Guidance for Industry
Analytical Procedures and Methods Validation Chemistry, Manufacturing, and Controls Documentation
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Draft Guidance
This guidance document is being distributed for comment purposes only.
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U.S. Department of Health and Human Services
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Center for Biologics Evaluation and Research (CBER)
August 2000
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TABLE OF CONTENTS
I. INTRODUCTION
II. BACKGROUND
III. TYPES OF ANALYTICAL PROCEDURES
IV. REFERENCE STANDARDS
V. METHODS VALIDATION FOR INDS
VI. CONTENT AND FORMAT OF ANALYTICAL PROCEDURES FOR NDAS, ANDAS, BLAS, AND PLAS
VII. METHODS VALIDATION FOR NDAS, ANDAS, BLAS, AND PLAS
VIII. STATISTICAL ANALYSIS
IX. REVALIDATION
X. METHODS VALIDATION PACKAGE: CONTENTS AND PROCESSING
XI. METHODOLOGY
ATTACHMENT ANDA, ANDA, BLA, AND PLA SUBMISSION CONTENTS
ATTACHMENT B METHODS VALIDATION PROBLEMS AND DELAY
REFERENCES
GLOSSARY
FOOTNOTES
Guidance for Industry 1
Analytical Procedures and Methods Validation
Draft - Not for Implementation
This draft guidance, when finalized, will represent the Food and Drug Administrations current thinking on this topic. It does not create or confer any rights for or on any person and does not operate to bind FDA or the public. An alternative approach may be used if such approach satisfies the requirements of
the applicable statutes, regulations, or both. |
If you plan to submit comments on this draft guidance, to expedite FDA review of your comments, please:
- Clearly explain each issue/concern and, when appropriate, include a proposed revision and
the rationale and/or justification for the proposed change.
- Identify specific comments by line numbers; use the pdf version of the document whenever
possible.
- If possible, e-mail an electronic copy (Word or WordPerfect) of the comments you have
submitted to the docket to cunninghamp@cder.fda.gov.
I. INTRODUCTION
This guidance provides recommendations to applicants on submitting analytical procedures,2 validation data, and samples to support the documentation of the identity, strength, quality, purity, and potency of drug substances and drug products.3 This guidance is intended to assist applicants in assembling information, submitting samples, and presenting data to support analytical methodologies. The recommendations apply to drug substances and drug products covered in new drug applications (NDAs), abbreviated new drug applications (ANDAs), biologics license applications (BLAs), product license applications (PLAs), and supplements to these applications.4 The principles also apply to drug substances and drug products covered in Type II drug master files (DMFs). If a different approach is chosen, the applicant is encouraged to discuss the matter in advance with the center with product jurisdiction to prevent the expenditure of resources on preparing a submission that may later be determined to be unacceptable.
The principles of methods validation described in this guidance apply to all types of analytical
procedures. However, the specific recommendations in this guidance may not be applicable to certain
unique analytical procedures for products such as biological, biotechnological, botanical, or
radiopharmaceutical drugs. For example, many bioassays are based on animal challenge models,
immunogenicity assessments, or other immunoassays that have unique features that should be
considered when submitting analytical procedure and methods validation information. Furthermore,
specific recommendations for biological and immunochemical tests that may be necessary for
characterization and quality control of many drug substances and drug products are beyond the scope
of this guidance document. Although this guidance does not specifically address the submission of
analytical procedures and validation data for raw materials, intermediates, excipients, container closure
components, and other materials used in the production of drug substances and drug products,
validated analytical procedures should be used to analyze these materials. For questions on
appropriate validation approaches for analytical procedures or submission of information not
addressed in this guidance, applicants should consult with the appropriate chemistry review staff at
FDA.
This guidance, when finalized, will replace the FDA guidance for industry on Submitting Samples and
Analytical Data for Methods Validation (February 1987).
Table of Contents
II. BACKGROUND
Each NDA and ANDA must include the analytical procedures necessary to ensure the identity,
strength, quality, purity, and potency of the drug substance and drug product, including bioavailability
of the drug product (21 CFR 314.50(d)(1) and 314.94(a)(9)(i)). Data must be available to establish
that the analytical procedures used in testing meet proper standards of accuracy and reliability (21
CFR 211.165(e) and 211.194(a)(2)).
Methods validation is the process of demonstrating that analytical procedures are suitable for their intended use. The methods validation process for analytical procedures begins with the planned and
systematic collection by the applicant of the validation data to support the analytical procedures. The
review chemist evaluates the analytical procedures and validation data submitted in the NDA or
ANDA. On request from FDA, an NDA or ANDA applicant must submit samples of drug product,
drug substance, noncompendial reference standards, and blanks so that the applicant's drug substance
and drug product analytical procedures can be evaluated by FDA laboratories (21 CFR 314.50(e)
and 314.94(a)(10)). The FDA laboratory analysis demonstrates that the analytical procedures are
reproducible by laboratory testing. The review chemists and laboratory analysts determine the
suitability of the analytical procedures for regulatory purposes. FDA investigators inspect the
analytical laboratory testing sites to ensure that the analytical procedures used for release and stability
testing comply with current good manufacturing practices (CGMPs) (21 CFR part 211) or good laboratory practices (GLPs) (21 CFR part 58), as appropriate.
Each BLA and PLA must include a full description of the manufacturing methods, including analytical
procedures, that demonstrate that the manufactured product meets prescribed standards of safety,
purity, and potency (21 CFR 601.2(a) and 601.2(c)(1)(iv)). Data must be available to establish that
the analytical procedures used in testing meet proper standards of accuracy and reliability (21 CFR
211.194(a)(2)). For BLAs, PLAs, and their supplements, the analytical procedures and their
validation are submitted as part of the license application or supplement and are evaluated by the
review committee. Representative samples of the product must be submitted and summaries of results
of tests performed on the lots represented by the submitted sample must be provided (21 CFR
601.2(a) and 601.2(c)(1)(vi)). The review committee chair may request analytical testing by CBER
laboratory analysts to evaluate the applicant's analytical procedures and verify the test results.
All analytical procedures are of equal importance from a validation perspective. In general, validated
analytical procedures should be used, irrespective of whether they are for in-process, release,
acceptance, or stability testing. Each quantitative analytical procedure should be designed to minimize
assay variation.
Analytical procedures and validation data are submitted in the sections of the application on analytical
procedures and controls. Recommendations on information to be submitted are included in sections
III through IX and XI of this guidance. Information on submission of the methods validation
package to the NDA or ANDA and samples to the FDA laboratories is provided in section X.
Table of Contents
III. TYPES OF ANALYTICAL PROCEDURES
A. Regulatory Analytical Procedure
A regulatory analytical procedure is the analytical procedure used to evaluate a defined
characteristic of the drug substance or drug product. The analytical procedures in the U.S.
