BioWhittakerTM LAL Review

Published by Cambrex Bio Science Walkersville, Inc.

Spring 2003

Investigating Out of Specification (OOS) reports, and the values used for
any statistical calculation or analy-
Test Results and The LAL Test sis." Based on this definition,
where the anomalous result is not
By Ronald N. Berzofsky Ph.D. the reportable value, a laboratory
investigation may be adequate; an

I
n 1998, the FDA published the
draft guidance entitled "Investi-
gating Out of Specification (OOS)
Test Results for Pharmaceutical
Production" 1. Since then, the time
and energy devoted to investigating
perceived OOS results and other
anomalous results has expanded to
monumental proportion. It's time to
take a step back and review exactly
what the OOS guidance says and
how it relates to LAL test results.
As defined in the guidance, the term
OOS results includes all suspect
results that fall outside the specifica-
tions or acceptance criteria estab-
lished in new drug applications, offi-
cial compendia, or by the manufac-
turer. Careful attention to definitions
of specifications and acceptance
criteria can allow investigations of
anomalous results to be limited to
identifying the cause without
expanding the investigation need-
lessly to rule out unlikely product
failures. Lynn Torbeck, in his article
"Reportable Values for Out-of-Spec-
ification Test Results"2, suggests
that a clear and focused definition of
the term, reportable result, "can min-
imize the confusion … and clarify
the related issues of assay valida-
tion and setting specifications."
Mr. Torbeck defines a reportable
value as "the end result of the com-
plete measurement method as doc-
umented. It is the value compared
with the specification, the values
collected when the term replicates
is used, the values used for official
OOS investigation of an unlikely
product failure may not be neces-
sary.
If an assay test result can be invali-
dated based on clear evidence of
laboratory error, there is no need for
If an assay test result can
be invalidated based on
clear evidence of laboratory
error, there is no need for
further investigation.
further investigation. That being
said however, laboratory errors,
which prevent valid test results from
being generated, should be investi-
gated and resolved. The guidance
says...

“Laboratory error should be rela-

tively rare. Frequent errors suggest
BioWhittaker becomes Cambrex Bio Science Walkersville, Inc.
problem that might be due to inad-
equate training of analysts, poorly
BioWhittaker has taken on the name of our parent company Cambrex.
maintained or improperly calibrated
And while our name has changed, our BioWhittaker Endotoxin Detection
equipment, or careless work.
products haven't.
Whenever laboratory error is identi-
fied, the firm should determine the
• Stringent quality control over all products
source of that error and take cor-
• Technical experts committed to meeting your needs
rective action to ensure that it does
• Fast, reliable customer service
not occur again. To ensure compli-
• Innovative leaders in Endotoxin Detection
ance with the CGMP regulations,
the manufacturer also should main-
Cambrex is a global company dedicated to life sciences, sharing knowledge
tain adequate documentation of the
and resources to deliver better service and support. We appreciate the
corrective action.”
continued opportunity to meet your needs with the best quality products.
Continued on page 2
 BioWhittakerTM LAL Review 2

