PMID: 8587043Nov 1, 1995Paper

Characterization of erythropoietin dimerization

Journal of Pharmaceutical Sciences
A M DePaolisB G Sharma

Abstract

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-terminal 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 follo...Continue Reading

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Citations

Jul 20, 2005·Pharmaceutical Research·Annabelle P VillalobosGeorge A Heavner
Nov 5, 1998·Journal of Controlled Release : Official Journal of the Controlled Release Society·M MorlockG Winter
Mar 4, 1999·Journal of Controlled Release : Official Journal of the Controlled Release Society·H Sah
Jul 8, 1998·European Journal of Pharmaceutics and Biopharmaceutics : Official Journal of Arbeitsgemeinschaft Für Pharmazeutische Verfahrenstechnik E.V·B BittnerT Kissel
Jul 25, 2006·Protein Expression and Purification·Kornelia SchrieblKarola Vorauer-Uhl
Jun 16, 2009·Journal of Liposome Research·Stefanie StrobachKarola Vorauer-Uhl
Mar 21, 2018·BioDrugs : Clinical Immunotherapeutics, Biopharmaceuticals and Gene Therapy·Yeong Ran JeongGyun Min Lee

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