Pharmacopeia/National Formulary (USP/NF) are those legally recognized under section
501(b) of the Food, Drug, and Cosmetic Act (the Act) as the regulatory analytical procedures
for compendial items. For purposes of determining compliance with the Act, the regulatory
analytical procedure is used.
B. Alternative Analytical Procedure
An alternative analytical procedure is an analytical procedure proposed by the applicant for
use instead of the regulatory analytical procedure. A validated alternative analytical procedure
should be submitted only if it is shown to perform equal to or better than the regulatory
analytical procedure. If an alternative analytical procedure is submitted, the applicant should
provide a rationale for its inclusion and identify its use (e.g., release, stability testing), validation
data, and comparative data to the regulatory analytical procedure.
C. Stability-Indicating Assay
A stability-indicating assay is a validated quantitative analytical procedure that can detect
the changes with time in the pertinent properties of the drug substance and drug product. A
stability-indicating assay accurately measures the active ingredients, without interference from
degradation products, process impurities, excipients, or other potential impurities. If an
applicant submits a non-stability-indicating analytical procedure for release testing, then an
analytical procedure capable of qualitatively and quantitatively monitoring the impurities,
including degradation products, should complement it. Assay analytical procedures for
stability studies should be stability-indicating, unless scientifically justified.
Table of Contents
IV. REFERENCE STANDARDS
A. Types of Standards
A reference standard (i.e., primary standard) may be obtained from the USP/NF or other
official sources (e.g., CBER, 21 CFR 610.20). If there are questions on whether a source of
a standard would be considered by FDA to be an official source, applicants should contact
the appropriate chemistry review staff. When there is no official source, a reference standard
should be of the highest possible purity and be fully characterized.
A working standard (i.e., in-house or secondary standard) is a standard that is qualified
against and used instead of the reference standard.
B. Certificate of Analysis
A certificate of analysis (COA) for reference standards from non-official sources should be
submitted in the section of the application on analytical procedures and controls. For
standards from official sources, the user should ensure the suitability of the reference standard.
The standard should be stored correctly and used within the established use interval.
C. Characterization of a Reference Standard
Reference standards from USP/NF and other official sources do not require further
characterization. A reference standard that is not obtained from an official source should be of
the highest purity that can be obtained by reasonable effort, and it should be thoroughly
characterized to ensure its identity, strength, quality, purity, and potency. The qualitative and
quantitative analytical procedures used to characterize a reference standard are expected to
be different from, and more extensive than, those used to control the identity, strength, quality,
purity, and potency of the drug substance or the drug product. Analytical procedures used to characterize a reference standard should not rely solely on comparison testing to a previously designated reference standard.
Generally, this characterization information should include:
- A brief description of the manufacture of the reference standard, if the manufacturing
process differs from that of the drug substance. Any additional purification
procedures used in the preparation of the reference standard should be described.
- Legible reproductions of the relevant spectra, chromatograms, thin-layer
chromatogram (TLC) photographs or reproductions, and other appropriate
instrumental recordings.
- Data establishing purity. The data should be obtained by using appropriate tests, such
as TLC, gas chromatography (GC), high-pressure liquid chromatography (HPLC),
phase solubility analysis, appropriate thermometric analytical procedures, and others
as necessary.
- Appropriate chemical attribute information, such as structural formula, empirical
formula, and molecular weight. Information to substantiate the proof of structure
should include appropriate analytical tests, such as elemental analysis, infrared
spectrophotometry (IR), ultraviolet spectrophotometry (UV), nuclear magnetic
resonance spectroscopy (NMR), and mass spectrometry (MS), as well as applicable
functional group analysis. Detailed interpretation of the test data in support of the
claimed structure should be provided.
- A physical description of the material, including its color and physical form.
- Appropriate physical constants such as melting range, boiling range, refractive index,
dissociation constants (pK values), and optical rotation.
- A detailed description of the analytical procedures used to characterize the reference
standard.
For biotechnological/biological product reference standards, the recommendations on
characterization information above may apply and should be considered. However, additional
and/or different tests would be important to assess physicochemical characteristics, structural
characteristics, biological activity, and/or immunochemical activity. Physicochemical
determinations may include isoform, electrophoretic, and liquid chromatographic patterns, as
well as spectroscopic profiles. Structural characterization may include a determination of
amino acid sequence, amino acid composition, peptide map, and carbohydrate structure.
Biological and/or immunochemical activity should be assessed using the same analytical
procedures used to determine product potency. These can include animal-based, cell culture-based, biochemical, or ligand/receptor-binding assays. While these tests may be needed for complete characterization of certain reference standards, specific recommendations for
validation of biological and immunochemical tests are not contained in this guidance document.
Table of Contents
V. METHODS VALIDATION FOR INDs
For an investigational new drug, sufficient information is required in each phase of an investigation to
ensure proper identification, quality, purity, strength, and/or potency. The amount of information on
analytical procedures and methods validation necessary will vary with the phase of the investigation
(21 CFR 312.23(a)(7)).
For general guidance on analytical procedures and methods validation information to be submitted for
phase 1 studies, sponsors should refer to the FDA guidance for industry on Content and Format of
Investigational New Drug Applications (INDs) for Phase 1 Studies of Drugs, Including Well-Characterized, Therapeutic, Biotechnology-Derived Products (November 1995). General guidance regarding analytical procedures and methods validation information to be submitted for phase 2 or phase 3 studies will be provided in the FDA guidance for industry INDs for Phase 2 and 3 Studies of Drugs, Including Specified Therapeutic Biotechnology-Derived Products, Chemistry,
Manufacturing, and Controls Content and Format, when finalized (draft guidance published April 1999).
All analytical procedures should be fully developed and validation completed when the NDA, ANDA,
BLA, or PLA is submitted.
Table of Contents
VI. CONTENT AND FORMAT OF ANALYTICAL PROCEDURES FOR NDAs, ANDAs, BLAs, AND PLAs
Any analytical procedure submitted in an NDA, ANDA, BLA, or PLA should be described in
sufficient detail to allow a competent analyst to reproduce the necessary conditions and obtain results
comparable to the applicant=s. Aspects of the analytical procedure that require special attention
should be described. If the analytical procedure used is in the current revision of the USP/NF or other
FDA recognized standard references (e.g., AOAC International Book Of Methods) and the
referenced analytical procedure is not modified, a statement indicating the analytical procedure and
reference may be provided rather than a description of the method (21 CFR 211.194). A description
of analytical procedures from any other published sources should be provided, because the referenced
sources may not be readily accessible to the reviewer.
The following is a list of information that should typically be included in a description of an analytical procedure.
A. Principle
A statement of the principle of the analytical procedure should be included. For example, separation is based on isocratic reversed phase HPLC with detection by UV.
