bacterial endotoxin testing in the laboratory

Bacterial Endotoxin Testing in Bacterial Endotoxin Testing in the Laboratorythe Laboratory

Michael E. Dawson, Ph.D., RACDirector of Regulatory Affairs

• Endotoxin Test Methods in the Pharmacopeia

• Essentials of the USP Bacterial Endotoxins Test (BET) Chapter – for gel-clot and photometric methods

• Recent Proposed Revisions to the BET (USP and EP)

• The FDA Guideline of 1987 and the 1991 Guidance –resolving differences with the BET

• Other Test Methodologies– Recombinant factor C reagent– Cartridge method– Monocyte Activation Test (MAT; or in vitro pyrogen test)

Outline

• Gel-clot

Note: In this case the reagent is reconstituted with buffer containing pH indicator

Endotoxin Test Methods in the Pharmacopeia

• Photometric – endpoint and kinetic– Turbidimetric and chromogenic reagents

Endotoxin Test Methods in the Pharmacopeia

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• Preparatory Testing:• Confirmation of test performance – for each lot of LAL reagent

– Gel-clot method:» “Appropriate” negative controls - quadruplicate?» Four replicates of each of four concentrations of

standard endotoxin: 2λ, λ, ½λ and ¼λ (where λ, lambda, is the labeled sensitivity of the reagent)

» Confirm labeled sensitivity within a factor of two.

Essentials of the USP Bacterial Endotoxins Test (BET) Chapter

2λ λ ½λ ¼λ Neg. control

+ + ‐ ‐ ‐

+ + ‐ ‐ ‐

+ + ‐ ‐ ‐

+ + ‐ ‐ ‐

• Preparatory Testing:• Confirmation of test performance – for each lot of LAL reagent.

– Photometric methods:» “Appropriate” negative controls – triplicate?» Three replicates of each of at least three

concentrations of standard endotoxin, (≤ 10 x difference between concentrations)

» Absolute value of correlation coefficient, |r|, ≥ 0.980

Essentials of the USP Bacterial Endotoxins Test (BET) Chapter

• Preparatory Testing (cont.):– Test for interfering factors – specific to each sample type:

• Detect known amounts of endotoxin added to the sample.• Gel-clot

– Controls» Duplicate negative controls.» Duplicates of four concentrations of standard

endotoxin: 2λ, λ, ½λ and ¼λ- confirm labeled sensitivity.

– Sample» Quadruplicates of four concentrations of standard

endotoxin: 2λ, λ, ½λ and ¼λ in the selected concentration of product - confirm labeled sensitivity.

» Quadruplicates of the selected concentration of product (no added endotoxin).

Essential Elements of the USP Bacterial Endotoxins Test (BET) Chapter (cont.)

• Preparatory Testing (cont.):– Test for interfering factors by the gel-clot method:


Endotoxin RS in LAL reagent water (LRW)2λ λ ½λ ¼λ Neg. control+ + - - -+ + - - -

2λ λ ½λ ¼λ Product Neg. control

+ + - - -+ + - - -+ + - - -+ + - - -

Endotoxin RS in product (dilution ≤ MVD)

• Preparatory Testing (cont.):– Test for interfering factors:

• Photometric Methods– Controls

» Duplicate negative controls.» Duplicates of at least three concentrations of standard

endotoxin (≤ 10 x difference between concentrations).– Sample

» Duplicates of a concentration of standard endotoxin from the middle of the standard series prepared in the selected concentration of product – recover spike within 50 – 200% of nominal concentration.

» Duplicates of the selected concentration of product (no endotoxin added).

Essentials of the USP Bacterial Endotoxins Test (BET) Chapter (cont.)

• Preparatory Testing (cont.):– Test for interfering factors by photometric methods:

Example (excluding standards and negative controls)


Rep. ID Unspiked sample(EU/mL)

Spiked sample (0.004 EU/mL)

Spiked minus Unsp.(EU/mL)

Mean % Recov.

1A

<0.001 0.0044 0.0044105%

2 <0.001 0.0040 0.0040

• Test Procedure:– Gel-clot:

• Limits test:– Controls:

» Duplicate negative controls.» Duplicate 2λ positive controls (standard endotoxin at

a concentration of 2λ) – not full standard series.» Duplicate positive product controls (PPCs - test

sample containing an added standard endotoxin concentration of 2λ).

– Test sample (at a dilution not to exceed the maximum valid dilution).

– Pass if test is valid and sample tests negative.– Sample does not fail unless it tests positive at the MVD.


• Test Procedure:– Gel-clot limits test:


Endotoxin RS in LAL reagent water (LRW):Neg. control 1

2--

2λ pos. con. 12

++

Spiked sample (2λ) 1(PPC) 2

++

Unspiked sample 1(test) 2

--

Test of product (at validated dilution):

• Test Procedure:– Gel-clot :

• Assay (Quantitative Test in the harmonized Draft):– Controls:

» Duplicate negative controls.» Duplicates of four concentrations of standard

endotoxin: 2λ, λ, ½λ and ¼λ.» Duplicate positive product controls (the highest

concentration of test sample containing an added standard endotoxin concentration of 2λ).

