Capillary Zone Electrophoresis Method for a Highly Glycosylated and Sialylated Recombinant Protein: Development, Characterization and Application for Process Development

Abstract

A purity method based on capillary zone electrophoresis (CZE) has been developed for the separation of isoforms of a highly glycosylated protein. The separation was found to be driven by the number of sialic acids attached to each isoform. The method has been characterized using orthogonal assays and shown to have excellent specificity, precision and accuracy. We have demonstrated the CZE method is a useful in-process assay to support cell culture and purification development of this glycoprotein. Compared to isoelectric focusing (IEF), the CZE method provides more quantitative results and higher sample throughput with excellent accuracy, qualities that are required for process development. In addition, the CZE method has been applied in the stability testing of purified glycoprotein samples.

Rapid fingerprinting of a highly glycosylated fusion protein by microfluidic chip‐based capillary electrophoresis–mass spectrometry

“…A closer look at molecular masses of peak four and six revealed a loss of sialic acid in the stressed sample, consistent with shorter migration times. This observation is in agreement with previous studies using CZE for characterization of highly sialylated proteins [19]. Low level of acetylation was observed in both control and stressed samples, as shown in Supporting Information Fig.…”

Capillary Electrophoresis Separations of Glycans

“…This was demonstrated for a biological glycoprotein manufactured by Amgen. 124 Proteins were isolated using an isoelectric focusing separation prior to CE with UV–visible absorbance detection. Using an acidic background electrolyte, capillary coated with poly(vinyl alcohol), and reversed polarity, the product was separated into 9 peaks of different sialic acid composition.…”

Recent advances in protein analysis by capillary and microchip electrophoresis

“…158 The method has been shown to have excellent specificity, precision, and accuracy. The peaks separated by CZE were characterized using orthogonal methods, and peak identification was determined by a combination of MALDI-MS and high performance anion exchange chromatography.…”