OOS Continued from page 1 If the assay parameters are not in and see if the test results are out of
However, laboratory errors do not specification, invalidate the entire specification before invalidating the
automatically mean there has been assay. If there is no other error, analysis. The analyst should not
a product failure. the investigation is complete. generate any assay data that they
There is no valid test result which suspect will later be invalidated
The first step in evaluating any labo- constitutes a reportable value. because of laboratory error.
ratory test result is to review all Retest the original solutions unless
assay parameters to determine if the there is reason to suspect they were Before discarding test preparations
assay is valid. In advance of per- not prepared correctly. or standard preparations, analysts
forming any test or investigating any should check the assay data for
laboratory error, the laboratory If the product specific parameters compliance with written specifica-
should establish written assay and are not in specification, and retest- tions. However, the analyst should
product specifications. Include ing and resampling is permitted (see not automatically retest sample
specifications for the replicates of FDA Guideline, Validation the Limu- solutions without consulting with the
standards (%CV), standard curve lus Amebocyte Lysate Test as an supervisor. The investigation should
parameters (slope, Y intercept, coef- End-Product Release Test for be well documented and scientifical-
ficient of correlation), replicates of Human and Animal Parenterals, Bio- ly defensible. Retesting sample
product (%CV), PPC recovery and logics and Medical Devices), initiate solutions can be considered part of
product test results. Evaluate all retesting and/or resampling. If prod- the investigation, but should require
assay parameters first. In the event uct specific parameters continue to documentation and supervisory
of an error, reject the entire assay if be out of specification, you now sign-off.
any assay parameter is out of spec- must expand the investigation.
ification. If sample dilutions are to be stored
The OOS guidance specifies the and retested, validate the maximum
Based on the definition discussed responsibilities of the analyst. If the time test solutions may be stored
above, only reportable results have analyst suspects an error in the per- and still yield accurate results.
the potential of constituting an OOS; formance of a laboratory test, the Specify type of storage container
i.e. PPC recovery and product test analyst should immediately docu- and storage temperature. Use of
results. All the other assay parame- ment what happened and stop the endotoxin-spiked samples is recom-
ters that fall out of specification, if analysis. The analyst should not mended.
they are not reportable results, con- knowingly continue an analysis that
stitute a laboratory error. Product can be invalidated for an assignable The OOS guidance also specifies
quality is NOT an issue. cause. The analyst should not wait the supervisor's responsibilities.
Once an OOS result has been iden-
tified, the supervisor's assessment
should be objective and timely.
NEW RESEARCH PRODUCT AVAILABLE a) An immediate assessment
could include re-examination of
Earlier this year in our Winter 2002 LAL Review, we shared with you infor-
mation about our next generation of endotoxin detection products, the actual test solutions and
Recombinant Factor C. We are pleased to announce our research grade accessories used in the original
test kit, PyrogeneTM , will be available for sale at the beginning of 2003. analysis.
This test method is a single end-point assay that measures the fluores- b) The supervisor should discuss
cence generated by the enzymatic cleavage of a synthetic substrate and the test method with the analyst.
has a linear detection range from 0.1-10.0 EU/ml. The Recombinant Fac- The supervisor should confirm
tor C assay has a better specificity for the detection of endotoxin and will analyst's knowledge of the correct
not detect beta (1,3)-glucan activity. procedure and the analyst's ability
to perform the procedure in the
Pyrogene TM can be ordered under part number 50-658U which contains correct manner.
192 tests per kit.
Using the TRENDING feature, a
For additional product information, please contact our Technical Services
Department at 1-800-654-4452 ext 7822, or to place an order, contact our
Customer Services Department at 1-888-403-8772.
Continued on page 4

