Unlocking Success: The Critical Steps in Analytical Test Development

In today's fast-paced industries, the development of reliable and robust analytical test methods is the key to ensuring product quality, safety, and regulatory compliance. Behind every breakthrough pharmaceutical or cutting-edge medical device lies a meticulously designed method that goes through critical steps to achieve accuracy, precision, and reliability.??

Join Cecilie Domar Villadsen , MSc in Medicinal Chemistry and Scientific Lead Consultant from the Emendo R&D team as she walks you through the pivotal milestones in the development of analytical test methods from idea to validation.?

Defining the Analytical Target Profile (ATP): Setting the Course for Success??

The journey starts with a clear destination. Defining the Analytical Target Profile (ATP) is the first step in creating an effective analytical test method. Identifying the performance characteristics, quality criteria, and regulatory requirements saves time and avoids mistakes later in development and validation.??

In other words, the ATP acts as a compass to ensure that the method is ultimately fit for purpose. Specifically, that it has been developed, validated, and optimized to fulfill its intended use accurately and reliably in a particular application or industry.?

Method Selection: The Right Path to Precision ?

The selection of the most suitable analytical technique is a crucial decision. When the ATP has been drafted and reviewed by relevant subject matter experts, the evaluation of potential analytical methods can begin. The final decision should be based on:??

• Availability of in-house methods that could be used as is or with slight modifications (i.e., evaluation of potential matrix effects of a new formulation).??
• Availability of suitable pharmacopeia methods that can be used without modification (OBS a rationale is needed if a suitable pharmacopeial method exists but is not used).??
• Availability of related pharmacopeia method that can be modified to be fit for the purpose. Carefully consider the difficulty of modification, robustness, and validation hereafter.??
• If no apparent and suitable test method is available, development of an entirely new method is needed (external inspiration e.g., publications could aid the process).??

When selecting a method, it is also advisable to consider the parameters listed below, although they should not be the driving force behind the choice:?

• Available equipment and cost/timeline of implementing new equipment.??
• Risk assessment for each considered Test Method.??
• Difficulty of the method and availability of qualified personnel.??
• Robustness of the method.??
• Safety concerns for laboratory personnel.?
• Suitability for testing by regulatory authorities, if relevant.??

Method Development: Variation Control in 4 Steps?

Once the method is selected, it's time to embark on the journey of development and optimization. This iterative process involves fine-tuning parameters. It is therefore highly advisable to understand how different parameters influence the result. A thorough understanding will drive robustness of the method but also aid problem solving and criticality assessment if deviations occur after implementation of the method.??

We recommend performing a structured variation reduction process (variation control chart) to control systematic and random variation of the method before starting the development and further optimization attempts.?

1. Start with a detailed map of the method (including all supporting activities such as buffer preparation etc.). This will be used for optimizing assay performance and robustness, as well as identify steps in the process that influence bias or precision. ?
2. For each step, determine parameters influencing the performance of the step and final method result (i.e., the mean and standard deviation). Examples of parameters influencing methods: a) Compositions of reagents: different columns, lot variations in reagents/kits etc. b) Sample handling: extraction time, temperature, agitation intensity, pipetting technique. c) Instrument Setup (tolerances): temperature, flow rate, wavelength detection.??
3. Describe the potential impact or influence of the different parameters (variation, contamination etc.) including severity, probability, and detectability.?
4. Classify all parameters influencing the method by nominating all parameters by: a) Constant: Parameters which can be held constant (i.e., is there a setting at which they contribute to a desirable outcome?). b) Noise: Parameters which are too difficult, expensive, or unimportant to control. c) Experimental: Important or critical parameters which must be investigated to understand their impact via e.g., DoE runs.??

Method Development: Optimization and Robustness?

During development the analytical test method is optimized to achieve acceptable performance. Both the future validation parameters and robustness should be assessed during development. Robustness testing strengthens the method's resilience against variations, enhancing its applicability and reproducibility in different laboratory settings.?

It is recommended to follow the process below for developing and optimizing the method:?

1. Perform pre-test using known components and settings (including parameters classified as “Constant”) to create a baseline of method performance before you start optimization.?
2. It is advised not to evaluate results obtained during development on pre-determined acceptance criteria. The acceptance criteria should be set after pre-validation/qualification, based on the capabilities of the test method.?
3. For parameters classified as “Experimental” (i.e., Important or critical parameters which must be investigated), set up a series of experiments to identify the right parameter set points. ?
4. As many parameters are interdependently influenced by each other it is important not only to test and optimize one parameter at a time. It is recommended to perform multifactorial setups like DoE.??

Method Development: Assay Optimization and Data Analysis?

In the development of analytical test methods, assay optimization plays a key role in achieving superior method performance. This critical step focuses on fine-tuning assay parameters to improve sensitivity, specificity, and overall efficiency. By optimizing the assay, the full potential of the method can be realized, ensuring accurate and reliable results.?

When developing assays, it is important to look at data to draw the right conclusions. Simple statistical analysis is useful to understand your data. We recommend looking at distribution, controls charts, box and scatter plots. ?

Optimization of assay performance is also a matter of asking the right questions:?

• Do we have stable results???
• What is our biggest problem???
• How is our problem solved???

Method Qualification: Setting the Acceptance Criteria??

Acceptance criteria define the thresholds that must be met for the method to be considered acceptable for its intended use. By setting these criteria, we can objectively evaluate and assess the method's performance against predefined benchmarks.?

Note that acceptance criteria are not mentioned in ICH Q2. At Emendo R&D we recommend using validation accept criteria relative to the product specification tolerance:??

• Tolerance = Upper Specification Limit (USL) – Lower Specification Limit (LSL)??
• How much of the specification tolerance is “consumed” by the analytical method???
• How does the method contribute to OOS events when releasing a product???

Using validation acceptance criteria relative to the product specification tolerance enhances the method's applicability and relevance to the specific product being analyzed. It ensures that the analytical test method is appropriately validated and validated to demonstrate its ability to reliably assess the product's quality attributes within the specified tolerance limits.?

Method Validation: Confirmation of fitness for purpose?

Method validation is a collection of data, which confirms that the requirements for the specific analytical method and intended use is fulfilled. During validation the method is confirmed to be fit for purpose, in other words, suitable and reliable for solving a particular analytical problem.??

We recommend consulting ICH Q2 R1 for guidance on validation requirements and parameters to include in the validation. ?

And remember, the expected level of validation is increasing during the drug product development phase towards marketing approval. Analytical methods used for characterization studies do not necessarily require validation, but they need to be scientifically sound and appropriate (qualified).??

Cecilie Domar Villadsen , Scientific Lead Consultant in Emendo R&D?

For more information on analytical test development and validation, or other Emendo R&D services, visit emendo.com/emendo-rd/?