parenteral preparations
except radiopharmaceuticals
All parenteral routes except
Intrathecal
5.0

18. (1)A new batch of LAL ORchange in the experimental conditions.
(2)Select NID (non-interfering dilution).
(3)N.B: Don’t use the 1st two fold dilution which performs properly.
ideal concentration is the 2nd or 3rd positive PPC
3.Validation of the LAL Test should include :
(3.1) Confirmation of Lysate Sensitivity () :GM = antilog (Σe/f)
(3.2) Inhibition and Enhancement Tests (2 phases):
2. Calculation of MVD = E.L ×Concentration of sample / ()
MVD:Maximum allowable dilution of a specimen at which the
E.L can be determined,
If the undiluted specimen is inhibitory, the product can be diluted,
but not exceeding MVD.
Lysate sensitivity (): Minimum amount of endotoxin for clot
formation under standard conditions.

19. 19
Should be performed on clean, endotoxin free sample
Phase II (Inhibition Enhancement / Validation Study) :
Performed on one batch to determine a compatible test dilution.
Two identical series of product dilutions (twofold dilutions), one spiked
with 2λ, and one left unspiked. The result of Phase I will tell you the
non‐interfering dilution (NID) of the product, which is used for Phase II.
The non‐interfering dilution is the first set of PPC that shows a gel.
Two identical series of endotoxin dilutions bracketing λ; One
prepared in LRW and the other in product diluted to the proposed test
dilution.
Successful validation requires that both series confirm label claim
(Geometric mean) within +/‐one two‐fold dilution.
Phase I (Preliminary Screening /Interference Assay):
Validation of Gel Clot LAL Method

20. 1. Prepare Standards or Standard Series and Add to Reaction Tubes
•Reconstitute CSE as per Manufacturers instructions,
•15 min vortex
•From 1 EU/ml stock prepare 2λ, λ, 0.5λ,0.25λ
standards
•from CSE vial (2λpositive control)
•Pipette 100µl of each control into duplicate tubes
mL ofLRW Volume added to LRW Endotoxin concentration
9.5mL 0.5 mL of 1000 EU/mL 50 EU/mL ---
4.5 mL 0.5 mL of 50Eu/mL 5 EU/mL ---
3 mL 1 mL of 5 EU/mL 1.25 EU/mL 40
1 mL 1 mL of 1.25 EU/mL 0.625 EU/mL 20
4 mL 1 mL of 0.625 EU/mL 0.125 EU/mL 4
2 mL 2 mL of 0.125 EU/mL 0.0625 EU/mL 2
2 mL 2 mL of 0.0625 EU/mL 0.03125 EU/mL 
2 mL 2 mL of 0.03125 EU/mL 0.015625 EU/mL 0.5
2 mL 2 mL of 0.015625 EU/mL0.0075EU/mL 0.25
Dilution Series for Use with Lysate Sensitivity of 0.03 EU/mL
Performing Gel Clot LAL Method
21

21. 2.Prepare Samples at Correct Dilution and Pipette into Reaction Tubes
•All tubes were labelled for: 2PWC,
•negative LRW water control (NWC),
•each sample dilution included undiluted (NPC)
•each sample dilution included undiluted (PPC)
•performed in two replicates.
•Preliminary Screening /Interference Assay:
•All tubes were labelled for: 2PWC,
•negative LRW water control (NWC),
•A standard curve in each batch of product was prepared as follows:
•1ml sample at 2 x test dilution+1ml LRW = negative control.
•1ml sample at 2 x test dilution+1ml 4= test dilution at 2
•1ml sample at 2 x test dilution + 1ml 2= test dilution at .
•1ml sample at 2 x test dilution + 1ml = test dilution at ½.
•1ml sample at 2 x test dilution + 1ml ½= test dilution at ¼.
•performed in two replicates.
•Inhibition Enhancement / Validation Study:

22. 3. Add Positive Product Controls
•Direct Hot spiking method:
•10µl of 20λCSE concentration.

23. Add Lysate to Reaction Tubes
•Add lysate before placing tubes in
incubator
•Do not run lysate down side of reaction
tube
•Ensure adequate gentle mixing of lysate
and sample
•The LAL-endotoxin Rx is optimized in the
range of pH 6.0-8.0.
•incubate for 60 minutes (+/-2 mins)
•Invert Tube in Smooth Motion
Gel Clot
(Positive)

24. 24
•Positive water control series (PWC) must confirm lambda ().
•Positive product control series (PPC) must confirm lambda ().
•Both negative water (NWC) and sample (NPC) controls not gel.
•Once a non-interfering dilution (NID) had been identified, Phase II is
performed.
•The endpoint of the standard curves in water and product were not greater
than one twofold dilution either side of the lysate sensitivity and the endpoint
of the standard curve in the product is not greater than a twofold difference to
the endpoint of the standard in water.
VALIDITY CRITERIA

25. 25
Microplate Absorbance Reader
i. Gel clot (0.03 EU/mL):
a) Gel clot (Limit test)
b) Gel clot (Semi-quantitative test)
ii. Photometric (0.001-0.005 EU/mL):
a) Turbidimetric (End-Point)
b) Turbidimetric (Kinetic)
c) Chromogenic (End-point)
d) Chromogenic (Kinetic)
Methods of Endotoxin detection
Positive gel clot

