PREDICTING THE PERFORMANCE OF GEL-FILTRATION CHROMATOGRAPHY OF PROTEINS

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 dc = 0.75-10.8 cm, column length Z = 10-80 cm, particle diameter dp = 10-17 μm) was chosen as a model separation system. In the calculation method, the maximum sample feed volume VF,M that satisfies a specified purity ratio QP and revocery ratio QR for a given column length Z and particle diameter dP 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 dP 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 dP 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. © 1990.