Shrivastava Anuj, Rathore Anurag S
Department of Chemical Engineering, Centre of Excellence for Biopharmaceutical Technology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India.
Pharm Res. 2025 Jun 6. doi: 10.1007/s11095-025-03874-8.
This study investigates the stability and kinetics of degradation when monoclonal antibodies (mAbs) process intermediates are stored in commonly used Protein A elution buffers, including citrate, acetate, and glycine, at varying pre-existing aggregates levels (low: 1-5%, moderate: 5-15% and high: 15-25%) at 4°C and 30°C to simulate standard and worst-case conditions.
mAb samples were subjected to thermal stress to achieve different levels of initial aggregates. The pre-aggregated samples were then incubated in different buffers at 4°C and 30°C to assess aggregation rates and stability. Aggregates were quantified using dynamic light scattering (DLS) integrated with machine learning (ML).
At 30°C, half-life reductions for citrate, acetate, and glycine buffers were 6.30-fold, 6.48-fold, and 9.64-fold, respectively, compared to 4°C, with glycine buffer offering the best stability, while citrate buffer provides the least. At higher initial aggregate levels, half-lives decreased by 2.15-, 1.95-, and 1.73-fold for citrate, acetate, and glycine buffers, respectively, compared to lower initial aggregates. Second-order kinetics dominated in samples having lower initial aggregate levels, while first-order kinetics prevailed in medium and high initial aggregate levels. Glycine buffer at 4°C with low initial aggregates achieved the highest half-life of 129 days, whereas citrate buffer at 30°C with high initial aggregate exhibited the lowest stability, with a half-life of 3.5 days.
The findings highlight the significance of using an optimal buffer system and appropriate storage conditions for in-process intermediates during mAb manufacturing to have a robust process that delivers safe and efficacious biotherapeutic products.
本研究调查了单克隆抗体（mAb）工艺中间体在常用的蛋白A洗脱缓冲液（包括柠檬酸盐、醋酸盐和甘氨酸）中，于4°C和30°C下，在不同的预先存在的聚集体水平（低：1 - 5%，中：5 - 15%，高：15 - 25%）下储存时的降解稳定性和动力学，以模拟标准和最坏情况条件。
mAb样品经受热应力以达到不同水平的初始聚集体。然后将预先聚集的样品在4°C和30°C下在不同缓冲液中孵育，以评估聚集速率和稳定性。使用与机器学习（ML）相结合的动态光散射（DLS）对聚集体进行定量。
在30°C时，柠檬酸盐、醋酸盐和甘氨酸缓冲液的半衰期分别比4°C时降低了6.30倍、6.48倍和9.64倍，甘氨酸缓冲液稳定性最佳，而柠檬酸盐缓冲液稳定性最差。与较低的初始聚集体水平相比，在较高的初始聚集体水平下，柠檬酸盐、醋酸盐和甘氨酸缓冲液的半衰期分别降低了2.15倍、1.95倍和1.73倍。在初始聚集体水平较低的样品中，二级动力学占主导，而在中等和高初始聚集体水平下，一级动力学占主导。4°C下低初始聚集体的甘氨酸缓冲液半衰期最长，为129天，而30°C下高初始聚集体的柠檬酸盐缓冲液稳定性最低，半衰期为3.5天。
研究结果突出了在mAb生产过程中，为工艺中间体使用最佳缓冲系统和适当储存条件的重要性，以确保生产出安全有效的生物治疗产品的稳健工艺。
