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.
RYMAN J T, MEIBOHM B. Pharmacokinetics of Monoclonal Antibodies[J]. CPT Pharmacometrics Syst Pharmacol, 2017,6(9):576-588.
CROMWELL M E, HILARIO E, JACOBSON F. Protein aggregation and bioprocessing[J]. AAPS J, 2006,8(3):e572-e579.
BRADER M L, ESTEY T, BAI S, et al. Examination of thermal unfolding and aggregation profiles of a series of developable therapeutic monoclonal antibodies[J]. Mol Pharm, 2015,12(4):1005-1017.
WANG W, SINGH S, ZENG D L, et al. Antibody structure, instability, and formulation[J]. J Pharm Sci, 2007,96(1):1-26.
HARRISON J S, GILL A, HOARE M. Stability of a single-chain Fv antibody fragment when exposed to a high shear environment combined with air-liquid interfaces[J]. Biotechnol Bioeng, 1998,59(4):517-519.
TASCHNER N, MULLER S A, ALUMELLA V R, et al. Modulation of antigenicity related to changes in antibody flexibility upon lyophilization[J]. J Mol Biol, 2001,310(1):169-179.
BRAUN A, KWEE L, LABOW M A, et al. Protein Aggregates Seem to Play a Key Role Among the Parameters Influencing the Antigenicity of Interferon Alpha (IFN-α) in Normal and Transgenic Mice[J]. Pharml Res, 1997,14(10):1472-1478.
ROBINSON M J, MATEJTSCHUK P, BRISTOW A F, et al. Tm-Values and Unfolded Fraction Can Predict Aggregation Rates for Granulocyte Colony Stimulating Factor Variant Formulations but Not under Predominantly Native Conditions[J]. Mol Pharm, 2018,15(1):256-267.
ERICSSON U B, HALLBERG B M, DETITTA G T, et al. Thermofluor-based high-throughput stability optimization of proteins for structural studies[J]. Anal Biochem, 2006,357(2):289-298.
SVILENOV H, MARKOJA U, WINTER G. Isothermal chemical denaturation as a complementary tool to overcome limitations of thermal differential scanning fluorimetry in predicting physical stability of protein formulations[J]. Eur J Pharm Biopharm, 2018,125:106-113.
JOHNSON C M. Differential scanning calorimetry as a tool for protein folding and stability[J]. Arch Biochem Biophys, 2013,531(1-2):100-109.
MAHLER H C, FRIESS W, GRAUSCHOPF U, et al. Protein aggregation: pathways, induction factors and analysis[J]. J Pharm Sci, 2009,98(9):2909-2934.
MANNO M, CRAPARO E F, PODESTA A, et al. Kinetics of different processes in human insulin amyloid formation[J]. J Mol Biol, 2007,366(1):258-274.
SHIRE S J, SHAHROKH Z, LIU J. Challenges in the development of high protein concentration formulations[J]. J Pharm Sci, 2004,93(6):1390-1402.
SALUJA A, FESINMEYER R M, HOGAN S, et al. Diffusion and sedimentation interaction parameters for measuring the second virial coefficient and their utility as predictors of protein aggregation[J]. Biophys J, 2010, 99(8):2657-2665.
BRUMMITT R K, NESTA D P, CHANG L, et al. Nonnative aggregation of an IgG1 antibody in acidic conditions: part 1. Unfolding, colloidal interactions, and formation of high-molecular-weight aggregates[J]. J Pharm Sci, 2011,100(6):2087-2103.
CONNOLLY B D, PETRY C, YADAV S, et al. Weak interactions govern the viscosity of concentrated antibody solutions: high-throughput analysis using the diffusion interaction parameter[J]. Biophys J, 2012,103(1):69-78.
LEHERMAYR C, MAHLER H C, MADER K, et al. Assessment of net charge and protein-protein interactions of different monoclonal antibodies[J]. J Pharm Sci, 2011,100(7):2551-2562.
SHI S, UCHIDA M, CHEUNG J, et al. Method qualification and application of diffusion interaction parameter and virial coefficient[J]. Int J Biol Macromol, 2013,62:487-493.
HE F, WOODS C E, BECKER G W, et al. High-throughput assessment of thermal and colloidal stability parameters for monoclonal antibody formulations[J]. J Pharm Sci, 2011,100(12):5126-5141.
PAUL M, VIEILLARD V, DA SILVA LEMOS R, et al. Long-term physico-chemical stability of diluted trastuzumab[J]. Int J Pharm,2013,448(1):101-104.
PAUL M, VIEILLARD V, JACCOULET E, et al. Long-term stability of diluted solutions of the monoclonal antibody rituximab[J]. Int J Pharm, 2012,436(1-2):282-290.
PAUL M, VIEILLARD V, ROUMI E, et al. Long-term stability of bevacizumab repackaged in 1mL polypropylene syringes for intravitreal administration[J]. Ann Pharm Fr, 2012,70(3):139-154.
QI W, ZENG Y, ORGEL S, et al. Preformulation study of highly purified inactivated polio vaccine, serotype 3[J]. J Pharm Sci, 2014,103(1):140-151.
OYAMA H, KOGA H, TADOKORO T, et al. Relation of Colloidal and Conformational Stabilities to Aggregate Formation in a Monoclonal Antibody[J]. J Pharm Sci, 2020,109(1):308-315.