LALreview LAL REVIEW: PUBLISHED BY LONZA 2008 Issue 3

EDITOR’S COLUMN

What is the most expensive endotoxin

test that you run in your QC lab? If I were
to guess, I would say the retest. How of-
ten do you find that a failure is due to an
entire lot of your product being contami-
nated with endotoxin? How often is a fail-
ure resolved by a retest and determined
to be a laboratory-based error?

My colleague Ruth Noe from the UK has

written an article for this issue of the LAL
Review that addresses different causes
of endotoxin test failures. She has also in-
cluded ideas to avoid some of the causes.

One of her comments includes avoiding

unexpected changes to PPC recoveries
Dedicated and calibrated pipettes can reduce variability
that are due to sample storage containers
and procedures. Do you have documenta-
tion that shows the level of endotoxin in a
product does not change over time when
The Most Expensive dosage route, methods of production, and raw
materials used. Rationalization projects with-
samples are held in a particular type of
container at a particular temperature?
Test is a Retest in the QC department typically look at docu-
mentation, number of representative samples
By Ruth Noe, UK and Ireland Sales Manager to test (especially for in-process and raw ma-
Have you screened your sample contain-
terial testing where regulations are not clear),
er for endotoxin content and any interfer-
In the last few years, pharmaceutical and methods, equipment, reagents, consumables
ence properties?
biopharmaceutical companies have been un- and technical support suppliers, training, and
der tremendous pressure to reduce running Out of Specification (OOS) and Out of Trend
If the answer to those questions is no, you
costs. As many patents expire, the previously (OOT) reporting and investigation proce-
may want to think about having your con-
exclusive market for many drug products has dures.
tainers tested or purchasing pre-tested
containers. Our Endotoxin Testing Service been opened to generic manufacturers able to
manufacture at much lower cost and having The harmonized Bacterial Endotoxin Test
can screen your sample containers. In
little or no research and development pipeline (BET) (United States, European and Japanese
addition, we offer depyrogenated, labeled
to fund. In response, many companies have Pharmacopeias) is required for all final prod-
glass sample containers that can help
now completed or are currently conducting ra- ucts which come into direct contact with the
alleviate testing failures due to sample
tionalization projects looking at every aspect bloodstream such as parenteral drugs and
container contamination as well as other
of production including raw materials sourc- some medical devices. The BET is a biologi-
pre-screened accessory products.
ing and testing, upstream and downstream cal assay and as such is more variable than
processing, in-process monitoring, clean- most chemistry-based QC assays. It is very
ing, fill and finish, packaging and, of course, important to adequately design and validate
Maribeth Donovan Janke, Ph.D.
support functions such as Quality Assurance your BET routine at the outset to reduce the
Sr. Product Manager
(QA) and Quality Control (QC). possibility of additional variability causing un-
necessary assay failures and retests.
Within the QC structure, the range of tests
depends on the final product characteristics, Taking steps to reduce the number of retests
appropriate regulations that apply, intended is desirable. When circumstances occur such
1 LALreview