B. Sampling
The number of samples (e.g., vials, tablets) selected, how they are used (i.e., as individual or
composite samples), and the number of replicate analyses per sample should be described.
C. Equipment and Equipment Parameters
A listing of all equipment (e.g., instrument type, detector, column type, dimensions) should be
included, as well as a list of equipment parameters (e.g., flow rate, temperatures, run time,
wavelength settings). A drawing representing the experimental configuration (e.g., illustrating
positions for a spray pattern analytical procedure) should be provided, when appropriate.
D. Reagents
A list of reagents and their grades (e.g., USP/NF, American Chemical Society (ACS)
Analytical Reagent) should be included. If in-house or modified commercial reagents are
used, directions for their preparation should be included. Unstable or potentially hazardous
reagents should be identified, and storage conditions, directions for safe use, and usable shelf
life for these reagents should be specified.
E. System Suitability Testing
System suitability test parameters and acceptance criteria are based on the concept that the
equipment, electronics, analytical operations, and samples to be analyzed constitute an
integrated system. System suitability testing ensures that the system is working properly at the
time of analysis. Appropriate system suitability criteria should be defined and included in the
analytical procedure.
All chromatographic analytical procedures should include system suitability testing and criteria.
Parameters typically used in system suitability evaluations are defined and discussed in the
CDER reviewer guidance on Validation of Chromatographic Methods (November 1994).
System suitability testing is recommended as a component of any analytical procedure, not just
those that involve chromatographic techniques. Regardless of the type of analytical
procedure, testing should be used to confirm that the system will function correctly
independent of the environmental conditions. For example, titration analytical procedures
should always include the evaluation of a blank (commonly referred to as a blank titration).
F. Preparation of Standards
Procedures for the preparation of all standard solutions (e.g., stock, working standard solutions, internal standards) should be included.
G. Preparation of Samples
Sample preparation for individual tests should be clearly described. Specific details should be
provided for unusual sample preparations (e.g., solid-phase extraction, derivatization).
H. Procedure
A step-by-step description of the procedure should be provided. The description should
include, where appropriate, equilibration times, injection sampling sequence, and system
suitability or start-up parameters. Unusual hazards should be identified.
I. Calculations
Representative calculations, with a tabulation defining all symbols and numerical factors, and
specific instructions for the calculation of degradation products and impurities should be
included. Any mathematical transformations or formulas used in data analysis should be
described in detail. These may include logarithmic transformations used to obtain a linear
relationship from exponential data, or the use of multiple order regression analyses.
J. Reporting of Results
1. General
The format used to report results (e.g., percent label claim, weight/weight,
weight/volume, parts per million (ppm)) including the specific number of significant
figures to be reported should be provided.
2. Impurities Analytical Procedures
The name and location/identifier (e.g., retention time (RT), relative retention time
(RRT)) of impurities and the type of impurity (e.g., process, degradant, excipient
degradant) should be included in the analytical procedures for impurities in the drug
substance and drug product. The detection limit (DL) or quantitation limit (QL)
should be stated, as appropriate. The DL or QL can be set using the drug substance's
detection response.
Reporting of organic impurities should cover (1) specified identified impurities by name, (2) specified unidentified impurities by location/identifier, (3) any unspecified impurities, and (4) total impurities. The total organic impurities for the drug product or drug substance is the sum of all impurities equal to or greater than their individual QL. See recommendations regarding appropriate QLs in FDA impurities guidances (see references). Inorganic impurities and residual solvents should also be addressed.
For the drug product, drug substance process impurities may be excluded from
reporting if an acceptable rationale is provided in the sections on analytical procedures
and controls. Drug product impurities from the drug product manufacturing process,
packaging, and labeling should be addressed.
The above reporting information may not be strictly applicable to all products (e.g.,
biological, biotechnological, botanical, radiopharmaceutical drugs), but any significant
process and product-related impurities should be determined and reported.
Table of Contents
VII. METHODS VALIDATION FOR NDAs, ANDAs, BLAs, AND PLAs
A. Noncompendial Analytical Procedures
In an NDA, ANDA, BLA, or PLA, data must be submitted to establish that the analytical
procedures used in testing meet proper standards of accuracy and reliability (21 CFR
211.194(a)(2)). Methods validation is the process of demonstrating that analytical
procedures are suitable for their intended use. At the time of submission, the NDA, ANDA,
BLA, or PLA should contain methods validation information to support the adequacy of the
analytical procedures.
The International Conference on Harmonisation (ICH) guidance Q2A Text on Validation of
Analytical Procedures (March 1995) and Q2B Validation of Analytical Procedures:
Methodology (November 1996) provide recommendations on validation of analytical
procedures. Analytical procedures outside the scope of the ICH guidances should still be
validated.
1. Validation Characteristics
Applicants should submit information on the validation characteristics of their
proposed analytical procedures (see ICH Q2A and ICH Q2B). Although not all of
the validation characteristics are needed for all types of tests (see section VII.A.3),
typical validation characteristics are:
- Accuracy
- Precision (repeatability and intermediate precision)
- Specificity
- Detection limit
- Quantitation limit
- Linearity
- Range
- Robustness
2. Other Methods Validation Information
Methods validation information should also include:
a. Robustness
Robustness, a measure of the analytical procedure's capability to remain unaffected by
small but deliberate variations, is described in ICH Q2A and Q2B. Such testing
should be performed during development of the analytical procedure and the data
discussed and/or submitted. In cases where an effect is observed, representative
instrument output (e.g., chromatograms) should be submitted.
b. Stress Studies
Degradation information obtained from stress studies (e.g., products of acid and base
hydrolysis, thermal degradation, photolysis, oxidation) for the drug substance and for
the active ingredient in the drug product should be provided to demonstrate the
specificity of the assay and analytical procedures for impurities. The stress studies
should demonstrate that impurities and degradants from the active ingredient and drug
product excipients do not interfere with the quantitation of the active ingredient. Stress
studies are described in various FDA guidances relating to the stability of drug
products (see references).
The design of the stress studies and the results should be submitted to the stability
section of the application. Representative instrument output (e.g., chromatograms)
and/or other appropriate data (e.g., degradation information obtained from stress
studies) should be submitted in the sections on analytical procedures and controls.
c. Instrument Output/Raw Data
i. Organic Impurities
Representative data should be submitted to support an assessment of the
organic impurities. Representative data for residual solvents are generally not
needed. Instrument output and the raw numerical values (e.g., peak area)
with appropriate identification and labeling (e.g., RT for chromatographic peaks, chemical shift (d) and coupling constant (J) for NMR) should be provided. The impurity profile should be assessed at the quantitation limit and the instrument output provided. Additional information should be provided to
confirm that the impurity profile is adequately characterized. For example, a
representative chromatogram using detection at a low wavelength, such as
25 nm, and double the proposed total run time could be submitted to
support the specificity of the analytical procedure.
