Isoforms analysis of recombinant human erythropoietin by polarity‐reversed capillary isoelectric focusing

Recombinant human erythropoietin (rhuEPO) has been extensively used as a pharmaceutical product for treating anemia in the clinic. Glycosylation of rhuEPO was crucial for affecting biological activity, immunogenicity, and pharmacokinetics. Because of the heterogeneity of glycan, the structure of rhu...

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Bibliographic Details
Published in:Electrophoresis 2020-12, Vol.41 (23), p.2055-2061
Main Authors: Ren, Ting‐jun, Zhang, Xiao‐xia, Li, Xiang, Chen, Hong‐xu
Format: Article
Language:English
Subjects:
Ammonium hydroxide
Anemia
Anolytes
Biological activity
Capillary isoelectric focusing
Electrophoresis, Capillary - methods
Erythropoietin
Erythropoietin - analysis
Erythropoietin - chemistry
Glycan
Glycoprotein
Heterogeneity
Humans
ICH guideline
Isoelectric focusing
Isoelectric Focusing - methods
Isoform
Linear Models
Pharmacology
Protein Isoforms
Quality control
Recombinant Proteins - analysis
Recombinant Proteins - chemistry
Reproducibility of Results
Sensitivity and Specificity
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Summary:Recombinant human erythropoietin (rhuEPO) has been extensively used as a pharmaceutical product for treating anemia in the clinic. Glycosylation of rhuEPO was crucial for affecting biological activity, immunogenicity, and pharmacokinetics. Because of the heterogeneity of glycan, the structure of rhuEPO was complex with several isoforms. Characterization of isoforms was important for quality control of rhuEPO. Here, an improved cIEF method has been established and validated. A polarity‐reversed focusing step was used by reversing both the polarity of the voltage and the catholyte and anolyte vials. A weak base (100 mM ammonium hydroxide solution) was used as a chemical mobilizer to make the acidic bands mobilize stably to the detection window. Compared with CZE method in European Pharmacopoeia, the numbers of isoforms and their peak area percentage were highly consistent. Better reproducibility and higher resolution have been obtained by the improved cIEF method. Moreover, in improved cIEF method, the isoelectric points (pI) of each isoform can be calculated and used for identification. It was also the first time that the cIEF method was fully validated for rhuEPO analysis according to the International Conference on Harmonization (ICH) guidelines.
ISSN:0173-0835
1522-2683
DOI:10.1002/elps.202000165