Qualification, Validation, and Verification

“These Pharmacopeial procedures are not by themselves designed to ensure that a batch of product is sterile or has been sterilized. This is accomplished primarily by validation of the sterilization process or of the aseptic processing procedures.”

Does it suit its purpose? The foregoing is clearly not an exhaustive list of the manners in which validation has been defined. It does appear that a recurring theme among the various definitions pertains to demonstrating that the method or process is suitable for its intended use. In this article, consider validation to be the demonstration that a method or process is suitable for its intended purpose. Accepting that, it is imperative that the intended purpose of a method or process is clearly stated at the outset of the validation. An example of the importance of such a statement can be found in Chapter ‹71› “Sterility Tests” (1). It states that “the following procedures are applicable for determining whether a Pharmacopeial article purporting to be sterile complies with the requirements set forth in the individual monograph with respect to the test for sterility.” The next paragraph states

“With proper design (see section IV.C.1), and reliable mechanisms to transfer process knowledge from development to commercial production, a manufacturer should be able to validate the manufacturing process. In a quality system, process validation provides initial proof, through commercial batch manufacture, that the design of the process produces the intended product quality (7). “

The same definition is provided in other FDA documents, such as Guideline on Sterile Drug Products Produced by Aseptic Processing. FDA document Guidance for Industry: Quality Systems Approach to Pharmaceutical Current Good Manufacturing Practice Regulations provides this definition:

FDA provides a definition of validation in numerous documents. One such document, Guidance for Industry: Analytical Procedures and Methods Validation Chemistry, Manufacturing, and Controls Documentation says “methods validation is the process of demonstrating that analytical procedures are suitable for their intended use” (5). There also are numerous documents defining validation within the context of processes. From FDA’s Guideline on General Principles of Process Validation:

However, it is important to remember that the main objective of validation of an analytical procedure is to demonstrate that the procedure is suitable for its intended purpose (4).

“Validation of an analytical procedure is the process by which it is established, by laboratory studies, that the performance characteristics of the procedure meet the requirements for the intended analytical applications.”

Definitions. Numerous documents provide definitions of validation. A dictionary definition (3) of validation includes “…the process of determining the degree of validity of a measuring device,” and for validate: “to make legally valid,” with synonyms “verify, substantiate.” Clearly, the synonyms do not distinguish between validation and verification, so let us now turn to definitions provided by other sources. USP chapter ‹1225› “Validation of Compendial Procedures” provides the following:

Qualification of analytical instrumentation is essential for accurate and precise measurement of analytical data. If the instrumentation is not qualified, ensuring that the results indicated are trustworthy, all other work based upon the use of that instrumentation is suspect. For the purposes of this article, the assumption will be made that the foundation of validation and verification work to follow is based solidly upon well-qualified instrumentation.

A recent issue of the Pharmacopeial Forum (2) had a diagram from a proposed General Chapter ‹1058› “Analytical Instrument Qualification” that was intended to show “…four critical components involved in the generation of reliable and consistent data (quality data).” From most to least critical, the components were quality-control check samples, system suitability tests, analytical methods validation, and analytical instrument qualification. In this article, consider system-suitability tests to be the same as verification. Why this usage should be acceptable will be explained. This article will consider validation and verification in detail. Reference to analytical instrument qualification is made. For further discussion of the top tier, “Quality Control Check Samples,” refer to Chapter ‹1058› “Analytical Instrument Qualification” (1).

The terms qualification, validation, and verification occur numerous times in US Pharmacopeia 29 (1). Qualification is found in Chapters ‹1035› “Biological Indicators for Sterilization,” ‹1043› “Ancillary Materials Cell, Gene, and Tissue-Engineered Products,” ‹1046› “Cell and Gene Therapy Products,” and ‹1119› “Near-Infrared Spectrophotometry,” among others. Validation appears in ‹1225› “Validation of Compendial Procedures,” ‹1223› “Validation of Alternative Microbiological Methods,” ‹1010› “Analytical Data—Interpretation and Treatment,” ‹1043› “Ancillary Materials for Cell-, Gene-, and Tissue-Engineered Products,” ‹1117› “Microbiological Best Laboratory Practices,” ‹1120› “Raman Spectrophotometry,” and many others. Verification appears in ‹1010› “Analytical Data—Interpretation and Treatment,” ‹1035› “Biological Indicators for Sterilization,” ‹1035› “Biological Indicators for Sterilization,” ‹85› “Bacterial Endotoxins Test,” and others. The terms also are present in documents from the US Food and Drug Administration, the Environmental Protection Agency (EPA), and the International Conference on Harmonization (ICH). Given the numerous definitions for the three terms, this article in part is intended to provide an approach to fostering more consistency in the usage of the terms.