Horseshoe Crab Named Delaware's
State Marine Animal
By Abigail Bradley
Editor’s Note: Abigail is now a freshman at the University of Delaware majoring in
biology. She plans to pursue a graduate degree in marine science and wants to
continue working on horseshoe crab preservation issues.
E
ver since I was a little child, I
have been interested in horse-
shoe crabs and have been for-
tunate enough to live near the
Delaware Bay where I grew to learn a
lot about them. When my mother
and I walked along the beach, we
would always look for stranded
horseshoe crabs and return them to
the water. I didn't know the impor-
tance of horseshoe crabs at the time;
I just didn't want to see the stranded
ones die. Then, I attended Coast
Day, a celebration of the Delaware
coast at the University of Delaware's
College of Marine Studies in Lewes. I
asked an oceanographer if returning
stranded horseshoe crabs to the
water was okay or if it was interfering
with the ecosystem. He said it was
not only okay, but that we should be
doing this because horseshoe crabs
were valuable creatures and were in
decline. This oceanographer, inci-
dentally, Dr. Doug Miller, would be the
man I wound up working for at the
College of Marine Studies during my
junior and senior years of high
school.
His remarks spurned my interest in
horseshoe crabs, and I began doing
research on them, as well as partici-
pating in the annual horseshoe crab
survey that the College of Marine
Studies holds. In ninth grade, I was
interested in designing a science lab
project involving horseshoe crabs.
When I described the type of experi-
ment I wanted to work on (testing dif-
ferent types of water for the presence
of endotoxin using LAL), Dr. Bill Hall
at the University of Delaware's Col-
lege of Marine Studies suggested I
contact BioWhittaker. When I called
BioWhittaker four years ago, I was
delighted to be put in touch with Greg
Green who helped explain some of
BioWhittakerTM LAL Review
the science to me and provided me
with supplies to work on the experi-
ment. This is what gave me my start
on horseshoe crab research. For the
rest of high school, I did all of my sci-
ence projects on horseshoe crabs.
I decided to start some horseshoe
crab science projects focusing on
which environmental factors influ-
ence their nest site selection. I
measured beach profile, sediment
grain size, and water temperature
and salinity at five beach sites off the
Delaware Bay. In 2001, I won a
national DuPont Science Challenge
Award (one of 50 winners out of over
13,000 entrants) and the President's
Environmental Youth Award. I also
received first place at the Sussex
County Science Fair, second at the
EPA Science Fair, and third at the
Delaware Valley Regional Science
Fair.
A few months ago, my mother
noticed a road in Lewes named
"Munchy Branch Lane." Such an
odd name for a new street got us
thinking about the way in which
communities choose new names for
streets, buildings, etc. That is when
it dawned on us that nothing is
named after the horseshoe crab in
Delaware, yet, in my opinion, they
are this state's most valuable
resource.
I then wrote an email to Governor
Minner suggesting that she consider
naming the horseshoe crab as
Delaware's state marine animal. She
told me the first step was to contact a
state representative and that she
would be "looking for the legislation."
I then contacted Representative John
Schroeder, who also happens to be
the brother of my Cape Henlopen
3
High School biology teacher, Rob
Schroeder. I provided Rep. Schroed-
er with a PowerPoint demonstration
presenting the value of horseshoe
crabs, not just to Delawareans, but to
all Americans. Not only are they 500
million year old creatures that
deserve our respect, but also there is
probably not a single American who
has NOT benefited from a vaccine,
an injectable antibiotic, an intra-
venous solution, insulin, or a medical
device that's been tested with LAL. I
...there is probably not a
single American who has
NOT benefited from a vac-
cine, an injectable antibiotic,
an intravenous solution,
insulin, or a medical device
that's been tested with LAL.
also researched state symbols and
learned that not a single other state
has a "marine animal" as one of its
symbols (some have marine mam-
mals, others have fish, crustaceans,
etc.)
Rep. Schroeder introduced the bill
and when it came time to be voted
on, I spoke before the House of Rep-
resentatives about the importance of
horseshoe crabs to pharmaceuticals
and ecology, and emphasized how
the Delaware Bay is home to more
horseshoe crabs then anywhere else
in the world. The bill passed the
House and the Senate unanimously
and was signed into law by the Gov-
ernor on June 25, 2002.
The same day the bill was signed, I
was invited to visit the Delaware
Aquatic Resources Education Cen-
ter where Dr. Ron Berzofsky of
BioWhittaker was giving a presenta-
tion. What a delight to meet in per-
son a representative of BioWhittak-
er, the organization that helped
launch my career in HSC research.
Who knows … maybe after college
and graduate school, I'll be fortunate
enough to work at BioWhittaker
someday too!

