Analytical Method Validation for Quality Assurance and Process Validation Professionals

Abstract

Method validation is a critical activity in the pharmaceutical industry. Validation data are used to confirm that the analytical procedure employed for a specific test is suitable for its intended purposes. These results demonstrate the performance, consistency, and reliability of the analytical method. This paper summarizes the requirements of method validation and data generation to document the evidence which demonstrates the suitability for its intended use. It also discusses the critical validation parameters required based on the ICH guidelines and various pharmacopeia as the procedure must be fully validated before being used for release or stability testing. A change control program must be in place to evaluate the impact of any change to the method from the parameters that were validated. A comparison of the terms-method validation, verification, and transfer- is also introduced. The objective of this paper is to provide a guide to Quality Assurance (QA) and Validation Professionals to better assess the method validation data provided by analytical scientists rather than to perform the method validation activity themselves.

Introduction

Manufacturers in the pharmaceutical and biotechnology industry continue to develop and commercialize challenging formulations of pharmaceutical products to meet medical needs. Ensuring the quality of active pharmaceutical ingredients (API) and the drug products continue to be a challenge for the quality unit as effective analytical methods are necessary to assess the materials and finished products at release and during stability. Therefore, the validation of analytical methods is very critical. Quality assurance and validation professionals should consider the validation status of analytical procedures and the impact on the validation of any proposed change when streamlining operating costs and reducing time to market are desired.

Many publications have explained in detail method validation activities throughout the drug development process. This paper bears the objective to guide QA and Validation Engineers on the method validation topic. It concentrates on the chromatographic procedures of chemical assay and impurities of small molecules drug products.

The content provides:

• A review of various guidelines applicable to the validation of analytical procedures to monitor pharmaceutical products
• The use of analytical testing to support pharmaceutical quality systems
• A look at key validation parameters that characterize analytical procedures
• A discussion of analytical lifecycle management
• A peek at method performance through system suitability testing.

Regulatory Expectations of Method Validation

Method validation is a critical activity and is a requirement of Good Manufacturing Practices (GMPs). Several guidelines are available from the Food and Drug Administration (FDA) as well as global organizations such as ICH and WHO. Many regulations from the US, Europe, and Japan mirror the ICH guidelines; thus, they are not mentioned here.

Section 21 Code of Federal Regulations (CFR) 211. 165 (e) Testing and Release for Distribution requires “The accuracy, sensitivity, specificity, and reproducibility of test methods employed by the firm shall be established and documented. Such validation and documentation are accomplished per Section 211.194(a) Laboratory Records.¹ This regulation is kept general for the manufacturer to develop more specific standard operating procedures (SOPs) with more detailed instructions to fit better in individual organizations. In November 2005, the ICH issued the latest version of Q2 (R1) Validation of Analytical Procedures: Text and Methodology, in which they combined the ICH Q2A and ICH Q2B to describe the validation parameters and directions to perform validation exercises.² This guideline accompanies the 21CFR 211 regulations as well as it provides specific instructions to guide method validations. This guideline serves the pharmaceutical industry well as it somewhat drives the consistency of the evaluation of method validation. In the pharmaceutical industry, most of the method validation information focuses on separation techniques due to their ability to quantify individual components in complex mixtures across a wide dynamic range. These technologies focus on quantitative assays as well as qualitative assays. In addition, the ICH guideline also stipulates method robustness to emphasize the ability of a method to tolerate deliberate variations in the operating parameters and ensure the performance of the method through its normal usage. In June 2018, ICH recognized the void and endorsed a plan to develop Q2 (R2) to include the analytical use of other analytical technologies such as spectroscopic and spectrometry data, and Q14 to address the analytical procedure development.³ Therefore, more change may be expected in the near future.

