Assessment of Incurred Sample Reanalysis for Macromolecules to Evaluate Bioanalytical Method Robustness: Effects from Imprecision

Abstract

Abstract. Incurred sample reanalysis (ISR) is recommended by regulatory agencies to demonstrate reproducibility of validated methods and provide confidence that methods used in pharmacokinetic and toxicokinetic assessments give reproducible results. For macromolecules to pass ISR, regulatory recommendations require that two thirds of ISR samples be within 30% of the average of original and reanalyzed values. A modified Bland-Altman (mBA) analysis was used to evaluate whether total error (TE), the sum of precision and accuracy, was predictive of a method's passing ISR and to identify potential contributing parameters for ISR success. Simulated studies determined minimum precision requirements for methods to have successful ISR and evaluated the relationship between precision and the probability of a method's passing ISR acceptance criteria. The present analysis evaluated ISRs conducted for 37 studies involving ligand-binding assays (LBAs), with TEs ranging from 15% to 30%. An mBA approach was used to assess accuracy and precision of ISR, each with a threshold of 30%. All ISR studies met current regulatory criteria; using mBA, all studies met the accuracy threshold of 30% or less, but two studies (5%) failed to meet the 30% precision threshold. Simulation results showed that when an LBA has ≤15% imprecision, the ISR criteria for both the regulatory recommendation and mBA would be met in 99.9% of studies. Approximately 71% of samples are expected to be within 1.5 times the method imprecision. Therefore, precision appears to be a critical parameter in LBA reproducibility and may also be useful in identifying methods that have difficulty passing ISR.

Analysis of Imprecision in Incurred Sample Reanalysis for Small Molecules

“…At 5% IS CV, a positive bias of up to 12% has no significant effect on passing rate (>99%); therefore, the data are not included ISR failure with an increase in sample size at 15.5, 17.5, and 20% CV (13). Also, the in silico results presented in the current paper are in line with those reported for macromolecules (14).…”

“…Currently, this ISR criterion is routinely used in bioanalysis and has been adopted by regulatory agencies (9-11). There have been studies in the past that have compared the current acceptance criterion with alternate acceptance criteria (12)(13)(14). The current study attempts to assess the sensitivity of the ISR acceptance criterion, specifically its ability to discriminate between low and high IS imprecision during study sample analysis and address the effect of sample size on ISR study acceptance.…”

“…Simulations were done using a procedure similar to that reported by Thway et al (14). Simulations were conducted using SAS, version 9.3 (SAS Institute, Inc., Cary, NC).…”

“…Each simulated ISR study has a sample size between 20 and 300 paired samples (20,40,60,80,100,120,140,160,180,200,250,300) and IS CV between 5 and 20% (5,10,11,12,13,14,14.5,15,and 20% CV). Ten thousand studies were simulated at each IS CV and ISR sample size combination.…”

“…Recently, Thway et al (2011) investigated the effect of imprecision on ISR analysis for macromolecules (14).…”