Biological and physicochemical characterization of recombinant human erythropoietins fractionated by Mono Q column chromatography and their modification with sialyltransferase

Ten erythropoietin (EPO) fractions differing in sialic acid content, ranging from 9.5 to 13.8 mol mol(-1) of EPO, were obtained from baby hamster kidney cell-derived recombinant human EPO by Mono Q column chromatography. The mean pI values of the EPO fractions determined by IEF-gel electrophoresis systematically shifted from 4.11 to 3.31, coinciding with the sialic acid content, without a change in the constitution of asialo N-linked oligosaccharides of each fraction. Although a linear relationship between the in vivo bioactivity and the sialic acid content of the fractionated samples was observed until 12.1 mol mol(-1) of EPO, there was no further increase in their activity over 12.4 mol mol(-1) of EPO. On the other hand, an inverse relationship between the in vitro bioactivity and sialic acid content of EPO was observed. Also, we showed that the in vivo bioactivity of some fractions with low sialic acid contents was increased after treatment with alpha 2,6-sialyltransferase, but the in vivo bioactivity of the other fractions with high sialic acid contents was either decreased or not affected.