During the years there has been concern that the tests for sterility as provided in Chapter ‹71› are not adequate to prove that a batch of product is sterile. As stated previously, the tests in Chapter ‹71› were intended only to show that a Pharmacopeial article is sterile. Such a demonstration constitutes a necessary but not sufficient condition for sterile pharmacopeial articles. If one were to validate an alternative procedure for that in Chapter ‹71›, it would not be necessary to develop one that is intended to demonstrate sterility of an entire lot of product.

In addition, it is appropriate that the conditions are provided under which the validation was performed. Given that there are essentially countless variations on experimental conditions, product matrix effects, and so forth, a validation cannot reasonably expect to address all such permutations. For example, Method 3 in the section of Chapter ‹1047› “Biotechnology-Derived Articles—Tests”, which addresses assays for total protein, indicates in a note:

“[Do not use quartz (silica) spectrophotometer cells: the dye binds to this material. Because different protein species may give different color response intensities, the standard protein and test protein should be the same.] There are relatively few interfering substances, but detergents and ampholytes in the test specimen should be avoided. Highly alkaline specimens may interfere with the acidic reagent (1).”

Therefore, given the following from FDA’s Guide to Inspections of Pharmaceutical Quality Control Laboratories: “Methods appearing in the USP are considered validated and they are considered validated if part of an approved ANDA” (8), the use of Method 3 would be valid if the conditions stated are met in testing the material of interest. The same FDA document states “For compendial methods, firms must demonstrate that the method works under the actual conditions of use,” which, for the sake of this article, will be considered verification. Chapter ‹1047› provides several other procedures, all also validated, that could be considered given test material that does not satisfy the conditions for Method 3.

Remember the purpose. It is important to bear in mind the purpose of the method to be validated. If the method is intended to serve as an alternative to a pharmacopeial method, then one must establish its equivalence to the pharmacopeial method in terms of the end result. Remember that the purpose of a method in the pharmacopeia is to determine whether the pharmacopeial article (for which a monograph exists in the pharmacopeia) satisfies the requirements in the monograph. If instead the purpose behind the use of a pharmacopeial method is for a purpose other than demonstrating that the article complies with monograph requirements (for example, imagine that total organic carbon is to be determined using Chapter ‹643› “Total Organic Carbon”), it is not necessary to perform the validation relative to the pharmacopeial results. This means that the validation should be conducted relative to the specific purpose for which it is intended. Also implicit in this is the use of a nonpharmacopeial method to determine something for which a pharmacopeial method exists, but again for purposes unrelated to satisfying a monograph requirement. In such a case, it is unnecessary to consider validating the method relative to that in the pharmacopeia.

Verification

If the use of the term validation is restricted to mean the demonstration of suitability of a method or process for its intended purpose, and the term verification for the demonstration that the previously validated method is suitable for use given specific experimental conditions that may or may not be appropriate given the conditions present during the validation, the terminological situation may be clarified.

These actual conditions include specific ingredients or products, specific laboratory personnel, equipment, and reagents. There are, however, instances in the literature where this distinction is not maintained. Consider the dictionary definition given previously for validation and its use of verification as a synonym for validation. Further muddying of the waters occurs when phrases such as “system suitability tests” (see Figure 1 and the system-suitability section in Chapter ‹621› “Chromatography”). The phrase also appears in the “Suitability of the Counting Method in the Presence of Product” section of Chapter ‹61› “Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests”, the “Suitability of the Test Method” section of Chapter ‹62› “Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms”, and the “Validation Test” section of Chapter ‹71› “Sterility Tests.” (1). In all cases, the intention is to ensure that the validated method will work under the specific conditions the analyst plans to use.

Figure 1

This means that a chromatographic system can deliver resolution and reproducibility on par with the system used during validation. For the two microbiology test chapters for nonsterile products, one must show that microbial growth in the presence of the article to be tested is not hindered. This is because the method depends on unencumbered microbial growth for it to work. In other words, a condition established in validating the method initially was unhindered microbial growth. The use of “validation test” in Chapter ‹71› is unfortunate because the intention was again to demonstrate that microbial growth is not hindered, as indicated in the following text:

“If clearly visible growth of microorganisms is obtained after the incubation, visually comparable to that in the control vessel without product, either the product possesses no antimicrobial activity under the conditions of the test or such activity has been satisfactorily eliminated. The test for sterility may then be carried out without further modification.”

It may be advantageous, and more consistent, for the text in Chapter ‹71› to be changed to “Suitability of the Test Method,” if not to “Verification of the Test Method.” The latter change also may be appropriate for Chapters ‹61› and ‹62›, given that what is being assessed is the verification that the actual test conditions relative to those established during the validation permits the proper functioning of the method. Given the harmonized status of these three chapters, such changes, although possible, would certainly take longer to become official.

