Analytical method validation (AMV) is required in the biopharmaceutical industry for all methods used to test final containers
(release and stability testing), raw materials, in-process materials, and excipients.
AMV is also required for test methods that are used to validate the process prior to process validation. This article reviews
current regulatory guidelines and the critical elements of analytical method development (AMD) that should be finalized before
Analytical methods are non-microbiological procedures which are used to test final containers, raw materials, in-process materials,
and excipients for release or to determine stability. Guidance for microbiological test method validation is reviewed in the
Parenteral Drug Association (PDA) Technical Report No. 33 and is outside of the scope of this guide.2 The International Conference on Harmonisation (ICH)'s Q2A and Q2B and the United States Pharmacopoeia's USP 28 <1225> provide
basic guidance.3-5 However, following just these guidelines may not provide sufficient evidence that a method is suitable for product release.6,7 FDA provides guidance on some of the issues that are not currently covered elsewhere.8,9
In addition to compliance, a biopharmaceutical manufacturer must consider how the acceptance criteria for the process validation
and all corresponding test methods are connected to the statistically derived product specifications that support acceptable
product release-to-reject ratios.6,7 This article addresses three major deficiencies in the available guidelines:
- the lack of clear expectations concerning when to qualify, validate, and verify test methods
- the lack of clear expectations concerning when certain method performance aspects should be covered (AMD or AMV)
- the lack of guidance for setting AMV acceptance criteria that directly impact product specifications and predicted product
release-to-reject ratios, which could ultimately impact patients and firms.
Qualify, Validate, or Verify?
The US Code of Federal Regulation 21 CFR 211.194(a)(2)) does not require recognized standard reference methods that have
already been validated and published in a standard reference (such as USP 28) to be fully revalidated.1,5 However, a biopharmaceutical firm must verify the suitability of these validated and published methods for their specific
product and laboratory environment.5 The verification of the suitability of methods under actual conditions of use is acceptable in the following cases:
- the methods are published in the current revision of the USP
- the methods are published in the Association of Official Analytical Chemists (AOAC) Book of Methods or another recognized
- the methods are detailed in an approved new drug application and the referenced method is not modified.1
In addition to the validation of new methods and the verification of previously validated methods, there is a third category,
analytical method qualification (AMQ), which can be used under appropriate circumstances. However, before AMQ can be used,
the exact scope or applicability and the detailed definitions for all three AMV categories must appear in a firm's written
procedures. Table 1 summarizes the general expectations for the applicability and requirements for using one of these three
Table 1: Applicability and Requirements for Analytical Method Qualification, Validation, and Verification
Which Performance Criteria?
The final AMV document must include evidence that a particular test procedure is suitable for its intended use. Formal
method validation studies should include all relevant ICH Q2A/B AMV characteristics listed in Table 2. The AMV protocol includes
all scientifically justified and logical step-by-step validation studies. Whenever a test method is developed and will be
used for drug development from Phase IIb onward, material for lot release, raw materials, in-process, or excipients, the AMD
studies should contain a series of critical elements. The evaluation of AMD characteristics should precede AMV and should
be summarized in an AMD report.6,7 All AMD data should ideally be generated in a GLP/GMP environment using qualified equipment operated by qualified personnel,
properly documented, summarized in an AMD report, and approved by quality assurance (QA). These results can be used as a qualified
method for material tested before Phase IIb.10 This also ensures that all AMD/AMQ data and results (summarized later in the AMV protocol) are valid from a compliance perspective.
Critical method performance criteria that should be evaluated during AMD are described below. The main argument for evaluating
these criteria during the AMD phase is to ensure that a test system is truly optimized before AMV. The validation process
should not be a trial-and-error effort but rather a formal process demonstrating that all AMV acceptance criteria are readily
Table 2: Validation Characteristics Per ICH Q2A and Q2B
Assay performance criteria such as accuracy and specificity are defined for analytical methods in current guidelines and should
always be covered during AMV according to assay classification.3,4 The validation characteristics for each test method category are listed in Table 2 and briefly described below. The five
ICH Q2A/B test categories are listed across the top row of Table 2, and the corresponding product specifications which would
trigger the selection of one of these five categories are listed in the second row. For example, a test that reports levels
of a characterized impurity should be validated as a quantitative limit test, and the AMV should include a formal evaluation
of a total of seven characteristics.12,13 On the other hand, a qualitative limit test for an impurity will have product specifications reported as less than a particular
pre-set limit. The actual impurity concentrations are not reported; only the detection limit (DL) and specificity are required
AMV characteristics. We are not quantitatively reporting this result, so we are not required to evaluate the quantitative
Accuracy is usually demonstrated by spiking an accepted reference standard into the product matrix. Percent recovery (observed/expected
x 100%) should ideally be demonstrated over the entire assay range using multiple data points for each selected analyte concentration.
Demonstrating accuracy is mostly affected by how well the reference standard is characterized and how well systematic sample
preparation errors are controlled. Data generated for determining accuracy may be used for other validation characteristics,
such as repeatability precision, linearity, assay range, and quantitation limit.
Repeatability precision indicates how precise the test results are under ideal conditions (same sample, operator, instrument, and day). Repeatability
precision should be demonstrated over the entire assay range, as with accuracy. Accuracy data may be used.