For quantitation purposes, the response factor of the drug substance may be
used for impurities without a reference standard. In cases where the response
factors are not close, this practice may still be acceptable, provided a
correction factor is applied or the impurities are, in fact, being overestimated.
Acceptance criteria and analytical procedures used to estimate identified or
unidentified impurities often are based on analytical assumptions (e.g.,
equivalent detector response). Assumptions should be discussed and justified.
ii. Drug Substance
Data should be submitted showing the separation and detection of impurities
using spiked or stress samples. Complete impurity profiles as graphic output
(e.g., chromatograms) and raw data (e.g, integrated peak areas) of
representative batches should be submitted in the sections on analytical
procedures and controls for the drug substance. For ANDAs and related
submissions, appropriate information for the batches used in the biobatch or
submission batch should be provided. All responses (e.g., peaks) should be
labeled.
The analytical procedure used should be capable of differentiating changes, if
any, between past and present batches. The quantitation limit and the type of
organic impurity (e.g., degradant, process impurity) should be stated. The
analytical procedure number, batch number, manufacturing date and site, and
date of analysis should be provided.
iii. Drug Product
Information such as instrument output (e.g., chromatograms) and raw data
(e.g., integrated peak areas) from representative batches under long-term and
accelerated stability conditions, and stressed samples should be submitted in
the sections on analytical procedures and controls of the drug product. For
ANDAs and related submissions, appropriate information for the biobatch or
submission batch should be provided. References to the raw data (e.g.,
chromatograms) should be included in the stability section of the application.
At a minimum, the submission should include instrument output and raw data for release testing and at the latest available time point for the same batch. All responses (e.g., peaks) should be labeled and identified. In addition, the
analytical procedure number, batch number of the drug product, manufacturing date, date of analysis, source and batch number of drug substance, manufacturing site, and container/closure information should be provided. The analytical procedures used should be capable of differentiating changes, if any, between past and present batches. The quantitation limit and the type (e.g., degradant, leachables from packaging) should be reported. Multiple methodologies can be used.
If process impurities from the drug substance and excipients with their related
impurities are not reported in the impurities analytical procedure, the potential
locations/identifier (e.g., RT, RRT) of these compounds should be described
and listed in the analytical procedure.
3. Recommended Validation Characteristics for Types of Tests
Table 1 is a summary of the validation characteristics that should be addressed during
validation of different types of analytical procedures. The same methodology can be
used for several purposes. The validation information should support the intended
purpose of the test. For example, if Raman spectroscopy is the methodology selected
to quantitate polymorphic forms as impurities, or chiral HPLC for enantiomeric
impurities, the recommended validation characteristics in Table 1 under quantitative
testing for impurities would apply. However, if Raman spectroscopy or chiral
HPLC are used for the purpose of identification or as specific tests, the recommended
validation characteristics listed for those types of tests would apply.
Table 1. Recommended Validation Characteristics of the Various Types of Tests.
Type of Tests / Characteristics |
Identification |
Testing for Impurities |
Assay Dissolution (Measurement Only), Content/Potency |
Specific Tests |
Quantitative |
Limit |
Accuracy |
- |
+ |
- |
+ |
+4 |
Precision-Repeatability |
- |
+ |
- |
+ |
+4 |
Precision-Intermediate Precision |
- |
+1 |
- |
+1 |
+4
|
Specificity |
+2 |
+ |
+ |
+5 |
+4 |
Detection Limit |
- |
-3 |
+ |
- |
- |
Quantitation Limit |
- |
+ |
- |
- |
- |
Linearity |
- |
+ |
- |
+ |
- |
Range |
- |
+ |
- |
+ |
- |
Robustness |
- |
+ |
-3 |
+ |
+4 |
NOTE:
- Signifies that this characteristic is not normally evaluated.
+ Signifies that this characteristic is normally evaluated.
1 In cases where reproducibility has been performed, intermediate precision is not needed.
2 Lack of specificity for an analytical procedure may be compensated for by the addition of a second
analytical procedure.
3 May be needed in some cases.
4 May not be needed in some cases.
5 Lack of specificity for an assay for release may be compensated for by impurities testing.
a. Identification
Identification analytical procedures may include tests such as IR, differential scanning
calorimetry (DSC), X-ray diffraction (XRD), UV, and HPLC retention time. A
specific identification test should be included for the active ingredient whenever
possible. In cases where a nonspecific identification analytical procedure is proposed
for the active ingredient, two independent analytical procedures are generally
sufficient, if justified. For other identification tests (e.g., a chiral HPLC retention time
as confirmation for the presence of an enantiomer, chloride test for a counterion) a
single test is acceptable. This concept of the number of identification tests is
applicable to both the drug substance and drug product.
b. Impurities
The validation characteristics under quantitative testing for impurities, as described
in Table 1, apply, regardless of which methodology is used to quantitate impurities. If
the same analytical procedure is proposed as a limit test, validation characteristics
under limit testing for impurities will apply.
c. Assay
Assay includes the content of the active ingredient, preservative (if used), and
measurement of content in dissolution and content uniformity samples.
d. Specific Tests
Specific tests to control the drug substance, excipient, or drug product can include
tests such as particle size analysis, droplet distribution, spray pattern, dissolution
(excludes measurement), optical rotation, and methodologies such as DSC, XRD, and
Raman spectroscopy. The validation characteristics may differ for the various
analytical procedures. For example, accuracy, repeatability, intermediate precision
and robustness should be evaluated for molecular size distribution gel permeation
chromatography (GPC).
B. Compendial Analytical Procedures
The suitability of a compendial analytical procedure must be verified under actual conditions of
use (21 CFR 211.194(a)(2)). Information to demonstrate that USP/NF analytical procedures
are suitable for the drug product or drug substance should be included in the submission.
Information on the specificity, intermediate precision, and stability of the sample solution
should be included. Compendial assay analytical procedures may not be stability-indicating,
and this should be considered when developing the specification (see section III.C). For
compendial items, additional analytical procedures, such as impurities or osmolality, may be
requested to support the quality of the drug product or drug substance. These additional
analytical procedures should be validated (see section VII.A).
Table of Contents
VIII. STATISTICAL ANALYSIS
A. General
Methods validation includes an assessment of the adequacy of the analytical procedure.
Statistical analysis (e.g., linear regression analysis, relative standard deviation) of methods
validation data is often used to demonstrate the validity of the method. The statistical procedures for the analysis of the validation data should be determined prior to the start of any
validation study. The procedure followed, including the amount of data to collect and the
criteria used in determining the acceptability of the analytical procedure, should be specified.
The raw methods validation data and statistical procedures used to analyze the raw data
should be provided and discussed in the sections on analytical procedures and controls. All
statistical procedures used in the analysis of the data should be based on sound principles and
be suitable for evaluating the dataset.
