GLP TOPICS
Characterization and
Management of Test and
Control Articles
BBS UNITED, ALEX HAYDEN/GETTY IMAGES
Jeff Morgan
“GLP Topics” addresses topics associated with good laboratory practice
requirements. We intend this column to be a useful resource for daily work
applications. The key objective for this column: Useful information.
Reader comments, questions, and suggestions are needed to help us fulfill
our objective for this column. Please send your comments and suggestions to
column coordinator Cindy Green at [email protected] or to managing editor Susan Haigney at [email protected].
EXECUTIVE SUMMARY
Good laboratory practices (GLPs) define the basic requirements for
characterizing test and control articles used in clinical trials and studies (1). Additional information follows this regulation with regard to
storage, distribution, identification, and documentation requirements.
However, the focus of these regulations does not detail the most expeditious ways to perform these activities or define the extent of characterization not only required by the regulations, but for the needs of the
sponsor. This discussion provides additional insight into the mechanisms for performing compliant and useful characterization of test and
control articles.
INTRODUCTION
Clinical, and even subclinical, testing is a jungle fraught with peril. The
research pathway to achieving a prototype device or an investigational
new drug eventually leads to the inevitable and pivotal point where it
is tested for safety, effectiveness, and efficacy. This pivotal point should
be approached with a degree of confidence if control articles have been
established and the treatment of test articles has been defined; if not,
then the peril increases.
GLPs were initiated when errors by food producers and drug manufacturers resulted in harm to the general public. Because of these errors, the
federal government took control of the food and drug industry under the
umbrella of the US Food and Drug Administration. GLPs were developed and published as 21 CFR 58 to regulate food and color additives,
animal food additives, human and animal drugs, medical devices for
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GLP TOPICS
human use, biological products, and electronic products to ensure the quality and integrity of the safety
data filed with FDA for marketing approval.
This article explores the requirements for characterizing, handling, storing, and documentation requirements for test and control articles with special
emphasis on GLPs.
EVERYTHING NEEDS A REFERENCE POINT
Every drug or device is designed to have an effect,
and that effect is measurable in some way. This is
the basis for a control article. The term article wears
many hats. For an in-vitro diagnostic, it may be a
blood sample or synthetic matrix containing the
diagnostic analyte. For a drug, it may be a metabolite, a purified powder, or an active biological ingredient. If the device delivers a type of treatment,
energy, or effect, the target of that treatment—or
surrogate means of measurement of the effect—is
the control article. The control article provides the
basis for comparison with a test article and is the
yardstick against which the effects are evaluated.
On the opposite side of the study is the test
article, which can be a clinical sample of blood or a
biological substance, a measurement of the level of
the drug at its target site, or a measurement of the
effect of the drug or device. For example, a drug
designed to control hypertension may be pure,
may be easily synthesized, and may meet all of the
needs for low toxicity and side effects. But if blood
pressure is unaffected in the study (test) population,
the drug is a failure. Proper characterization of the
control article—in this case the antihypertensive
drug—could assist in investigating the source of
the failure, whether the root cause is a manufacturing, labeling, or dosing error. The control article
serves as a failsafe but only if its composition,
strength, purity, and storage conditions are known
and carrier compounds are defined to the extent
that the synergy or antagonism of such compounds
are explored.
IT’S NEVER TOO LATE
Establishing control articles should not be an event
that is put on hold until time for clinical studies.
54 Journal of GXP Compliance
This statement sounds obvious, but it happens
in industry more frequently than it should. The
requirements of GLP specify that the identity,
strength, purity, and composition or other characteristic, which appropriately defines the test
or control article, shall be determined and documented for each batch. How do you know that you
have a reliable control article? If the control article
has a traceable standard, the standard must be
compared to the control article in the early stages
of the development of the drug or device. Test and
control articles may be obtained from a commercial
supplier, from a sponsor, or may be prepared by
synthesis or other production methods.