Preventive Maintenance on readers can avoid assay failures
that a retest is in order, a firm should have
appropriate OOS protocols that clearly define
the procedure for retesting. There should be a
rationale behind the retest that is defendable.
One cannot test into compliance. Regulatory
agencies have issued guidances to help a
firm define an OOS procedure.
Variability in the BET
There are many points of variability for any
test system: the equipment, consumables,
reagents, environment, technician, documen-
tation of procedures, and results analysis. For
the BET, it is critical that the equipment used
is installed, validated and maintained appro-
priately. The laboratory environment is also
critical and should be assessed during equip-
ment installation and validation to ensure
factors such as temperature, humidity, light,
air handling, cleanliness, and cross contami-
nation risk are managed within acceptable
limits. Equipment such as pipettes should
be periodically cleaned and calibrated to en-
sure accurate volume dispensing and limit
contamination of the test system. Ideally pi-
pettes should be dedicated to endotoxin test-
ing and certainly not used in any potentially
live bacterial assay preparation.
Consumables and Reagents
The consumables used in the BET according to
the Pharmacopeia must be “free of detectable
endotoxin and of interfering effects for the
test”, the test being defined as the method
LALreview 2
in use. The selection of consumables should
ideally be confirmed prior to the Installation,
Operational and Performance Qualification
(IQ/OQ/PQ) testing of the system and not be
changed unless absolutely necessary. Ge-
neric consumables suppliers may be able to
provide consumables such as 96-well plates
and tips which are certified as apyrogenic or
pyrogen-free. However, one should check the
limit for the endotoxin load to assure it is ap-
propriate for the sensitivity of the endotoxin
detection method to be used and that inter-
fering factors are tested for as required by
the Pharmacopeia. The endotoxin detection
reagent suppliers can usually supply appro-
priately certified consumables required for
the BET methods using their own reagent sys-
tems.
It should be noted that as the Limulus Ame-
bocyte Lysate (LAL) reagents for the BET are
biologically sourced, no two lots will react
exactly the same. However, carefully con-
trolled manufacture, formulation and QC test-
ing will ensure that the reaction with known
endotoxin standards is within an acceptable
range. Furthermore, reagents from different
suppliers differ in manufacture and formu-
lation. For this reason it is suggested to use
the consumables recommended, tested and
certified by the supplier of the reagents in
use. Due to lot to lot variability of endotoxin
detection consumables, some suppliers sug-
gest that the customer test each new lot of
critical consumables such as the 96-well
plates or gel clot tubes with the current in-use
lot of reagents using the Initial Qualification
test method. One should also ensure that the
consumables in use are compatible with the
equipment being used. For instance, the right
tips are used with the appropriate pipettes.
Regulatory agencies, such as the U.S. FDA,
have set acceptable limits for various testing
criteria of the BET methods. For instance, the
allowable recovery percent for a Positive Prod-
uct Control (PPC) is 50 to 200% of the known
amount of endotoxin introduced into the test
well. Setting tighter specifications in an inter-
nal Standard Operating Procedure (SOP) than
those recommended by the reagent manufac-
turer or a regulatory agency may be setting
your test up for failure.
Technician Training
Following installation and validation of the
BET system with the chosen reagents and
consumables, it is critical to ensure techni-
cian training and procedure documentation
are adequate to further reduce variability.
Ideally, technicians should be trained on all
aspects of bacterial endotoxin testing such
as correct use of pipettes, understanding of
the reagent and consumable system, use of
software (if appropriate), equipment calibra-
tion procedures, management of preparation
area, assessment of results, critical impact
of test results on manufacturing and product
release, completion of documentation, Out of
Specification and Out of Trend investigation
procedures, and reporting structures.
Assuming you have appropriate IQ/OQ/PQ
and servicing procedures for your equipment
system, adequate control of the laboratory
environment, good sourcing of reagents and
consumables, comprehensive technician
training programs and well documented test
and OOS/OOT procedures, incidences requir-
ing repeat testing should be limited but will
not be eradicated. There are two very differ-
ent types of BET failures: product indepen-
dent and product dependent. Both have very
different impacts on the final result and ac-
tion to be taken.
Product Independent Failures
Product independent failures, also termed
“assay failures”, can be blank reactions, fail-
ure to meet standard curve specifications or
failure of the percent Correlation of Variation