B. Comparative Studies
Comparative studies are performed to evaluate intermediate precision (e.g., different
equipment, analysts, days). Comparative studies are also used to evaluate between
laboratory variability (i.e., reproducibility) when an analytical procedure is used in more than
one laboratory or to compare and evaluate the precision and accuracy of two analytical
procedures (e.g., regulatory analytical procedure and an alternative analytical procedure).
When comparative studies are performed, homogeneous samples from the same batch should
be used, if feasible. Comparative results should be statistically analyzed and discussed and
any bias explained.
C. Statistics
For information on statistical techniques used in making comparisons, as well as other general
information on the interpretation and treatment of analytical data, appropriate literature or texts
should be consulted (see references).
Table of Contents
IX. REVALIDATION
When sponsors make changes in the analytical procedure, drug substance (e.g., route of synthesis), or
drug product (e.g., composition), the changes may necessitate revalidation of the analytical
procedures. Revalidation should be performed to ensure that the analytical procedure maintains its
characteristics (e.g., specificity) and to demonstrate that the analytical procedure continues to ensure
the identity, strength, quality, purity, and potency of the drug substance and drug product, and the
bioavailability of the drug product. The degree of revalidation depends on the nature of the change.
When a different regulatory analytical procedure is substituted (e.g., HPLC for titration), the new
procedure should be validated (see section VII).
If during each use an analytical procedure can meet the established system suitability requirements only
with repeated adjustments to the operating conditions stated in the analytical procedure, the analytical
procedure should be reevaluated, amended, and revalidated, as appropriate.
FDA intends to provide guidance in the future on postapproval changes in analytical procedures.
Table of Contents
X. METHODS VALIDATION PACKAGE: CONTENTS AND PROCESSING
Part of the methods validation process may include FDA laboratory analysis to demonstrate that an
analytical procedure is reproducible by laboratory testing. A methods validation package (see X.A)
and samples (see X.B) will be needed for this process.
A. Methods Validation Package
The methods validation package will usually include information copied from pertinent sections
of the application. To aid the review chemist, these copies should retain the original pagination
of the application sections.
For ANDA and NDA products, the archival copy and extra copies of the methods validation
packages should be submitted with the application. For ANDAs and related supplemental
applications, one archival copy and two extra copies of the methods validation package
should be submitted. For NDAs and related supplemental applications, one archival copy and
three extra copies should be submitted. For BLAs and PLAs, a separate methods validation
package need not be submitted. Information similar to that specified here should be included
in the BLA or PLA submission.
The methods validation package should include:
1. Tabular List of All Samples to Be Submitted
The list should include the lot number, identity (with chemical name and structure
where required for clarity), package type and size, date of manufacture, and quantity
of the samples.
2. Analytical Procedures
A detailed description of each of the analytical procedures listed in the specifications
should be submitted. The description should be sufficient to allow the FDA laboratory
analysts to perform the analytical procedure (see section VI).
3. Validation Data
Appropriate validation data to support the analytical procedures should be submitted.
Individual values as well as summary tables should be provided. Representative
instrument output and raw data and information regarding stress studies should be
included (see section VII).
4. Results
The results obtained by the applicant for the submitted samples should be provided.
Alternatively, COAs could be submitted. The dates of analysis should be stated.
5. Composition
The components and composition of the drug product should be provided.
6. Specifications
The specifications for the drug substance and the drug product should be included.
7. Material Safety Data Sheets
The applicant should include material safety data sheets (MSDSs) for all samples,
standards, and reagents (29 CFR 1910.1200(g)). As appropriate, MSDSs should be
provided for other materials used in the analytical procedures listed in the methods
validation package. In the case of toxic or hazardous materials, MSDSs should be
posted on the outside of the package to facilitate safe handling.
B. Selection and Shipment of Samples
On request from CDER, an NDA or ANDA applicant must submit samples of drug product,
drug substance, noncompendial reference standards, and blanks, so that the suitability of the
applicant's drug substance and drug product analytical procedures can be evaluated by FDA
laboratories (21 CFR 314.50(e) and 314.94(a)(10)). For BLAs and PLAs, representative
samples of the product must be submitted, and summaries of the results of tests performed on
the lots represented by the submitted sample must be provided (21 CFR 601.2(a) and
601.2(c)(1)(vi)).
For CDER products, the number of sets of samples that should be submitted for methods
validation will be identified in the instructions forwarded to the applicant by the FDA
laboratory. In general, the quantity of samples in each set should be double the amount
needed to carry out the testing as performed by the applicant. Along with the drug substance
and the drug product samples, the applicant should submit internal standards, non-USP
reference standards, samples of impurities, degradation products, and unusual reagents. A set
of samples will be shipped to each assigned laboratory.
For biological products, CBER should be consulted on the submission of samples and
supporting materials.
Unless specified differently by the reviewer, samples from any batch, preferably samples from an aged batch, may be selected for NDAs and NDA supplemental applications. The submitted drug product samples should be from a batch made with the proposed market formulation. For ANDAs and appropriate supplements, a sample of the finished product from
a batch being used to support approval of the submission should be used. If a sample is selected from a batch not described in the application, an amendment containing a copy of the batch record and certificate of analysis should be provided to the ANDA. For supplements that do not require submission and review of an exhibit batch record and associated data, any
commercial batch may be submitted. For biological products, samples from several consecutively manufactured batches should be submitted.
The drug product should be supplied in its original packaging. Bulk substances (e.g., drug
substances, impurities, excipients) should be stored in opaque nonreactive containers. To
prevent breakage during shipping, the samples should be adequately packaged in a sturdy
container. Samples shipped from outside the United States should contain the appropriate
customs forms to reduce delay in delivery.
If special storage precautions (e.g., freezing, use of an inert gas blanket) are required to
protect sample integrity, arrangements should be made in advance with the validating
laboratory for scheduled direct delivery. If a sample is toxic or potentially hazardous, the
container should be prominently labeled with an appropriate warning and precautionary
handling instructions.
C. Responsibilities of the Various Parties
1. Applicant
In the sections of the application on analytical procedures and controls, the applicant
should provide a name, address, telephone number, and facsimile number so that
samples can be requested. If this information is not provided, the contact person and
address listed in the NDA, ANDA, BLA, or PLA submission will be used.
The methods validation packages should be compiled and submitted with the NDA or
ANDA submission. For BLAs and PLAs, a separate methods validation package
need not be submitted.
When an FDA laboratory contacts the applicant for samples, the applicant should
provide FDA laboratories with the samples within 10 working days. With the
exception of sample delivery arrangements, all communications concerning validation
at the FDA laboratories should be made through or with the knowledge of the review
chemist for CDER applications, or the BLA/PLA committee chair for CBER
applications.
