Nemergut Michal, Žoldák Gabriel, Schaefer Jonas V, Kast Florian, Miškovský Pavol, Plückthun Andreas, Sedlák Erik
Department of Biophysics, P.J. Šafárik University, Jesenna 5, Košice, 041 54, Slovakia.
Department of Biophysics, Institute of Molecular and Cellular Biophysics, Technical University of Munich, James-Franck-Str. 1, Garching, D-85748, Germany.
Protein Sci. 2017 Nov;26(11):2229-2239. doi: 10.1002/pro.3278. Epub 2017 Sep 6.
Monoclonal antibodies of the immunoglobulin G (IgG) type have become mainstream therapeutics for the treatment of many life-threatening diseases. For their successful application in the clinic and a favorable cost-benefit ratio, the design and formulation of these therapeutic molecules must guarantee long-term stability for an extended period of time. Accelerated stability studies, e.g., by employing thermal denaturation, have the great potential for enabling high-throughput screening campaigns to find optimal molecular variants and formulations in a short time. Surprisingly, no validated quantitative analysis of these accelerated studies has been performed yet, which clearly limits their application for predicting IgG stability. Therefore, we have established a quantitative approach for the assessment of the kinetic stability over a broad range of temperatures. To this end, differential scanning calorimetry (DSC) experiments were performed with a model IgG, testing chaotropic formulations and an extended temperature range, and they were subsequently analyzed by our recently developed three-step sequential model of IgG denaturation, consisting of one reversible and two irreversible steps. A critical comparison of the predictions from this model with data obtained by an orthogonal fluorescence probe method, based on 8-anilinonaphthalene-1-sulfonate binding to partially unfolded states, resulted in very good agreement. In summary, our study highlights the validity of this easy-to-perform analysis for reliably assessing the kinetic stability of IgGs, which can support accelerated formulation development of monoclonal antibodies by ranking different formulations as well as by improving colloidal stability models.
免疫球蛋白G（IgG）型单克隆抗体已成为治疗许多危及生命疾病的主流疗法。为了在临床上成功应用并获得良好的成本效益比，这些治疗性分子的设计和配方必须保证长时间的长期稳定性。加速稳定性研究，例如通过热变性，极有可能在短时间内实现高通量筛选活动，以找到最佳的分子变体和配方。令人惊讶的是，尚未对这些加速研究进行经过验证的定量分析，这显然限制了它们在预测IgG稳定性方面的应用。因此，我们建立了一种定量方法来评估在广泛温度范围内的动力学稳定性。为此，使用一种模型IgG进行差示扫描量热法（DSC）实验，测试离液序列高的配方和扩展的温度范围，随后通过我们最近开发的IgG变性三步连续模型进行分析，该模型由一个可逆步骤和两个不可逆步骤组成。基于8-苯胺基萘-1-磺酸盐与部分未折叠状态的结合，将该模型的预测结果与通过正交荧光探针法获得的数据进行关键比较，结果非常吻合。总之，我们的研究突出了这种易于执行的分析方法在可靠评估IgG动力学稳定性方面的有效性，这可以通过对不同配方进行排名以及改进胶体稳定性模型来支持单克隆抗体的加速配方开发。