– Sample:» A series of twofold dilutions of test sample (greatest

dilution not to exceed the maximum valid dilution).– Pass if test is valid and sample contains < endotoxin limit

(quantify by multiplying the endpoint dilution factor by λ.


• Test Procedure:– Gel-clot Assay


Endotoxin RS in LAL reagent water (LRW)2λ λ ½λ ¼λ Neg. control

+ + - - -+ + - - -

Dilution: 1x 2x 4x 8xUnspiked 1 + + - -

2 + + - -Spiked (2λ) 1 +

2 +

Dilutions of product (1x = validated dilution)

If λ = 0.125 EU/mL and limit is 1 EU/mL, product contains 2 x 0.125 EU/mL = 0.25 EU/mL . Meets specification (must test negative at MVD of 8).

• Test Procedure:– Photometric methods:

• Same as the test for interfering factors.• Pass if test is valid and sample contains < endotoxin limit.


• In 2007 the USP and EP published proposed changes.– USP changes published in Pharmacopeial Forum 33(3).– EP changes published in PharmEuropa 19(2).

• Intent is to improve harmonization and clarity.• In the May-June, 2009 issue of Pharmacopeial Forum,

35(3) USP published the current version of the document for information purposes only - not for comments.

• The essentials of the chapters are unchanged.

Recent Proposed Revisions to the BET

• FDA Guideline, 1987– “Guideline on Validation of the Limulus Amebocyte Lysate

Test as an End-Product Endotoxin Test For Human and Animal Parenteral Drugs, Biological Products and Medical Devices”.

• FDA Interim Guidance, 1991– “FDA Interim Guidance for Human and Veterinary Drug

Products and Biologicals: Kinetic LAL Techniques.”– Inserted in the middle of the online version of the 1987

Guideline – in the medical devices section.

http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm070286.pd

The FDA Guideline of 1987 and the 1991 Guidance

• CBER licensed reagent should be used for validation, release and in-process testing - despite the title.

• Qualify technicians (use the LAL reagent qualification procedure) and assess the test laboratory variability.

• Allows for use of control standard endotoxin (CSE) standardized against reference standard endotoxin (RSE).

• Gel-clot method:– Confirm labeled sensitivity once per day– Then allows use of 2λ positive control for routine gel-clot

tests (not validation) after the first test of the day – different from the BET.


• Photometric methods:– Provides for product standard curves in which standard

endotoxin is diluted in product.– Provides for archived (stored) standard curves.

• Requires a positive control included with each test and spike recovery within +/- 25% to verify archived curve validity.

• Only specified for routine testing - not for validations.

• Gives guidance on when to repeat validation:– Change LAL manufacturer (not method): revalidate for 1 lot

of product.– Change test method: revalidate for 3 lots of product.– Change of LAL lot, manufacturing process or product

formulation, revalidation not required; PPC will (may) suffice. CAUTION: Consider revalidation for the latter two.


• Retest provisions– Allows for up to two retests.

IMPORTANT:• The FDA guidance documents were written before the

FDA Out of Specification (OOS) Guidance of 2006.• An investigation should be conducted before any retests.• Retests should be justified in accordance with an OOS

SOP. • A product should not be released based on the results of a

retest following an OOS result without justification.

FDA Guideline of 1987 and 1991 Guidance

Resolving Differences Between the BET and Guidance Documents

BET (USP and EP) FDA Guidance Resolution

Gel‐clot limits test No standard series ‐2λ positive control

Standards series for at least 1st test of day

Include standard series with 1st test of day*

Gel‐clot assay Standard series Standards series for at least 1st test of day

Include standard series with 1st test of day*

Photometric methods – PPC recovery

50 ‐200% +/‐ 25 % and +/‐50 %

50 ‐200% (but see LAL manufacturer’s instructions)

* If procedures state that test is being run per the USP or EP BET, note exceptions.

Consult appropriate regulations and guidances for your specific application.

Resolving Differences Between the BET and Guidance Documents (cont.)

BET (USP and EP) FDA Guidance Resolution

Photometric Methods – [PPC]

= a standard conc. at or near mid curve

4λ, 0.5 or 5 EU/mL = a standard conc. at or near mid curve

Photometric Methods –Archived curves

Not included Included – run positive control with each test with +/‐25% recovery.**

Validate as an alternative method.Include a positive control in each test. *

Photometric Methods – Product standard curves

Not included (but is in EP Guidelines)

Included – requires “clean” product

Validate as an alternative method.*

CSE Not included (is in EP Guidelines)

Included Validate as an alternative method.*

* If procedures state that the test is being run per the USP or EP BET, note exceptions.**For the chromogenic and endpoint turbidimetric methods, run a standards series for at least the

first test of the day.

