Bioprocessing Technology Institute (BTI), Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Republic of Singapore.
Sci Rep. 2024 Nov 29;14(1):29735. doi: 10.1038/s41598-024-80649-y.
The increasing demand for biotherapeutics has necessitated the evaluation of their critical quality attributes, one of which is glycosylation, an essential post-translational modification found on many biological molecules. In particular, the purification of N-glycans after their release from the proteins and derivatization is important in ensuring the removal of the deglycosylated protein, excess labelling reagents and salts for subsequent analysis. However, current methods of N-glycans purification are either expensive, laborious, time-consuming or not catered for high throughput analysis. To overcome these constraints, we developed a high throughput purification method for fluorescent derivatized N-glycans using cellulose functionalized magnetic beads (CMBs). We compared the method with two current purification methods, hydrophilic interaction chromatography solid phase extraction (HILIC-SPE) and gel filtration using human serum IgG (hsIgG) and bovine fetuin and assessed their reproducibility. The CMB method yielded highly similar glycan profiles to the two methods with very good precision. We then assessed the compatibility of the method to purify N-glycans derivatized with different fluorescent labels (RapiFluor MS, 2-aminobenzamide and procainamide). We also applied the methodology to analyse N-glycans in the biotherapeutic protein, recombinant alpha-1-antitrypsin (rAAT) which was modified with higher sialylation content. Importantly, the method successfully captured the differences in the glycan profiles between the modified and unmodified rAAT. Finally, we automated the method together with the digestion and labelling protocol onto a robotic liquid handler for high throughput glycosylation analysis. The versatility of the CMB method, together with its affordability and robustness, may provide an alternative workflow for the high throughput analysis of glycans in biotherapeutic modalities.
生物疗法的需求不断增加，这就需要评估其关键质量属性，其中之一是糖基化，这是许多生物分子上的一种重要翻译后修饰。特别是，在从蛋白质上释放 N-糖链并进行衍生化后，对 N-糖链进行纯化对于确保去除去糖基化的蛋白质、过量的标记试剂和盐以进行后续分析非常重要。然而，目前 N-糖链的纯化方法要么昂贵、费力、耗时，要么不适合高通量分析。为了克服这些限制，我们使用纤维素功能化磁性珠（CMB）开发了一种用于荧光衍生化 N-糖链的高通量纯化方法。我们将该方法与两种当前的纯化方法，亲水相互作用色谱固相萃取（HILIC-SPE）和使用人血清 IgG（hsIgG）和牛胎球蛋白的凝胶过滤进行了比较，并评估了它们的重现性。CMB 方法与两种方法产生的糖型谱非常相似，具有非常好的精密度。然后，我们评估了该方法与不同荧光标记（RapiFluor MS、2-氨基苯甲酰胺和普鲁卡因酰胺）衍生的 N-糖链的兼容性。我们还将该方法应用于分析经过更高唾液酸化修饰的生物治疗蛋白重组人α-1-抗胰蛋白酶（rAAT）的 N-糖链。重要的是，该方法成功地捕捉到了修饰和未修饰 rAAT 之间糖型谱的差异。最后，我们将该方法与消化和标记方案一起自动化到机器人液体处理机上，以进行高通量糖基化分析。CMB 方法的多功能性、可负担性和稳健性，可能为生物治疗模式中聚糖的高通量分析提供一种替代工作流程。
