Characterization of Native Reversible Self-Association of a Monoclonal Antibody Mediated by Fab-Fab Interaction

Abstract

The native reversible self-association of monoclonal antibodies has been associated with high viscosity, liquid-liquid, and liquid-solid phase separation. We investigated the native reversible self-association of an IgG1, which exerts this association even at low protein concentrations, in detail to gain further understanding of this phenomenon by extensive characterization of the association as a function of multiple factors, namely pH, temperature, salt concentration, and protein concentration. The nature of the self-association of the full-length IgG1 as well as the corresponding Fab and Fc fragment was studied by viz. size exclusion chromatography combined with multiangle light scattering, batch dynamic and static light scattering, analytical ultracentrifugation, small angle X-ray scattering, asymmetric flow field flow fractionation coupled with multiangle light scattering, and intrinsic fluorescence. We rationalized the self-association as a combination of hydrophobic and electrostatic interactions driven by the Fab fragments. Finally, we investigated the long-term stability of the IgG1 molecule.

Conformational Ensembles of Antibodies Determine Their Hydrophobicity

“…Taken together, these findings indicate that protonation states are not the primary source of error in our hydrophobicity calculations. On the other hand, the abovementioned study by Gentiluomo et al (8) found a strong influence of the pH on aggregation. However, we note that hydrophobicity and aggregation propensity are different properties.…”

“…A major problem in the development process is the inherent tendency of some concentrated protein solutions to form aggregates. This problem may be influenced by various factors such as temperature, mechanical stress, and pH ( 7 , 8 , 9 ). Aggregation can be reversible or irreversible and covalent or noncovalent, and the resulting aggregates may be soluble or insoluble ( 8 ).…”

“…This problem may be influenced by various factors such as temperature, mechanical stress, and pH ( 7 , 8 , 9 ). Aggregation can be reversible or irreversible and covalent or noncovalent, and the resulting aggregates may be soluble or insoluble ( 8 ). Furthermore, multiple mechanisms of antibody aggregation have been discussed in literature ( 7 , 10 ), indicating that various factors contribute to antibody aggregation.…”

Detection of Isopeptide Bonds in Monoclonal Antibody Aggregates

“…The localization of the interface sites can be driven by both amino acid sequence and higher order structure of the therapeutic. There are examples of dimer formation being driven by Fab-Fab, Fc-Fc and Fab-Fc interactions (15)(16)(17)(18)(19). However, the behavior of dimers with regards to driving aggregation of mAbs is poorly understood.…”

Opalescence Measurements: Improvements in Fundamental Knowledge, Identifying Sources of Analytical Biases, and Advanced Applications for the Development of Therapeutic Proteins

“…, and liquid-liquid phase separation 5,6 . At the root of these phenomena are complex protein-protein interactions (PPI) that are governed by the intrinsic molecular properties of the protein molecules themselves 7 , as well as the extrinsic solution and environmental conditions like pH, temperature, protein concentration and the presence of excipients. While there are many different instruments available to the pharmaceutical scientist for measuring opalescence, the specific capabilities, optical configurations and calibration practices can vary considerably and are not always evident to the end user.…”