Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians University, Butenandtstrasse 5-13, Munich D-81377, Germany.
Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians University, Butenandtstrasse 5-13, Munich D-81377, Germany.
J Pharm Sci. 2020 Jun;109(6):2048-2058. doi: 10.1016/j.xphs.2020.03.011. Epub 2020 Mar 16.
Understanding the formulation features that ensure sufficient stability during long-term storage is critical for developing next-generation therapeutic proteins. In this work, we investigate the physical stability of a bispecific antibody (Bis-mAb) in 12 different formulation conditions. Isothermal chemical denaturation with urea indicates a higher resistance to denaturant-induced unfolding when pH is increased from 5.0 to 6.5 but shows minor influence from the buffer type and ionic strength. Dynamic and static light scattering are used to derive the interaction parameter (k) and second virial coefficient (A), respectively. These two parameters indicate that Bis-mAb exhibits highest colloidal stability in formulations containing 10 mM histidine buffer without added sodium chloride. Further, we observe that the highest relative monomer yield (RMY) after isothermal refolding, that is the highest refoldability, from urea is measured for the low ionic strength histidine formulations. Finally, we show long-term stability data on all 12 Bis-mAb formulations after storage at 4 °C and 25 °C for 12 months. The least amount of soluble aggregates and subvisible particles were detected in the Bis-mAb formulations with the highest colloidal stability and refoldability from urea. We suggest that the optimization of these two features is crucial for obtaining physically stable formulations of Bis-mAb.
了解确保长期储存期间充分稳定性的配方特点对于开发下一代治疗性蛋白质至关重要。在这项工作中，我们研究了 12 种不同配方条件下双特异性抗体 (Bis-mAb) 的物理稳定性。等度化学变构用脲表明，当 pH 从 5.0 增加到 6.5 时，对变性剂诱导的展开具有更高的抵抗力，但缓冲液类型和离子强度的影响较小。动态和静态光散射分别用于推导相互作用参数 (k) 和第二维里系数 (A)。这两个参数表明，Bis-mAb 在不含氯化钠的 10 mM 组氨酸缓冲液的配方中表现出最高的胶体稳定性。此外，我们观察到，从脲中进行等温复性后，最高的相对单体产率 (RMY)，即最高的复性率，是在低离子强度的组氨酸配方中测量的。最后，我们展示了所有 12 种 Bis-mAb 配方在 4°C 和 25°C 下储存 12 个月后的长期稳定性数据。在具有最高胶体稳定性和从脲中复性能力的 Bis-mAb 配方中，检测到的可溶性聚集体和亚可见颗粒最少。我们建议优化这两个特性对于获得 Bis-mAb 的物理稳定配方至关重要。
