**4. Qualification and validation of release and stability methods**

Qualification should be performed prior to method implementation in the Quality laboratories to ensure the integrity of the data provided on the Certificate of Analysis for clinical lots. In most cases, at early stages of clinical development, only one sample type requires qualification because, in general, the drug substance (DS) and drug product (DP), for which specifications are established requiring testing in the Quality labs, often have the same composition (formulation). If this is not the case, a technical assessment should be made of whether differences in the matrix have the potential to impact the qualification results and, if so, a strategy for verifying the qualification status of the two sample types, relative to each other, should be devised. For example, full qualification of the DS can be followed by a matrix verification for the DP, generally in the form of a repeat of the specificity and precision evaluation.

Analytical Method Validation for Biopharmaceuticals 121

In addition to the stability indicating properties of the method, the assessment of sample stability can be considered as a pre-requisite to method validation. Sample stability can be divided into two activities – an evaluation of sample storage conditions prior to analysis, and assessment of the stability of prepared samples while waiting for analysis. Samples are often stored frozen after collection and thawed prior to analysis; in these instances, sample integrity should be assessed over a minimum of one freeze-thaw cycle for each sample type (preferably more than one cycle in most cases). Sample stability after preparation and before analysis (e.g., time spent in an auto-sampler) should be evaluated to determine the maximum duration of an assay (sequence). Details of sample handling should be included

Method validation confirms the performance characteristics demonstrated during method qualification and demonstrates the suitability of the method for commercial use. This confirmation effort should follow a pre-approved protocol with clear and justifiable acceptance criteria. In the context of the analytical lifecycle, the key components of method

1. The experimental design of method validation should mimic the qualification design, and acceptance criteria should be linked to the target expectations used in the qualification experiments. In the absence of such rigor, validation experiments become exploratory

3. Validation acceptance criteria should only include the objective parameters from the qualification to avoid any subjective interpretations, which could impact the outcome of the confirmatory validation studies. For example, frequently during qualification studies, scientists expect that the residual from linear regression does not show any bias (trend). Since the community has not adopted a uniform measure of the bias (which is frequently based on visual evaluation), it is not advisable to include such a requirement

Validation studies should be executed for sample types that will be routinely tested in GMP environments to make decisions about product disposition. This typically includes the

Sample types listed on all release and stability specifications (intermediates, drug

Samples associated with process controls and in-process decision points.

research and run the risk of undermining the results of the method qualification. 2. Similarly to the qualification (in most cases), the validation acceptance criteria are set based on the type of method and should not differ from target expectations. When qualification target expectations are not met during a qualification study, the rationale for re-evaluation of the acceptance criteria should be proposed in the qualification summary. Setting acceptance criteria for the precision of a method frequently causes confusion, anxiety, and inconsistency in practice. For validation studies, requirements for a reporting interval aligned with the specification for precision studies provide excellent guidance for setting the acceptance criteria for precision and other

in the validation protocol.

validation are as follows:

performance characteristics.

in the validation acceptance criteria.

substance and drug product);

following sample types:

Method validation is typically completed before process validation in adherence with cGMP procedures outlined in ICH Q2R1(ICH 2005) . Method validation for release and stability methods can be considered as the pivotal point in the method lifecycle because it justifies the use of the method in commercial settings to guide decisions about product disposition and lot stability. In addition, the validation activity provides a defined point of transfer of ownership of the methods from the development organization to the commercial (operations) organization. Typically, these activities are initiated after the sponsor has made a commitment to commercialize the drug candidate (which generally occurs after positive feedback from clinical trials).

ICH Q2R1 specifies that method validation has three components: assessment of performance characteristics, demonstration of robustness and system suitability. It should be noted that industry practice dictates that method qualification also evaluates these three components, with a noticeable difference, in that while validation has a formal protocol and pre-defined acceptance criteria for the performance characteristics, method qualification does not. It is a good practice to adopt general target expectations for method qualification as a means of evaluating the outcome of exploratory work on performance characteristics. The expectation should reflect the desired characteristics for the methods with respect to precision, range, QL, etc. If the method does not meet these expectations, the method should be re-developed and/or optimized. Recently, many companies have adopted the practice of developing and qualifying multiproduct methods that can be used for more than one product within specific molecular classes, such as monoclonal antibodies. In such instances, verification of performance could be adequate instead of full qualification studies once the method has undergone full qualification for the first molecule of the specified class.