OOS Continued from page 2
graph summarizing historical prod-
uct results may be helpful in identi-
fying a trend towards unacceptable
test performance. Typically 3 out of
4, or 4 out of 5 values all on the
same side of the mean indicate a
trend. Values outside the 3 stan-
dard deviation upper control limit
(UCL) or lower control limit (LCL)
clearly indicate atypical and unex-
pected results.
If the product test result is out of
specification or suspect, analyze
historical endotoxin results. Does
the data indicate a trend towards
suspect or unsatisfactory results?
If the PPC recovery is out of speci-
fication, a graph summarizing a par -
ticular analyst's past performance
compared to other analysts may be
helpful in identifying a problem
associated with an analyst depend -
ent assay technique or dedicated
accessory, i.e., micropipette.
c) The supervisor should
examine the Reaction Times
obtained in the analysis and iden-
tify anomalous or suspect infor -
mation.
d) The supervisor should con-
firm performance of the instru-
ment.
e) The supervisor should
determine that the appropriate
reference standards, reagents
and other solutions were used
and that they met quality control
specifications.
f) The supervisor should eval-
uate the performance of the test-
ing method to ensure that it is
performing according to the stan-
dard expected on method valida-
tion data.
g) The supervisor should doc-
ument and preserve evidence of
the initial assessment.
When clear evidence of laboratory
error exists, laboratory testing
results should be invalidated.
BioWhittakerTM LAL Review
When evidence for laboratory evi-
dence remains unclear, a failure
investigation should be conducted
to determine what caused the unex-
pected results.
Laboratory Investigation Phase
A number of practices are used dur-
ing the laboratory phase of an
investigation. These include: (1)
retesting a portion of the original
sample(s), (2) testing a specimen
from the collection of a new sample
from the batch, (3) resampling test-
ing data, and (4) using outlier test-
ing.
Retesting
Retesting implies re-assay of the
original sample. Situations where
retesting is indicated include inves-
tigating test instrument malfunc-
tions or identifying a possible sam-
ple handling integrity problem, for
example, a suspected dilution error.
Generally, retesting is neither spec-
ified nor prohibited by approved
applications or by the compendia.
However, FDA generally does not
permit the substitution of one test
result for another without invalidat-
ing the suspect test for an assigna -
ble cause. The laboratory investiga-
tion should uncover the cause
whenever possible.
Note: The FDA Guideline on the
Validation of the Limulus Amebo-
cyte Lysate Test as an End-Product
Endotoxin Test for Human and Ani-
mal Parenteral Drugs, Biological
Products, and Medical Devices
allows for retesting. Retesting is
performed using twice the original
number of replicates.
Decisions to retest should be based
on the objectives of the testing and
sound scientific judgment. Retest-
ing often should be performed by an
analyst other than the one who per-
formed the original test. Therefore,
have at least two analysts validated
(competing Initial Qualification Test)
to perform the LAL test prior to
needing a second analyst to retest
suspect samples.
4
Resampling
Resampling involves analyzing a
specimen from the collection of a
new sample from the batch.
The FDA Guideline on the Valida-
tion of the Limulus Amebocyte
Lysate Test as an End-Product
Endotoxin Test for Human and Ani-
mal Parenteral Drugs, Biological
Products, and Medical Devices
allows for resampling, specifically
when the repeat testing on pooled
samples indicates the presence of
endotoxin. In such case, an addi-
tional ten units from the lot are test-
ed individually.
Averaging
Averaging test data (individual
determinations) can be a valid
approach, but its use depends upon
the sample and the purpose of the
test. If the sample can be assumed
to be homogeneous using averages
can provide a more accurate result.
In the case of microbiological
assays, the USP prefers the use of
averages (of individual determina-
tions) because of the innate vari-
ability of the biological test system.
Reliance on averages (of reportable
values) has the disadvantage of
hiding variability among individual
test results (individual reportable
values). For this reason, unless
averaging is specified by the test
method or adequate written investi-
gation procedures, all individual test
results (individual reportable val-
ues) should be reported.
It should be noted that a test might
consist of replicates (of individual
values) to arrive at a result
(reportable value). This determina-
tion is considered one test and one
result. The use of replicates (of
individual determinations) should
be included in the written, test
methodology. Unexpected variation
in replicate determinations should
trigger investigation and documen-
tation requirements.
Continued on page 5