(%CV) to meet acceptable limits for standards.
Product independent failures are often tech-
nician dependent and require a simple retest
with the original test parameters. The inves-
tigation is laboratory based and although de-
lay in product results and release may occur,
involvement of the production and packaging
team is normally minimal.
Blank and Negative Control Failures
Blank reactions and negative control failures
are one of the most common causes of assay
failures and there are many actions one can
take to limit occurrence:
— Avoid environmental contamination of the
well by dust, hair, skin cells and other par-
ticulates during pipetting by use of ad-
equate personal protective equipment
and cleaning routines, when appropriate.
— Old air conditioning units and old vortex
mixers can shed particulates; if possible,
locate the reader and preparation area
away from these items.
— Reduce the possibility of endotoxin con-
tamination of the blanks or negative
controls during the preparation of posi-
tive controls and standards by careful
management of preparation area and
good pipetting techniques. Use of “no
fly” zones can reduce contamination,
meaning used pipette tips should not
travel over the blank wells or negative
control tubes during assay preparation.
— Limit endotoxin contamination of the sys-
tem from Gram-negative organisms by
adequate cleaning, good timing of assay
preparation and use of dedicated pipettes.
— Use of certified consumables as recom-
mended by reagent manufacturer further
reduces the possibility of contamination.
— SOPs should contain clear procedures for
preparation and pipetting of samples
and controls into the reaction vessels
which should be followed closely. In par-
ticular, take care to use calibrated pipettes
with the correct fitting tips and good pi-
petting technique at this stage.
— Do not cut short the vortex mixing steps
as this can compromise the standard
curve as well as the difference between
the blank and lowest standard endotoxin
concentration.
— Take care not to contaminate the LAL re-
agent water used for blanks by airborne
contaminants or cumulative contamina-
tion from incorrectly sourced or used tips.
Use a fresh bottle of water as frequently
as you feel comfortable. Some users will
use a fresh bottle for each assay; others
will use the entire bottle to the last drop.
Standard Curve and Positive Control
Failures
When the test results fail to produce a mean-
ingful standard curve or positive control reac-
tion, it is often due to technician or pipette
related error. Errors such as incorrect Control
Standard Endotoxin (CSE) reconstitution,
poor preparation of controls and incorrect pi-
petting are common causes of positive control
and standard curve failures. Again, one should
ensure that correctly calibrated pipettes and
appropriate tips are used in the preparation
A clean work area can reduce contamination
of the standards as this can cause variability.
Distraction during preparation and plating
is often the cause of technician error rather
than training or competency. Some laborato-
ries employ a “no entry” or “no interruption”
rule when the BET is being prepared. Although
gross environmental or system contamina-
tion can cause large spot contaminations
in the positive control or standards causing
failure of %CV limits or mismatched pairs, this
is rare and the most likely cause is pipetting
error. Use of a multichannel pipette for lysate
addition in 96-well plate endotoxin test meth-
ods may reduce variability compared to use
of repeat pipettors.
Hot Wells and Spot Contaminations
Despite following all the procedures above to
avoid assay contamination, it is well known
and accepted in various pharmaceutical QC
workgroups that “hot wells” in 96-well plate
assays and spot contaminations do still oc-
cur. Although the exact cause of a “hot well”
is not clear, it is usually seen as a replicate
that has an unusually high level of apparent
endotoxin signal compared to other replicates
of the sample. The blanks or negative controls
or samples that are expected to be “clean”
would be the most likely points at which “hot
wells” or spot contaminations are seen, as
there is no deliberate endotoxin dispensed
into these wells to cause the positive reac-
tions which would otherwise mask these low
level contamination reactions. Some firms
have reported a phenomenon called “cold
wells” where one replicate reacts more slowly
in the presence of endotoxin than the other
replicate samples.
For 96-well plate endotoxin detection methods
in particular, this is compounded by the fact
that the manufacturers of the 96-well plates
do not manufacture the plates specifically for
bacterial endotoxin testing. Rather, they are
made usually for cell culture work. The BET
reagent supplier will assess each batch, cer-
tifying those which pass the acceptable en-
dotoxin and interfering factor limits. The only
action that can be taken in these cases is a
simple retest under the same conditions. If a
lot of plates is determined to be the cause of
multiple retests, the best solution is to simply
source an alternative lot of plates from the
supplier.