Standard industry practice dictates that methods used to assess drug substance and drug product stability should show that they are able to detect changes in quality attributes. This can be demonstrated in forced degradation studies on the appropriate sample types using conditions known to impact protein quality, such as elevated temperature, pH extremes, and incubation with oxidizing agents such as hydrogen peroxide to induce molecular changes such as aggregation, deamidation, peptide bond cleavage and protein oxidation.

In addition to the stability indicating properties of the method, the assessment of sample stability can be considered as a pre-requisite to method validation. Sample stability can be divided into two activities – an evaluation of sample storage conditions prior to analysis, and assessment of the stability of prepared samples while waiting for analysis. Samples are often stored frozen after collection and thawed prior to analysis; in these instances, sample integrity should be assessed over a minimum of one freeze-thaw cycle for each sample type (preferably more than one cycle in most cases). Sample stability after preparation and before analysis (e.g., time spent in an auto-sampler) should be evaluated to determine the maximum duration of an assay (sequence). Details of sample handling should be included in the validation protocol.

120 Analytical Chemistry

feedback from clinical trials).

**4. Qualification and validation of release and stability methods** 

generally in the form of a repeat of the specificity and precision evaluation.

Qualification should be performed prior to method implementation in the Quality laboratories to ensure the integrity of the data provided on the Certificate of Analysis for clinical lots. In most cases, at early stages of clinical development, only one sample type requires qualification because, in general, the drug substance (DS) and drug product (DP), for which specifications are established requiring testing in the Quality labs, often have the same composition (formulation). If this is not the case, a technical assessment should be made of whether differences in the matrix have the potential to impact the qualification results and, if so, a strategy for verifying the qualification status of the two sample types, relative to each other, should be devised. For example, full qualification of the DS can be followed by a matrix verification for the DP,

Method validation is typically completed before process validation in adherence with cGMP procedures outlined in ICH Q2R1(ICH 2005) . Method validation for release and stability methods can be considered as the pivotal point in the method lifecycle because it justifies the use of the method in commercial settings to guide decisions about product disposition and lot stability. In addition, the validation activity provides a defined point of transfer of ownership of the methods from the development organization to the commercial (operations) organization. Typically, these activities are initiated after the sponsor has made a commitment to commercialize the drug candidate (which generally occurs after positive

ICH Q2R1 specifies that method validation has three components: assessment of performance characteristics, demonstration of robustness and system suitability. It should be noted that industry practice dictates that method qualification also evaluates these three components, with a noticeable difference, in that while validation has a formal protocol and pre-defined acceptance criteria for the performance characteristics, method qualification does not. It is a good practice to adopt general target expectations for method qualification as a means of evaluating the outcome of exploratory work on performance characteristics. The expectation should reflect the desired characteristics for the methods with respect to precision, range, QL, etc. If the method does not meet these expectations, the method should be re-developed and/or optimized. Recently, many companies have adopted the practice of developing and qualifying multiproduct methods that can be used for more than one product within specific molecular classes, such as monoclonal antibodies. In such instances, verification of performance could be adequate instead of full qualification studies once the method has undergone full qualification for the first molecule of the specified class.

Standard industry practice dictates that methods used to assess drug substance and drug product stability should show that they are able to detect changes in quality attributes. This can be demonstrated in forced degradation studies on the appropriate sample types using conditions known to impact protein quality, such as elevated temperature, pH extremes, and incubation with oxidizing agents such as hydrogen peroxide to induce molecular changes such as aggregation, deamidation, peptide bond cleavage and protein oxidation.

Method validation confirms the performance characteristics demonstrated during method qualification and demonstrates the suitability of the method for commercial use. This confirmation effort should follow a pre-approved protocol with clear and justifiable acceptance criteria. In the context of the analytical lifecycle, the key components of method validation are as follows:


Validation studies should be executed for sample types that will be routinely tested in GMP environments to make decisions about product disposition. This typically includes the following sample types:

