PDF(Pubmed)
Abstract：
Characterization of highly glycosylated biopharma-ceuticals by mass spectrometry is challenging because of the huge chemical space of coexistent glycoforms present. Here, we report the use of an array of HPLC-mass spectrometry-based approaches at different structural levels of released glycan, glycopeptide, and hitherto unexplored intact glycoforms to scrutinize the biopharmaceutical Myozyme, containing the highly complex lysosomal enzyme recombinant acid α-glucosidase. The intrinsic heterogeneity of recombinant acid α-glucosidase glycoforms was unraveled using a novel strong anion exchange HPLC-mass spectrometry approach involving a pH-gradient of volatile buffers to facilitate chromatographic separation of glycoforms based on their degree of sialylation, followed by the acquisition of native mass spectra in an Orbitrap mass spectrometer. Upon considering the structures of 60 different glycans attached to seven glycosylation sites in the intact protein, the large set of interdependent data acquired at different structural levels was integrated using a set of bioinformatic tools and allowed the annotation of intact glycoforms unraveling more than 1,000,000 putative intact glycoforms. Detectable isoforms also included several mannose-6-phosphate variants, which are essential for directing the drug toward its target, the lysosomes. Finally, for the first time, we sought to validate the intact glycoform annotations by integrating experimental data on the enzymatically dissected proteoforms, which reduced the number of glycoforms supported by experimental evidence to 42,104. The latter verification clearly revealed the strengths but also intrinsic limitations of this approach for fully characterizing such highly complex glycoproteins by mass spectrometry.
摘要：
由于共存糖型的巨大化学空间，通过质谱表征高度糖基化的生物药物具有挑战性，即异源糖蛋白变体。这里,我们报告了一系列基于HPLC-MS的方法在不同结构水平的聚糖释放，糖肽，和迄今为止尚未开发的完整糖型来仔细检查生物制药Myozyme®，含有高度复杂的溶酶体酶重组酸性α-葡萄糖苷酶。使用新型强阴离子交换（SAX）-HPLC-MS方法揭示了重组酸α-葡萄糖苷酶糖型的内在异质性，该方法涉及挥发性缓冲液的pH梯度，以根据糖型的唾液酸化程度促进糖型的色谱分离，然后在Orbitrap质谱仪中获取天然质谱。在考虑60个不同的聚糖连接到完整的蛋白质中的七个糖基化位点的结构，使用一组生物信息学工具整合了在不同结构水平上获得的大量相互依赖的数据，并允许完整糖型的注释解开超过1,000,000个推定的完整糖型。可检测的同工型还包括几种甘露糖-6-磷酸变体，这对于将药物引向其目标-溶酶体至关重要。最后,第一次,我们试图通过整合酶解剖的蛋白质形式的实验数据来验证完整的糖型注释，这将实验证据支持的糖型数量减少到42,104。后一种验证清楚地揭示了这种方法通过质谱法充分表征这种高度复杂的糖蛋白的优势和内在局限性。
