Solution pH That Minimizes Self-Association of Three Monoclonal Antibodies Is Strongly Dependent on Ionic Strength

Abstract

Monoclonal antibodies display highly variable solution properties such as solubility and viscosity at elevated concentrations (>50 mg/mL), which complicates antibody formulation and delivery. To understand this complex behavior, it is critical to measure the underlying protein self-interactions that govern the solution properties of antibody suspensions. We have evaluated the pH-dependent self-association behavior of three monoclonal antibodies using self-interaction chromatography for a range of pH values commonly used in antibody formulations (pH 4.4-6). At low ionic strength (<25 mM), we find that each antibody is more associative at near-neutral pH (pH 6) than at low pH (pH 4.4). At high ionic strength (>100 mM), we observe the opposite pH-dependent pattern of antibody self-association. Importantly, this inversion in self-association behavior is not unique to multidomain antibodies, as similar pH-dependent behavior is observed for some small globular proteins (e.g., ribonuclease A and α-chymotrypsinogen). We also find that the opalescence of concentrated antibody solutions (90 mg/mL) is minimized at low ionic strength at pH 4.4 and high ionic strength at pH 6, in agreement with the self-interaction measurements conducted at low antibody concentrations (5 mg/mL). Our results highlight the complexity of antibody self-association and emphasize the need for systematic approaches to optimize the solution properties of concentrated antibody formulations.

Engineered Autonomous Human Variable Domains

“…Conformational stability is primarily governed by solvent-shielded residues within the domains or at domain interfaces and is critical for long-term antibody activity. The colloidal stability of human dAbs, which is arguably the main obstacle holding back the field [78, 79], arises from intermolecular self-interactions mediated by solvent-exposed residues that are commonly located in CDRs [80]. Strategies for improving colloidal stability include altering solution properties such as pH and ionic strength, and the use of excipients [67, 77].…”

Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody

“…can either increase or decrease the extent of RSA of mAbs, depending on the distinct nature of the non-covalent interactions for an individual mAb formulated under specific conditions. 18,19 A variety of analytical tools have been used to characterize the RSA of proteins, and related effects on solution properties, including dynamic light scattering (DLS), 21 composition-gradient multi-angle static light scattering, 20,21 isothermal titration calorimetry, 15 surface plasmon resonance, 22 proton magnetic relaxation dispersion, 23 nuclear magnetic resonance, 24 fluorescence resonance energy transfer, 25 mass spectrometry (MS), 26 self-interaction nanoparticle spectroscopy, 27 size-exclusion chromatography, 28 analytical ultracentrifugation, 29 small angle X-ray scattering, 30 and atomic force microscopy. 31 Most of these measurements provide reliable data only at low-to-moderate protein concentrations (»1-20 mg/mL).…”

A systematic multitechnique approach for detection and characterization of reversible self-association during formulation development of therapeutic antibodies

“…19,25 Because the oligomeric and monomeric species exist in equilibrium with each other, formulation efforts such as optimization of protein concentration, pH, and ionic strength can shift the equilibrium toward the monomeric species. 26,27 A variety of analytical techniques are available to detect and characterize self-association. These techniques include but are not limited to proton magnetic relaxation dispersion, 28,29 surface plasmon resonance, 30,31 isothermal titration calorimetry, 32 nuclear magnetic resonance, 33 fluorescence energy transfer, 34 mass spectrometry, 35 self-interaction nano-particle spectroscopy, 36 analytical ultracentrifugation (AUC), 20,37 dynamic light scattering (DLS), and composition-gradient multiangle static light scattering (CG-MALS).…”