Multi-Dimensional Stability Analysis of Monoclonal Antibodies
GUO Sha, ZHANG Feng, YU Chuan-fei, WU Gang, CUI Yong-fei, WANG Lan*
Division of Monoclonal Antibody Products, Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Product, National Institutes for Food and Drug Control, Beijing 102629, China
Abstract：OBJECTIVE To explore the significance of stability evaluation of monoclonal antibodies (mAbs), an important part of druggability of mAbs, from the aspects of structural stability, colloidal stability and weak intermolecular interaction. METHODS Taking trastuzumab, rituximab and bevacizumab as examples, the stability characteristics of the three mAbs were explored from different aspects, thus to demonstrate various dimensions of stability evaluation of mAbs. Firstly, the structural stability of antibody molecules was evaluated by detecting the melting temperature (Tm) through intrinsic fluorescence (IF). Secondly, static light scattering technique (SLS) was used to predict the colloidal stability of mAbs. Subsequently, isothermal stability tests (60 ℃, 48 h) were used to monitor the stability performance of the samples in real time. Lastly, dynamic light scattering technique (DLS) was used to predict protein aggregation tendency from the point of weak intermolecular interaction of dissolved proteins. RESULTS IF detection showed that the first melting temperature (Tm1) of the three mAbs were all above 65 ℃, indicating that their structures were stable. SLS detection showed that trastuzumab had the best colloidal stability since no obvious aggregation signal was detected at 95 ℃, while rituximab and bevacizumab showed obvious aggregation at about 70 ℃. The isothermal stability test also showed similar results. The application of DLS techniques showed that the second virial coefficient (B22) and diffusion interaction coefficient (KD) of trastuzumab were positive (3.19×10-3, 65.46), indicating better long term colloidal stability, rituximab was suboptimal, while the B22 and KD of bevacizumab were both negative (-1.44×10-4,-6.46), suggesting that the molecules were prone to be self-associated. CONCLUSION In this paper, the contents, levels and depth of the stability evaluation of mAbs are characterized by various techniques and discussed from multi-aspects, which provides reference for antibody molecular screening, formulation optimization, different combinations between antibody molecules and formulations, stability evaluation and so on.
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2021, Vol. 56 
