(M1530-01-04) Resolving Adsorption in Urine Based Kidney Biomarker Assays

Purpose: This study aimed to address the challenges of developing bioanalytical methods for detecting six kidney injury biomarkers in human urine, in alignment with FDA guidance. These biomarkers — Clusterin (CLU), Cystatin-C (Cys-C), Kidney Injury Molecule-1 (KIM-1), N-acetyl-β-D-glucosaminidase (NAG), Neutrophil Gelatinase-Associated Lipocalin (NGAL), and Osteopontin (OPN) — are recommended for use when renal tubular injury is a concern, with levels measured pre- and post-dose.

Methods: According to the Qualification Decision and Executive Summary issued by the FDA, the recommended pre-analytical process includes immediate centrifugation of urine samples at 2,000g for 10 minutes at room temperature, aliquoting into cryotubes, and freezing at -70°C, with shipment on dry ice. Desalting was reported as necessary for CLU, Cys-C, and KIM-1. Most biomarkers exhibited poor short-term stability at room temperature and low freeze-thaw stability. Addition of protease inhibitors was suggested to improve stability for Cys-C and KIM-1. During method development, the need for sample desalting was investigated, sample stability was assessed, and adsorption-related losses were addressed under various storage conditions, with different additives tested.

Results: Contrary to earlier reports, desalting was not required for any of the six biomarkers, as comparable concentrations were observed in both desalted and non-desalted urine samples. While protease inhibitors were reported to improve stability in previous studies, no quantitation issues were observed that would justify their inclusion, and thus no further testing with protease inhibitors was conducted. Apparent instability was instead attributed to adsorption, especially for CLU, Cys-C, KIM-1, NAG, and OPN. Various storage containers (polypropylene, silanized glass, and LoBind tubes) were tested and additives (Tween-20 and BSA) were evaluated. Adsorption was effectively prevented by treating urine samples with 0.2% Tween-20 in standard polypropylene tubes, with no adverse impact on assay performance (Table 1). Using this approach, all six biomarker methods were validated and short-term stability (6 hours at room temperature; 3 hours for OPN), consistent performance over three freeze-thaw cycles (2 cycles for OPN), and long-term stability for up to seven months at -70°C were established (Table 2).

Conclusion: Based on the data from method development, neither desalting nor the use of protease inhibitors were necessary for reliable quantification of the six kidney injury biomarkers in urine. Sample instability observed in previous reports could be attributed to adsorption, which was effectively resolved through the use of 0.2% Tween-20 in standard polypropylene tubes (see Table 1). These findings provide a simplified and robust workflow for evaluating renal safety biomarkers.


Table 1. Adsorption Summary for Kidney Biomarkers


Table 2. Stability Summary for Kidney Biomarkers (% of Theoretical Concentration)