New Drug Development Center, Osong Medical Innovation Foundation, 123, Osongsaengmyeong-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungbuk, Republic of Korea.
New Drug Development Center, Osong Medical Innovation Foundation, 123, Osongsaengmyeong-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungbuk, Republic of Korea.
Talanta. 2019 Jun 1;198:105-110. doi: 10.1016/j.talanta.2019.01.093. Epub 2019 Jan 25.
N-glycans influence the activity of antibody drugs such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Thus, glycan profiling is considered a critical quality attribute (CQA) and requires routine and comprehensive monitoring. In this report, we validate the new glycan profiling method called Rapi-Fluor method, which reduced the sample preparation time and increased the FLR and MS intensities compared with conventional 2-AB method. Optimized glycan release, labeling, hydrophilic interaction liquid chromatography (HILIC) enrichment, and HILIC separation resulted in low variation and short preparation time. The method evaluated for human IgG standard varied from 100 µg/mL to 4000 µg/mL in 25 µL of water. The determination of coefficient (r > 0.9992), recovery (88.992% ~ 111.198%), limit of detection (LOD < 193.274 µg/mL), limit of quantification (LOQ < 585.679 µg/mL), and precision (Intra-day < 2.317%RSD and Inter-day < 4.287%RSD) were evaluated with four major glycans from antibody drugs. In addition, the method was used for glycan profiling of five different commercial antibodies. The method yielded precise results for IgG glycan analysis and demonstrated effective glycan profiling of commercial antibody drugs.
N-糖基化会影响抗体药物的活性，如抗体依赖的细胞介导的细胞毒性（ADCC）和补体依赖的细胞毒性（CDC）。因此，聚糖分析被认为是关键质量属性（CQA），需要进行常规和全面的监测。在本报告中，我们验证了一种新的聚糖分析方法，称为 Rapi-Fluor 方法，与传统的 2-AB 方法相比，该方法缩短了样品制备时间，并提高了荧光比率（FLR）和质谱（MS）强度。经过优化的聚糖释放、标记、亲水相互作用色谱（HILIC）富集和 HILIC 分离，使得方法具有低变异性和短的制备时间。该方法对人 IgG 标准品的评估范围为 25µL 水中 100µg/mL 至 4000µg/mL。在 4 种主要的抗体药物糖中，该方法的相关参数评估结果为：决定系数（r>0.9992）、回收率（88.992%~111.198%）、检测限（LOD<193.274µg/mL）、定量限（LOQ<585.679µg/mL）和精密度（日内<2.317%RSD，日间<4.287%RSD）。此外，该方法还用于 5 种不同的商业抗体的聚糖分析。该方法对 IgG 聚糖分析的结果精确，能够有效地对商业抗体药物进行聚糖分析。
