Characterization of Erythropoietin Dimerization

□ Recombinant human erythropoietin (rHuEPO) is a monomeric glycoprotein hormone when stored under refrigeration. Higher temperatures and various formulation conditions have shown the monomer to partially dimerize followed by aggregation to higher molecular weight species. To protect the product against aggregation, it is critical to understand this dimerization mechanism. The formation of the dimer was analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry, endoproteinase LysC mapping, and N-termlnal sequencing. The dimer was shown to have an average molecular mass of 53.5 kDa, vs 27.8 kDa for the monomer. The dimer to monomer ratio of 1.9 instead of 2 seemed to be caused by some loss of sialic acids during the purification. The LysC map of the dimer also showed two new peptide peaks not present In the monomer. The sequencing of the new peptides revealed the presence of two types of EPO dimers. The data indicate the dimerization mechanism to involve an initial reduction of the Cys7-Cys161 disulfide bond and subsequent random reoxidation of the free thiols across two EPO molecules. The absence of free thiols in the dimer was confirmed by a fluorescent thiol probe. The higher molecular weight aggregation followed from random intermolecular reoxidation of Cys7 and Cys161.