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Identification of Methionine Oxidation in Human Recombinant Erythropoietin by Mass Spectrometry: Comparative Isoform Distribution and Biological Activity Analysis Publisher Pubmed



Hedayati MH1 ; Norouzian D2 ; Aminian M3 ; Teimourian S4 ; Ahangari Cohan R2 ; Khorramizadeh MR5
Authors
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Authors Affiliations
  1. 1. Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
  2. 2. Department of Pilot Nanobiotechnology, Pasteur Institute of Iran, Tehran, Iran
  3. 3. Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
  4. 4. Department of Medical Genetics, Iran University of Medical sciences, Tehran, Iran
  5. 5. Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, University of Medical Sciences, Tehran, Iran

Source: Preparative Biochemistry and Biotechnology Published:2017


Abstract

Background: Oxidative degradation of human recombinant erythropoietin (hrEPO) may occur in manufacturing process or therapeutic applications. This unfavorable alteration may render EPO inefficient or inactive. We investigated the effect of methionine/54 oxidative changes on the amino acid sequences, glycoform distribution and biological activity of hrEPO. Methods: Mass spectrometry was applied to verify the sequence and determine the methionine oxidation level of hrEPO. Isoform distribution was studied by capillary zone electrophoresis method. In vivo normocythemic mice assay was used to assess the biological activity of three different batches (A, B, and C) of the proteins. Results: Nano-LC/ESI/MS/MS data analyses confirmed the amino acid sequences of all samples. The calculated area percent of three isoforms (2–4 of the 8 obtained isoforms) were decreased in samples of C, B, and A with 27.3, 16.7, and 6.8% of oxidation, respectively. Specific activities were estimated as 53671.54, 95826.47, and 112994.93 mg/mL for the samples of A, B, and C, respectively. Conclusion: The observed decrease in hrEPO biological activity, caused by increasing methionine oxidation levels, was rather independent of its amino acid structure and mainly associated with the higher contents of acidic isoforms. © 2017 Taylor & Francis.