Stability Testing in Biological Matrix: When Does a Failed Stability Compromise Study Data?
A bioanalytical method for drug V in human plasma has been validated, including stability in plasma at room temperature for 6 hours, at 4°C for 24 hours, and at −80°C for 12 months. During a clinical study, samples were collected and stored at −80°C for up to 18 months before analysis. The study protocol allowed storage up to 24 months, but the validated long-term stability only covers 12 months. The lab analyzed all samples in a single batch after 18 months of storage. QC samples (spiked at three levels) were prepared fresh and analyzed with the study samples; all QCs were within ±10% of nominal. Calibration curves were acceptable. The study samples showed concentrations consistent with expected pharmacokinetic profiles. However, a post-hoc review revealed that the long-term stability data at 12 months showed a −10% change at the low QC (5 ng/mL) and −8% at the mid QC (50 ng/mL), which were within the ±15% acceptance criterion but indicate a trend of degradation. The lab wants to report the data without further stability testing.
1. Assess whether the 18-month storage period is adequately covered by the stability data. What are the risks of reporting the data?
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The validated long-term stability covers only 12 months at −80°C. The study samples were stored for up to 18 months, which exceeds the validated period by 6 months. Although the 12-month stability data met acceptance criteria (±15% change), the observed −10% change at the low QC suggests a degradation trend. Extrapolating beyond the validated period is not acceptable without additional stability data. The risk is that the study samples may have degraded beyond 15% by 18 months, especially at low concentrations, leading to underestimation of drug levels. The fact that QCs (freshly prepared) passed does not address storage stability of incurred samples. Therefore, the data are not supported by stability data covering the actual storage duration. Reporting the data without further stability testing could lead to inaccurate pharmacokinetic conclusions. Regulatory guidelines require that the storage period of study samples be covered by validated stability data. If not, a stability waiver or additional testing is needed. The lab should either: (1) Perform a long-term stability study at −80°C for at least 18 months (preferably 24 months) using incurred samples or spiked samples, and demonstrate that the change is within ±15%; or (2) If the study is retrospective, conduct a stability analysis using incurred samples stored for the same duration (e.g., re-assay a subset after additional storage) to bridge the gap. Until then, the data should be considered provisional.
2. The lab proposes to use the 12-month stability data and the fact that the 18-month samples gave results consistent with the expected PK profile as justification. Critically evaluate this proposal.
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Using consistency with expected PK profiles as justification is not scientifically valid because it is circular: the expected profile may be based on the same data or on assumptions. Consistency does not prove stability; degradation could be uniform across samples and still produce a plausible profile, especially if the degradation is proportional. Moreover, the 12-month stability data show a trend of degradation at low concentrations, making it likely that at 18 months the change could exceed 15%. The proposal lacks a quantitative basis. To justify reporting, the lab would need to demonstrate that the change at 18 months is within acceptance criteria. This could be done by: (1) Extending the stability study to 18 months using spiked samples and showing that the change is ≤15%; (2) Performing a stability comparison using incurred samples stored for 18 months and re-assaying them after additional storage or using a different method; (3) Applying a correction factor if degradation is characterized and consistent, but this is generally not acceptable for regulated studies. The lab should not report the data as definitive without addressing the stability gap. A conservative approach is to report the data with a caveat that the storage period exceeded validated stability, and to initiate a stability study to confirm. However, for regulatory submissions, such data may be rejected. The best course is to perform additional stability testing and, if necessary, re-analyze samples if sufficient volume remains. If not, the study may need to be repeated or the data flagged as exploratory.