One should be careful in using the phrase “hot
well” to simply explain an unexpected single
well reaction whatever the location. A firm
3 LALreview
should be able to defend that the result is not
due to other possible sources of contamina-
tion, such as pipetting error, spot contamina-
tion, water used for the control, pipette tip,
etc., before concluding that a “hot well” is the
cause.
Product Dependent Failures
Product dependent failures, also termed
“sample” or “product failures”, would include
mismatched replicate reactions, failure in %
Positive Product Control (PPC) recovery and
rarely, but most critically, failure to meet the
endotoxin limit assigned to each individual
product tested. Product dependent failure
is quite a different type of investigation and
would typically involve a full OOS/OOT investi-
gation due to the potential impact on produc-
tion and the final product.
Mismatched Replicate Reactions
Pharmaceutical products will typically con-
tain no detectable endotoxin and therefore no
%CV for the sample replicates is calculated in
photometric methods. In instances where de-
tectable endotoxin is seen in replicate sample
wells, a %CV is calculated and if it is outside
acceptable limits, it should be investigated. It
is possible to have one sample replicate react
and others show no detectable endotoxin. In
cases where this single reaction is very close
to the lowest standard reaction time, this
could simply be that the endotoxin content
of the sample is very close to the sensitivity
of the assay and the calculated endotoxin re-
sult passes the endotoxin limit of the product.
In cases where a single reaction causes the
sample to fail the endotoxin limit, a retest and
re-sampling may be required. Single cell or
tube reaction may be due to “hot wells”, spot
contamination during assay preparation or
pipetting error.
In those samples where %CV in either sample
or sample PPC wells can be calculated and is
above the acceptable limit, they can initially
be treated as a product independent failure.
Getting it right the first time saves resources
As this is potentially caused by technician
error or equipment failure, a simple retest of
the sample should be conducted in the first
instance. The most common cause of %CV
failure is pipetting error, “hot wells” or spot
contamination and usually on retest the %CV
passes. In kinetic endotoxin detection meth-
ods, %CV failure can also be due to air bubbles
in the well which are read at the baseline
(time = 0) and then disperse or pop invalidat-
ing the baseline read. Usually any air bubbles
in wells are dispersed by the shake prior to
the baseline read, but any viscous product
being tested may cause problems. For test-
ing of any viscous products with low MVD, it
is recommended to use a kinetic chromo-
genic method as this is less affected by the
variability in optical densities caused by air
bubbles due to the larger signal to noise ratio.
Air bubbles can be avoided by not “blowing
out” tips during pipetting. Viscous samples
can also cause issues with mismatched pairs
in gel clot testing and again dilution or use of
a kinetic chromogenic method would be rec-
ommended in this case. Sample homogeneity
can cause issues with %CV and is seen more
commonly when testing biological products
or products with difficult dissolution proper-
ties. Always ensure the sample preparation
SOP is very clear to avoid variability between
technicians and good mixing and dissolution
of the sample. For %CV failures, the number
of retests required to pass the product var-
ies depending on how this type of failure is
assigned. Most laboratories would simply
retest once, but some would assign this as
a full product failure and retest a number of
repeats. Whatever decision is made based on
%CV product failure retests, it should be ratio-
nalized, investigated and well documented
for audit purposes.
Positive Product Control Failures
The PPC is used as an inhibition and enhance-
ment control in an endotoxin test. There are
different causes for the PPC recovery to be
out of specification, some that can be re-
solved with a retest and others that may
require more significant investigation. On oc-
casion the PPC recovery may fail due to the
mathematics of the standard curve analysis
used. Linear regression is commonly used for
kinetic assay data analysis. Putting a straight
line through inherently curved data can cause
as much as 30% over-prediction error. This
level of error is most commonly found at the
point on the standard curve where the PPC
would be back-predicted. Using polynomial
regression to generate the standard curve
and the PPC recovery may correct a failure
due to mathematics.
If the retest also fails PPC recovery, this may
indicate a change in the final product which
could ultimately impact manufacturing de-
sign. Changes in PPC status for a product may
be caused by changes in sampling technique
or vessel, raw material or raw material sup-
plier, cleaning routines, process flow com-
ponents such as a filter bed, formulation, or
simply the temperature and time at which the
sample is stored. To avoid issues with sample
