Zhang Cheng, Karu Kersti, Dalby Paul A
Department of Biochemical Engineering, University College London, Gower Street, London WC1E 6BT, U.K.
Department of Chemistry, University College London, 20 Gordon Street, London WC1H 0AJ, U.K.
Mol Pharm. 2025 Sep 1;22(9):5389-5399. doi: 10.1021/acs.molpharmaceut.5c00358. Epub 2025 Aug 7.
The hinge region of antibody fragments plays a crucial role in their stability and aggregation properties. In this study, we investigated the effects of hinge truncations on the thermal stability and aggregation propensity of the A33 Fab antibody fragment. Eight Fab variants were engineered by introducing stop codons to truncate 1-8 residues at the hinge region (heavy chain residues 221-228). These variants were then expressed, purified, and characterized in terms of stability and aggregation propensity using SDS-PAGE, SEC-HPLC, LC-MS, and thermal stability assays. Our findings demonstrate that truncating the hinge region can enhance the thermal stability and reduce the aggregation of Fab fragments, and that progressive truncations identified an optimal hinge length for stability. Notably, the 227TGA variant exhibited a significant 14.5% reduction in aggregation rate compared to the wild type, without compromising thermal stability. By contrast, 221TGA removed all of the hinge and reduced the aggregation rate by 13%, but also decreased the thermal stability. These results suggest that hinge truncation is a promising strategy for improving the developability of therapeutic antibody Fab fragments by mitigating some of the stability issues associated with aggregation.
抗体片段的铰链区在其稳定性和聚集特性中起着关键作用。在本研究中，我们研究了铰链区截短对A33 Fab抗体片段热稳定性和聚集倾向的影响。通过引入终止密码子在铰链区（重链残基221 - 228）截短1 - 8个残基，构建了8种Fab变体。然后使用SDS - PAGE、SEC - HPLC、LC - MS和热稳定性测定对这些变体进行表达、纯化，并在稳定性和聚集倾向方面进行表征。我们的研究结果表明，截短铰链区可以提高Fab片段的热稳定性并减少其聚集，并且逐步截短确定了一个稳定的最佳铰链长度。值得注意的是，与野生型相比，227TGA变体的聚集速率显著降低了14.5%，而不影响热稳定性。相比之下，221TGA去除了所有铰链区，聚集速率降低了13%，但也降低了热稳定性。这些结果表明铰链区截短是一种有前景的策略，可通过缓解与聚集相关的一些稳定性问题来提高治疗性抗体Fab片段的可开发性。
