Bacterial Endotoxin Testing in

the Laboratory

Michael E. Dawson, Ph.D., RAC
Director of Regulatory Affairs

Outline

• 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)
Endotoxin Test Methods in the
Pharmacopeia
• 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
Density
Optical

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

2λ λ ½λ ¼λ Neg. control
+ + ‐ ‐ ‐
+ + ‐ ‐ ‐
+ + ‐ ‐ ‐
+ + ‐ ‐ ‐

Essentials of the USP Bacterial

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

Endotoxin RS in product (dilution ≤ MVD)

Product Neg.
2λ λ ½λ ¼λ
control
+ + - - -
+ + - - -
+ + - - -
+ + - - -
Essentials of the USP Bacterial
Endotoxins Test (BET) Chapter (cont.)
• 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  Spiked  Spiked  Mean 

sample sample  minus   % 
(EU/mL) (0.004  Unsp. Recov.
EU/mL) (EU/mL)
1 <0.001 0.0044 0.0044
A 105%
2 <0.001 0.0040 0.0040
Essential Elements of the USP Bacterial
Endotoxins Test (BET) Chapter (cont.)
• 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.

Essential Elements of the USP Bacterial

Endotoxins Test (BET) Chapter (cont.)
• Test Procedure:
– Gel-clot limits test:

Endotoxin RS in LAL reagent water (LRW):

Neg. control 1 -
2 -
2λ pos. con. 1 +
2 +

Test of product (at validated dilution):

Spiked sample (2λ) 1 +
(PPC) 2 +
Unspiked sample 1 -
(test) 2 -
Essentials of the USP Bacterial
Endotoxins Test (BET) Chapter (cont.)
• 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.

Essentials of the USP Bacterial

Endotoxins Test (BET) Chapter (cont.)
• Test Procedure:
– Gel-clot Assay
Endotoxin RS in LAL reagent water (LRW)
2λ λ ½λ ¼λ Neg. control
+ + - - -
+ + - - -

Dilutions of product (1x = validated dilution)

Dilution: 1x 2x 4x 8x
Unspiked 1 + + - -
2 + + - -
Spiked (2λ) 1 +
2 +
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).
Essentials of the USP Bacterial
Endotoxins Test (BET) Chapter (cont.)

• Test Procedure:
– Photometric methods:
• Same as the test for interfering factors.
• Pass if test is valid and sample contains < endotoxin limit.

Recent Proposed Revisions

to the BET

• 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.
The FDA Guideline of 1987 and
the 1991 Guidance

• 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/GuidanceComplianceRegulatoryInfo
rmation/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.
The FDA Guideline of 1987 and the 1991
Guidance
• 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.

FDA Guideline of 1987 and 1991

Guidance
• 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.
Resolving Differences Between the
BET and Guidance Documents

BET (USP and EP) FDA Guidance Resolution

Gel‐clot limits test No standard series ‐ Standards series for  Include standard 
2λ positive control at least 1st test of  series with 1st test of 
day day*
Gel‐clot assay Standard series Standards series for  Include standard 
at least 1st test of  series with 1st test of 
day day*
Photometric  50 ‐200% +/‐ 25 % and +/‐ 50 ‐200% (but see 
methods – PPC  50 % LAL manufacturer’s 
recovery 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  = a standard conc. at  4λ, 0.5 or 5 EU/mL = a standard conc. at 
Methods – [PPC] or near mid curve or near mid curve
Photometric  Not included Included – run  Validate as an 
Methods – positive control with  alternative method.
Archived curves each test with +/‐ Include a positive 
25% recovery.** control in each test. *
Photometric  Not included (but is in  Included – requires  Validate as an 
Methods – Product  EP Guidelines) “clean” product alternative method.*
standard curves
CSE Not included (is in EP  Included Validate as an 
Guidelines) 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.
Other Test Methodologies

• 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:

rFC Method (cont.)

• 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.

Other Test Methodologies:

rFC Method (cont.)

• 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.
Other Test Methodologies:
Cartridge 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:

Monocyte Activation Test (MAT)

• 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:
MAT (cont.)

• 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.
Other Test Methodologies:
MAT (cont.)
• 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.”

Conclusions

• 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