Lal presentation

LAL: Choice of Test MethodLAL: Choice of Test Method

ByByTim SandleTim Sandle

Bio Products LaboratoryBio Products Laboratory

IntroductionIntroduction

How did we get here?How did we get here? Main test methods - Gel-Clot, Turbidimetric Main test methods - Gel-Clot, Turbidimetric

and Chromogenicand Chromogenic Advantages and disadvantages of each Advantages and disadvantages of each

methodmethod Comparison between the main methodsComparison between the main methods

Introduction - LAL TestIntroduction - LAL Test LAL Test - for performing BETLAL Test - for performing BET Alternative to Pyrogen (Rabbit) during 1980sAlternative to Pyrogen (Rabbit) during 1980s USP 1980; FDA Guide 1987; Ph. Eur. 1988; USP 1980; FDA Guide 1987; Ph. Eur. 1988;

BP 1989 (Gel-clot test)BP 1989 (Gel-clot test) To meet the requirements for the Bacterial To meet the requirements for the Bacterial

Endotoxin Test in Ph. Eur. 2.6.14 / USP Endotoxin Test in Ph. Eur. 2.6.14 / USP <85><85>

Introduction - LAL TestIntroduction - LAL Test

Main Test Methods:Main Test Methods:Test Method Covered

Gel-clot

Kinetic turbidimetric

Kinetic end-point*

Kinetic chromogenic

Kinetic end-point*

Non-EP / USP e.g. wet proteincolorimetric

X

Introduction - LAL Test Introduction - LAL Test

Different methods, Different methods, same principlessame principles» LimulusLimulus amebocyte lysate reagent from horse shoe amebocyte lysate reagent from horse shoe

crabscrabs» LAL detecting endotoxinLAL detecting endotoxin» Detection based on natural clotting mechanism Detection based on natural clotting mechanism

(Levin and Bang, 1968)(Levin and Bang, 1968)» Tests utilise the clotting cascadeTests utilise the clotting cascade

Introduction - LAL TestIntroduction - LAL Test

Different methods, Different methods, same principlessame principlesEndotoxin

Factor C Active Factor C ß-(1,3)-D-Glucan

Factor B Active Factor B------------------------Active Factor G Factor G

Proclotting Enzyme Clotting Enzyme

Coagulogen Coagulin

Gel Formation

Gel -clot LAL TestGel -clot LAL Test

PrinciplePrinciple» LAL can be purified to be of different sensitivities LAL can be purified to be of different sensitivities

so a clot = probability of a number of Endotoxin so a clot = probability of a number of Endotoxin Units (EU) in a given sampleUnits (EU) in a given sample

Limit or semi-quantitative through dilution Limit or semi-quantitative through dilution seriesseries

Gel -clot LAL TestGel -clot LAL Test

MethodMethod» Slide spot, micro-plate or tubeSlide spot, micro-plate or tube

Tube methodTube method» Water bath or hot block (37Water bath or hot block (37ooC +/- 1C +/- 1ooC)C)» Reaction tube: 0.1 ml lysate + 0.1 ml sampleReaction tube: 0.1 ml lysate + 0.1 ml sample» One hour incubation (+/- 2 minutes)One hour incubation (+/- 2 minutes)» Invert tube through 180Invert tube through 180oo - check for gelation - check for gelation

Gel -clot LAL TestGel -clot LAL Test

EP / USP testingEP / USP testing– More complicatedMore complicated

» Positive controlsPositive controls» Negative controlsNegative controls» Endotoxin standard curve (confirm label claim)Endotoxin standard curve (confirm label claim)» Positive product controls (spiked samples)Positive product controls (spiked samples)» Semi-quantitative through two-fold dilutionsSemi-quantitative through two-fold dilutions

Gel -clot LAL TestGel -clot LAL Test

Advantages of the testAdvantages of the test» Easy to performEasy to perform» As a qualitative test - quick and simpleAs a qualitative test - quick and simple» Inexpensive Inexpensive » Low equipment costsLow equipment costs» Good for simple products or waterGood for simple products or water» Reference method for USP / EPReference method for USP / EP

Gel -clot LAL TestGel -clot LAL Test Disadvantages to the testDisadvantages to the test

» Quantitation is difficultQuantitation is difficult» Fixed incubation timeFixed incubation time» InterferenceInterference» Limited ‘limit of detection’Limited ‘limit of detection’» Margin of errorMargin of error» No automationNo automation» VibrationVibration» SubjectiveSubjective» Compliance issuesCompliance issues

Photometric methodsPhotometric methods Turbidimetric and Chromogenic TechniquesTurbidimetric and Chromogenic Techniques Many similaritiesMany similarities

» Use spectrophotometryUse spectrophotometry» Use a standard curve (r = 0.980)Use a standard curve (r = 0.980)» Increased throughputIncreased throughput» Wider ranges of quantitationWider ranges of quantitation» As kinetic methods - single stepsAs kinetic methods - single steps» Reduced margins of errorReduced margins of error