There are many ways to perform the comparison
to the traceable standard, including chemical characterization, purity, chromatographic profile, titer,
activity, or potency. However, if the drug or device
is on the cutting edge with no traceable standard,
the game becomes increasingly difficult to play. The
following scenarios demonstrate.
Case 1: The New Analyte
Imagine that a university has found a new biological marker for a disease that results in deterioration
of an organ. A rise in the marker has correlated
with early prediction of the disease, and treatment
is available. Imagine further that the university is
seeking a commercial partner to develop a reliable in-vitro diagnostic assay for FDA approval and
potential worldwide marketing. What is the first
order of business: engage a patent attorney, develop
a prototype test, engage sales and marketing to plan
a strategy, hire additional staff, go public, or none of
the above?
The correct answer is “none of the above.”
The first step should be to characterize the new
analyte. Chances are that in preliminary studies,
extensive characterization of the test article was
deemed to be unnecessary. Determine the purity
of the substance. If the substance is not pure, then
purify it through a traditional chemical method.
High performance liquid chromatography (HPLC)
is a good place to start (see Table I). But also be
aware that in some cases, the largest quantity that
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is purified does not represent the best quality.
Therefore, during the purification process, the
pre- and post-peaks may actually represent the
target. Take care to purify sufficient material to
perform additional characterization, testing, and
most importantly, sufficient material to preserve as
a future reference.
The next step in characterization is to determine
the composition of the new analyte. The extent of
characterization required is subjective and will be
determined by the general nature of the analyte. A
chemical entity can undergo mass spectrometry;
antibodies can be characterized by their activity
and immunoglobulin type. The take-home here
is that the time to do this is as soon as possible,
because well-characterized starting material will be
the fastest path to success.
It is extremely important that any and all analytical methods used in characterization of the control
(and test) article are subjected to a rigorous validation to assess the precision, accuracy, repeatability
and linearity. This activity is always dependent
on an approved written protocol with acceptance
criteria, documented training of the individuals
performing the validation, and a thorough quality
review of the results. Include checks to evaluate
the routine use of methods including the impact of
instruments, different technicians, etc.
Case 2: The Magic Machine
Imagine a medical device that stimulates regeneration of pancreatic cells and has met preliminary design verification. The device is now ready to test on
a small number of subjects in a clinical study. The
study has stipulated that success will be assessed
by study of periodic levels of insulin, glucagon,
and somatostatin. There are potentially two control
articles: a defined and well-characterized prototype
instrument to which the manufacture of all test machines is measured, and clinical laboratory controls
with known quantities and predictable stability
of the analytes used to test the results. The details
are all in the design of the study, but must use a
control that faithfully produces expectable analytical results.
THE GOLDEN SPIKE
Once characterization of the control article is
complete, this material, analyte, condition, drug, or
device is the reference to which everything else is
compared. It provides an anchor by which to judge
changes during development and assess efficacy
during a clinical study. This is much easier to accomplish if a so-called gold standard of a substance
is sequestered and used to evaluate and qualify new
lots of drug, antibody, or protein. The going gets
tougher in the device realm, especially when assessing the efficacy of treatment by an instrument or
energy-imparting device.
While most GLP studies involve human samples
and nonclinical laboratory studies, devices and
physiological responses that cannot be measured
in a human sample do exist. In these cases, the
best control article may be a well-defined prototype
instrument. This would be a device where component parts have been defined by edge-of-failure testing or by a design-of-experiments (DOE) approach
during which key components are interchanged or
substituted to define the specific conditions where
the device operates optimally.
For example, the quality and tolerance of capacitors or transistors may be tested in the prototype by
a DOE matrix that will characterize the most favorable parts to faithfully replicate the desired physical
or physiological effects and, therefore, provide the
desired efficacy of the device. The conundrum is
ensuring that the physical or physiological effects
are representative of true outcomes, which is beyond the scope of this discussion.