26. 26
Validation of Gel Clot LAL Method:
1. Determination of MVD
K= 350 EU/dose, M= 600 mL (6X10 mL + 540 mL WFI), E.L for WFI =
0.25EU/ml.
Therefore: K=0.25EU/ml X 540 mL= 135 EU; 350-135 EU= 215 EU;
E.L= 215EU/60mL= 3.58EU/mL, =0.03EU/mL, Conc.= 1mL/mL;
Therefore MVD=3.58EU/mL/0.03EU/mL = 1:114
mL of LRW mL of sample added to LRW
or BD100 dispersing agent
Final dilution factor
4.5 mL 0.5 mL (original sample) 1:10
1mL 1mL (of 1:10 sample dilution) 1:20
1mL 1mL (of 1:20 sample dilution) 1:40
1mL 1mL (of 1:40 sample dilution) 1:80
4.5 mL 0.5 mL (of 1:10 sample dilution) 1:100

27. 27
Validation of Gel Clot LAL Method:
Replicates 2  /2 /4 Negative
control
Endpoint Log
endpoint
Mean logs
0.0625
Eu/mL
0.03125
Eu/mL
0.0156
Eu/mL
0.0078
Eu/mL
LRW
1 + + - - - 0.03125 -1.522 -1.44
2 + + - - - 0.03125 -1.522
3 + + - - - 0.03125 -1.522
4 + - - - - 0.0625 -1.204
Replicates 2  /2 /4 Negative
control
Endpoint Log
endpoint
Mean logs
0.0625
Eu/mL
0.03125
Eu/mL
0.0156
Eu/mL
0.0078
Eu/mL
LRW
1 + + - - - 0.03125 -1.522 -1.52
2 + + - - - 0.03125 -1.522
3 + + - - - 0.03125 -1.522
4 + + - - - 0.0625 -1.522
2. Confirmation of labelled lysate sensitivity : =0.0312 Eu/mL
Forthe1
st
lotnumber,usingtheequationGM=antilog(Σe/f)=0.0362EU/mL
Forthe2ndlotnumber,usingtheequationGM=antilog(Σe/f)=0.0301EU/mL

28. 28
Validation of Gel Clot LAL Method:
3. Phase I: Preliminary Screening /Interference Study:
Sample dilutionundiluted1:101:201:401:801:100
Unspiked (NPC) ++ ++ ++ ++ ++ ++
Spiked (PPC) ++ ++ ++ ++ ++ ++
A.ThefollowingtestdilutionswerepreparedbydilutionuptocalculatedMVDinLRWafter
heatingat70-80°Cfor10minsfollowedbycentrifugationat2000rpmfor10minutes.
Negative Water Control (NWC) = --
Positive Water Control (PWC) = ++
B.ThefollowingtestdilutionswerepreparedbydilutionuptocalculatedMVDinBD100
(dispersingagent)withvortexingfor5mins,centrifugationat2000rpmfor10minutes.
Sample dilutionundiluted1:101:201:401:801:100
Unspiked (NPC) ++ ++ ++ ++ ++ ++
Spiked (PPC) ++ ++ ++ ++ ++ ++
Negative Water Control (NWC) = --
Positive Water Control (PWC) = ++
C.Heatingat70-80°Cfor10mins.Inaddition,theLALreagentwasrehydratedwith
Endotoxin-SpecificBufferSolution(BG-120)toblockβ-D-GlucansandLAL-RM.
Sample dilutionundiluted1:101:201:401:801:100
Unspiked (NPC) ++ ++ ++ -- -- --
Spiked (PPC) ++ ++ ++ ++ ++ ++
Negative Water Control (NWC) = --
Positive Water Control (PWC) = ++
Non-interferingdilution(NID)is1:40

29. 29
Validation of Gel Clot LAL Method:
3. Phase II: Inhibition Enhancement/ Validation Study:
Replicates2  /2 /4 Negative
Control
EndpointLogendpointMean
logs
0.0625
Eu/mL
0.03125
Eu/mL
0.0156
Eu/mL
0.0078
Eu/mL
LRW
1 + + - - - 0.03125 -1.522 -1.36
2 + + - - - 0.03125 -1.522
3 + - - - - 0.0625 -1.204
4 + - - - - 0.0625 -1.204
Replicates 2  /2 /4 Negative
control
Endpoint Log endpoint Mean
logs
0.0625
Eu/mL
0.03125
Eu/mL
0.0156
Eu/mL
0.0078
Eu/mL
LRW
1 + + - - - 0.03125 -1.522 -1.44
2 + + - - - 0.03125 -1.522
3 + - - - - 0.0625 -1.204
4 + + - - - 0.03125 -1.522
In Sample Matrix (Endotoxin/product) :
GM = antilog (Σe/f) = 0.0433 Eu/mLIn Water Matrix (Endotoxin/LRW):
GM = antilog (Σe/f) = 0.0306 Eu/mLGMofbothsample&watermatriceswasachievedwiththesameefficiency

30. 30
Conclusion:
EstablishmentandValidationoftheLimulusAmebocyte
Lysateforthequalitycontrolofthepharmaceuticalproducts
especiallybiologicalonesisrecommended.
GoodManufacturingPracticesalliedtoQualityAssurance
andQualityControlareimportantcontrolstoensurefinal
productquality.

31. 31
My Special Thanks
CEO Dr.Nabil Elbablowy IPC Manager Dr. Ashraf Talaat
Supervisors:
Prof .Dr .Hamadllah Hafez Zeden Prof .Dr .Magdy Ali Amin
Dr. Aymen Samir Yassin Dr. Mohamed Reda Diab