Effects of Protein Conformation, Apparent Solubility, and Protein–Protein Interactions on the Rates and Mechanisms of Aggregation for an IgG1Monoclonal Antibody

Abstract

Non-native protein aggregation is a key degradation pathway of immunoglobulins. In this work, the aggregation kinetics of an immunoglobulin gamma-1 monoclonal antibody (IgG1 mAb) in different solution environments was monitored over a range of incubation temperatures for up to seven months using size exclusion chromatography. Histidine and citrate buffers with/without sodium chloride were employed to modulate the mAb's conformational stability, solubility (in the presence of polyethylene glycol, PEG), and protein-protein interactions as measured by differential scanning calorimetry, PEG precipitation, and static light scattering, respectively. The effect of these parameters on the mechanism(s) of mAb aggregation during storage at different temperatures was determined using kinetic models, which were used to fit aggregation data to determine rate constants for aggregate nucleation and growth processes. This approach was used to investigate the effects of colloidal protein-protein interactions and solubility values (in PEG solutions) on the mechanisms and rates of IgG1 mAb aggregation as a function of temperature-induced structural perturbations. Aggregate nucleation and growth pathways for this IgG1 mAb were sensitive to temperature and overall conformational stability. Aggregate growth, on the other hand, was also sensitive to conditions affecting the solubility of the mAb, particularly at elevated temperatures.

Effect of Polysorbate 20 and Polysorbate 80 on the Higher-Order Structure of a Monoclonal Antibody and Its Fab and Fc Fragments Probed Using 2D Nuclear Magnetic Resonance Spectroscopy

“…These results imply that despite exhibiting lower T onset for conformational changes at pH 5.8, Fab aggregates less at pH 5.8 compared to pH 8.0, indicating that the colloidal stability is playing a major role in Fab aggregation. These results are quite consistent with recent studies which indicated that the colloidal stability can account for the lack of a clear relationship between the conformational stability and aggregation of mAbs 49,50 .…”

“…In the first study on the aggregation behavior of ten different IgG1 antibodies 49 , there was no absolute trend between the conformational stability and aggregation of the IgG1 molecules, implying that colloidal stability plays a major role in IgG1 aggregation. In the second study 50 , changing the solution pH from 6.5 to 4.5 reduced the thermal stability of IgG1; however, less protein aggregation was observed at pH 4.5, because of the increased colloidal stability of the protein. Decreasing pH to acidic values increases the net charge on the protein surface leading to increased repulsive interactions between individual protein molecules, thus leading to an increase in the colloidal stability of the protein.…”

Physicochemical Stability of Monoclonal Antibodies: A Review

“…17 Independently from any other factor, proteins have a certain propensity to self-associate, which has been shown to be particularly implicated in aggregates growth. 66 This phenomenon seems to be governed by electrostatic interactions end by dipole moments between protein surfaces, 67 and might engage preferentially Fab-Fab interactions. 68 Self-association highly impacts solution viscosity and is dependent on the ionic strength of the solution.…”

Stress Factors in mAb Drug Substance Production Processes: Critical Assessment of Impact on Product Quality and Control Strategy

“…[11][12][13] The protein is exposed to various buffer and salt solutions necessitated by the chromatographic and filtration steps of the process, which can also induce some aggregate formation. [14][15][16][17] Many of the process steps involve filtration, a process that creates a complex set of stress conditions including shear effects, cavitation, localized high concentrations of protein, and interactions at the filter membrane surface. This can even result in the formation of a gel layer 18 which is then sloughed out into the bulk as aggregates and can also act as a nucleus for further aggregate growth.…”