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

Abstract

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.

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications—Updated and completely revised edition

“…CIEF and iCIEF have matured into techniques that are routinely applied in analytical laboratories, especially in the biopharmaceutical industry. Exemplary applications include, but are not limited to, monoclonal antibodies [192,[209][210][211][212][213][214][215], bispecific antibodies [216][217][218], ADCs [219,220], PEGylated proteins [221,222], glycoproteins, in particular erythropoietin [213,223,224], and fusion proteins [213,225]. Because this subsection is part of the former "proteins" section, which was extended to all biopharmaceuticals, it should not go unmentioned that the range of applications has been expanded beyond proteins and includes oligosaccharides [226], LNPs and related mRNA vaccines [227][228][229][230], virus-like particles [231,232], poliovirus [230,233], adeno-associated virus [230,[234][235][236][237], papillomavirus [238], and bacteria and yeast cells [230].…”

“…For example, Kristl and Stutz found 50 mM glutamic acid pH 10.5 best suited for the mobilization of ovalbumin [284]. Ren et al used 100 mM ammonium hydroxide as anolyte replacement for the mobilization of the acidic erythropoietin and under CIEF conditions with reversed polarity to mirror the pH gradient [224]. Mobilization through the addition of salt to the catholyte fell out of favor and is not used anymore.…”

“…Urea is still one of the most used additives to prevent precipitation [192, 209–211, 216, 219, 223, 224, 267, 268]. However, several other substances have proven their usefulness.…”

Applications of capillary electrophoresis for biopharmaceutical product characterization

“…A CZE based method for separation of deamidation products, and a CGE method for molecular weight transformation products were developed. Other hormones such as human chorionic gonadotropin, parathyroid hormone, and recombinant human erythropoietin were also characterized for their quality attributes like pI, isoforms, stability, plasma proteolysis, and potency using CE-based methods [115][116][117][118][119][120][121][122][123][124][125][126][127].…”

Immunoaffinity Capillary Electrophoresis in the Era of Proteoforms, Liquid Biopsy and Preventive Medicine: A Potential Impact in the Diagnosis and Monitoring of Disease Progression

“…Proteomics should ideally be the driver for improving diagnostics and will serve as a primary method for detection of molecular changes indicative of disease states. Numerous laboratories have reported a plethora of assays using capillary electrophoresis in conventional format and in microfluidic format for detection and quantification of several proteoforms [254][255][256][257][258][259][260][261][262][263][264][265][266][267]. However, in order for proteoforms to reach their full clinical potential, it is necessary to be able to accurately detect and quantify near all proteoforms in biological fluids using advanced and sophisticated analytical instrumentation, such as IACE.…”