Short communication
Optimization and qualification of capillary zone electrophoresis method for glycoprotein isoform distribution of erythropoietin for quality control laboratory

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Abstract

The European Pharmacopoeia (Ph. Eur.) monograph for Erythropoietin Concentrated Solution describes a capillary zone electrophoresis method for identification of recombinant human erythropoietin. However, this method has shown poor reproducibility due to inadequate capillary conditioning. We have modified the Ph. Eur. method to make it more robust and suitable for the quality control laboratory for the analysis of epoetin alfa and epoetin alfa after formulation with polysorbate 80. This study qualified the modified method by showing improved robustness and reproducibility. The study also characterized and qualified a secondary standard of epoetin alfa as a substitute for the primary standard, Ph. Eur. erythropoietin Biological Reference Preparation, which is available in limited supply. Four sets of analyses were performed to assess repeatability, intermediate precision, and the secondary standard. The results showed that the modified method is suitable for its intended purpose to test epoetin alfa and formulated epoetin alfa samples. The epoetin alfa secondary standard is a suitable substitute for the primary standard. Further, we developed a procedure for the removal of polysorbate 80 from formulated epoetin alfa, allowing the material to be analyzed by the modified Ph. Eur. method.

Introduction

Erythropoietin (EPO) is a glycoprotein hormone that regulates erythropoiesis, or red blood cell production [1]. EPO is produced by the liver and kidney [2]. Capillary electrophoresis (CE) has been one of the widely used tools for the direct analysis of glycoform distribution of glycoprotein [3]. Several CE methods have been developed for the separation of EPO glycoforms [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. Watson and Yao developed the first capillary zone electrophoresis (CZE) method using a fused-silica capillary with weakly acidic electrolyte solution containing 1,4-diaminobutane (DAB) [5]. After the re-examination of this method by Kinoshita et al., the method was found to be troublesome due to significant loss on resolution after several injections, and the life of the capillary was limited [3]. Lopez-Soto-Yarritu et al. proposed a higher DAB concentration, and the migration time was controlled, but the zone broadening was significantly increased, and the peak areas varied due to protein adsorption onto the capillary wall [9]. European Pharmacopoeia (Ph. Eur.) has published a monograph describing the separation of glycoforms of EPO by CZE [4]. However, the Ph. Eur. method was prone to poor reproducibility [11]. Sanz-Nebot et al. optimized the Ph. Eur. method by adjusting the pH value of separation electrolyte and capillary length in order to achieve better resolution for the separation of erythropoiesis-stimulating protein. Although the intraday reproducibility was improved, the inter-day reproducibility was relatively high, and not all the isoforms were completely baseline resolved [12]. Benavente et al. made further improvements on the CZE method and proposed a multivariate calibration method using partial least-squares (PLS) in order to characterize binary mixtures of two types of recombinant human erythropoietin (epoetin alfa and beta) [14]. Although the reproducibility was improved, the %RSD of migration times for all the peaks still failed to meet the system suitability acceptance criterion of <2% required by Ph. Eur. Other CE techniques such as isoelectric focusing (cIEF) method have also been developed for EPO analysis [16] but it is not required by Ph. Eur. [4]. Therefore, it was not studied and compared in this work. After the European Department for the Quality of Medicine first published the specifications for the CZE method in the 2002 European Pharmacopoeia, the EPO manufacturers have been required to adopt it as a quality control (QC) method for product release.

The objective of this study was to modify the current Ph. Eur. method to make it suitable for routine QC testing of EPO drug substance. The root cause for poor reproducibility of the CZE method was studied and identified. The study also qualified a secondary standard as a substitute for primary standard, which is available in limited supply from Ph. Eur. In addition, the study evaluated a strategy for the removal of polysorbate 80 from EPO formulated with polysorbate 80. Formulations containing polysorbate 80 interferes with the analysis of isoforms of EPO in CE separation, and the desalting procedure using molecular mass cut-off filter [4] is unable to remove polysorbate 80.

Section snippets

Chemicals and reagents

All chemicals used in the preparation of buffers and solutions were of ACS reagent or electrophoresis grade. Acetic acid (HAc) (glacial), anhydrous sodium acetate (NaAc), 1,4-diaminobutane (DAB), sodium chloride (NaCl), sodium citrate dihydrate, citric acid, tris-(hydroxymethyl)aminomethane hydrochloride (Tris–HCl), tris-(hydroxymethyl)aminomethane (Tris-base), and N-tris-(hydroxymethyl)methylglycine (tricine) were supplied by Sigma–Aldrich (St. Louis, MO, USA). Sodium hydroxide solutions (1 M

Restoration of the fused-silica capillary surface

Fused silica possesses different surface silanol groups—isolated, vicinal, and geminal—that are responsible for the pH-dependent charge on the capillary surface [17]. At pH > 3, the silanol groups will ionize, resulting in a negative charge on the capillary wall. In solutions containing ions, cations will migrate to the negatively charged wall forming an electrical double layer (EDL) described by the Stern–Gouy–Chapman model [18]. When an electrical potential is applied to the capillary, the

Conclusions

The CZE method for Ph. Eur. monograph for erythropoietin concentrated solution has been modified. The modifications made to the Ph. Eur. method include the use of secondary standard in place of EPO BRP, the conditioning of the capillary, and the removal of polysorbate 80 for formulated epoetin alfa samples. Improved repeatability and intermediate precision were obtained. The method is suitable for use in quality control laboratories.

Junge Zhang Ph.D. candidate, Chemistry Department, Drexel University, Philadelphia, PA. M.S. University of Louisiana at Monroe, Monroe, LA. Senior Research Scientist, 2002—present. Analytical Development, BIO Pharmaceutical Development, Centocor Research and Development, Malvern, PA.

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    Junge Zhang Ph.D. candidate, Chemistry Department, Drexel University, Philadelphia, PA. M.S. University of Louisiana at Monroe, Monroe, LA. Senior Research Scientist, 2002—present. Analytical Development, BIO Pharmaceutical Development, Centocor Research and Development, Malvern, PA.

    Utpal Chakraborty Ph.D. Associate Director, Analytical Development, BIO Pharmaceutical Development, Centocor Research and Development, Malvern, PA.

    Annabelle P. Villalobos Associate Director, Analytical Development, BIO Pharmaceutical Development, Centocor Research and Development, Radnor, PA.

    John M. Brown Ph.D. Director, BIO Pharmaceutical Development, Centocor Research and Development, Raritan, NJ.

    Joe P. Foley Professor, Drexel University. B.S., Centre College of Kentucky,1978. Ph.D., University of Florida, 1983. National Research Council Postdoctoral Fellow, National Institute of Standards and Technology, 1983–85. Assistant Professor of Chemistry, Louisiana State University, 1985–91. Associate Professor of Chemistry, Louisiana State University, 1991. Associate Professor of Chemistry, Villanova University, 1991–97. Professor of Chemistry, Villanova University,1997–98. Professor of Chemistry, Drexel University, 1998—present. Visiting Professor of Chemistry, University of Frankfurt (Germany), 2006–07. Editorial boards: Bioanalysis, June 2008—present. Electrophoresis, October 2007—present. The Analyst, 1991–2000. Journal of Microcolumn Separations, 1991–2001. Analytical Communications, 1996–1999.

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