Despite the popularity of targeted and immune therapies, the number of studies dealing with the quantitation of aggregates for Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved mAb and related products are still very scarce in literature. In this work, 30 therapeutic proteins including monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), Fc-fusion proteins and a bi-specific antibody (bsAb) were investigated using size exclusion chromatography (SEC). Their levels of high molecular weight species (HMWS) were experimentally estimated between 0.1% and 13.1%. Except for blinatumomab, etanercept and pembrolizumab, the HMWS amount for the other antibodies was well below the limit of 5% usually set a specification for therapeutic mAbs in the biopharmaceutical industry. The main chromatographic peak shape of 24 therapeutic antibodies and the NIST mAb [1] was found suitable (0.8 < As < 1.5) with a generic SEC method involving potassium-based salts mobile phase. Conversely, only acidic therapeutic proteins (pI < 7) could be successfully analyzed with a mass spectrometry (MS) compatible mobile phase containing 100 mM ammonium acetate. This study aimed to provide HMWS data for 30 therapeutic proteins covering a wide range of physico-chemical properties with molecular weights between 54 and 153 kDa, pI values comprised between 6.1 and 9.4 and hydrophobic interaction chromatography (HIC) retention factors ranging from 1.2 to 6.0 for the mAbs.

尽管靶向和免疫疗法很受欢迎，但有关食品和药物管理局（FDA）和欧洲药品管理局（EMA）批准的mAb及其相关产品的聚集体定量研究的文献仍然很少。在这项工作中，使用尺寸排阻色谱（SEC）研究了30种治疗性蛋白质，包括单克隆抗体（mAb），抗体-药物偶联物（ADC），Fc融合蛋白和双特异性抗体（bsAb）。通过实验估计它们的高分子量物质（HMWS）含量在0.1％和13.1％之间。除blinatumomab，依那西普和pembrolizumab以外，其他抗体的HMWS含量远低于通常在生物制药行业中为治疗性mAb设定的5％的极限。发现24种治疗性抗体和NIST mAb [1]的主要色谱峰形状适用于涉及钾基盐流动相的通用SEC方法（0.8 <As <1.5）。相反，仅酸性治疗性蛋白质（p 使用包含100 mM乙酸铵的质谱（MS）兼容流动相可以成功地分析I < 7）。这项研究旨在为30种治疗蛋白提供HMWS数据，这些蛋白涵盖广泛的理化性质，分子量在54至153 kDa之间，p I值在6.1至9.4之间，疏水相互作用色谱（HIC）保留因子在1.2至单克隆抗体为6.0。