4 LALreview
storage, holding studies for each product type
should be conducted over time at varying
storage temperatures in the sample vessels
currently in use. A maximum holding time
for samples before testing and storage tem-
perature range should be assigned for each
product type. Endotoxin content as well as
PPC recovery differences over time and tem-
perature ranges should be monitored during
the holding study. Some laboratories will as-
sign a holding time for both refrigeration and
freezing of samples to allow some flexibility.
If your retest passes, technician error, spot
contamination of the sample dilutions or con-
sumable contamination may be assigned and
the result accepted within documented retest
acceptance criteria. If the retest fails the PPC
recovery, re-sampling or sourcing of an alter-
native sample aliquot should be conducted
to rule out sample contamination with inter-
fering factors. This test should be conducted
under the same conditions as the original
sample. Depending on the equivalence of the
resample to the original sample, decisions
can be made about any lots of product the
positive result may impact, any cleaning that
may be required and any changes that may
need to be made to the manufacturing pro-
cess. It is rare for a product PPC failure to be
repeated on retest or resampling as they are
normally attributed to technician error, but
when it does, the impact on the final product
can be very costly.
Endotoxin Limit Failures
Endotoxin limit failure of a product is the least
desirable outcome of your BET. One should
approach the investigation with a clear pro-
cedure in place if laboratory or sampling er-
ror cannot be ruled out initially. The sample
should be retested and if it passes, the initial
failure may be attributed to contamination of
the sample during sample preparation. If the
retest also fails, then re-sampling should be
conducted according to defined procedures
appropriate to the sample being tested. Trend-
ing of the endotoxin results of the product
should be conducted to ascertain if there is
an upward trend rather than a sudden spike
of endotoxin in the system which may give
some idea of why the failure occurred. If the
re-sampling test passes, then it could be
assumed that the original sample became
contaminated during sampling or assay
preparation and the outcome was therefore a
laboratory error rather than product failure. If
the resample test fails, then decisions should
be made regarding the impact on the final
product and production.
What is a “Normal” or “Acceptable”
Failure Rate?
Upon analysis of the failure data from a num-
ber of laboratories, it is clear that what is seen
as “normal” failure rates for one laboratory will
be quite differently perceived by another labo-
Reduce Assay Failures with Lonza Products
Product Type Size
200, 300, 1000 μl,
Pipette Tips 96 tips per tray
LAL Reagent Grade Plates 96-well plates
Reagent Reservoirs — 10 per pkg
Glass Sample Containers,
10 ml
depyrogenated
For Part Numbers and Pricing, please contact your local Lonza Sales Office.
ratory. It is therefore very difficult to state ac-
ceptable failure limits for bacterial endotoxin
testing as there are so many variables which
have different impacts in different laborato-
ries. For example, a laboratory testing clearly
defined, validated pharmaceutical products
using a kinetic technique would usually have
a much lower failure rate than a research
laboratory conducting infrequent testing of
biologically sourced research samples us-
ing an endpoint chromogenic technique. The
potential for laboratory based assay failures
does appear to increase when new techni-
cians are being trained, there is a change in
method or product types being tested, or the
environment is altered in some way. Trending
is the key. It is good practice to monitor as-
say and product failures for each laboratory
so that any significant changes may be real-
ized quickly, corrective action be taken in a
proactive manner and preventive actions put
in place to limit further occurrence.
Your local Lonza Product or Scientific Support
Specialist would be more than happy to dis-
cuss further your assay failure rates and help
troubleshoot to improve this.
Quantity
5 trays per pkg
50 plates per case
25 or 100 per box
Single (Europe only)
5 LALreview
Lonza Walkersville, Inc.
8830 Biggs Ford Road
Walkersville, MD 21793

Contact Information
North America
Customer Service: 800-638-8174
Scientific Support: 800-521-0390
E-mail: scientific.support@lonza.com
Online Ordering: www.lonza.com
International
Contact your local Lonza Distributor
Customer Service: 301-898-7025, ext. 2322
Fax: 301-845-8291
E-mail: scientific.support@lonza.com
Lonza Walkersville, Inc.
Walkersville, MD 21793
Unless otherwise noted, all trademarks herein are
marks of the Lonza Group or its affiliates.
© Copyright 2008, Lonza Walkersville, Inc.
All rights reserved. A-LAL-2008-3, 12/08

Europe International Offices

Customer Service: 32 (0) 87 321 611
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E-mail: scientific.support.eu@lonza.com
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