The key here is to define the control article as
completely as possible, and use the information obtained by a comparison to test articles to determine
the safety, effectiveness, and medical outcome.
THE CASE OF THE DISAPPEARING TEST ARTICLE
Test articles seem to be fairly straightforward at first
glance. A blood sample is taken from a test subject;
physiological parameters are obtained from the test
subject; a diagnostic test is performed to determine
whether the effect of the drug or device meets
expectations. But without thorough knowledge of
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GLP TOPICS
TABLE I: Characterization methods and key activities for test and control articles.
Activity and Responsibility
Procedural Step
Test and control article uniformity and
stability—study personnel
Determine the uniformity of test and control article concentration (homogeneity) and the
chemical stability prior to or during the study in order to ensure adequate quality and
integrity of the dosing formulation.
Screen individual formulation stability to ensure that samples taken will not be subjects of degradation.
Analytical requirements—study personnel
Develop and validate methods with sufficient selectivity, accuracy, precision, and
sensitivity to enable test and control article concentrations to be determined in the
formulations.
Identify methods capable of detecting the test and control article in a range of concentrations in the carrier used.
Establish a reference standard and define the characteristics with regard to purity
and identity. This reference standard can be a chemically-defined material or a wellcharacterized test article that is used in the formulation.
Use methods that are stability indicating. The techniques used must be able to detect
instability in the event it occurs. Stability indicating methods include methods such as
HPLC and gas chromatography (GC).
Sampling and stability assessment—study
personnel
Establish procedures for sampling to ensure that samples taken are representative of
the formulation.
Maintain records of the quantity removed to ensure accountability for all test and control articles and formulations.
Sample sufficient quantities to allow for duplicate analyses.
Remove samples from several areas of the bulk material. Typically, the top, side, and
bottom should be sampled.
Analyze each of the three samples separately. The test results from the different samples should not demonstrate any significant difference.
Remove additional samples over time to evaluate stability of the text mixture.
Continue the stability surveillance throughout the anticipated study period.
the proper management of the test article, unpleasant
surprises may arise.
Using the in-vitro diagnostic example, the analyte
may be stable as a control article in a simple buffer
or even in an aqueous solution. It may be stable at
room temperature for many months or years under
those conditions. But the naturally-occurring target
analyte may not have a long shelf life under the conditions from its biological source.
Therefore, the proper collection, handling, and
storage conditions of the test article must be established or characterized. An analyte in a blood
sample may be bound to a carrier protein as it travels
56 Journal of GXP Compliance
through the body, and its concentration may be
stabilized in-vivo. But once the sample is removed
from the body, it is possible that the analyte could
be subjected to conditions where it could become
degraded by enzymes, light, or microorganisms. The
analyte might be absorbed by a protein unrelated to
its carrier, adsorbed to the container in which it is
stored, or otherwise damaged; therefore, its levels
could become reduced or the analyte could be rendered totally undetectable. This may appear within
hours or even weeks after improper storage and handling conditions. Samples often “die” without notice
because of failure to test the stability of the sample
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under various conditions that are intended to cover a
multitude of problems, if properly investigated.
It is important that test articles, no matter what
their origin, be subjected to many conditions,
collected by several means, preserved under various conditions, and stressed to the limit by a DOE
approach. This includes whether the best sample is
whole blood, serum, or plasma; which anticoagulant
produces the most stable sample; whether blood
must be immediately separated into cellular and
noncellular components; whether the analyte best
tolerates storage at 2-8°C, -20°C or whether ultralow
temperatures produce stable test articles. Some analytes are even susceptible to multiple freeze-thaws
and, therefore, must be aliquotted and used once
after thawing.
Without this level of diligence, not only a few, but
potentially all test articles could be rendered useless.
Worse yet, the incorrect clinical result could lead
to an incorrect assumption about the efficacy of the
study, squandering time and resources.
