During drug development programs, bioanalytical assays are validated and used to quantify drugs and their metabolites in samples from a variety of different biological matrices. Incurred sample reanalysis (ISR) has recently become an accepted way to assess the quality of bioanalytical assays and is widely used within the pharmaceutical industry and by contract research organizations for this purpose. ISR occurs when samples obtained from an in vivo study are reanalyzed to demonstrate that the assay is reproducible. This article provides a brief overview of some recent guidelines and white papers describing ISR that can assist in establishing a robust ISR program.
Bioanalytical method validation
In pharmacokinetic studies, bioanalytical method validation is crucial to minimizing random error and systematic bias to ensure the quality of the analytical results. Validation of a bioanalytical assay, according to good laboratory practice (GLP) requirements, requires the preparation of standard and quality control (QC) samples by spiking the biological matrix with authenticated analytical reference standard of known identity and purity. In addition, other characteristics of the assay are quantified and include aspects such as long-term, short-term, benchtop, and freeze-thaw stability, recovery of analytes and any internal standards, ion suppression effects, etc. These types of assessments are outlined in the US Food and Drug Administration’s (FDA) bioanalytical method validation guidance in 2001.1
Incurred sample reanalysis
Following the publication of the FDA’s official guidance for bioanalytical method validation, some uncertainties still remained in relation to process guidelines due to differing interpretations of the FDA guidance, especially for large-molecule assays. Many of these questions were addressed during the third American Association of Pharmaceutical Scientists (AAPS)/FDA bioanalytical workshop and conference. The concept of incurred sample reanalysis was established in the conference report2, which stated that performance of spiked standards and QCs may not adequately mimic that of study samples from dosed subjects (incurred samples). The reproducibility and accuracy of the assay may be evaluated by the bioanalysis of incurred samples and is a supplement to the usual pre-study bioanalytical method validation activities. This report indicated that incurred sample reanalysis is a necessity for both nonclinical and clinical studies and it provided a general framework and rationale for ISR. It also described the types of studies in which to perform ISR, but left many practical details about how comparisons should be calculated and evaluated to the discretion of the scientist.
A subsequent paper by Rocci et al. addressed some of the practical and scientific questions concerning ISR procedures such as the types of studies to perform ISR, the number and timing of samples to be reanalyzed, and a novel statistical approach to the assessment of acceptable reproducibility.3
A follow-up workshop was convened to provide a forum for consensus building about incurred sample reproducibility for both large and small molecules. The June 2009 report provides recommendations for ISR assessment, studies that should be tested, selection of samples, and acceptance criteria.4
Among the highlights were a consensus that individual samples, not pooled samples, should be used for ISR testing. The analysis should be conducted on the same number of replicates (singlet, duplicate, etc.) as the original analysis. Participants agreed that the number of samples repeated for ISR analysis should be 5-10% of the total sample size, with 5% being suitable for larger studies. Selecting fewer samples from more subjects is better than full PK profiles, and the samples should be analyzed near the Tmax and near the end of the elimination phase. The acceptance criteria for small molecules (non-ligand binding assays) states that two-thirds of the repeat samples should agree within 20%, and for ligand-binding assays, two-thirds of the repeat samples should agree within 30%. Results from or reference to ISR assessments should be included in the report of the study from which samples were taken. Finally, an SOP or study plan is crucial to the proper conduct of ISR.
The most recent paper published in October 2009, representing the European Bioanalysis Forum, presents an alternate viewpoint on selected aspects of ISR.5 Namely, it points out changes in the evolution of ISR philosophy over time introduced a new concept that failed ISR may be due to either poor assay methodology or poor bioanalytical execution, and made recommendations for the frequency of ISR bioanalysis and selection of studies.
Laboratory solutions for ISR
Considerable scientific judgment and care is required both in the preparation of the entire ISR process and in the interpretation and follow-up of ISR results. The choice of a data processing system for bioanalytical support needs to take into account the factors and choices described above. While there are many laboratory info
Watson LIMS analyzes calibration curves from standards and back-calculates concentrations for QCs and unknowns. Configurable parameter flags alert the user to acceptability criteria. Color-coding enhances visual inspection of results. (Source: Thermo Fisher Scientific)
rmation management systems (LIMS) available for pharmaceutical work, a dedicated bioanalytical data system may help to facilitate data processing and ISR sample selection and reporting.
In order to perform ISR calculations and reporting in a reliable and consistent manner, scientists need a solution that ensures data consistency with ease of use; specifically, they require a solution that integrates both laboratory instrumentation and software tools. A purpose-built bioanalytical data system, like Thermo Scientific Watson LIMS, provides users with the necessary workflow for the generation of analytical runs, and the importing, analysis, review, and reporting of data and subsequent export of results to external systems. The latest release of Watson provides out-of-the-box functionality for the selection and tagging of ISR samples, a wide variety of calculations that are specific to ISR and the original numerical values, and built-in template reporting, including five methods of user-configurable %Difference calculations and overall study assessment of ISR results for each analyte. The design of the ISR calculations and reporting functionality inside Watson was influenced by input from several contributors.6-8
Selective reaction monitoring (SRM) using a triple stage quadrupole mass spectrometer coupled to a high performance liquid chromatograph, or LC/MS, is the most common chromatographic method for bioanalysis. When coupled with Thermo Scientific TSQ series mass spectrometers, Watson can now acquire LC/MS data directly from the instrument. The raw data is stored in an Oracle database, allowing integrated chromatogram review, re-integration, and approval within the secured, audit-trailed Watson application.
A carefully designed ISR program provides additional data to assure confidence in the reliability and reproducibility of a validated method for nonclinical and clinical study samples. ISR can lead to continuous review and improvement practices for the laboratory performing bioanalytical assays.
About the Author
Dr. Joel Usansky has over 15 years experience in the pharmacokinetics and drug metabolism field with Sanofi-Aventis, Allergan, and Schering-Plough. He holds a PhD from the University of Manchester, UK.
1. US Department of Health and Human Services, FDA (CDER and CVM): Guidance for Industry. Bioanalytical Method Validation. Rockville, Md. May 2001.
2. Viswanathan CT, et al. Workshop/conference report—quantitative bioanalytical methods validation and implementation: best practices for chromatographic and ligand binding assays. AAPS J. 2007;9(1):E30-E42.
3. Rocci ML Jr, Devanarayan V, Haughey DB, Jardieu P. Confirmatory Reanalysis of Incurred Bioanalytical Samples. AAPS J. 2007; 9(3): E336–E343.
4. Fast DM, et al. Workshop Report and Follow-Up – AAPS Workshop on Current Topics in GLP Bioanalysis: Assay Reproducibility for Incurred Samples – Implications of Crystal City Recommendations. AAPS J. 2009;11(2),238–241.
5. Timmerman P, Luedtke S , van Amsterdam P, Brudny-Kloeppel M, Lausecker B. Incurred sample reproducibility: views and recommendations from the European Bioanalytical Forum. Bioanalysis. 2009;1(6),1049-1056.
6. Moran J. Approaches to Demonstrating Reproducibility and Implications for Watson. Presented at Thermo Informatics World User Group Meeting Oct. 2006.
7. Hillewaert V. Incurred sample reproducibility. Presented at Thermo Informatics World User Group Meeting Oct. 2007.
8. Weigl P. , Bansal S. Processing of Incurred Samples Reanalysis. Presented at Thermo Informatics World User Group Meeting Oct. 2007.
This article was published in Drug Discovery & Development magazine: Vol. 12, No. 10, November/December 2009, pp. 37-38.
Filed Under: Drug Discovery