In July 2015, the Food and Drug Administration (FDA) also issued the revision of Guidance Document “Analytical Procedures and Methods Validation of Drugs and Biologics.” As the previous version is an adaptation of the ICH Q2 guidelines, this final document is a more condensed version, of which much information found in the ICH is removed and replaced with ICH references. Building on the ICH guidelines, the FDA guidance recommends that the intended purpose and scope of the analytical method be considered in the selection of the analytical instrumentation and methodology. An analytical method development section is added to the validation report to explain the selection of method parameters based on the intended purpose and the analytical procedure.⁴ In addition, this final guidance document also gives the reference to the US Pharmacopeia (USP) General Chapters Validation of Analytical Procedures <1225>, Verification of Compendial Monographs <1226>, and Transfer of Analytical Methods <1224>.^5-7

The validation parameters found in the ICH Q2 (R1) and the USP General Chapter <1225> are similar, with the exception that robustness testing is not part of the validation of <1225> because it is recommended to be studied as part of method development. Robustness testing is the study of small but deliberate changes to the method conditions that were optimized during development. These conditions are often interrelated; thus, parameters can be studied one at a time or using an experimental design. Robustness sometimes is viewed as part of the pre-validation experiment rather than part of validation.

The method categories of the two guidelines are similar; however, the USP <1225> also includes the category of procedures to measure product performance, such as the dissolution method for solid dosage forms.⁵ Most of these guidelines were written for the traditional dosage forms of small molecule type of API.

Validation Parameters and their Impact on the Analytical Procedure

Method Development

When a need for an analytical procedure is identified, relevant information should be gathered prior to the method development phase. Such information may include known chemical structures, analyte concentration, solubility, stability, sample matrix, spectroscopic properties, and other physical and chemical characteristics of the API and other components of interest. The intended purpose of the method and the fitness of use must be understood. Most importantly, the complexity of the sample preparation, the choice of analytical instrumentation, the length of the analysis, the availability of reagents or standards should be considered during the method design phase to ensure method efficiency in a routine Quality Control (QC) environment. Other factors are the cost of solvents or materials and the cost of waste. The sample throughput should also be evaluated. A white paper discusses in detail a simple five-step process to develop an HPLC analytical method for assay and impurities.8 Data from method development will serve the basis to establish the validation parameters and suitable acceptance criteria.

Method Validation Parameters

The following list includes validation parameters to be studied for an HPLC quantitative analytical assay, specifically chromatographic procedures. Not all parameters are necessary when other types of analytical technologies are used.

• Accuracy
• Precision
• Specificity
• Detection Limit
• Quantitation Limit
• Linearity and range
• Ruggedness
• Robustness

Similarly, the checklist in Table 1 will help to evaluate the validability of a method, specifically a chromatographic method, when preparing the validation protocol. Not all parameters apply to other types of methods; however, it can be used as a template.

Specificity

Specificity is the ability to assess the analyte unequivocally in the presence of components which may be expected to be present, and it plays a critical role in the method validation.² The following citations from warning letters are typical for lack of specificity requirement:

• Specificity is not determined during method validation, nor is it required in the procedure.
• Unidentified HPLC peaks found during stability testing of the biobatch/validation lots were not identified nor evaluated.
• Filters used to filter dissolution aliquots have not been validated to assure there is no loss upon filtration or interaction between the filter and the drug product.

Several factors can impact the specificity of the method that may require additional study or revalidation. The intrinsic stability of the API, its interaction with the excipients, changes to the manufacturing process, composition of the dosage form, raw materials, packaging components, or storage or handling of the finished products. These factors may cause unintended changes to the final products and generate undesirable degradation products.

For methods that are intended to monitor the quality of the product over time, specificity studies include forced degradation studies and stress testing. As an example, for products stored at room temperature, this stress testing should be done with drug substance and drug product samples exposed to high heat (>40°C), humidity (>75%), and exposed to light.⁹ The drug substances are also exposed to acid, base, and oxidation stress. An approximately 5-20% loss of active ingredient at the most stringent conditions is the goal of stress studies.8 For products stored in refrigerated or frozen conditions, the stress studies should be evaluated on a case-to-case basis.

