Determination of Purity of Antitoxin/Antiserum Products by High Performance Size Exclusion Chromatography

OBJECTIVE To establish a high performance size exclusion chromatography(HPSEC) method for the purity determination of antitoxin/antiserum products and to be applied to determine the purity of 52 samples. METHODS TOSOH TSKgelG3000SWxl column was used, the 0.2 mol·L^-1 phosphate buffer(pH 7.0) containing 1% isopropanol were used as mobile phase. The flow rate was 0.6 mL·min^-1, the detection wavelength was 280 nm, the injection volume was 20 μL, and the column was kept at room temperature. The relative percentage of each component was calculated by peak area normalization. RESULTS One batch of antitoxin and one batch of antiserum were selected, in the protein concentration ranges from 1.2-24 mg·mL^-1, the peak areas of F(ab')₂, high molecular weight impurities and small molecular weight impurities had good linear relationship with the corresponding protein concentration(n=5, r>0.999 0), repeatability RSD was 0.4%-0.9%(n=6). The limits of quantitation of F(ab')₂ were 0.1 μg and 0.2 μg, the limits of detection of F(ab')₂ were 0.02 μg and 0.1 μg, the sample solution was stable at room temperature within 12 h. CONCLUSION The HPSEC can be applied to determine the purity of antitoxin and antiserum products.