Protein aggregation and mitigation strategy in low pH viral inactivation for monoclonal antibody purification
| Autor: | Chao Huang, Zizhuo Xing, Sanchayita Ghose, Xuankuo Xu, Yuanli Song, Weixin Jin, Zheng Jian Li |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
Immunology
CHO Cells Protein aggregation Fluorescence spectroscopy 03 medical and health sciences chemistry.chemical_compound Colloid Protein Aggregates 0302 clinical medicine Cricetulus Dynamic light scattering Protein Domains Report Immunology and Allergy Animals Humans Surface charge Homology modeling 030304 developmental biology Protein Unfolding 0303 health sciences Antibodies Monoclonal Hydrogen-Ion Concentration Monomer chemistry 030220 oncology & carcinogenesis Biophysics Titration |
| Zdroj: | MAbs |
| Popis: | Significant amounts of soluble product aggregates were observed during low-pH viral inactivation (VI) scale-up for an IgG4 monoclonal antibody (mAb IgG4-N1), while small-scale experiments in the same condition showed negligible aggregation. Poor mixing and product exposure to low pH were identified as the root cause. To gain a mechanistic understanding of the problem, protein aggregation properties were studied by varying critical parameters including pH, hold time and protein concentration. Comprehensive biophysical characterization of product monomers and aggregates was performed using fluorescence-size-exclusion chromatography, differential scanning fluorimetry, fluorescence spectroscopy, and dynamic light scattering. Results showed IgG4-N1 partially unfolds at about pH 3.3 where the product molecules still exist largely as monomers owing to strong inter-molecular repulsions and favorable colloidal stability. In the subsequent neutralization step, however, the conformationally changed monomers are prone to aggregation due to weaker inter-molecular repulsions following the pH transition from 3.3 to 5.5. Surface charge calculations using homology modeling suggested that intra-molecular repulsions, especially between CH2 domains, may contribute to the IgG4-N1 unfolding at ≤ pH 3.3. Computational fluid dynamics (CFD) modeling was employed to simulate the conditions of pH titration to reduce the risk of aggregate formation. The low-pH zones during acid addition were characterized using CFD modeling and correlated to the condition causing severe product aggregation. The CFD tool integrated with the mAb solution properties was used to optimize the VI operating parameters for successful scale-up demonstration. Our research revealed the governing aggregation mechanism for IgG4-N1 under acidic conditions by linking its molecular properties and various process-related parameters to macroscopic aggregation phenomena. This study also provides useful insights into the cause and mitigation of low-pH-induced IgG4 aggregation in downstream VI operation. |
| Databáze: | OpenAIRE |
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