The analytical method must be shown to be stability-indicating for testing of finished product stability samples. The method must show that all main components, impurities, and any potential degradation products can be measured without interferences from the placebo or the matrix. For stability-indicating methods relying on separation techniques (e.g., chromatography, electrophoresis), peak purity needs to be demonstrated using appropriate orthogonal detection (e.g., photodiode array, mass spectrometry).^10,11

Precision

The precision of a method is the degree of repeated measurements under the same conditions over the operating range of concentrations. The method precision can take place at different levels. For a typical HPLC for assay and impurities measurement, several levels of precision should be studied. (Figure 1)

Repeatability is the variability of the measurements of the same sample. This parameter is studied for system precision and method precision. (1) System precision, of which the same sample preparation (typically 100% concentration) is injected several times, and (2) method precision, of which a sample is prepared several times (typically n=6). The system precision evaluates the reliability of the analytical system to precisely measure the component while the method precision takes into account also the variability of the sample preparation.

Intermediate Precision is the variability obtained when samples are analyzed by different analysts, on different days, and with different sets of instruments. For a validation that may have been done over five years ago, this parameter may not have been studied; therefore, the procedure does not meet the current requirements of method precision.

Reproducibility is the variability obtained when samples are analyzed to qualify different laboratories. Method transfer studies in which the testing is performed on the same set of samples by the transferring and receiving laboratories will satisfy method reproducibility requirements. Different approaches to conduct method transfer are listed in the USP General Chapter - Transfer of Analytical Methods <1224>.⁷

Accuracy

The accuracy of a method is the capability of the method to come close to the quantity’s true value within the working range of concentrations. There are several ways to determine the accuracy of a method. Many regulatory authorities recommend that a reference standard is for accuracy.

Testing against a Reference Standard: This experiment is typically done using a reference standard. The accuracy is the recovery of the quantity added to the solution.

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Complex samples: This approach is when the active ingredient is spiked onto the placebo, which is a mixture of excipients. The results with and without a placebo are compared to determine if there is any interference in the measurement of the component of interest from the placebo.

Standard addition: This is used when the material already contains a small amount of the component of interest - for example, the method to measure residual solvents. This approach can also be used if a clean matrix/placebo is not available.

Comparison with a reference method: This approach is used when a reference method is available. In this approach, test data from testing of selected samples using the new method is compared to the test data obtained from testing of the same samples using the reference method (true value).

In most cases, external standardization is used for the calculation; thus, linearity must be demonstrated together with accuracy and precision over the range of working concentrations.

Detection Limit/Quantitation Limit (DL/QL)

These parameters are studied when quantitation of a low level of a component of interest is needed - for example, an analytical method for impurities or degradation products. The DL/QL levels can be determined based on the signal-to-noise ratio or based on the standard deviation of the response and the slope. These values should also be verified with the concentration determined. For methods measuring the impurities or degradation product, a verification of the QL should be included in the system suitability testing section for method performance.

Ruggedness and Robustness

The ruggedness, also referred to as intermediate precision, measures the method performance under normal variability, while the robustness measures the method performance with small but deliberate changes. The ruggedness is usually addressed in the precision study (intermediate precision).

Robustness

While precision assesses the typical variations in the normal operation of a procedure, robustness measures deliberate variations (e.g., flow rate +/- 20%, column length, mobile phase composition, pH detector wavelength, sonication time). These changes can be studied individually or with an experimental design. The full set of system suitability parameters are evaluated for robustness. Several references discussed these parameters in detail. The changes in sample preparation schemes must also be studied, such as shaking time, extraction time, fi ltering.^11,12

Solution stability is a critical component of robustness testing. It should be studied for all solutions used in the analytical procedure such as standard, sample, system suitability solution, mobile phases, reagents, etc. Companies should also have a general SOP for the storage of reagents or solvents, of which a ‘use-by’ date should also be determined. Robustness should be evaluated for all components of interests, including the elution and quantitation of the impurities and potential degradation products.

Content of the Analytical Method

The content of the analytical method is listed in the FDA guidance of the industry issued in 2018. An analytical method should contain the following sections:^2,4

• Principle/Scope
• Apparatus/Equipment
• Operating Parameters
• Reagents/Standards
• Sample Preparation
• Standards Solution Preparation
• Procedure
• System Suitability
• Calculations
• Data Reporting
• Reference Standards and Materials

Reference Standards and Materials

Reference standards play a critical role in any analysis. In HPLC analyses, reference standards are used for identification and quantitation. Each company must have an SOP to guide the qualification, distribution, and maintenance of reference standards. Reference standards must be fully characterized, and the impurities profiles must be determined and tracked using appropriate testing. Companies can use commercially available reference standards or qualify their secondary standards for volume testing.

