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.