The same cautions provided at the end of the section on validation are applicable here. If a method in use previously was derived from a pharmacopeial method but used for a purpose other than satisfying monograph requirements, it is not necessary to adopt a revised method in the pharmacopeia when it becomes official. It is therefore not necessary to reverify the suitability of your test article to the revised method. Likewise, the use of a nonpharmacopeial method for purposes other than satisfying a monograph requirement when a pharmacopeial method exists of potential relevance does not necessitate reverification.

General requirements for validation

There are numerous documents that describe the general approach to a validation process. They describe several characteristics (data elements in Chapter ‹1225›) that may be examined during validation, with specific sets selected based upon the nature of the test method. A brief description of these characteristics is provided herein using the characteristics as outlined in the IC Harmonization Harmonized Tripartite Guideline, Validation of Analytical Procedures: Text and Methodology.

Accuracy is a determination of how close the measured value is (in the case of an analytical method) to the true value. As such, one might define accuracy of method as equal to true value plus error. Error may contain both the systematic error (bias) and imprecision of measurement. With the potential error possible, it is important to include a means of reflecting the “true value” as closely as possible. For many compendial tests, this involves the use of a reference standard. Because a method is expected to be useful over a range of true values, the accuracy should be assessed over the expected range of values to which the method is to be applied. As stated previously, the validation should also state the conditions under which the accuracy was determined. Because it is not possible to determine all possible sets of conditions for which a compendial assay might be applicable, accuracy may need to be verified before use of a validated method. The concept of accuracy is more problematic for microbiological assays.

The precision of a method determined during validation should be representative of the repeatability (reproducibility) of the method. As was the case for the determination of accuracy, it should be determined over the expected range of articles to be measured, and the conditions used during the validation should be clearly stated. As for accuracy, the use of reference standards is common because the goal of the assessment of precision is to determe method repeatability without introducing unknown variance as a result of different test articles or test articles drawn from a heterogeneous source. The latter point also complicates the validation of microbiological assays.

Specificity refers to the ratio of false positives to false negatives. A highly specific method would have a very low ratio, given that it should be able to detect the article of interest present in very low quantities in the presence of much higher quantities of similar but not identical articles. As stated previously, specificity should be determined over the expected range of usage for the method, and conditions used during the validation should be clearly stated.

Linearity, in essence, refers to the existence of a direct relationship between the quantity of article contained in the sample being analyzed and the measured value resulting from the analysis. It is not the purpose of this article to delve into statistical intricacies pertaining to data transformation, the use of linear or nonlinear regression techniques, residual analysis, and so forth. Currently, it is sufficient that an assay purporting to be quantitative in nature must have a demonstrable quantitative relationship between the quantity of material of interest contained in the sample and the measured response.

Range is directly related to linearity, and ties in accuracy and precision as well. It represents the lowest and highest quantities of material of interest contained within the samples under analysis that provide data with acceptable accuracy, precision, and linearity.

Detection limit represents the least amount of material of interest contained within the sample under analysis that produces a signal exceeding the underlying noise. No assertions pertaining to accuracy, precision, and linearity are necessary at this level of material of interest. For example, if a method is validated to have a detection limit of 3 ng of total protein using Method 3 (1), then a sample containing 3 ng would elicit a signal discernible from underlying noise. It would not be possible to state from such data alone whether there was in fact an exact quantity ng of protein in the sample, only that there were at least 3 ng.

Quantitation-limit determination is more demanding in that currently it is necessary to establish the minimum quantity of material of interest contained within the sample that produces a signal that lies within the linear range of data. That is to say, the quantitation limit represents the lowest end of the range.

Intermediate precision (ruggedness in USP Chapter ‹1225› [1]) pertains to the establishment of “…the effects of random events on the precision of the analytical procedure” (4). Referring to the previous discussion under accuracy pertaining to error components, intermediate precision considers random error introduced by such factors as specific equipment, analysts, laboratories, days, and so forth. It is not meant to include systematic error (bias).

Robustness is probably most directly related to the consideration of conditions under which a validated method is shown to be suitable. This text is very useful in considering robustness:

“If measurements are susceptible to variations in analytical conditions, the analytical conditions should be suitably controlled or a precautionary statement should be included in the procedure. One consequence of the evaluation of robustness should be that a series of system suitability parameters (e.g., resolution test) is established to ensure that the validity of the analytical procedure is maintained whenever used (4).”

General requirements for verification

One question that may be asked of the compendia is whether a method provided as official (in the compendia or supplements) requires validation. USP Chapter ‹1225› states:

“…users of analytical methods described in the USP-NF are not required to validate accuracy and reliability of these methods, but merely verify their suitability under actual conditions of use (1).”

