Duivelshof Bastiaan L, Denorme Steffy, Sandra Koen, Liu Xiaoxiao, Beck Alain, Lauber Matthew A, Guillarme Davy, D'Atri Valentina
Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU-Rue Michel-Servet 1, 1211 Geneva, Switzerland.
School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel-Servet 1, 1211 Geneva, Switzerland.
Pharmaceutics. 2021 Oct 20;13(11):1744. doi: 10.3390/pharmaceutics13111744.
The identification and accurate quantitation of the various glycoforms contained in therapeutic monoclonal antibodies (mAbs) is one of the main analytical needs in the biopharmaceutical industry, and glycosylation represents a crucial critical quality attribute (CQA) that needs to be addressed. Currently, the reference method for performing such identification/quantitation consists of the release of the -glycan moieties from the mAb, their labelling with a specific dye (e.g., 2-AB or RFMS) and their analysis by HILIC-FLD or HILIC-MS. In this contribution, the potential of a new cost- and time-effective analytical approach performed at the protein subunit level (middle-up) was investigated for quantitative purposes and compared with the reference methods. The robustness of the approach was first demonstrated by performing the relative quantification of the glycoforms related to a well characterized mAb, namely adalimumab. Then, the workflow was applied to various glyco-engineered mAb products (i.e., obinutuzumab, benralizumab and atezolizumab). Finally, the glycosylation pattern of infliximab (Remicade) was assessed and compared to two of its commercially available biosimilars (Remsima and Inflectra). The middle-up analysis proved to provide accurate quantitation results and has the added potential to be used as multi-attribute monitoring method.
治疗性单克隆抗体（mAb）中所含各种糖型的鉴定和准确定量是生物制药行业的主要分析需求之一，糖基化是一个需要解决的关键关键质量属性（CQA）。目前，进行此类鉴定/定量的参考方法包括从mAb中释放聚糖部分，用特定染料（如2-AB或RFMS）对其进行标记，并通过亲水相互作用液相色谱-荧光检测（HILIC-FLD）或亲水相互作用液相色谱-质谱（HILIC-MS）进行分析。在本研究中，研究了一种在蛋白质亚基水平（中上游）进行的新的具有成本效益和时间效益的分析方法用于定量目的的潜力，并与参考方法进行了比较。该方法的稳健性首先通过对与一种特征明确的mAb（即阿达木单抗）相关的糖型进行相对定量来证明。然后，将该工作流程应用于各种糖基工程化的mAb产品（即奥妥珠单抗、贝那利珠单抗和阿替利珠单抗）。最后，评估了英夫利昔单抗（类克）的糖基化模式，并将其与两种市售生物类似药（雷米西和英夫利妥）进行了比较。中上游分析证明能够提供准确的定量结果，并且具有用作多属性监测方法的额外潜力。
