Quantitative determination of the beta-2-microglobulin monomer by gel filtration chromatography

Abstract

Beta-2-microglobulin (B2M) is an important material in dialysis-related amyloidosis research. Unfortunately, the quantitative detection of the B2M monomer is difficult during B2M production. In this study, we established a method for the detection of the B2M monomer and its dimer in solution via gel filtration chromatography. The B2M powder was dissolved in 0.01 mol/L phosphate-buffered solution (PBS, pH 7.2-7.4) with a final mass concentration of 0.5 g/L. The B2M sample was analyzed on a TSKgel SuperSW2000 column (30 cm×4.6 mm, 4 μm) by an Agilent 1200 Series HPLC using 0.01 mol/L PBS (pH 7.2-7.4) as the mobile phase at a flow rate of 0.5 mL/min. The temperature of the column was 25℃, and the detection wavelength was 280 nm. The purities of the B2M monomer and the B2M dimer were tested. A series of concentrations of B2M monomer solutions were prepared to create a standard working curve. The standard working curve of the B2M monomer had a good linear relationship (coefficient of correlation (r2) was 0.9948). The limit of quantitation for the B2M monomer in PBS was 0.08 g/L (S/N=10). The recoveries were 85.0%-96.7% with relative standard deviations of 1.7%-3.3% at spiked levels of 0.10-0.30 g/L. The quantification of the B2M monomer was undisturbed by the B2M dimer, which can form during the B2M purification process. This determination method is simple, stable, and reliable for the determination of the B2M monomer in B2M industrial production.