Szabo Miklos, Sarkozy Daniel, Szigeti Marton, Farsang Robert, Kardos Zsofia, Kozma Adam, Csanky Eszter, Chung Doo Soo, Szekanecz Zoltan, Guttman Andras
Borsod Academic County Hospital, Miskolc, Hungary.
Horváth Csaba Memorial Laboratory of Bioseparation Sciences, Research Center for Molecular Medicine, Faculty of Medicine, Doctoral School of Molecular Medicine, University of Debrecen, Debrecen, Hungary.
Front Bioeng Biotechnol. 2022 Mar 8;10:839374. doi: 10.3389/fbioe.2022.839374. eCollection 2022.
Coronavirus Disease 2019 (COVID-19) is a major public health problem worldwide with 5-10% hospitalization and 2-3% global mortality rates at the time of this publication. The disease is caused by a betacoronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The receptor-binding domain (RBD) of the Spike protein expressed on the surface of the virus plays a key role in the viral entry into the host cell the angiotensinconverting enzyme 2 receptor. Neutralizing monoclonal antibodies having the RBD as a target have the ability to inhibit angiotensin-converting enzyme 2 (ACE2) receptor binding, therefore, prevent SARS-CoV-2 infection, represent a promising pharmacological strategy. Bamlanivimab is the first anti-spike neutralizing monoclonal antibody, which got an emergency use authorization from the FDA for COVID-19 treatment. Albeit, bamlanivimab is primarily a neutralizing mAb, some of its effector function related activity was also emphasized. The effector function of antibody therapeutics is greatly affected by their linked carbohydrates at the conserved Fc region, possibly influenced by the manufacturing process. Various capillary gel electrophoresis methods are widely accepted in the biopharmaceutical industry for the characterization of therapeutic antibodies. In this paper we introduce a capillary gel electrophoresis based workflow for ) size heterogeneity analysis to determine the presence/absence of the nonglycosylated heavy chain (NGHC) fragment (SDS-CGE); ) capillary gel isoelectric focusing for possible -glycosylation mediated charge heterogeneity determination, e.g., for excess sialylation and finally, ) capillary gel electrophoresis for glycosylation profiling and sequencing. Our results have shown the presence of negligible amount of non-glycosylated heavy chain (NGHC) while 25% acidic charge variants were detected. Comprehensive -glycosylation characterization revealed the occurrence of approximately 8.2% core-afucosylated complex and 17% galactosylated linked oligosaccharides, suggesting the possible existence of antibody dependent cell mediated cytotoxicity (ADCC) effector function in addition to the generally considered neutralizing effect of this particular therapeutic antibody molecule.
2019年冠状病毒病（COVID-19）是一个全球性的重大公共卫生问题，在撰写本文时，其住院率为5%-10%，全球死亡率为2%-3%。该疾病由一种名为严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的β冠状病毒引起。病毒表面表达的刺突蛋白的受体结合域（RBD）在病毒进入宿主细胞（血管紧张素转换酶2受体）过程中起关键作用。以RBD为靶点的中和单克隆抗体具有抑制血管紧张素转换酶2（ACE2）受体结合的能力，因此可预防SARS-CoV-2感染，是一种有前景的药理学策略。巴瑞替尼单抗是首个抗刺突中和单克隆抗体，已获得美国食品药品监督管理局（FDA）用于COVID-19治疗的紧急使用授权。尽管巴瑞替尼单抗主要是一种中和性单克隆抗体，但其一些与效应器功能相关的活性也得到了强调。抗体治疗药物的效应器功能在很大程度上受其保守Fc区域连接的碳水化合物影响，可能受生产工艺影响。各种毛细管凝胶电泳方法在生物制药行业中被广泛用于治疗性抗体的表征。在本文中，我们介绍了一种基于毛细管凝胶电泳的工作流程，用于：）大小异质性分析，以确定非糖基化重链（NGHC）片段的存在与否（SDS-CGE）；）毛细管凝胶等电聚焦，用于可能的N-糖基化介导的电荷异质性测定，例如用于过量唾液酸化，最后，）毛细管凝胶电泳用于糖基化谱分析和测序。我们的结果显示非糖基化重链（NGHC）的含量可忽略不计，同时检测到25%的酸性电荷变体。全面的N-糖基化表征揭示了约8.2%的核心岩藻糖基化复合物和17%的半乳糖基化连接寡糖的存在，这表明除了这种特定治疗性抗体分子通常被认为的中和作用外，可能还存在抗体依赖性细胞介导的细胞毒性（ADCC）效应器功能。
