A sialylation-sensitive cell-based in vitro bioassay for erythropoietin; incorporation of the galactose-binding Erythrina crista-galli lectin.

The in vivo biological activity of erythropoietin (Epo) is dependent on its being adequately sialylated. Current in vitro bioassays for Epo do not correlate with the in vivo bioassays as the former do not take into account the role the liver plays in clearing desialylated glycoproteins from the circulation. Here we describe a sialylation-sensitive cell-based Epo bioassay. In the first instance, Epo activity in vitro was measured using proliferation of AS-E2 cells, and in vivo using the polycythaemic mouse bioassay. Activity in vivo was progressively abolished by controlled desialylation, whereas activity in vitro was essentially unaffected. Incorporation of an incubation step with a solid-phase galactose-binding lectin (Erythrina crista-galli), effectively mimicking passage through the liver in vivo, renders the in vitro bioassay sensitive to desialylation, such that Epo desialylated almost to completion had <10% of the activity of untreated Epo. These studies offer proof of principle, that rational manipulation of in vitro bioassays can allow prediction of activity in vivo without the use of live animals.

Relationships between the N-glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures.

Eight preparations of recombinant human erythropoietin (EPO) with differing isoform compositions were produced by using different culture conditions and purification procedures. The N-glycan structures of these EPOs were analysed using a recently developed profiling procedure and identified using matrix-assisted laser desorption ionization mass spectrometry. The specific activities of each of the EPOs were estimated by in vivo and in vitro mouse bioassays. The eight EPOs were found to differ in their isoform compositions (as judged by isoelectric focusing), their N-glycan profiles, and in their in vivo and in vitro bioactivities. N-glycan analyses identified at least 23 different structures among these EPOs, including bi-, tri- and tetra-antennary N-glycans, with or without fucosylation or N-acetyllactosamine extensions, and sialylated to varying degrees. Mass spectrometry also indicated the presence of N-glycans with incomplete outer chains, terminating in N-acetylglucosamine residues, and of molecular masses consistent with phosphorylated or sulphated oligomannoside structures. The tetrasialylated tetra-antennary N-glycan contents of the eight rEPOs were found to be significantly and positively correlated with their specific activities as estimated by mouse in vivo bioassay, and significantly and negatively correlated with their specific activities as estimated by mouse in vitro bioassay. It was concluded that the tetrasialylated tetra-antennary N-glycan content of EPO is a major determinant for its in vivo biological activity in the mouse.