Background/Purpose:

Protein biomarkers are widely used in clinical studies to assess drug efficacy, such as bone and cartilage turnover markers in osteoarthritis or rheumatoid arthritis clinical studies. In most of these clinical studies it is highly recommended to analyze as many visits, including all follow-up visits, at the same time, often requiring sample storage below -70°C for an extended period of time. Thus, determination of long-term stability of the biomarkers upon storage is critical.

Typically, long-term stability is measured in samples with a baseline assessment before storage in order to determine the nominal value. Subsequently, samples are assessed in the same condition at frequent intervals, covering different storage periods. However, this method requires numerous samples with a large volume, which is often challenging to obtain. Thus, we suggest an alternative approach by monitoring in-house quality control (QCs) as indication of analyte stability.

Methods:

We investigated the long-term stability of the most widely analyzed serum biomarkers in musculoskeletal related clinical trials: carboxyterminal of type 1 collagen (S-CTX-I), osteocalcin (S-OC), aminoterminal propeptide of type 1 collagen (S-PINP), and metalloproteinases 3 and 9 (S-Total-MMP-3 and S-MMP-9). At least 12 serum samples were assayed at baseline and re-assayed after a storage period. Subsequent results were compared to baseline.

Additionally, we monitored the performance of our in-house QC samples for the same biomarkers and compared the data with the stability data previously determined. Urinary carboxyterminal telopeptide of type 2 collagen (U-CTX-II) long-term stability was assessed in samples after QC monitoring to validate our approach. QC monitoring was also performed for serum cartilage oligomeric matrix protein (S-COMP), another cartilage turnover marker.

An analyte is considered stable when the coefficient of variation (CV) and the accuracy (variation from target value) of the QC results are inferior to ± 15%.

Results:

Conclusion:

Assay analysis of protein biomarkers for clinical trials require a very stringent and robust quality process, which is based on the use of independent, endogenous QC samples throughout the complete duration of the clinical trial. We proved that long-term stability for biomarkers can be assessed by monitoring in-house QCs as confirmed by the U-CTX-II results.

Thus, based on the data obtained during QC monitoring for S-COMP we suggest that this marker is stable up to 19 months below -70°C. However, these data should be confirmed by a standard validation stability test.