Effects of Monovalent Salt on Protein-Protein Interactions of Dilute and Concentrated Monoclonal Antibody Formulations

Abstract

In this study, we used sodium chloride (NaCl) to extensively modulate non-specific protein-protein interactions (PPI) of a humanized anti-streptavidin monoclonal antibody class 2 molecule (ASA-IgG2). The changes in PPI with varying NaCl (CNaCl) and monoclonal antibody (mAb) concentration (CmAb) were assessed using the diffusion interaction parameter kD and second virial coefficient B22 measured from solutions with low to moderate CmAb. The effective structure factor S(q)eff measured from concentrated mAb solutions using small-angle X-ray and neutron scattering (SAXS/SANS) was also used to characterize the PPI. Our results found that the nature of net PPI changed not only with CNaCl, but also with increasing CmAb. As a result, parameters measured from dilute and concentrated mAb samples could lead to different predictions on the stability of mAb formulations. We also compared experimentally determined viscosity results with those predicted from interaction parameters, including kD and S(q)eff. The lack of a clear correlation between interaction parameters and measured viscosity values indicates that the relationship between viscosity and PPI is concentration-dependent. Collectively, the behavior of flexible mAb molecules in concentrated solutions may not be correctly predicted using models where proteins are considered to be uniform colloid particles defined by parameters derived from low CmAb.

Characterizing Experimental Monoclonal Antibody Interactions and Clustering Using a Coarse-Grained Simulation Library and a Viscosity Model

“…Such interactions may not be accurately reflected by net PPI under dilute conditions and instead require measurements at high concentration that probe shorter length scales. Small-angle X-ray or neutron scattering (SAXS/SANS) quantifies S ( q ) across a wide range of length scales (∼5 to 500 nm) at high concentration and thus encodes information on short- and long-range interactions. − However, extraction of information about short-range attractions from experimental structure factors requires numerical simulations using models that approximate the shape of, and anisotropic interactions between, mAbs at a high concentration. ,− …”

Simulation of high‐concentration self‐interactions for monoclonal antibodies from well‐behaved to poorly‐behaved systems

“…However, at elevated protein concentrations, intermolecular distances decrease and, on average, proteins experience significant interactions with multiple neighboring proteins 20 . As such, although measurements at low protein concentration provide a fundamental quantity related to how proteins interact with one another, they are not generalizable predictors of high‐concentration behavior 19,22 …”

“…20 As such, although measurements at low protein concentration provide a fundamental quantity related to how proteins interact with one another, they are not generalizable predictors of high-concentration behavior. 19,22 To help address this, prior work used measurements of B 22 via SLS to parameterize relatively simple domain-level coarse-grained (CG) models for use in Monte Carlo (MC) molecular simulations to quantitatively predict self-interactions at high protein concentration that could be directly compared to experimental SLS data. 19,20,23 The predictions were found to be accurate for "simple-colloidal" net repulsions and mildly net attractive conditions.…”

Role of Domain–Domain Interactions on the Self-Association and Physical Stability of Monoclonal Antibodies: Effect of pH and Salt

“…Small-angle X-ray/neutron scattering (SAXS/SANS) have been widely used in recent years to characterize PPI directly from concentrated mAb formulations. ,,, Therefore, in this study, the PPI of NISTmAb molecules was also characterized using SAXS (Figure ). SAXS profiles of NISTmAb prepared in pH 6 buffer conditions are shown in Figure a and c. No upturn is observed in the low- q region from any of the SAXS profiles, indicating that there was no high-molecular-weight species formed in either 5 or 300 mM ionic strength at such a pH condition.…”

“…Small-angle X-ray/neutron scattering (SAXS/SANS) have been widely used in recent years to characterize PPI directly from concentrated mAb formulations. 26,34,55,56 Therefore, in this study, the PPI of NISTmAb molecules was also characterized using SAXS (Figure 7). SAXS profiles of NISTmAb prepared in pH 6 buffer conditions are shown in Figure 7a and c.…”