Predicting the performance of gel-filtration chromatography of proteins

Abstract

A method for determining the column and operating variables for the preparative separation of proteins by gel-filtration chromatography (GFC) of proteins is presented. The revocery of bovine serum albumin monomer from its dimer and higher molecular weight aggregates on high-performance GFC columns of various dimensions (column diameter d c = 0.75–10.8 cm, column length Z = 10–80 cm, particle diameter d p = 10–17 μm) was chosen as a model separation system. In the calculation method, the maximum sample feed volume V F,M that satisfies a specified purity ratio Q P and revocery ratio Q R for a given column length Z and particle diameter d P at a certain linear mobile phase velocity u was sought. The productivity P, defined as the amount of the recovered protein per unit column volume per unit time, was then calculated. The effects of Z and d P on the P— u relationship were examined. It was found that in general P increases with increasing u and the slope of the P— u curve becomes steep with decreasing d P and/or Z. A maximum in the P— u relationship was observed when the separation was difficult. The results show that it is not advantageous to employ larger particle diameter packings and/or a longe column in scaling-up. It is rather recommended that a short column packed with small gels be operated at relatively low flow-rates in the preparative GFC of proteins. It is also suggested that the calculation method presented is useful for scaling up GFC columns.