Rapid high-performance affinity chromatography on micropellicular sorbents.

Abstract

Short columns (30 x 4.6 mm I.D.), packed with 2-micron 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 micrograms 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 mM for elution, the subclasses of human IgG were separated at 40 degrees 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.