Solubility Challenges in High Concentration Monoclonal Antibody Formulations: Relationship with Amino Acid Sequence and Intermolecular Interactions

Abstract

The purpose of this work was to elucidate the molecular interactions leading to monoclonal antibody self-association and precipitation and utilize biophysical measurements to predict solubility behavior at high protein concentration. Two monoclonal antibodies (mAb-G and mAb-R) binding to overlapping epitopes were investigated. Precipitation of mAb-G solutions was most prominent at high ionic strength conditions and demonstrated strong dependence on ionic strength, as well as slight dependence on solution pH. At similar conditions no precipitation was observed for mAb-R solutions. Intermolecular interactions (interaction parameter, kD) related well with high concentration solubility behavior of both antibodies. Upon increasing buffer ionic strength, interactions of mAb-R tended to weaken, while those of mAb-G became more attractive. To investigate the role of amino acid sequence on precipitation behavior, mutants were designed by substituting the CDR of mAb-R into the mAb-G framework (GM-1) or deleting two hydrophobic residues in the CDR of mAb-G (GM-2). No precipitation was observed at high ionic strength for either mutant. The molecular interactions of mutants were similar in magnitude to those of mAb-R. The results suggest that presence of hydrophobic groups in the CDR of mAb-G may be responsible for compromising its solubility at high ionic strength conditions since deleting these residues mitigated the solubility issue.

Retracted: Molecular characterization of excipients’ preferential interactions with therapeutic monoclonal antibodies

“…[14] Human antibodies show limited conformational and colloidal stability, which often leads to challenges in manufacturing from the expression and purification steps to the final product formulation. [15][16][17][18] Substantial interest has been directed towards understanding the effects of excipients on proteins to identify ideal formulation recipes. [19] The wide-ranging effects of excipients remain the subjects of continuing studies, [20][21][22][23] including recent investigations of excipient-protein interactions.…”

An accelerated surface-mediated stress assay of antibody instability for developability studies

“…As a measure of colloidal stability and propensity to aggregation, we next determined the diffusion interaction parameter K D of each variant ( Figure 5 ), which is directly related to the second virial coefficient B 22 . 71 K D has been correlated with antibody solubility, 72 antibody solution viscosity 73 and aggregation propensity. 74 All determined K D values of the variants were negative and below −5.34 mL/g, suggesting that protein attraction was prevailing.…”

Toward Drug-Like Multispecific Antibodies by Design

“…Although solubility enhancements can be achieved with higher ionic strength formulations that prevent electrostatic association, these formulations can cause adverse reactions, such as detachment and retinal edema [ 151 ], which motivates the use of protein engineering strategies to improve solubility. While CDRs are known to play a dominant role in antibody stability and affinity, they may also be large contributors to solubility [ 90 , 91 , 93 , 99 , 125 , 152 , 153 , 154 ]. As such, CDR engineering is a major focus in the development of antibodies with favorable solubility in concentrated formulations.…”