Xin Lun, Prorok Monika, Zhang Zhe, Barboza Guilherme, More Rahul, Bonfiglio Michael, Cheng Lv, Robbie Kevin, Ren Steven, Li Yunsong
BioDev Department WuXi Biologics USA, 1 Cedarbrook Dr, Cranbury, NJ, 08512, USA.
CMC Management, WuXi Biologics, Cranbury, NJ, USA.
Pharm Res. 2025 Jan;42(1):151-171. doi: 10.1007/s11095-024-03801-3. Epub 2025 Jan 17.
High concentration protein formulation (HCPF) development needs to balance protein stability attributes such as conformational/colloidal stability, chemical stability, and solution properties such as viscosity and osmolality.
A three-phase design is established in this work. In Phase 1, conformational and colloidal stability are measured by 384-well-based high-throughput (HT) biophysical screening while viscosity reduction screening is performed with HT viscosity screening. Collectively, the biophysical and viscosity screening data are leveraged to design the phase 2 of short-term stability study, executed using 96-well plates under thermal and freeze/thaw stresses. In phase 2, samples are analyzed by stability-indicating assays and processed with pair-wise Student's t-test analyses to choose the final formulations. In phase 3, the final formulations are then confirmed through a one-month accelerated stability in glass vials.
Using a model antibody A (mAb-A), the initial HT screening successfully established the 384-well based platform. A lead formulation was chosen from the second round based on statistical analyses and subsequently tested against the commercial formulation of mAb-A as a control. Compared to the control, the lead formulation reduced the viscosity of mAb-A by 30% and decreased subvisible particles after thermal stress by 80%.
HT biophysical screening in 384-well plates was demonstrated to effectively guide the rational design of a high-throughput stability screening study using 96-well plates. This platform enables the identification of a high concentration formulation within seven weeks within the first two phases of study that strategically balance stability with solution properties, thus achieving a rapid development of HCPF.
高浓度蛋白质制剂（HCPF）的开发需要平衡蛋白质的稳定性属性，如构象/胶体稳定性、化学稳定性，以及溶液性质，如粘度和渗透压。
本研究建立了一个三阶段设计。在第一阶段，通过基于384孔板的高通量（HT）生物物理筛选来测量构象和胶体稳定性，同时使用HT粘度筛选进行粘度降低筛选。综合利用生物物理和粘度筛选数据来设计第二阶段的短期稳定性研究，该研究在热应激和冻融应激条件下使用96孔板进行。在第二阶段，通过稳定性指示分析对样品进行分析，并采用成对学生t检验分析进行处理，以选择最终制剂。在第三阶段，然后通过在玻璃小瓶中进行为期一个月的加速稳定性试验来确认最终制剂。
使用模型抗体A（mAb-A），初始的HT筛选成功建立了基于384孔板的平台。基于统计分析从第二轮中选择了一种先导制剂，并随后将其与mAb-A的商业制剂作为对照进行测试。与对照相比，先导制剂使mAb-A的粘度降低了30%，并使热应激后的亚可见颗粒减少了80%。
在384孔板中进行的HT生物物理筛选被证明能有效地指导使用96孔板的高通量稳定性筛选研究的合理设计。该平台能够在研究的前两个阶段的七周内识别出一种高浓度制剂，该制剂在稳定性和溶液性质之间进行了战略平衡，从而实现了HCPF的快速开发。