This text is consistent with the proposal in this article that the term validation be reserved for the process whereby one determines if a given method is suitable for its intended purpose (which must be clearly defined), and that the term verification be reserved for the demonstration that the conditions under which the method is to be performed will be appropriate for the method.

Another question may be given that verification involves demonstrating that the conditions to be evaluated are suitable for use with the validated method, how does one go about assessing that? It should be evident that a subset of the determinations performed during the validation would be appropriate. Important conditions to consider include equipment, possible matrix effects (components included in the article to be tested that were not evaluated during the validation), and other conditions for which there is no clear indication provided in the method as to their suitability. A proposed new General Chapter ‹1226› “Verification of Compendial Procedures” (see reference 9 for a discussion of this chapter) provides some guidance as to how the verification process may be executed, but ultimately the user is responsible for selecting which of the characteristics (data elements) evaluated during the validation should be examined as part of the verification. The user should establish which of those validation characteristics are critical to the successful use of the validated method.

Summary

There has been some confusion about when an analytical method should be validated and when it should be verified. In fact, there have been occasions when the terms have been used interchangeably. It is suggested that the term validation be reserved for the process necessary to demonstrate that a method is suitable for its intended purpose. Effective validation begins with a proper statement of the purpose of the method. This statement should accompany the method validation report, and in some circumstances, such as with Chapter ‹71› “Sterility Tests” (1), the statement should appear in the text accompanying the method. Depending upon the degree to which robustness is assessed during the validation process, there may be a set of conditions determined that may be suitable for the use of the method, and conditions that are contraindicated. If such conditions have been established, it is helpful for them to accompany the text describing the method (for example, Method 3 in [9]).

The term verification should be reserved for the process whereby it is established that the conditions under which an article is to be tested by a validated method are indeed suitable for that method. The verification process might be considered to include a subset of the validation process, as suggested by Figure 1. The characteristics (data elements) of a validation process are contained in several documents, and which of these are incorporated in the validation should be appropriate to the method’s intended purpose (and spelled out in the validation protocol.) The characteristics from the validation that are assessed during the verification should be representative of the critical aspects of the method. An example of the verification of the range for Method 3 was provided. Given that verification, as described in this article, is intended to address the suitability of a particular set of conditions for use with a validated method, robustness is not likely to be important for the verification process.

For both validation and verification, one must remember the underlying purpose of the method. If the method is from the pharmacopeia and is intended to be used in demonstrating that a pharmacopeial article meets requirements (for which there is a monograph), the method is considered to be validated, and it would be necessary to verify that the test article is suitable for use with the method. If the method is from the pharmacopeia but is not intended for use in satisfying monograph requirements, it may need to be validated relative to the specific nonpharmacopeial purpose. If instead the method is not from the pharmacopeia but is intended to satisfy monograph requirements, it must be validated as providing equivalent results to the pharmacopeial method. Finally, if the nonpharmacopeial method is not intended to satisfy monograph requirements, it must be validated according to its specific purpose, and this would not require comparison to any pharmacopeial method.

David A. Porter is a pharmaceutical consultant with Vectech Pharmaceutical Consultants, Inc. (Farmington, MI), [email protected]

Submitted: Oct. 26, 2006. Accepted: Dec. 11, 2006.

Keywords: qualification, USP, validation, verification

References

1. United States Pharmacopeia 29—National Formulary 24, United States Pharmacopeial Convention, (2006).

1. Pharmacopeial Forum 32 (2), 595–604 (Mar.–Apr. 2006).

3. Webster’s Seventh New Collegiate Dictionary. (G. & C. Merriam Co., Springfield, MA, 1965).

4. International Conference on Harmonization Harmonized Tripartite Guideline, Validation of Analytical Procedures: Text and Methodology, (ICH, Geneva, Switzerland, 2005).

5. FDA, Center for Drug Evaluation and Research, Guidance for Industry: Analytical Procedures and Methods Validation Chemistry, Manufacturing, and Controls Documentation, (Rockville, MD, Aug. 2000).

6. FDA, Center for Drug Evaluation and Research, Guideline on General Principles of Process Validation, (Rockville, MD, May 1987).

7. FDA, Center for Biologics Evaluation and Research, Guidance for Industry Quality Systems Approach to Pharmaceutical Current Good Manufacturing Practice Regulations, (Rockville, MD, Sept. 2006).

8. FDA, Office of Regulatory Affairs, Guide to Inspections of Pharmaceutical Quality Control Laboratories, (Rockville, MD, July 1993).

9. Pappa et al., “Development of a New USP General Information Chapter: Verification of Compendial Procedures.” Pharm. Technol. 30 (3), 164–169 (2006).