Understanding mAb aggregation during low pH viral inactivation and subsequent neutralization

Abstract

Monoclonal antibodies (mAbs) and related recombinant proteins continue to gain importance in the treatment of a great variety of diseases. Despite significant advances, their manufacturing can still present challenges owing to their molecular complexity and stringent regulations with respect to product purity, stability, safety, and so forth. In this context, protein aggregates are of particular concern due to their immunogenic potential. During manufacturing, mAbs routinely undergo acidic treatment to inactivate viral contamination, which can lead to their aggregation and thereby to product loss. To better understand the underlying mechanism so as to propose strategies to mitigate the issue, we systematically investigated the denaturation and aggregation of two mAbs at low pH as well as after neutralization. We observed that at low pH and low ionic strength, mAb surface hydrophobicity increased whereas molecular size remained constant. After neutralization of acidic mAb solutions, the fraction of monomeric mAb started to decrease accompanied by an increase on average mAb size. This indicates that electrostatic repulsion prevents denatured mAb molecules from aggregation under acidic pH and low ionic strength, whereas neutralization reduces this repulsion and coagulation initiates. Limiting denaturation at low pH by D-sorbitol addition or temperature reduction effectively improved monomer recovery after neutralization. Our findings might be used to develop innovative viral inactivation procedures during mAb manufacturing that result in higher product yields. K E Y W O R D SANS fluorescence, downstream processing, monoclonal antibodies, protein aggregation, protein unfolding, viral inactivation

Effect of the fluorescent probes ThT and ANS on the mature amyloid fibrils

“…The hydrophobic probe ANS is commonly used in protein folding studies as a tool for the detection of the formation of partially folded intermediates with solvent-exposed hydrophobic clusters (see, e.g. [32][33][34][35]). This dye has low fluorescence quantum yield in polar environments, such as aqueous solutions, but its fluorescence is dramatically increased in nonpolar environments.…”

Characterisation of a novel ACE2-based therapeutic with enhanced rather than reduced activity against SARS-CoV2 variants

“…Finally, we achieved high expression yields for our ACE2 receptor decoy, using a non-optimised transient transfection system and single-step protein-A affinity purification. The stability of the construct at pH 3.5, while showing no change in its aggregation profile and binding capacity, lends to its suitability for antibody-like purification processes such as low pH viral inactivation 45 . We have also determined an optimal pH to enhance stability of the protein using a commonly adopted His buffer for clinical-stage monoclonal antibodies 46 , raising the Tm to 52°C.…”

Insights into the Conformation and Self-Association of a Concentrated Monoclonal Antibody using Isothermal Chemical Denaturation and Nuclear Magnetic Resonance

“…But, at pH 4, NMR analysis established that mAb1 was in a native state with greater polypeptide conformational freedom that we attributed to intramolecular repulsions. 6,31,45,46,52 We gained deeper insight into the dynamics of mAb1 that lead to aggregation at high concentrations by adding NMR to the characterization regimen. We also showed the utility of NMR in providing insight into the pH and concentration dependent microstructures of mAb1.…”

“…Interestingly, an earlier study of IgG1 stability in acidic conditions also reported lower aggregation rates attributed to charge-mediated repulsions, despite detecting greater exposed hydrophobic area due to greater conformational flexibility. 31 Hydrophobic interactions are also one of the major factors contributing to attractive PPI. 15,17,39,47 Although the hydrophobic surface of mAb1 is relatively unchanged over the pH range as shown by the homology modeling (Figure 2 and 1S), the impact of hydrophobic interactions may also vary with pH.…”

“…Partially disrupted conformations have exposed hydrophobic areas and are more aggregation prone especially at high concentrations when the mutual distances are ≤ 1 nm. 7,[16][17][18]30,31 Therefore, we investigated the reason for the relatively low aggregation rate of mAb1 at lower pH values between 4 and 5, across a wide concentration range, using NMR. NMR directly provided unequivocal information on the pH -dependent conformation of mAb1 with increasing concentration.…”