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(T_m)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T_m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T_m,重复检测5次。通过将阿达木单抗与制剂缓冲液的T_m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T_m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T_m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T_m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(T_agg)进行检测。结果: nanoDSF技术能够检测单抗样品的T_m,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,T_m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T_m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T_m图谱基本一致,T_m1和T_m3检测结果也没有明显差异,T_m2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T_m的同时检测起始聚集温度(T_agg),随着浓度升高,检测到的T_agg呈逐渐下降的趋势,这对T_m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。

Objective: To discuss the feasibility and potential application of nano-differential scanning fluorescence (nanoDSF)to detect the melting temperature(T_m) of adalimumab. Methods: Using nano-DSF technique to measure T_m 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 T_m, and the detection was repeated for 5 times. The specificity of nano-DSF was inspected by comparing the T_m results of adalimumab mAb and its formulation buffer. The precision of nano-DSF was obtained by evaluating the results of 5 T_m test results of adalimumab mAb at 6 different concentrations. The detection accuracy of nano-DSF technology was assessed by comparasion with the T_m 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) T_m. Besides, the initial aggregation temperature (T_agg) 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 T_m 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 T_m values were within 1%. Compared with the results of differential scanning calorimetry (DSC), nano-DSF results were 2-3 ℃ lower generally, but the T_m measured by the two methods had good correlations(r≥0.99).The T_m 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 T_m1 and T_m3 while T_m2 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 T_agg at the same time of T_m detection. The detected T_agg showed a trend of gradual decline with increasing concentrations, which played an important role in the interpretation of T_m 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.