OOS Continued from page 4
Each individual value, which is com-
bined to produce the reportable
value, does not need to meet the
specification of the reportable value
unless an establishment has defined
it as reportable in policies or proce-
dures. Even if these non-reportable
individual replicates do not conform
to the specification of the reportable
value, there is no product failure and
any investigation can be limited in
scope to the laboratory error. The
individual values should not be
reported, nor is their standard devia-
tion or % CV reported. Any variabil-
ity within these values is a laborato-
ry assay issue
In some cases, evaluating a series
of reportable values may be part of
the test procedure. If some of the
reportable values are OOS and
some are within specification and all
are within the documented variation
of the method, the passing results
should be given no more credence
PYROGENT® Ultra Returns!
Imagine running your gel clot assays
without ever having to use a vortex
mixer. How much time would you
save? The initial vortexing of the
CSE requires a minimum of 15 min-
utes, vortexing each standard
between dilutions involves another
minute, and additional vortexing
before adding standards to reaction
tubes equals a lot of preparation
time.
The problem is, proper vortexing is
critical to the LAL assay, right? Well,
not anymore. Cambrex is reintro-
ducing BioWhittaker's PYROGENT ®
Ultra, a gel clot kit configuration that
includes our proprietary Liquid
Endotoxin Standards that never
need vortexing! Each kit includes a
gel clot LAL reagent matched to a
series of pre-diluted liquid endotoxin
standards. The Liquid Endotoxin
BioWhittakerTM LAL Review
than the failing results, in the
absence of clear evidence that ana-
lytical error had occurred.
To use averaged reportable values
for assay reporting, all test results
should conform to specifications. All
reportable values must meet specifi-
cation or an OOS has occurred.
Averaging OOS reportable values
with in-specification reportable val-
ues without an investigation is NOT
acceptable.
Conclusion
In summary, laboratory errors, if
they occur, DO NOT constitute a
product failure. Industry experience
suggests that with appropriate train-
ing and validated procedures, labo-
ratory errors associated with the
LAL test are infrequent. These
errors must be investigated when
they occur, but the investigation can
be limited in scope. With clear defi-
nitions for reportable values, appro-
Standards never have to be vor-
texed! Simply select the dilution you
need, invert it for three seconds,
open, and use. There is no need to
vortex or make dilutions of the CSE.
You can have your assay underway
in less than five minutes.
In addition to saving time, you'll
experience a cost savings by not
having to use dilution tubes and
additional pipette tips. Most impor-
tantly, endotoxin dilution errors are
no longer a variable.
Each 200 test kit contains 4 x 50
test/vials of lysate, 5 x 5 ml/vials of
gel clot Liquid Endotoxin Standard
(2 8, 8, 0.5 8, 0.25 8, 20 8) and a
matched Certificate of Quality. Addi-
tionally, the 20 8 standard is includ-
ed to make spiking of the positive
product controls an easy step.
5
priate raw material specifications,
validated depyrogenation proce-
dures and controlled manufacturing
processes, product related out-of-
specification results (OOS) also
should not occur. In a well con-
trolled system, the time and energy
devoted to investigating out-of-spec-
ification results is at a true minimum.
References
1. "Guidance for Industry, Investigat-
ing Out of Specification (OOS) Test
Results for Pharmaceutical Produc-
tion," Federal Register 63 (189),
522276-52277 (1998).
2. Torbeck, Lynn D. "Reportable Val-
ues for Out-of-Specification Test
Results," Pharmaceutical Technology,
February 1999.
Gel clot LAL has been available for
over 25 years. Pyrogent® Ultra is a
major improvement in this assay
whose time has come.
The kits are available in the follow-
ing configuration:
Catalog Number Lysate Sensitivity
N403 0.03 EU/ml
N406 0.06 EU/ml
N412 0.125 EU/ml
For additional product information,
please contact our Technical Ser-
vices Department at 1-800-654-
4452 ext 7822, or to place an order,
contact our Customer Services
Department at 1-888-403-8772.
 BioWhittakerTM LAL Review Telephone:
Customer Service (888) 403-8772
Technical Service (800) 654-4452 x7822
Published by Cambrex Bio Science Walkersville, Inc.
In Maryland (301) 898-7025
FAX (301) 845-8708

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