Developability Assessment of Engineered Monoclonal Antibody Variants with a Complex Self-Association Behavior Using Complementary Analytical and in Silico Tools
Developability Assessment of Engineered Monoclonal Antibody Variants with a Complex Self-Association Behavior Using Complementary Analytical and in Silico Tools
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DOI：
10.1021/acs.molpharmaceut.8b00867
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发表时间：
2018-12-01
影响因子：
4.9
通讯作者：
Esfandiary, Reza
中科院分区：
文献类型：
--
作者：
Shan, Lu;Mody, Neil;Esfandiary, Reza
单克隆抗体（mAb）是复杂的分子结构。由于不期望的溶液性质，例如可逆自缔合、高粘度和液液相分离，它们通常容易受到开发挑战，特别是在高浓度下。除了配方优化，应用蛋白质工程可以提供替代的缓解策略。在发现阶段的蛋白质工程可以提供极大的好处，以优化分子特性，从而改善开发能力的概况。在这里，我们提出了一个案例研究，利用互补的分析和预测的计算机方法。我们已经系统地识别和重新设计了负责模型mAb自结合的有问题的残基，这些残基是由疏水性和静电相互作用的复杂组合驱动的。值得注意的发现包括疏水相互作用对自缔合的更主要贡献以及CDR区突变减轻自缔合的潜在可行性。工程突变面板使我们能够评估常用的可开发性筛选测定之间的潜在相关性，包括亲和捕获自相互作用纳米光谱法（AC-XPS），动态光散射（DLS）和PEG沉淀法的表观溶解度。此外，我们评估了实验测量和计算预测之间的相关性。CamSol是一种计算机计算工具，可以解释复杂的分子相互作用和邻近的热点，被认为是一种有效的筛选工具。我们的工作导致了重新设计的mAb变体，更适合高浓度液体制剂的开发。工程化的mAb表现出增强的体外和模拟的体内溶解度和降低的自缔合倾向，同时保持结合亲和力和热稳定性。
Monoclonal antibodies (mAbs) are complex molecular structures. They are often prone to development challenges particularly at high concentrations due to undesired solution properties such as reversible self-association, high viscosity, and liquid liquid phase separation. In addition to formulation optimization, applying protein engineering can provide an alternative mitigation strategy. Protein engineering during the discovery phase can provide great benefits to optimize molecular properties, resulting in improved develop ability profiles. Here, we present a case study utilizing complementary analytical and predictive in silico methods. We have systematically identified and reengineered problematic residues responsible for the self-association of a model mAb, driven by a complex combination of hydrophobic and electrostatic interactions. Noteworthy findings include a more dominant contribution of hydrophobic interactions to self-association and potential feasibility of mutations in the CDR regions to mitigate self-association. The engineered mutation panel enabled us to assess potential correlations among commonly utilized developability screening assays, including affinity capture self-interaction nanospectroscopy (AC-SINS), dynamic light scattering (DLS), and apparent solubility by PEG-precipitation. In addition, we evaluated the correlations between experimental measurements and computational predictions. CamSol, an in silico computational tool that accounts for complex molecular interactions and neighboring hotspots, was found to be an effective screening tool. Our work led to reengineered mAb variants, better suited for high-concentration liquid formulation development. The engineered mAbs exhibited enhanced in vitro and simulated in vivo solubility and reduced self-association propensity, while maintaining binding affinity and thermal stability.