2. Review Chemist
The review chemist will review the application to determine that the analytical
procedures are adequate to ensure the identity, strength, quality, purity, and potency
of the drug substance and/or drug product. Any changes in the methods resulting from
the review of the application may require resubmission of the methods validation
package. The review chemist, in coordination with the appropriate FDA laboratories,
will decide which analytical procedures are to be validated. Comments from the FDA
laboratories, if any, will be forwarded by the review chemist to the applicant on
completion of the studies by the laboratories.
3. FDA Laboratory
An FDA laboratory will contact applicants with instructions on the submission of
samples and the addresses to which samples should be mailed. The laboratory will
test the samples according to the submitted analytical procedures to determine
whether the analytical procedures are acceptable for quality control and suitable for
regulatory purposes. Results and comments will be forwarded to the review chemist
on completion of the studies.
4. Investigator
The investigator inspects the analytical laboratory testing sites where the release and
stability testing are performed to ensure that the analytical procedures are performed
in compliance with CGMP/GLP.
Table of Contents
XI. METHODOLOGY
Sections II through IX provide general information on the submission of analytical procedures and
methods validation information, including validation characteristics. Additional information on certain
methodologies is provided below.
A. High-Pressure Liquid Chromatography (HPLC)
The widespread use of HPLC analytical procedures and the multitude of commercial sources
of columns and packings frequently have created problems in assessing comparability. Many
of the following points may also apply to other chromatographic analytical procedures.
1. Column
The following characteristics are useful for defining a particular column and, if known,
should be included in the analytical procedure description. If method development has
indicated that columns from only one commercial source are suitable, this information should be included as part of the analytical procedure. If more than one column is suitable, a listing of columns found to be equivalent should be included.
a. Column Parameters
- Material: glass, stainless steel, plastic
- Dimensions: length, inner diameter
- Frit size
- Filter type
- Precolumn and/or guard column type, if used
b. Packing Material
- Particle type: size, shape, pore diameter
- Surface modification (e.g., bonded surface type, surface coverage, percent carbon, additional silylation)
- Recommended pH range for column use
2. System Suitability Testing
Each analytical procedure submitted should include an appropriate number of system
suitability tests defining the critical characteristics of that system. Criteria for all system
suitability testing should be provided. The system suitability tests listed below are
defined in CDER's reviewer guidance on Validation of Chromatographic Methods (November 1994).
- Tailing factor
- Relative retention
- Resolution
- Relative standard deviation (RSD)
- Capacity factor
- Number of theoretical plates
The RSD is normally performed at the beginning of the run. However, for assays with
lengthy run times or as otherwise justified by the applicant, the reported average may
be taken from injections at the beginning and end of the run, or at the beginning,
middle, and end of the run.
If an internal standard is used, the minimum acceptable resolution between the
internal standard and one or more active ingredients should be specified. If the
analytical procedure is used to control the level of impurities, the minimum resolution
between the active ingredient and the closest eluting impurity, or the two peaks
eluting closest to each other, should be given.
3. Operating Parameters
The sequence of injection of blanks, system suitability standards, other standards,
and samples should be defined. Flow rates, temperatures, and gradients should be
described.
Complete details should be provided for the preparation of the mobile phase,
including the order of addition of the reagents and the methods of degassing and
filtration. The effect of adjustments in mobile phase composition on retention times
should be included in the analytical procedure. The rationale for the use of
precolumns and/or guard columns should be provided and justified. Any special
requirements, such as the use of inert tubing or injection valves, should be specified.
B. Gas Chromatography (GC)
At a minimum, the following parameters should be included in the description of a GC
procedure. Additional parameters should be specified if required by the analytical procedure.
If method development has indicated that columns from only one commercial source are
suitable, this information should be included as part of the analytical procedure. If more than
one column is suitable, a listing of columns found to be equivalent should be included.
1. Column
- Column dimensions: length, internal diameter, external diameter
- Stationary phase
- Column material (e.g., silica, glass, stainless steel)
- Column conditioning procedure
2. Operating Parameters
- Gases: purity, flow rate, pressure
- Temperatures: column, injector, detector (including temperature program, if used)
- Injection (e.g., split, splitless, on-column)
- Detector
- Typical retention time and total run time
3. System Suitability Testing
Appropriate system suitability criteria should be defined and included in all analytical
procedures.
If an internal standard is used, the minimum acceptable resolution between the internal standard and one or more active ingredient should be specified. If the analytical procedure is used to control the level of impurities, the minimum resolution between the active ingredient and the closest eluting impurity, or the two peaks eluting closest
to each other, should be given.
The RSD is normally performed at the beginning of the run. However, for assays with
lengthy run times or as otherwise justified by the applicant, the reported average may
be taken from injections at the beginning and end of the run, or beginning, middle, and
end of the run.
C. Spectrophotometry, Spectroscopy, Spectrometry and Related Physical Methodologies
These analytical procedures include, but are not limited to, IR spectrophotometry, near IR
spectrophotometry (NIR), UV/visible spectrophotometry (UV/Vis), atomic emission and
atomic absorption, NMR, Raman spectroscopy, MS, and XRD.
Spectrometric analytical procedures may not be stability-indicating. The bias of the analytical
procedure should be evaluated by comparing it with a chromatographic procedure, where
appropriate. When manually operated equipment is used, the description of the analytical
procedure should include an acceptance criterion for the amount of time that may elapse
between sampling and reading. Appropriate system suitability and/or calibration testing is
recommended. Validation criteria should include specificity (demonstrating no interference of
placebo), linearity, repeatability, intermediate precision, and robustness.
D. Capillary Electrophoresis (CE)
At a minimum, the parameters listed below should be specified for a capillary electrophoretic
analytical procedure. Additional parameters may be included as required by the procedure.
If method development has indicated that capillaries from only one commercial source are
suitable, this information should be included as part of the analytical procedure. If more than
one capillary is suitable, a listing of capillaries found to be equivalent should be included.
1. Capillary
- Capillary dimensions: length, length to detector, internal diameter, external diameter
- Capillary material
- Capillary internal coating (if any)
2. Operating Parameters
- Capillary preparation procedure: procedure to be followed before the first use, before the first run of the day, before each run (e.g., flush with 100 millimolar sodium hydroxide, flush with running buffer)
- Running buffer: composition, including a detailed preparation procedure with the order of addition of the components
- Injection: mode (e.g., electrokinetic, hydrodynamic), parameters (e.g., voltage, pressure, time)
- Detector
- Typical migration time and total run time
- Model of CE equipment used
- Voltage (if constant voltage)
- Current (if constant current)
- Polarity (e.g., polarity of electrode by detector)
3. System Suitability Testing
Each analytical procedure should include the appropriate system suitability tests
defining the critical characteristics of that system. Other parameters may be included
at the discretion of the applicant.
