CPSA Digest 2003

Day 3

WeOA1

Impact on Drug Development

Three recent regulatory guidances have had an impact on how work is performed within a Good Laboratory Practice (GLP) compliant development setting.

• Food and Drug Administration (FDA) Guidance for Industry - Bioanalytical Method Validation (2001)
• "Drug Metabolites in Safety Testing" as published in Toxicology and Applied Pharmacology 182, 188-196 (2002)
• FDA Guidance for Industry on 21 CFR Part 11 - Electronic Records (2002)

Bioanalytical Method Validation
Bioanalytical method validation applies to the quantitative determination of drugs and/or metabolites in all biological matrices (e.g., blood, serum, plasma, urine, tissue, skin samples). The fundamental parameters for method validation include: accuracy; precision; selectivity; sensitivity; reproducibility; and stability.

Clarification on Full, Cross, and Partial Validation
While the FDA document provides "guidance" on method validation issues, there is occasionally a need for clarification and/or interpretation. Since laboratories function within a regulated environment, departmental procedures should be defined and documented by internal standard operating procedures (SOP). For example, clarification is needed on full validation, cross validation and partial validation.

• A full validation is performed for any new drug entity that has been recommended for development. Full validation is also required when there is a need to add metabolites to a validated method.
• Partial validation refers to modifications of a validated method. The scope of work is related to the extent of change.
• Cross validation across two or more bioanalytical methods compares all criteria for acceptance with the aim to show that regardless of which method is used, the results would be comparable. Cross validation reports are critical within NDA or related documentation when two or more bioanalytical methods are used to generate data within the same study or across different studies.

Partial Validation Examples

Method Change Method transfer between labs Method transfer between analysts Change in internal standard Change in instrument platform Change in sample processing Change in chromatography Change in anticoagulant

Number of Core Validation Runs 3 1 1-3 3 1-3 1-3 1

Incurred Samples
Common pooled quality control (QC) samples are used in three concentrations (low, medium and high) for bioanalytical methods. However, the FDA Guidance recommends use of incurred samples (i.e., cross validation should use real study samples). The use of incurred samples is necessary because an analyte may be well characterized and shown to be stable but there may be metabolites that are unstable. These metabolites may break down to a product that interferes (common ion) with the assay, thus the recommendation is to use incurred samples.

Reference Standards
An analytical assay is only as good as the reference standard used. Reference standards must be of known identity and purity, be identical to the analyte, and have a certificate of analysis, identified source, lot number and expiration date. Problems usually arise with metabolite standards (not analytical reference standards) because only a limited supply is available with limited authentication. Sometimes, synthesis is not possible and metabolites must be purified from a biological source. Laboratories can still operate in a GLP compliant manner as long as all the exceptions in the compliance statement are listed.

Selectivity and Recovery
Selectivity

• Analysis of blank matrix from six sources
• Acceptance criteria are not well defined in the FDA Guidance; define criteria, justify criteria as reasonable, and document in SOP
• Assessment of ion suppression

• Should be consistent, precise and reproducible for analyte and internal standard
• Should be conducted at three concentrations vs. non-extracted standards

Calibration Curve
The simplest model that adequately describes the concentration-response relationship should be used. Selection of weighting and use of a complex regression equation should be justified. In reality, most researchers choose a quadratic fit without starting out with the simplest model and justifying every change. The dynamic range of the assay is important; however, if there are three or four orders of magnitude, then a quadratic fit is preferred instead of a linear fit.

Stability Determinations

• Freeze/thaw stability
• Short-term temperature stability
• Long-term stability
• Stock solution stability
• Processed sample stability
• When compounds have potentially labile metabolites, the stability of analyte in matrix from dosed subjects (or species) should be confirmed

Repeat Analysis
Reassays should be done in triplicate if the sample volume allows. When concentrations are high, dilute with matrix and then assay. It is important to note that an SOP must be in place that clearly defines the procedure when samples are reanalyzed. The rationale for the repeat analysis and the reporting of the repeat analysis should be clearly documented. When data are reintegrated, be prepared to show the original data and the reintegrated data side by side.

Documentation
Reports should include summary tables of analytical runs of clinical or preclinical samples. Information documented should include: assay run identification, date and time of analysis, assay method, analysts, run start and stop times, significant equipment and material changes, and any potential issues or deviation from the established method. Further documentation consists of complete serial chromatograms from a minimum of 5% up to 20% of subjects from every study, with standards and QC samples from those analytical runs. Subjects whose chromatograms are to be submitted should be defined prior to the analysis of any clinical samples to avoid bias.

References
FDA Guidance for Industry - www.fda.gov/cder/guidance/4252fnl.htm

Return to Proceedings »