A Novel Approach for Quantitation of Nonderivatized Sialic Acid in Protein Therapeutics Using Hydrophilic Interaction Chromatographic Separation and Nano Quantity Analyte Detection

• Published in: Journal of pharmaceutical sciences Vol. 104; no. 1; pp. 15 - 24
• Main Authors: Chemmalil, Letha, Suravajjala, Sreekanth, See, Kate, Jordan, Eric, Furtado, Marsha, Sun, Chong, Hosselet, Stephen
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2015; Elsevier Limited
• Subjects: 2-diamino-4; 5-methylenedioxybenzene; Aerosols; Algorithms; Analytic Sample Preparation Methods; Chemistry, Pharmaceutical - methods; chromatography; Chromatography, High Pressure Liquid; Drug Contamination - prevention & control; glycoprotein; Glycoproteins - chemistry; HPLC; Hydrophobic and Hydrophilic Interactions; LC-MS; Limit of Detection; Models, Molecular; N-acetylneuraminic acid; N-Acetylneuraminic Acid - analysis; N-glycolylneuraninic acid; nano quantity analyte detector; Nanotechnology; Nephelometry and Turbidimetry; Particle Size; PolyGLYCOPLEX amide column 1; Recombinant Proteins - chemistry; Refractive index; Refractometry; Reproducibility of Results; LC-MS; nano quantity analyte detector; N-glycolylneuraninic acid; Refractive index; 2-diamino-4; chromatography; glycoprotein; 5-methylenedioxybenzene; PolyGLYCOPLEX amide column 1; HPLC; N-acetylneuraminic acid; PolyGLYCOPLEX amide column; 1
• ISSN: 0022-3549; 1520-6017
• DOI: 10.1002/jps.24093

This paper describes a novel approach for the quantitation of nonderivatized sialic acid in glycoproteins, separated by hydrophilic interaction chromatography, and detection by Nano Quantity Analyte Detector (NQAD). The detection technique of NQAD is based on measuring change in the size of dry aerosol and converting the particle count rate into chromatographic output signal. NQAD detector is suitable for the detection of sialic acid, which lacks sufficiently active chromophore or fluorophore. The water condensation particle counting technology allows the analyte to be enlarged using water vapor to provide highest sensitivity. Derivatization-free analysis of glycoproteins using HPLC/NQAD method with PolyGLYCOPLEX™ amide column is well correlated with HPLC method with precolumn derivatization using 1, 2-diamino-4, 5-methylenedioxybenzene (DMB) as well as the Dionex-based high-pH anion-exchange chromatography (or ion chromatography) with pulsed amperometric detection (HPAEC-PAD). With the elimination of derivatization step, HPLC/NQAD method is more efficient than HPLC/DMB method. HPLC/NQAD method is more reproducible than HPAEC-PAD method as HPAEC-PAD method suffers high variability because of electrode fouling during analysis. Overall, HPLC/NQAD method offers broad linear dynamic range as well as excellent precision, accuracy, repeatability, reliability, and ease of use, with acceptable comparability to the commonly used HPAEC-PAD and HPLC/DMB methods. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:15–24, 2015