TRUTH IN PURCHASING–TRUST BUT VERIFY
If test and control articles are purchased from a
commercial supplier, there are several caveats that
must be observed (Table II). All manufacturers are
not created equal. Every manufacturer has production tolerances of not only the test and control
target material, but in the matrix in which it is
supplied.
For example, if one is looking for human albumin as a control article, and it is important that the
preparation be essentially globulin-free, it is important to know what the term “essentially” means to
the manufacturer. Some manufactures will allow
microgram traces of globulin, while others’ tolerances can be into the milligram range per gram of
albumin. If globulin interferes with the analyte,
then it is beneficial to know what the manufacturer’s tolerances are. This is where requesting a
comprehensive certificate of analysis (CoA) can be
useful. Often a CoA focuses on the analyte at hand
and states the results of a few additional analytical tests. However, the manufacturer may actually
conduct important tests such as electrophoresis or
cross-reactivity that may define and provide critical
information about the analyte and the preparation.
FDA will expect adherence to 21 CFR 820.80(d)
as follows:
“Final acceptance activities. Each manufacturer shall establish and maintain procedures
for finished device acceptance to ensure that
each production run, lot, or batch of finished
devices meets acceptance criteria.”
If the reagents are past expiration or not even
standardized, there is no way to ensure the production lot meets any criteria. Therefore, there must be
defined procedures and specifications for raw materials used in testing that are not used in production.
This can be tricky when it is necessary to use a
chemical to test a chemical. It must be ensured that
all chemicals and reagents are handled properly and
are used before they expire.
ADDITIONAL POINTS TO CONSIDER
In addition to the actions described in Table II,
there will be differences in expectations for test and
control articles depending on where the study will
be conducted. The Organization for Economic Cooperation and Development (OECD) (2), with members from 34 countries, has substantial influence in
the European Union, South America, and the AsiaPacific region. The following are some differences
between FDA and OECD (3) requirements, which
could influence the characterization and labeling of
test and control articles:
•One of the key and important differences is
that FDA does not include a requirement for
reference articles (i.e., analytical standards).
OECD’s definition of a reference item can
include both control items and reference
items.
•FDA states that a storage container shall be
labeled with name, chemical abstract number
or code number, batch number, expiration date
if any, and storage condition if appropriate.
OECD does not require batch number.
•For control article characterization, FDA states,
“In those cases where marketed products are
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GLP TOPICS
TABLE II: Commercial suppliers of test and control articles points to consider.
Commercially supplied test and control articles (such as marketed pharmaceutical or biological products) should be produced by the
manufacturer under GMP conditions.
Documentation such as the certificate of analysis or the certificate of testing must be supplied along with the test or control article.
Selection of commercial sources should include availability (no backorders), availability of several lots and typical outdating profiles.
Supplier audits are worth the time invested.
used as control articles, such products will be
characterized by their labeling.” OECD does
not include this statement.
•For stability of test and control articles as
such or in mixtures, FDA states that this can
be determined before study initiation or concomitantly according to standard operating
procedures (SOPs). OECD states only that stability of the test item in the vehicle should be
determined.
•FDA states that if more than one component of
a mixture has an expiration date, the earliest
date shall be shown on the container. OECD
does not address this, but it would be the
expectation.
•The most significant difference is that FDA
does not address reference standards, to which
all characterization, stability, receipt, distribution, and disposal requirements apply under
the OECD GLPs, not counting the requirements
to their mixture with a carrier.
Control articles or reference substances as they
are called in the OECD principles are of utmost
importance because they are commonly used to
calibrate analytical instruments or are used as
clinical laboratory controls. The accuracy of the
reference substances also determines the accuracy
of the analytical method. In other words, if the
reference standard is inaccurate, the test results
will suffer.
The moral here is before beginning the test and
control article characterization process, know the
regulations of the country in which the study will
be conducted.
