Department of Chemistry, The University of Kansas, Lawrence, KS 66045 USA.
CMC Biologics Drug Product Development, AbbVie, North Chicago, IL 60061 USA.
J Pharm Sci. 2024 Aug;113(8):2065-2071. doi: 10.1016/j.xphs.2024.05.008. Epub 2024 May 17.
Antibody drug conjugates (ADCs) represent one of the fastest growing classes of cancer therapeutics. Drug incorporation through site-specific conjugation in ADCs leads to uniform drug load and distribution. These site-specific modifications may have an impact on ADC quality attributes including protein higher order structure (HOS), which might impact safety and efficacy. In this study, we conducted a side-by-side comparison between the conjugated and unconjugated mAb. In the ADC, the linker-pyrrolobenzodiazepine was site specifically conjugated to an engineered unpaired C215 residue within the F domain of the light chain. Differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) indicated a decrease in thermal stability for the C2 transition of the ADC. Size exclusion chromatography (SEC) analysis showed that conjugation of the mAb resulted in earlier aggregation onset and increased aggregation propensity after 4 weeks at 40 °C. Differential hydrogen-exchange mass spectrometry (HX-MS) indicated that upon conjugation, light chain residues 150-155 and 197-204, close to the conjugation site, showed significantly faster HX kinetics, suggesting an increase in backbone flexibility within this region, while heavy chain residues 32-44 exhibited significantly slower kinetics, suggesting distal stabilization of the mAb backbone.
抗体药物偶联物 (ADC) 是癌症治疗领域发展最快的一类药物。ADC 中的药物通过定点偶联进行结合，从而实现均一的药物负载和分布。这些定点修饰可能会影响 ADC 的质量属性，包括蛋白质高级结构（HOS），从而影响安全性和疗效。在这项研究中，我们对共轭和未共轭 mAb 进行了平行比较。在 ADC 中，连接子-吡咯并苯二氮䓬通过定点偶联到轻链 F 结构域中一个工程化的未配对 C215 残基上。差示扫描量热法（DSC）和差示扫描荧光法（DSF）表明 ADC 的 C2 转变热稳定性降低。尺寸排阻色谱（SEC）分析表明，mAb 的偶联导致在 40°C 下 4 周后更快地开始聚集，并且聚集倾向增加。差示氢交换质谱（HX-MS）表明，偶联后，靠近偶联点的轻链残基 150-155 和 197-204 的 HX 动力学明显加快，表明该区域的骨架柔性增加，而重链残基 32-44 的动力学明显减慢，表明 mAb 骨架的远端稳定化。
