Low-Flow Sheathless Capillary Electrophoresis鈥揗ass Spectrometry for Sensitive Glycoform Profiling of Intact Pharmaceutical Proteins

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• 作者：Rob Haselberg ; Gerhardus J. de Jong ; Govert W. Somsen
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：February 19, 2013
• 年：2013
• 卷：85
• 期：4
• 页码：2289-2296
• 全文大小：457K
• 年卷期：v.85,no.4(February 19, 2013)
• ISSN：1520-6882

文摘

Capillary electrophoresis coupled to time-of-flight mass spectrometry (CE鈥揟OF-MS) via a porous tip sheathless electrospray ionization (ESI) interface was studied for the characterization of pharmaceutical glycoproteins. To achieve optimal glycoform separation, background electrolytes of low pH were used in conjunction with a capillary with a neutral coating exhibiting near-zero electroosmotic flow. Crucial interfacing parameters, like ESI voltage and ESI tip-to-end plate distance, were optimized for very low flow rates (5 nL/min) in order to attain maximum sensitivity and stable performance. Under optimal conditions, the sheathless CE鈥揗S interface provided significantly increased ionization efficiencies for intact proteins and decreased ionization suppression leading to detection limits in the picomolar-range. Analysis of a sample of recombinant human interferon-尾 allowed the assignment of at least 18 glycoforms, plus a variety of deamidation, succinimide, and oxidation products, representing a considerable improvement over sheath-liquid CE鈥揗S. The sheathless CE鈥揗S system also proved highly suitable for the glycoprofiling of recombinant human erythropoietin, revealing 74 glycoforms in a 60-min run. In addition, oxidation and acetylation products were detected, overall resulting in assignment of more than 250 different isoforms. Semiquantitative glycoprofiles could be derived for both pharmaceutical proteins, with estimated glycoform concentrations analyzed ranging from 0.35 to 950 nM. These profiles may be very useful for quality control of biopharmaceuticals and their biosimilars.