58 Journal of GXP Compliance
MAINTENANCE AND DOCUMENTATION
The major requirements for control articles are listed in
Table III. In addition, the stability of each test or control
article should be determined. This can be done either
before study initiation or simultaneously according to
written SOPs that provide for periodic reanalysis of
each batch.
The test and control articles must be received, stored,
maintained, and accounted for throughout the study. A
tracking log or form should be developed for each item
and should include key information (Table IV).
Each storage container for a test or control article
should be labeled by name, chemical abstract number or code number, batch number, expiration date,
and, where appropriate, storage conditions necessary to maintain the identity, strength, purity, and
composition.
Storage containers should be assigned to a particular
test article for the length of the study and all accounting
records must be retained in study-specific files according to regulatory requirements.
CONCLUSIONS
The use of test and control articles in nonclinical laboratory studies has been established as a key component
to the study. But if incompletely or improperly characterized, the resultant data will be suspect at best and
will not reflect the truth of the safety, effectiveness, and
efficacy of the study article. Characterization must be
complete and accurate and, most importantly, traceable.
Weller best expressed the bottom line in 1988 (4):
“If experimental work is conducted in compliance with GLP, with or without the aid of computers, it should be possible for an inspector, maybe
four or five years hence, to look at the records of
the work and determine easily why, how, and by
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TABLE III: FDA requirements for test and control articles.
The identity, strength, purity, composition and other characteristics should be determined for each batch and documented.
Methods of synthesis, fabrication, or derivation of test and control articles should also be documented.
Copies of this documentation must be stored with the study data and must available for FDA inspection.
TABLE IV: Key items for test and control article accountability.
Code number of the test or control article that corresponds with protocol
Description of test or control article
Initial quantity of product received
Expiration date, if any
Storage conditions
Date placed into inventory
Removal of test or control articles from inventory, including date, quantity, reason, and location of usage. Initials of the individual
issuing the article, initials of the individual receiving the article. Any comments.
Verify that quantities used for sample retention, quantities damaged and found unusable, and expired material are recorded.
Review the return or destruction of test and control articles.
whom the work was done, who was in control,
what equipment was used, the results obtained,
any problems that were encountered and how they
were overcome.”
If the goal of the nonclinical study is to produce sustainable documentation and good science, then these
words certainly apply to test and control articles and
their thorough characterization.
ARTICLE ACRONYM LISTING
REFERENCES
Jeff Morgan, President and Principal of JWM Associates LLC, has
assisted both international and domestic medical device, biopharmaceutical companies, and clinical laboratories since 1998.
Jeff may be contacted at 253.208.3430 and [email protected].
1. FDA, 21 CFR 58.105, Title 21, Food and Drugs Chapter I, Food
and Drug Administration, Department of Health and Human
Services, Subchapter A, General, Part 58 Test and Control
Articles, Sec. 58.105 Test and control article characterization,
43 Federal Register 60013, Dec. 22, 1978, as amended at 52 FR
33781, Sept. 4, 1987; 67 FR 9585, Mar. 4, 2002.
2. Information about the Organization for Economic Co-operation
and Development can be found online at http://www.oecd.org/
3. OECD, Principles and Guidance for Compliance Monitoring, ISBN:
9789264012820, OECD Press, January 18, 2006.
4. D.L.M. Weller, “GLP and Quality Assurance,” Anal. Proc. 198, Vol
25, 199/200. GXP
CoA
Certificate of Analysis
FDA
US Food and Drug Administration
GLP
Good Laboratory Practice
OECD
Organization for Economic Co-operation and
Development
SOP
Standard Operating Procedure
ABOUT THE AUTHOR AND COLUMN COORDINATOR
Cindy Green, RAC, the column coordinator, has been working with regulated industry for nearly 35 years holding senior
positions in regulatory, quality assurance, and quality control for
several biotechnology and medical device companies. She is the
president of her own consulting company and can be reached by
e-mail at [email protected].
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