Turbidimetric LAL TestTurbidimetric LAL Test

Principle:Principle:» Links the rate of gelation (as turbidity) to determine Links the rate of gelation (as turbidity) to determine

endotoxin contentendotoxin content» Plotting turbidity (optical density) against endotoxin Plotting turbidity (optical density) against endotoxin

concentration from a series of standardsconcentration from a series of standards

End-point or kinetic methodEnd-point or kinetic method

Turbidimetric LAL TestTurbidimetric LAL Test

MethodMethod» Heated plate reader or heated tube reader (37Heated plate reader or heated tube reader (37ooC C

+/-1+/-1ooC)C)» SpectrophotometerSpectrophotometer» Computer softwareComputer software» Lysate sensitivity determined via curveLysate sensitivity determined via curve

Turbidimetric LAL TestTurbidimetric LAL Test

Advantages of the testAdvantages of the test» Real time measurementReal time measurement» Results can be ‘seen’Results can be ‘seen’» Reading is automated: objectivityReading is automated: objectivity» SoftwareSoftware» Less dilutions cf Gel-clot (in-use costs)Less dilutions cf Gel-clot (in-use costs)» Over-coming interferenceOver-coming interference

Turbidimetric LAL TestTurbidimetric LAL Test

Disadvantages of the testDisadvantages of the test» Turbid samplesTurbid samples» Samples with precipitationSamples with precipitation» Some biologicalsSome biologicals» Equipment costEquipment cost» Vibration, bubbles and background noiseVibration, bubbles and background noise» Technician expertiseTechnician expertise

Chromogenic LAL testChromogenic LAL test

PrinciplePrinciple» Uses the clotting cascade, but in a modified wayUses the clotting cascade, but in a modified way» Synthetic chromogenic substrate - pNA - in the Synthetic chromogenic substrate - pNA - in the

presence of LAL and endotoxin produces a yellow presence of LAL and endotoxin produces a yellow colour. Intensity of colour = relates to amount of colour. Intensity of colour = relates to amount of endotoxinendotoxin

End-point or kinetic methodEnd-point or kinetic method

Chromogenic LAL testChromogenic LAL test

MethodMethod» Heated plate reader or heated tube reader (37Heated plate reader or heated tube reader (37ooC C

+/-1+/-1ooC)C)» SpectrophotometerSpectrophotometer» Computer softwareComputer software» Lysate sensitivity determined via curveLysate sensitivity determined via curve

Chromogenic LAL testChromogenic LAL test

Advantages of the testAdvantages of the test» Fast, real time measurementFast, real time measurement» Results can be ‘seen’Results can be ‘seen’» Reading is automated: objectivityReading is automated: objectivity» SoftwareSoftware» Less dilutions cf Gel-clot (in-use costs)Less dilutions cf Gel-clot (in-use costs)» Over-coming interferenceOver-coming interference

Chromogenic LAL TestChromogenic LAL Test

Disadvantages of the testDisadvantages of the test» Coloured samplesColoured samples» Equipment and reagent costEquipment and reagent cost» Technician expertise / variabilityTechnician expertise / variability

ComparisonComparison

What’s similar?What’s similar?» Use the same / similar principleUse the same / similar principle» Use LALUse LAL» Use tube or micro-plateUse tube or micro-plate» Require endotoxin standardsRequire endotoxin standards» Meet Regulatory requirements - FDA, MCA, EP, Meet Regulatory requirements - FDA, MCA, EP,

USP, JPUSP, JP

ComparisonComparison

How do they compare?How do they compare?Method Gel-clot Kinetic

TurbidimetricKineticChrmogenic

Typical lowerdetection limit

0.03 EU / mL 0.001 Eu / mL 0.005 EU / mL

Typical upperdetection limit

Fixed 100 EU / mL 50 EU / mL

Technicianinvolvement

High Medium Medium

Ease of use Low Medium High

ComparisonComparison

How do they compare?How do they compare?Method Gel-clot Kinetic

TurbidimetricKineticChrmogenic

Test robustness Medium Medium High

Equipment cost Low High Medium

Reagent cost Medium Medium High

ComparisonComparison

How do I choose?How do I choose?– Gel-clot method?Gel-clot method?– Turbidimetric method?Turbidimetric method?– Chromogenic method?Chromogenic method?– End point or kinetic?End point or kinetic?

SummarySummary

Brief introduction to the LAL testBrief introduction to the LAL test The three main methodsThe three main methods Advantages and disadvantages of eachAdvantages and disadvantages of each Comparison between themComparison between them

SummarySummary

Final choice…Final choice…– It’s up to you:It’s up to you:

Your budgetYour budget Your application - water, raw materials, in-process Your application - water, raw materials, in-process

samples or final productssamples or final products Volumes to be testedVolumes to be tested Material to be tested - turbid, coloured, precipitate, Material to be tested - turbid, coloured, precipitate,

inhibitioninhibition Endotoxin limitEndotoxin limit Degree of complianceDegree of compliance

Thank you for your timeThank you for your time