安全监测

纳米级差示扫描荧光法在阿达木单抗热分析中的应用*

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  • 1.中国食品药品检定研究院,北京 102629;
    2.国家卫生健康委员会生物技术产品检定方法及其标准化重点实验室, 北京 102629;
    3.国家药品监督管理局生物制品质量研究与评价重点实验室,北京 102629;
    4.烟台大学药学院,烟台 264005;
    5.甘肃省药品检验研究院,兰州 730070
第一作者 郭 莎 Tel:(010)53852177;E-mail:guosha@nifdc.org.cn
贾 哲 Tel:(010)53852199;E-mial:jiajia123163@163.com
**Tel:(010)53852159;E-mail:wanglan@nifdc.org.cn

修回日期: 2022-12-14

  网络出版日期: 2024-06-24

基金资助

*国家科技重大专项重大新药创制自身免疫疾病治疗的单抗类生物类似药质量评价和国家标准品建立(2018ZX09736008-006);2020年国家药品标准提高项目课题单抗生物类似药质控方法的研究与优化(2020S08)

Application of nano-differential scanning fluorescence inthermal analysis of adalimumab*

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  • 1. National Institutes for Food and Drug Control, Beijing 102629, China;
    2. NHC Key Laboratory of Verification Methods and Standardization of Biotech Products, Beijing 102629, China;
    3. NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, Beijing 102629, China;
    4. School of Pharmacy, Yantai University, Yantai 264005, China;
    5. Gansu Institute for Drug Control, Lanzhou 730070, China

Revised date: 2022-12-14

  Online published: 2024-06-24

摘要

目的: 探讨纳米级差示扫描荧光法(nanoDSF法)检测阿达木单抗熔解温度(Tm)的可行性及潜在应用。方法: 采用nanoDSF技术对0.25、0.5、1、5、10、50 mg·mL-1 6个不同浓度下的阿达木单抗Tm进行检测,检测过程中参数设置:升温区间为25~95 ℃,升温速率为1 ℃·min-1,收集内源性荧光信号测得Tm,重复检测5次。通过将阿达木单抗与制剂缓冲液的Tm检测结果进行比较,确认方法的特异性;通过6个不同浓度下阿达木单抗5次Tm检测结果的离散度评估,得到方法的精密度;通过将0.25、0.5、1 mg·mL-1 3个浓度下阿达木单抗的Tm检测结果与相应浓度下的差式扫描量热法(DSC法)检测结果进行比较,对nano DSF技术的检测准确性进行评价;进行上述方法学验证后,将该技术应用于阿达木单抗原研药与6种生物类似药Tm的相似性评价。此外,本研究还通过收集抗体分子在升温过程中的散射光信号,对6个不同浓度阿达木单抗的起始聚集温度(Tagg)进行检测。结果: nanoDSF技术能够检测单抗样品的Tm,而相应制剂缓冲液则并无特征性曲线,说明该方法具有特异性;nanoDSF技术检测0.25、0.5、1、5、10、50 mg·mL-1 6个不同浓度的样品,每个样品各检测5次,Tm值检测结果间RSD均在1%以内,说明方法具有较好的精密度;与DSC法的结果相比,nanoDSF普遍低2~3℃,但2种方法测量的Tm具有较好的相关性,r在0.99以上;nano DSF技术测得0.25、0.5、1、5、10、50 mg·mL-1 6个不同浓度阿达木单抗的Tm图谱基本一致,Tm1Tm3检测结果也没有明显差异,Tm2有随着浓度升高而降低的趋势。在阿达木单抗原研药和生物类似药的相似性研究中,nanoDSF法与DSC法的检测结果间也是相关的;此外,在检测Tm的同时检测起始聚集温度(Tagg),随着浓度升高,检测到的Tagg呈逐渐下降的趋势,这对Tm检测结果的解释起到一定的提示作用。结论: nano DSF技术具有较好的特异性、精密度、准确性和较广的浓度适用范围,适用于原研药与生物类似药热稳定性的相似性研究等。

本文引用格式

郭莎, 贾哲, 贺鹏飞, 田向斌, 于传飞, 武刚, 崔永霏, 刘春雨, 王兰 . 纳米级差示扫描荧光法在阿达木单抗热分析中的应用*[J]. 药物分析杂志, 2023 , 43(1) : 153 -161 . DOI: 10.16155/j.0254-1793.2023.01.18

Abstract

Objective: To discuss the feasibility and potential application of nano-differential scanning fluorescence (nanoDSF)to detect the melting temperature(Tm) of adalimumab. Methods: Using nano-DSF technique to measure Tm of adalimumab mAb at 6 different concentrations including 0.25, 0.5, 1, 5, 10, 50 mg·mL-1, parameters were set during the detection process: the temperature range was 25-95 ℃, the rate of temperature elevation was 1 ℃·min-1, the intrinsic fluorescence signal was collected to measure Tm, and the detection was repeated for 5 times. The specificity of nano-DSF was inspected by comparing the Tm results of adalimumab mAb and its formulation buffer. The precision of nano-DSF was obtained by evaluating the results of 5 Tm test results of adalimumab mAb at 6 different concentrations. The detection accuracy of nano-DSF technology was assessed by comparasion with the Tm detection results of adalimumab mAb with the differential scanning calorimetry (DSC) under 3 concentrations including 0.25, 0.5, 1 mg·mL-1. In the bases of method evaluation above, the technology was applied to evaluate the similarity between adalimumab mAb reference biotherapeutic product (RBP) and 6 similar biotherapeutic products (SBPs) Tm. Besides, the initial aggregation temperature (Tagg) of adalimumab mAb under 6 different concentrations was also detected by collecting the scattered light signals of antibody molecules during the heating process. Results: nano-DSF technique could specifically identify Tm of adalimumab but there was no characteristic curve of corresponding formulation buffer, indicating the specificity of this method. Besides the nano-DSF technique had good precision in detecting 6 samples with different concentrations of 0.25, 0.5, 1, 5, 10, 50 mg·mL-1 and each sample was detected 5 times, and the RSDs of Tm values were within 1%. Compared with the results of differential scanning calorimetry (DSC), nano-DSF results were 2-3 ℃ lower generally, but the Tm measured by the two methods had good correlations(r≥0.99).The Tm maps of adalimumab mAb with different concentrations of 0.25, 0.5, 1, 5, 10, 50 mg·mL-1 were basically the same by nano-DSF, and there was no obvious difference in the results of Tm1 and Tm3 while Tm2 had a tendency to decrease with increa-sing concentrations. In the similarity study of adalimumab mAb RBP and SBPs, the results of nano-DSF and DSC were also consistent.In addition, detecting the Tagg at the same time of Tm detection. The detected Tagg showed a trend of gradual decline with increasing concentrations, which played an important role in the interpretation of Tm detection results. Conclusion: nano-DSF technology has satisfactory specificity, precision, accuracy and wide concentration application range, thus ensuring the suitability in the thermal stability similarity study of RBP and SBPs, etc.

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