Performance of the Chromatographic Method

Typically, the system suitability is established according to USP General Chapter <621>. Validation data are used to establish the acceptance criteria for the system suitability test (SST). It is recommended that system suitability should be designed with commercially available reagents, solvents, or standards.

When the method is transferred to a Quality Control (QC) or a contract lab, the critical parameters of the methods must be well studied to understand the performance of the method at the receiving lab. It is recommended that SST data be evaluated and trended to detect potential deviations prior to system failure.¹³

Validation by Phases

Several global guidelines require that full validation be completed for the analytical method prior to the testing of the first registration batch. Method validation is also expected during drug development. However, the expectation at the early phase of method qualification is more relaxed than in the later phases (IIb or III) or when the fi rst registration batch is manufactured. These phase-appropriate expectations are appropriate because the analytical method is evolving and based on the risk involved. Critical parameters should be studied to ensure that the test is suitable to release and monitor clinical supplies. The current trend of validation activities is a risk-based approach to determine the critical validation parameters at each phase of development. In the early development phases, the sample matrix is frequently changing due to the frequent changes in the drug substance manufacturing process and the drug product formulation. Therefore, analytical methods are frequently being updated and re-validated to ensure the suitability of the method for its intended use. In addition, the number of testing samples is small and performed by a few analysts in a single lab. Therefore, limited validation may be appropriate in the early phases. As the manufacturing process and formulation are decided in late development, complete method validation is required. Figure 2 lists a typical flow of validation parameters to be studied based on the phase of product development.¹⁴

Changes to the Analytical Methods

Many regulatory guidance documents indicate that validation is a process, which makes the validation report a living document. It is important to understand that the analytical procedure used for a specific test must be proven suitable for its intended purpose and demonstrated through validation. The validity of a specific method must be demonstrated in the laboratories; therefore, method validation must be fully completed prior to being used routinely in the Quality Control laboratories for release or stability testing. Whenever the method or the test sample is changed outside of the original scope of the method, validation must be reviewed to ensure the procedure continues to function as intended. Different method validation parameters may be reevaluated to accommodate the change. These changes are tracked with a change control program and properly characterized. Table 2 lists some specific changes that may cause method revalidation.^12,15

This program will evaluate each change that could impact the method validity, discuss deviations, track the corrective and preventive actions, and evaluate the method performance. Risk assessment should be integrated into the revalidation for the selection of validation parameters to ensure the intended use of the method is met. Whether the activity is validation, revalidation, or verification, the activities should be performed under an approved protocol with appropriate acceptance criteria to assure that the analytical method is fi t-for-purpose and stays in compliance.¹⁶

If the evaluation determines that the change is within the scope of the method, no additional validation is needed. This evaluation and its conclusion should be documented. The system suitability test parameters should also be reviewed and redefined, if necessary.

Verification of Compendial Monographs

For standard methods recognized by organizations such as The American Society for Testing and Materials (ASTM), International Standard Organization (ISO), United States Pharmacopeia (USP), which has already been validated by the method’s sponsor, validation is not necessary by the second user. However, verification is necessary, and the validation parameters should be evaluated to determine which additional experiments are necessary to ensure that the compendial method is suitable for the test sample. USP General Chapter <1226> contains recommendations to be used for different types of methods.6 In most cases, the manufacturer should evaluate the analytical method to ensure that the method can be used for their material, and the system suitability requirements should be verified under the actual conditions of use. The most critical parameter is the specificity to assure that the method is specific for the material tested, and all impurities/degradation products are accounted for. In many cases, the compendial method may need to be adjusted for use in a routine analytical laboratory. While method validation is the qualification of an analytical method using a well-qualified sample lot, verification is a qualification of a material of interest using a well-qualified standard method. The verification plan should be done under a protocol, and a report will be assembled to document the verification data generated.

Method Lifecycle Management

Analytical data are used to determine the quality of the drug substance and drug products; for that reason, method performance should be reviewed and trended every time that the procedure is used. Therefore, method validation is an ongoing process. Figure 3 illustrates the method lifecycle. The analytical procedure is developed, qualified, and validated according to the intended use of the method. Once the method is implemented, it should be reviewed periodically and optimized to assure that it is sufficiently robust for routine use over a long period.

