Institute of Chemistry, Martin-Luther-Universität Halle-Wittenberg, von-Danckelmann-Platz 4, DE-06120, Halle, Saale, Germany.
Pharmaceutical Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorferstrasse 65, DE-88397, Biberach, Germany.
Pharm Res. 2020 Apr 1;37(4):78. doi: 10.1007/s11095-020-02792-1.
Predicting thermal protein stability is of major interest in the development of protein-based biopharmaceuticals. Therefore, this study provides a predictive tool for determining transition enthalpies, which can be used for ranking different proteins according to their thermal stability.
Unfolding and aggregation profiles of eight different therapeutic monoclonal antibodies (mAbs) of type G, isotype 1 were investigated. The unfolding profiles were determined by intrinsic fluorescence (IF) spectroscopy and differential scanning calorimetry (DSC). A three-state unfolding fitting model was used to determine thermodynamic parameters for macromolecular multi-domain mAbs in IF experiments, like the van't Hoff enthalpy change (∆H) and the entropy change (∆S) of the unfolding event. The derived values were compared to thermodynamic parameters obtained directly by calorimetry. Moreover, differences in the Fab enthalpies were used to predict aggregation behavior and protein thermal stabilities. To do so, the liquid-formulated mAbs were investigated exemplarily by size exclusion chromatography (SEC) after accelerated thermal-induced stress conditions.
Comparing the thermodynamic parameters derived from IF spectroscopy and DSC resulted in similar values. Data generated by thermal-induced stress at 40°C show similar stability ranking as postulated through the Fab enthalpies for mAbs in two different formulations, while at 25°C a meaningful ranking is not possible, because distinct differences in the thermal stability cannot be observed. The additional consideration of Fab enthalpies to predict the 40 °C SEC ranking seems to be more reliable compared to the use of exclusively the melting temperatures or aggregation onset temperatures and times.
We show that thermodynamic profiling can help predicting unfolding and aggregation properties of therapeutic mAbs at 40°C. Therefore, analyzing thermodynamic unfolding parameters is a useful and supportive tool discriminating thermal stability profiles of mAbs for further pharmaceutical development and clinical studies.
预测热蛋白稳定性在基于蛋白质的生物制药开发中具有重要意义。因此，本研究提供了一种预测工具，用于确定转变焓，可以根据热稳定性对不同蛋白质进行排序。
研究了 8 种不同类型 G、同种型 1 的治疗性单克隆抗体（mAb）的解折叠和聚集曲线。通过内源荧光（IF）光谱法和差示扫描量热法（DSC）测定了展开曲线。使用三态展开拟合模型来确定 IF 实验中大分子多结构域 mAb 的热力学参数，如展开事件的范特霍夫焓变（ΔH）和熵变（ΔS）。将推导的值与直接通过量热法获得的热力学参数进行比较。此外，还比较了 Fab 焓的差异，以预测聚集行为和蛋白质热稳定性。为此，通过尺寸排阻色谱（SEC）在加速热诱导应激条件下对液体配方的 mAb 进行了示例研究。
比较 IF 光谱法和 DSC 得出的热力学参数得出了相似的值。在 40°C 下进行热诱导应激产生的数据显示出与两种不同配方的 mAb 的 Fab 焓假设的相似稳定性排序，而在 25°C 下则不可能进行有意义的排序，因为无法观察到明显的热稳定性差异。与仅使用熔融温度或聚集起始温度和时间相比，将 Fab 焓额外考虑在内来预测 40°C 的 SEC 排序似乎更可靠。
我们表明，热力学分析可以帮助预测治疗性 mAb 在 40°C 下的展开和聚集特性。因此，分析热力学展开参数是一种有用的支持工具，可以区分 mAb 的热稳定性谱，为进一步的药物开发和临床研究提供参考。