If an internal standard is used, the minimum acceptable resolution between the internal
standard and one or more active ingredient should be specified. If the analytical
procedure is used to control the level of impurities, the minimum resolution between
the active ingredient and the closest eluting impurity, or the two peaks eluting closest
to each other, should be given.
E. Optical Rotation
Optical rotation is used for the measurement of stereochemical purity. Visual polarimeters rely
on a monochromatic source, which traditionally was sodium D, but has expanded to virtually
any wavelength.
If measurements are to be made at a wavelength other than sodium D, an explanation for
selecting the wavelength should be given, along with a comparison of the specific rotation at
sodium D and the wavelength to be used. Circular dichroism (CD) spectra may suffice for this
purpose. In addition to the provisions of USP <781>, procedures for measurement of
specific rotation should include the solvent, concentration, and, for aqueous solutions, the pH
to which the solution should be adjusted. The conditions and equipment should be shown to
be suitable to confirm the stereochemical identity of a racemate or an enantiomer.
The enantiomeric purity can be expressed as enantiomeric excess (e.e.), using the following
formula as an example:
e.e. = 100% * {{M} - [m]}/{[M] + [m]}
where [M] and [m] are the concentrations of the major and minor enantiomers, respectively.
This yields values of zero for a racemate and 100 percent for a pure enantiomer. An
intermediate concentration gives intermediate values; for example, 97:3 would give an e.e. of
9 percent.
Appropriate system suitability and/or calibration testing is recommended. Validation criteria
should include specificity, and intermediate precision.
F. Methodologies Relating to Particle Size Analysis
Particle size analysis is an important element for quality control and regulatory evaluation of
certain drug substances and drug products. The normal concepts of validation may differ for
particle size methodologies as compared to other analytical methodologies such as HPLC.
However, a standard mixture may be used for calibration.
Particle size evaluation can include characteristics of size, morphology, surface, and population
of particles. The following parameters are useful for describing particle size analysis for
characterization of drug substances and drug products.
1. Particle Size Methods
Types of particle size methods include, but are not limited to:
a. Nonfractionation methods that evaluate an entire population of particles
- Microscopy (optical, electron)
- Light scattering (dynamic, photon correlation, laser diffraction)
- Electrozone sensing
- Photozone sensing
b. Fractionation methods that use physical techniques to separate particles on the basis of size
- Sieving
- Cascade impactor
- Sedimentation
- Size exclusion chromatography
2. Calibration and Validation Characteristics
To ensure proper instrument operation, the system should be calibrated according to the manufacturer's and/or the laboratory's specification, as appropriate.
The methods validation usually involves evaluation of intermediate precision and
robustness. Assurance should be provided that the data generated are reproducible
and control the product's quality. See additional information in sections V and VII.
G. Dissolution
The equipment used for dissolution is covered by USP <711> or USP <724>. The
dissolution procedure description and validation should include the following.
1. Dissolution Medium
A brief discussion of the reasons for selecting the medium.
2. Procedure
A dissolution test consists of a dissolution procedure and method of analysis
(automated on-line analysis or manual sampling followed by HPLC analysis). The
written procedure should cover the following items:
- Apparatus
- Preparation of standard
- Preparation of sample
- Method of analysis (e.g., UV, HPLC)
- Sampling procedure (e.g., intervals, filtration, handling of samples, dilutions)
- Calculations
- Acceptance criteria
Regardless of the method of analysis, system suitability criteria should be described.
Blank and standard solution spectra or chromatograms should be included.
3. Validation Characteristics
Both the dissolution procedure and the method of analysis should be validated.
The time needed for the completion of the sample analysis should be stated in the
procedure. Data should be submitted to support the stability of the dissolution sample
during the procedure. If filters are used on-line or during sample preparation,
appropriate recovery studies should be performed and documented and any bias
should be addressed.
H. Other Instrumentation
1. Noncommercial Instrumentation
FDA encourages the development and use of the most appropriate instrumentation.
However, the use of rare or exotic systems not only places an undue burden on the
regulatory laboratory, but also may delay the validation process.
When noncommercial instrumentation is used, the instrumentation should be capable
of being constructed from commercially available components at a reasonable cost, if
possible. For unique methodologies or instrumentation requiring contract fabrication,
the applicant's cooperation with the FDA laboratories in helping facilitate duplication
of the analytical procedure is important. In addition to design and equipment
specifications, complete performance assessment procedures should be provided.
Such systems may be found suitable for regulatory use.
2. Automated Analytical Procedures
The use of automated analytical procedures, although desirable for control testing,
may lead to delay in regulatory methods validation because FDA laboratories have to
assemble and validate the system before running samples. To avoid this delay,
applicants should demonstrate the equivalence of a manual procedure to the
automated procedure based on the same principle whenever possible.
ATTACHMENT A
NDA, ANDA, BLA, AND PLA SUBMISSION CONTENTS
The information relating to analytical procedures and methods validation that should be submitted in
NDAs, ANDAs, BLAs, and PLAs is identified below with a cross-reference to the section of this
guidance that provides recommendations and/or discussion on the topics.
Information that should be included in the analytical procedures and controls sections
Reference standard information |
Section IV |
Analytical procedures |
Section III, VI |
Validation data |
Section VII |
Stress studies |
Section VII.A.2.c |
Instrument output/raw data for impurities |
Section VII.A.2.b |
Statistical analysis |
Section VIII |
Revalidation, as needed |
Section IX |
Information that should be included in the methods validation package5
Contents of the MV Package |
Section XI |
Representative instrument output/data for stress studies |
Section VII.A.2.c |
Representative instrument output and raw data for initial and oldest sample of a batch |
Section VII.A.2.b |
Information that should be included in the stability section
Stress study designs and results |
Section VII.A.2.b |
Reference (volume and page number of submission) to instrument output and raw data
submitted to the section
dedicated to analytical procedures and controls |
Section VII.A 2.c |
Table of Contents
ATTACHMENT B
METHODS VALIDATION PROBLEMS AND DELAY
Listed below are examples of common problems that can delay successful validation.
- Failure to provide a sample of a critical impurity, degradation product, internal standard, or
novel reagent
- Failure to submit well-characterized reference standards for noncompendial drugs
- Failure to provide sufficient detail or use of unacceptable analytical procedures. For example:
- Use of arbitrary arithmetic corrections
- Failure to provide system suitability tests
- Differing content uniformity and assay analytical procedures without showing
equivalence factors for defining corrections as required by the current USP chapter <905> - Uniformity of Dosage Units
- Failure to submit complete or legible data. For example:
- Failure to label instrument output to indicate sample identity
- Failure to label the axes
- Inappropriate shipping procedures. For example:
- Failure to properly label samples
- Failure to package samples in accordance with product storage conditions
- Inadequate shipping forms (e.g., missing customs form for samples from outside the
United States)
- Failure to describe proper storage conditions on shipping containers
Table of Contents
REFERENCES
FDA Documents6
Guidance for Industry: ANDAs: Impurities in Drug Products (Draft, December 1998).
