Rapid high-performance affinity chromatography on micropellicular sorbents

Abstract

Short columns (30 × 4.6 mm I.D.), packed with 2-μm fluid-impervious silica microspheres with surface-bound Protein A or a lectin were used for fast separation and quantitation of immunoglobulins and glycoproteins by biospecific interaction chromatography. With stepwise elution, the total analysis time including column reequilibration did not exceed 3 min. In the assay of IgG with a stepwise change in pH best results were obtained with citrate buffer, which facilitated not only fast but also very sensitive analysis. The calibration curve was linear in the range 0.5–40 μg of human IgG. By using morpholinoethanesulfonic acid—4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-acetic acid buffer with a linear decrease in pH from 6.0 to 4.0 and an increase in magnesium chloride concentration to 200 m M for elution, the subclasses of human IgG were separated at 40°C above pH 4.0 in 3 min. Micropellicular concanavalin A and wheat germ agglutinin were used for rapid affinity chromatography of horseradish peroxidase and fetuin, respectively. The results suggest that micropellicular affinity sorbents afford fast and sensitive high-performance liquid chromatographic analysis by biospecific interaction chromatography. Although developed primarily for rapid analysis, the micropellicular Protein A exhibited unexpectedly high adsorption capacity ( e.g., 4.5 mg human IgG per ml of wet bed volume). This suggests that such columns could be employed in preparative protein chromatography as well.