Department of Analytical Chemistry, Stockholm University, 10691, Stockholm, Sweden.
J Am Soc Mass Spectrom. 2013 Jul;24(7):1053-63. doi: 10.1007/s13361-013-0623-z. Epub 2013 Apr 30.
The serum clearance rate of therapeutic antibodies is important as it affects the clinical efficacy, required dose, and dose frequency. The glycosylation of antibodies has in some studies been shown to have an impact on the elimination rates in vivo. Monitoring changes to the glycan profiles in pharmacokinetics studies can reveal whether the clearance rates of the therapeutic antibodies depend on the different glycoforms, thereby providing useful information for improvement of the drugs. In this paper, a novel method for glycosylation analysis of therapeutic antibodies in serum samples is presented. A microfluidic compact-disc (CD) platform in combination with MALDI-MS was used to monitor changes to the glycosylation profiles of samples incubated in vitro. Antibodies were selectively purified from serum using immunoaffinity capture on immobilized target antigens. The glycans were enzymatically released, purified, and finally analyzed by MALDI-TOF-MS. To simulate changes to glycan profiles after administration in vivo, a therapeutic antibody was incubated in serum with the enzyme α1-2,3 mannosidase to artificially reduce the amount of the high mannose glycoforms. Glycan profiles were monitored at specific intervals during the incubation. The relative abundance of the high mannose 5 glycoform was clearly found to decrease and, simultaneously, that of high mannose 4 increased over the incubation period. The method can be performed in a rapid, parallel, and automated fashion for glycosylation profiling consuming low amounts of samples and reagents. This can contribute to less labor work and reduced cost of the studies of therapeutic antibodies glycosylation in vitro and in vivo.
治疗性抗体的血清清除率很重要，因为它会影响临床疗效、所需剂量和给药频率。在一些研究中，抗体的糖基化被证明会影响体内的消除率。在药代动力学研究中监测聚糖谱的变化，可以揭示治疗性抗体的清除率是否取决于不同的糖型，从而为药物的改进提供有用的信息。本文提出了一种用于血清样本中治疗性抗体糖基化分析的新方法。采用 MALDI-MS 联用的微流控光盘（CD）平台，监测体外孵育样品中聚糖谱的变化。采用固定化靶抗原的免疫亲和捕获法，从血清中选择性地纯化抗体。将聚糖用酶释放、纯化，最后用 MALDI-TOF-MS 分析。为了模拟体内给药后糖型谱的变化，将治疗性抗体与α1-2,3 甘露糖苷酶在血清中孵育，人为地减少高甘露糖糖型的数量。在孵育过程中，在特定时间间隔监测聚糖谱。在孵育过程中，高甘露糖 5 型的相对丰度明显降低，同时高甘露糖 4 型的相对丰度增加。该方法可快速、平行、自动进行聚糖谱分析，消耗的样品和试剂较少。这有助于减少体外和体内治疗性抗体糖基化研究的劳动力和成本。