Consult appropriate regulations and guidances for your specific application.

• Recombinant factor C (rFC) Reagent– Produced using a genetically modified insect cell line.– Only uses the first step of the natural clotting cascade (from

the Asian horseshoe crab Carcinoscorpius rotundicauda)..

Other Test Methodologies

• Does not incorporate factor B and the clotting enzyme.• In the natural reagent the additional steps amplify the

reaction and result in the extraordinary sensitivity of LAL.• Is a fluorometric assay.

– Compensates for the absence of the amplifying steps –fluoroscopy is much more sensitive than spectroscopy.

• Substrate incorporates a fluorophore (analogous to the chromophore in chromogenic methods).

• Test read in a fluorometer (typically a plate reader).

Other Test Methodologies:rFC Method (cont.)

• Methodology– Prepare samples and standards as for any other photometric

assay – add to microplate.– Preincubate the plate is for at least 10 minutes.– Mix reagent

• Recombinant enzyme solution, buffer and the fluorogenic substrate in a 1:4:5 ratio (the reagent mixture cannot be stored).

– Read as an endpoint test after a one hour incubation.– Is linear over a range of 0.01 to 10 EU/mL (or a narrower

range for improved linearity).– Standard line is constructed from the log of the Δ relative

fluorescence units (RFU) regressed on the log standard endotoxin concentration.


• FDA has elected not to license recombinant reagent.

• FDA Guideline specifies the use of LAL reagent licensed by CBER.

• The BET in USP 32 specifies reagent “… which has been prepared and characterized for use as an LAL Reagent.”

• The EP and the pending revision to the USP specify use of LAL “… manufactured in accordance with the regulations of the competent authority.”

• Validate rFC reagent as an alternative method.


• Cartridges preloaded with chromogenic LAL reagent – four channels– Two unspiked channels– Two channels containing added endotoxin spike (PPC).

• Run in either a single cartridge portable unit or a multi-cartridge bench top unit.

• Relies upon an archived curve– No positive control to verify curve validity - or negative

control.• Not able to run the standards specified in the BET for

photometric tests.• Validate as an alternative method.

Other Test Methodologies:Cartridge Method

• Samples are incubated overnight with blood or a cell suspension.

• Pyrogens stimulate monocytes to produce cytokines.• Test for cytokine by ELISA.• New EP chapter on the Monocyte Activation Test

(2.6.30)• Draft published in Ph.Eur. July 2008.• Published in EP supplement 6.7 in September, 2009 – for

implementation in April, 2010.

• Detects non-endotoxin pyrogens as well as endotoxin.• Results are given in endotoxin equivalent units (EEU) –

uses an endotoxin standard.

Other Test Methodologies:Monocyte Activation Test (MAT)

• EP chapter allows flexibility in the way the test is run.• There are disagreements about the best way to run the

MAT:– Fresh whole blood vs. peripheral blood mononuclear cells

(PBMCs) vs. cryopreserved blood vs. monocyte like cell line.

– Pooled vs. unpooled blood from donors (response varies from donor to donor).

– Cytokine to measure – IL-6 vs. IL-1β etc.

Other Test Methodologies:MAT (cont.)

• Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM)– Evaluated MATs potential for replacing the rabbit pyrogen

test (RPT).• Assessed the validation status of five in vitro test methods

proposed for pyrogen testing of pharmaceuticals and other products.

– Concluded:• None of these test methods can be considered a complete

replacement for the RPT.• Methods can be considered for use to detect Gram-negative

endotoxin in human parenteral drugs on a case-by-case basis -

– Subject to validation for each specific product to demonstrate equivalence to the RPT.


• Expert panel advising ICCVAM:– “The Panel also felt that the lack of direct parallel testing in

rabbits with the products tested in the validation study was a significant limitation to the study design.”

• FDA, in a letter of April 24, 2009, stated:– “FDA concurs with the ICCVAM recommendations that

none of the in vitro pyrogen test methods can be considered as a complete replacement for the rabbit pyrogen test (RPT) without additional product-specific information. FDA also endorses the recommendation that these in vitro pyrogen test methods may be considered on a case by case basis for the detection of Gram negative endotoxin in parenteral drugs, subject to product-specific validation.”


• The Bacterial Endotoxins Test (BET) chapter in the USP is the primary reference for endotoxin testing.

• There are no fundamental changes in the coming revisions to the BET (USP and EP).

• The FDA Guideline of 1987 and the 1991 Guidance offer recommendations in areas not addressed in the USP –and some contradictions.

• Other test methods not addressed in the pharmacopeia may be of value – validate as alternative methods.

M. Dawson, Associates of Cape Cod, Inc., October, 2009

Conclusions