To support the product testing, validation must be studied carefully to understand the critical parameters. The performance of the method must be tracked, and trend analysis should be done at regular intervals to proactively prevent unintended changes to the method that could impact the monitoring of the drug product. Table 3 gives a template to plan for the validation activities of an analytical procedure.

While most of the ICH guidelines focus mainly on early phases of development and prepare for registration, ICH Q12 provides regulatory and technical considerations to manage the lifecycle of pharmaceutical products using a risk-based and science-based concept.¹⁷ In essence, this guideline provides the framework to allow flexible regulatory approaches to post-approval changes to the pharmaceutical product using the information collected through product development. The product lifecycle consists of three stages: (1) process development where the product profile is established and the critical quality attributes are determined, (2) risk management, used to develop the design space and establish the control strategies for the product, and (3) managing the product lifecycle including continual improvement.^17-19

In ICH Q12, the lifecycle concept is being applied to analytical method validation. Figure 4 illustrates the three steps to manage the analytical procedure. This framework will allow the maintenance of the method performance through development, qualification, and continual verification. During the method development, the critical attributes of the method are determined with a focus on an analytical target profile.^17-21 Once the method parameters are understood, the procedure is qualified using a validation protocol, and data are documented in the validation report. Once the procedure is successfully validated or verified, the procedure must be followed through the lifecycle of the product to assure that it remains fi t for its intended purpose. If the procedure needs adjustment consistently every time it is used, it should be reevaluated and amended for additional ruggedness, as appropriate.

Over the product lifecycle, new information may surface that initiate additional method development or validation. New technologies or changes to the product target may also trigger additional development to ensure the method continues to be reliable to monitor the product quality. Method performance must be reviewed periodically to anticipate unintended changes and prevent failures. The availability of aged samples can be critical for comparative studies; thus, a sufficient amount should be retained to support the method validation lifecycle.^18,19

Audit and Inspection

Analytical methods are the tools to monitor the quality of drug substance and drug products; therefore, validation is critical to assure that the method is reliable. Method validation is one of the critical areas that will be included in any quality audit or inspection. The validation report is usually scrutinized against the current industry practices for compliance. Table 4 shows an example to evaluate the method validation report of a solid formulation. The list contains all validation parameters and the recommendations from ICH Q2(R1). It can be revised to accommodate different types of method validation or different company’s Standard Operating Procedures (SOPs). Manufacturers are encouraged to review their validation activities, especially if validation activities were conducted prior to 2012. A review of the change control is also advisable because many minor changes over many years could have an imperceptible impact on the performance of the analytical procedure.^11,15

Summary

Analytical procedures are used to monitor the product quality, such as the potency of the finished product, or to detect the presence of new unknown impurities; therefore, validation of analytical procedures is a critical process in pharmaceutical drug manufacturing. Manufacturers must have a rigorous process to assure the validity of the procedure used because an uncontrolled process can lead to product investigations and product recalls. Management of the method validation lifecycle is critical to ensure all the changes to the analytical method are tracked and trended effectively because unintended changes to the product quality may pose a potential risk or unknown harm to patient safety.

Acknowledgments

The author expresses her gratitude to the following colleagues for their time and efforts to provide a technical review of the manuscript: Kin Tang, Ph.D. of Genentech, Oscar Liu, Ph.D. of Silver Spring Scientific, LLC, and Anne Aubry, Ph.D. retired.

Author Biography

Kim Huynh-Ba is the Managing Director of Pharmalytik Consulting (www.pharmalytik.com). She has almost 30 years of experience in Quality Systems, Strategic Drug Development, Validation, and Stability Sciences. She is a member of the Council of Experts of US Pharmacopeia, chairing the Small Molecules 4 Expert Committee. Kim is also an Adjunct Professor at Temple University and Illinois Institute of Technology, teaching Quality Audit, Good Manufacturing Practices, ICH quality guidelines, and Pharmaceutical Analysis. Kim has over 30 publications and made over 200 presentations nationally and globally.

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