Site-Specific Characterization and Absolute Quantification of Pegfilgrastim Oxidation by Top-Down High-Performance Liquid Chromatography–Mass Spectrometry

The characterization and absolute quantification of protein biopharmaceuticals and their product-related impurities, e.g., oxidation variants, is essential due to their potential impact on biological activity and immunogenicity. Here, we present site assignment and absolute quantification of oxidation variants of pegfilgrastim, a poly­(ethylene glycol) modified recombinant human granulocyte-colony stimulating factor. Pegfilgrastim stressed with 1.0% hydrogen peroxide served as a model protein for developing a top-down high-performance liquid chromatography-mass spectrometry (HPLC-MS) platform that allowed direct site assignment of Met122, Met127, and Met138 oxidation within a total analysis time of 30 min. Three different absolute quantification methods, namely, UV absorption spectroscopy, full-scan MS, and all-ion fragmentation (AIF) MS were compared. Additionally, the monitoring of all generated fragment ions or selected sets of fragment ions were evaluated for the AIF method. Linearity of calibration curves from 5.0 to 25 ng μL–1, 25 to 250 ng μL–1, and 100 to 1000 ng μL–1 was confirmed. The AIF method achieved a lower limit of detection of 0.85 ng μL–1 and a lower limit of quantification of 2.54 ng μL–1. On the basis of the comparison of relative standard deviations of interday measurements, AIF was concluded to be the method of choice for concentrations up to 50 ng μL–1, and UV measurements should be carried out above this concentration. Finally, an expired pegfilgrastim batch was analyzed as a a real biopharmaceutical sample to confirm the feasibility of our approach for monitoring low levels of oxidation variants.

蛋白质生物制剂及其产物相关杂质（如氧化变异体）的表征与绝对定量至关重要，因为这些杂质可能对生物活性与免疫原性产生潜在影响。本研究针对聚乙二醇修饰的重组人粒细胞集落刺激因子——培非格司亭（pegfilgrastim）的氧化变异体，开展了位点定位与绝对定量研究。经1.0%过氧化氢胁迫处理的培非格司亭作为模型蛋白，用于构建自上而下高效液相色谱-质谱联用（HPLC-MS）分析平台，该平台可在30分钟总分析时长内直接完成Met122、Met127及Met138位点氧化的定位。本研究对比了三种不同的绝对定量方法，分别为紫外吸收光谱法、全扫描质谱法以及全离子碎裂（AIF）质谱法。此外，针对AIF法，本研究还评估了对所有生成的碎片离子或选定的碎片离子组的监测策略。验证了浓度范围5.0~25 ng·μL⁻¹、25~250 ng·μL⁻¹以及100~1000 ng·μL⁻¹下校准曲线的线性关系。AIF法的检出限低至0.85 ng·μL⁻¹，定量限低至2.54 ng·μL⁻¹。通过对比日间测量的相对标准偏差，本研究得出结论：当样品浓度不高于50 ng·μL⁻¹时，优先选用AIF法；当浓度高于该阈值时，则应采用紫外吸收光谱法进行定量。最后，本研究选取一批过期的培非格司亭样品作为真实生物制剂样本进行分析，验证了本方法用于监测低水平氧化变异体的可行性。