Abstract：
Glycosylation is a crucial attribute for biotherapeutics with significant impacts on quality, stability, safety, immunogenicity, pharmacokinetics, and efficacy. Therefore, to ensure consistent glycosylation, a systematic review of biotherapeutics is absolutely required including the variable glycan structure (micro-heterogeneity) and different occupancy at individual site (macro-heterogeneity) from drug design to upstream and downstream bioprocesses. Various methods have been used for glyco-characterization of biotherapeutics at the glycan, glycopeptide, and intact protein levels. In particular, intact protein analysis is considered a facile and rapid glycoform monitoring approach used throughout the product development lifecycle to determine suitable glycosylation lead candidates and reproducible product quality. However, intact glycoform characterization of diverse and complex biotherapeutics with multiple N- and O-glycosylation sites can be very challenging. To address this, a robust analytical platform that enables rapid and accurate characterization of a biotherapeutics with highly complex multiple glycosylation using two-step intact glycoform mass spectrometry has been developed. We used darbepoetin alfa, a second-generation EPO bearing multiple N- and O-glycosylation sites, as a model biotherapeutics to obtain integrated information on glycan heterogeneity and site occupancy through step-by-step MS of intact protein and enzyme-treated protein. In addition, we performed a comparative assessment of the heterogeneity from different products, confirming that our new method can efficiently evaluate glycosylation equivalence. This new strategy provides rapid and accurate information on the degree of glycosylation of a therapeutic glycoprotein with multiple glycosylation, which can be used to assess glycosylation similarity between batches and between biosimilar and reference during development and production.
摘要：
糖基化是生物治疗的关键属性，对质量有重大影响，稳定性,安全,免疫原性,药代动力学，和功效。因此,为了确保一致的糖基化，绝对需要对生物治疗进行系统的审查，包括可变的聚糖结构（微观异质性）和从药物设计到上游和下游生物过程的各个部位的不同占有率（宏观异质性）。各种方法已被用于生物治疗剂在聚糖的糖表征，糖肽，和完整的蛋白质水平。特别是,完整的蛋白质分析被认为是在整个产品开发生命周期中使用的一种简便且快速的糖型监测方法，以确定合适的糖基化前导候选物和可重复的产品质量。然而,具有多个N-和O-糖基化位点的多样且复杂的生物治疗剂的完整糖型表征可能是非常具有挑战性的。为了解决这个问题，已经开发了一个强大的分析平台,该平台能够使用两步完整糖型质谱快速和准确地表征具有高度复杂的多重糖基化的生物治疗剂.我们用了darbepoetinalfa,第二代EPO带有多个N-和O-糖基化位点，作为模型生物治疗剂，通过完整蛋白质和酶处理蛋白质的逐步MS获得有关聚糖异质性和位点占有率的综合信息。此外,我们对不同产品的异质性进行了比较评估，证实了我们的新方法可以有效地评估糖基化等效性。这种新策略提供了关于具有多重糖基化的治疗性糖蛋白的糖基化程度的快速而准确的信息，它可用于评估开发和生产过程中批次之间以及生物仿制药与参比之间的糖基化相似性。
