In-Depth Comparison of Lysine-Based Antibody-Drug Conjugates Prepared on Solid Support Versus in Solution
In-Depth Comparison of Lysine-Based Antibody-Drug Conjugates Prepared on Solid Support Versus in Solution
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DOI：
10.3390/antib7010006
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发表时间：
2018-03-01
期刊：
影响因子：
4.7
通讯作者：
Owen, Shawn C.
中科院分区：
文献类型：
--
作者：
Arlotta, Keith J.;Gandhi, Aditya V.;Owen, Shawn C.
抗体药物偶联物是一种快速发展的靶向化疗药物。随着公司和研究人员开始开发新的抗体-药物偶联物（ADC）候选物，高通量方法将变得越来越普遍。在这里，我们使用先进的表征技术来评估两种曲妥珠单抗- dm1 (T-DM1) adc；一种用蛋白A固定化生产，另一种用溶液生产。在液相色谱-质谱法（LC/MS）确定有效载荷位置和分布后，分别使用差示扫描量热法（DSC）、动态光散射法（DLS）、拉曼光谱法（Raman spectroscopy）和等温滴定量热法（ITC）对其热稳定性、热诱导聚集、三级结构和结合亲和力进行了表征。DSC和DLS分别观察到抗体C(H)2结构域的热稳定性和聚集开始温度的微小差异。然而，用拉曼光谱观察到的二级和三级结构没有显著差异，或者用ITC测量的结合亲和力。基于赖氨酸的ADC偶联产生一个天生的异质群体，可以在敏感的表征技术的结果中产生显著的可变性。这些adc的表征表明在热稳定性方面存在名义上的差异，但在三级结构或结合亲和力方面没有差异。我们的研究结果使我们得出结论，在蛋白质A固定后合成的基于赖氨酸的adc，在小规模的偶联中很常见，与大规模的基于溶液的方法生产的等效adc高度相似。
Antibody drug conjugates are a rapidly growing form of targeted chemotherapeutics. As companies and researchers move to develop new antibody-drug conjugate (ADC) candidates, high-throughput methods will become increasingly common. Here we use advanced characterization techniques to assess two trastuzumab-DM1 (T-DM1) ADCs; one produced using Protein A immobilization and the other produced in solution. Following determination of payload site and distribution with liquid chromatography-mass spectrometry (LC/MS), thermal stability, heat-induced aggregation, tertiary structure, and binding affinity were characterized using differential scanning calorimetry (DSC), dynamic light scattering (DLS), Raman spectroscopy, and isothermal titration calorimetry (ITC), respectively. Small differences in the thermal stability of the C(H)2 domain of the antibody as well as aggregation onset temperatures were observed from DSC and DLS, respectively. However, no significant differences in secondary and tertiary structure were observed with Raman spectroscopy, or binding affinity as measured by ITC. Lysine-based ADC conjugation produces an innately heterogeneous population that can generate significant variability in the results of sensitive characterization techniques. Characterization of these ADCs indicated nominal differences in thermal stability but not in tertiary structure or binding affinity. Our results lead us to conclude that lysine-based ADCs synthesized following Protein A immobilization, common in small-scale conjugations, are highly similar to equivalent ADCs produced in larger scale, solution-based methods.