Guidance for Industry: ANDAs: Impurities in Drug Substances (February 2000).
Guidance for Industry: CMC Content and Format of INDs for Phase 2 and 3 Studies of Drugs,
Including Specified Therapeutic Biotechnology-Derived Products (Draft, December 1997).
Guidance for Industry: Content and Format of Investigational New Drug Applications (INDs)
for Phase 1 Studies of Drugs, Including Well-Characterized, Therapeutic, Biotechnology-
derived Products (February 1995).
Guidance for Industry: Investigating Out of Specification (OOS) Test Results for Pharmaceutical
Production (Draft, September 1998).
Guidance for Industry: Stability Testing of Drug Substances and Drug Products (Draft, June
1998).
Guidance for Industry: Submission of Chemistry, Manufacturing, and Controls Information for
Synthetic Peptide Substances (November 1994).
Guidance for Industry: Submitting Documentation for the Stability of Human Drugs and
Biologics (February 1987).
Reviewer Guidance: Validation of Chromatographic Methods (November 1994).
FDA CDER MAPP 5221.1 Requesting Methods Validation for ANDAs (November 1998).
International Conference on Harmonization Guidances
ICH Q1A: Stability Testing of New Drug Substances and Products (November 1994)
ICH Q1B: Photostability Testing of New Drug Substances and Products (November 1996)
ICH Q1C: Stability Testing for New Dosage Forms (May 1997)
ICH Q2A: Text on Validation of Analytical Procedures (March 1995)
ICH Q2B: Validation of Analytical Procedures: Methodology (May 1997)
ICH Q3A: Impurities in New Drug Substances (January 1996)
ICH Q3B: Impurities in New Drug Products (May 1997)
ICH Q3C: Impurities: Residual Solvents (December 1997)
ICH Q5C: Quality of Biotechnological Products: Stability Testing of Biotechnological/Biological Products (July 1996)
ICH Q6A: Specifications: Test Procedures and Acceptance Criteria for New Drug Substances
and New Drug Products: Chemical Substances (Draft (Step 2) November 1997)
ICH Q6B: Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products (March 1999)
U.S. Pharmacopeia/National Formulary
Chapter <621> Chromatography; US Pharmacopeia 23, United States Pharmacopeial Convention, Inc., Rockville MD: 1994
Chapter <781> Optical Rotation, US Pharmacopea 23, United States Pharmacopeial Convention, Inc., Rockville, MD: 1994
Chapter <1225> Validation of Compendial Methods; US Pharmacopeia 23, United States Pharmacopeial Convention, Inc., Rockville MD: 1994
Interpretation and Treatment of Analytical Data; USP Pharmacopeial Forum, United States Pharmacopeial Convention, Inc., Rockville MD: 1994, Volume 24, Number 5, pp. 7051 - 7056
Other
Miller, J.C., J.N. Miller, and E. Horwood, Statistics for Analytical Chemistry, 3rd edition, Prentice
Hall, 1993.
Saunders, B.D., and R.G. Trapp, Basic and Clinical Biostatistics, 2nd edition, Appleton and Lange, 1994.
Table of Contents
GLOSSARY
Acceptance Criteria: Numerical limits, ranges, or other suitable measures for acceptance of the results of analytical procedures.
Active moiety: The molecule or ion, excluding those appended portions of the molecule that cause the drug to be an ester, salt (including a salt with hydrogen or coordination bonds), or other
noncovalent derivative (such as a complex, chelate, or clathrate) of the molecule, responsible for the
physiological or pharmacological action of the drug substance (21 CFR 314.108(a)). The active
moiety is the entire molecule or ion, not the active site.
Detection Limit: The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be detected, but not necessarily quantitated as an exact value.
Drug Product: A finished dosage form, for example, a tablet, capsule, or solution that contains a drug substance, generally, but not necessarily, in association with one or more other ingredients (21 CFR 314.3(b)).
Drug Substance/Active Ingredient: An active ingredient that is intended to furnish pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease or to affect the structure or any function of the human body. The active ingredient does not include intermediates used in the synthesis of such ingredient. The term includes those components that may
undergo chemical change in the manufacture of the drug product and be present in the drug product in
a modified form intended to furnish the specified activity or effect (21 CFR 210.3(b)(7) and
314.3(b)).
Placebo (or Blank): A dosage form that is identical to the drug product except that the drug substance is absent or replaced by an inert ingredient or a mixture of the drug product excipients
quantitatively equivalent to those found in the drug product dosage form.
Quantitation Limit: The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be quantitatively determined with suitable precision and accuracy. The
quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices,
and is used particularly for the determination of impurities and/or degradation products.
Reagent: For analytical procedures, any substance used in a reaction for the purpose of detecting, measuring, examining, or analyzing other substances.
Specification: The quality standards (i.e., tests, analytical procedures, and acceptance criteria) provided in an approved application to confirm the quality of the drug substances, drug products,
intermediates, raw materials, reagents, and other components including container closure systems, and
in-process materials.
Spiking: The addition of a small known amount of a known compound to a standard, sample, or placebo, typically for the purpose of confirming the performance of an analytical procedure or the
calibration of an instrument.
Stability-Indicating Assay: A validated quantitative analytical procedure that can detect the changes with time in the pertinent properties (e.g., active ingredient, preservative level) of the drug substance and drug product. A stability-indicating assay accurately measures the active ingredients
without interference from degradation products, process impurities, excipients, or other potential
impurities.
Working Standard: A standard that is qualified against and used instead of the reference standard (also known as in-house or secondary standard).
Table of Contents
Footnotes:
1 This guidance has been prepared by the Analytical Methods Technical Committee of the Chemistry, Manufacturing, and Controls Coordinating Committee (CMC CC) in the Center for Drug Evaluation and Research (CDER) and the Center for Biologics Evaluation and Research (CBER) at the Food and Drug Administration (FDA).
2 Analytical procedure is interchangeable with method or test procedure.
3 The terms drug substance and drug product, as used in this guidance, refer to human drugs and biologics.
4 Sponsors preparing investigational new drug applications (INDs) should also consider the recommendations in this guidance. However, the amount and depth of the information that should be submitted to support an IND depends in large part on the phase of the investigation and the specific testing proposed in humans (see section V).
5 For BLAs and PLAs, a separate methods validation package need not be submitted. Information similar to what is listed here should be included in the BLA or PLA submission.
6 Draft guidances have been included for completeness only. As draft documents, they are not intended to be implemented until published in final form.
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