Preparative HPLC of Soybean Trypsin Inhibitor Using Large Particle Diameter Supports

Abstract

Two systems were developed to purify Soybean Trypsin Inhibitor (STI) using anion exchange chromatography. Both systems demonstrated that large diameter particles (30 and 55 μm) could be used effectively for protein purification. Preparative samples of 455 mg and 7.2 g were processed to yield highly purified products. Preparative purifications were achieved either on an analytical instrument with an analytical (0.46 × 30 cm) column or on a large scale system with a preparative (4.8 × 50 cm) column. Sample introduction by frontal loading appeared to lead to some protein-protein displacement, thus allowing for some pre-gradient fractionation and enrichment of the more strongly retained STI on the column. This is referred to as an “overfed” system. Throughput is discussed and used to evaluate the different systems. An apparent lack of resolution in preparative scale chromatograms did not necessarily indicate an absence of fractionation. It was possible to isolate protein products of high purity with high throughput values using larger diameter supports.