The Relationship between Protein–Protein Interactions and Liquid–Liquid Phase Separation for Monoclonal Antibodies

Abstract

Being able to predict and control concentrated solution properties for solutions of monoclonal antibodies (mAbs) is critical for developing therapeutic formulations. At higher protein concentrations, undesirable solution properties include high viscosities, opalescence, particle formation, and precipitation. The overall aim of this work is to understand the relationship between commonly measured dilute solution parameters, the reduced osmotic second virial coefficient b 22 and the diffusion interaction parameter k D and liquid−liquid phase separation, which occurs at higher protein concentrations. For globular proteins such as lysozyme or γB-crystallin, the location of the liquid−liquid coexistence curve is controlled by the net protein−protein interaction, which is related to b 22 . Because many mAbs undergo reversible self-association due to forming highly directional interactions, it is not known if b 22 can be used as a reliable predictor for LLPS since increasing the anisotropy in the interaction potential causes phase separation to occur at much stonger net protein−protein attractions or lower values of b 22 . Here, we map the coexistence curves for three mAbs, referred to as COE-01, COE-07, and COE-19, in terms of b 22 and k D values. The measurements are carried out at a low salt condition near the pI, where protein−protein interactions are expected to be anisotropic due to the presence of electrostatic attractions, and under salting-out conditions at high ammonium sulfate concentrations, which is expected to reduce the anisotropy by screening electrostatic interactions. We also show that deviations from a linear correlation between b 22 and k D can be used as an indicator of reversible self-association. Each of the mAbs under salting-out conditions follows the correlation supporting the hypothesis that protein−protein interactions are nonspecific, while deviations from the correlation occur for COE-01 and COE-19 under low salt conditions indicating the mAbs undergo reversible self-association. For five out of the six conditions, the onset of phase separation, as reflected by the reduced virial coefficient at the critical point b 22 c occurs in a small window −1.6 > b 22 c > −2.3, which is similar to what has been observed for lysozyme and for bovine γB-crystallin. Under low salt conditions, b 22 c ≈ −5.1 for COE-19, which we previously showed to self-associate into small oligomers. Our findings suggest that under conditions where mAb interactions are weakly anisotropic, such as occur at high salt conditions, phase separation will begin to occur in a small window of b 22 . Deviations from the window can occur when mAbs undergo reversible self-association, although this is not always the case and likely depends upon whether or not highly directional interactions are passivated in the oligomer formation. We expect fitting LLPS measurements to simplified interaction models for mAbs will provide additional insight into the nature of the protein−protein interactions and guide their development for calculating ...

Combined Effects of Pressure and Ionic Strength on Protein–Protein Interactions: An Empirical Approach

“…The calculated b 2 * for the AS-free solution matches well with prior measurements of ovalbumin . Mapping interaction data in terms of the reduced coefficient b 2 * rather than B 22 allows for a comparison of interaction strength with data in the literature for other protein systems. , …”

Correlating Surface Activity with Interface-Induced Aggregation in a High-Concentration mAb Solution

“…Much effort has been directed toward understanding the impacts of formulating high-concentration drugs on stability and protein particle formation. Previous studies include the development of experimental and molecular simulation techniques to understand how intermolecular protein interactions impact clustering and aggregation at high protein concentrations. ,− , However, most studies involving high concentrations of protein therapeutics are devoted to studying bulk solutions. Thus, the interfacial behavior of protein films formed in concentrated mAb solutions is still not well understood.…”

Protein Association in Solution: Statistical Mechanical Modeling

“…The importance of this discovery is that it allows a multi-protein action (crystallization) to be simplified through modeling, only requiring understanding of the first step, namely, the protein-protein pair interaction, measured by the second virial coefficient. Other binary coefficients, such as k D , obtained from dynamic light scattering [54,55] and the Huggins viscosity coefficient [56], have proven to be very informative as well. For example, it has been shown that, as with B 22 , k D is correlated with the cloud point temperature, which determines the two-phase region in protein solutions [57].…”