Reusch Dietmar, Haberger Markus, Maier Bernd, Maier Maria, Kloseck Ronny, Zimmermann Boris, Hook Michaela, Szabo Zoltan, Tep Samnang, Wegstein Jo, Alt Nadja, Bulau Patrick, Wuhrer Manfred
a Pharma Biotech Development Penzberg; Roche Diagnostics GmbH ; Penzberg , Germany.
MAbs. 2015;7(1):167-79. doi: 10.4161/19420862.2014.986000.
Immunoglobulin G (IgG) crystallizable fragment (Fc) glycosylation is crucial for antibody effector functions, such as antibody-dependent cell-mediated cytotoxicity, and for their pharmacokinetic and pharmacodynamics behavior. To monitor the Fc-glycosylation in bioprocess development, as well as product characterization and release analytics, reliable techniques for glycosylation analysis are needed. A wide range of analytical methods has found its way into these applications. In this study, a comprehensive comparison was performed of separation-based methods for Fc-glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods were compared for precision, accuracy, throughput and other features; special emphasis was placed on the detection of sialic acid-containing glycans. Seven, non-mass spectrometric methods were compared; the methods utilized liquid chromatography-based separation of fluorescent-labeled glycans, capillary electrophoresis-based separation of fluorescent-labeled glycans, or high-performance anion exchange chromatography with pulsed amperometric detection. Hydrophilic interaction liquid chromatography-ultra high performance liquid chromatography of 2-aminobenzamide (2-AB)-labeled glycans was used as a reference method. All of the methods showed excellent precision and accuracy; some differences were observed, particularly with regard to the detection and quantitation of minor glycan species, such as sialylated glycans.
免疫球蛋白G（IgG）可结晶片段（Fc）糖基化对于抗体效应功能（如抗体依赖性细胞介导的细胞毒性）及其药代动力学和药效学行为至关重要。为了在生物工艺开发以及产品表征和放行分析中监测Fc糖基化，需要可靠的糖基化分析技术。多种分析方法已应用于这些领域。在本研究中，对用于IgG生物制药Fc糖基化谱分析的基于分离的方法进行了全面比较。在2个不同日期对一种治疗性抗体参考物质进行了6次分析，并比较了这些方法的精密度、准确性、通量和其他特性；特别强调了含唾液酸聚糖的检测。比较了七种非质谱方法；这些方法利用基于液相色谱的荧光标记聚糖分离、基于毛细管电泳的荧光标记聚糖分离或带有脉冲安培检测的高效阴离子交换色谱法。以2-氨基苯甲酰胺（2-AB）标记聚糖的亲水相互作用液相色谱-超高效液相色谱法作为参考方法。所有方法均显示出优异的精密度和准确性；观察到了一些差异，特别是在次要聚糖种类（如唾液酸化聚糖）的检测和定量方面。
