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Área de trabajo: Biotecnología, Farmacéutica.

Título: Separación de Isoformas de Sialilación de Anticuerpos Monoclonales Intactos por Cromatografía de Intercambio Iónico por gradiente de pH.

Título original: Separation of Intact Monoclonal Antibody Sialylation Isoforms by pH Gradient Ion-Exchange Chromatography.

Autor: Dai Zhenyu,1 Xu Qun,1 Lina Liang,1 and Jeffrey Rohrer2 1Thermo Fisher Scientific, Shanghai, People’s Republic of China; 2Thermo Fisher Scientific, Sunnyvale, CA, USA.

Introduction

Glycosylated proteins—including erythropoietins, monoclonal antibodies (MAbs), and various hormones— constitute a large portion of major approved therapeutic biological drugs. Sialic acid, usually attached at terminal positions of glycan molecules, is important to many biological processes such as cell recognition and migration. Sialic acids also have significant effects on the properties of therapeutic proteins, especially on the circulation half-life of the protein. For example, the circulation half-life of sialylated rhEPO is 5.6 h, whereas that of nonsialylated rhEPO is 1.4 min.1 Thus, monitoring protein glycosylation, including sialylation, is important for both glycoprotein characterization and quality control purposes.

Common approaches for monitoring glycosylated proteins include separating intact protein glycoforms and profiling oligosaccharides. Oligosaccharide profiling (both fluorescently labeled or unlabeled) requires releasing the glycan from the protein, whereas intact protein glycoforms monitoring is convenient and straightforward, and isolated protein glycoforms can be subjected to further characterization. A high-performance liquid chromatography (HPLC) method with higher resolution to separate protein glycoforms with different states of sialylation is needed. Because of the extra negative charge provided by sialic acid, anion-exchange chromatography is the ideal technique for resolving differently sialylated proteins. A recent report shows that pH-gradient-based ion-exchange chromatography (IEC) provides better resolution than routine salt-gradient-based IEC, especially for separation of protein isoforms.2

 

Equipment

• UltiMate 3000 Dual BioRS LC system, including:

– DGP-3600BM Biocompatible Dual-Gradient Micro Pump (P/N 5042-0066)

– WPS-3000TBFC Thermostatted Biocompatible Pulled-Loop Well Plate Autosampler with Integrated Fraction Collection (P/N 5825-0020)

– TCC-3000SD Thermostatted Column Compartment (P/N 5730-0010)

– DAD-3000 Diode Array Detector (P/N 5730-0010) with 13 μL flow cell • Thermo Scientific™ Dionex™ Chromeleon™ Chromatography Data System (CDS) software, version 7.2

 

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