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

Abstract

Attractive protein−protein interactions in concentrated monoclonal antibody (mAb) solutions may lead to the formation of clusters that increase viscosity. Here, we propose an analytical model that relates mAb solution viscosity to clustering by accounting for the contributions of suboptimal mAb packing within a cluster and cluster fractal dimension. The influence of short-range, anisotropic attractions and longrange Coulombic repulsion on cluster properties is investigated by analyzing the clustersize distributions, cluster fractal dimensions, radial distribution functions, and static structure factors from a library of coarse-grained molecular dynamics simulations. The library spans a vast range of mAb charges and attractive interactions in solutions of varying ionic strength. We present a framework for combining the viscosity model and simulation library to successfully characterize the attraction, repulsion, and clustering of an experimental mAb in three different pH and cosolute conditions by fitting the measured viscosity or structure factor from small-angle X-ray scattering. At low ionic strength, the cluster-size distribution is impacted by strong charges, and both the viscosity and net charge or structure factor and net charge must be considered to deconvolute the effects of short-range attraction and long-range repulsion.

Subclass Effects on Self-Association and Viscosity of Monoclonal Antibodies at High Concentrations

“…The calibration of the chips was verified at the start of each session by obtaining the η of 50–90% glycerol solution . The low shear η was measured for all systems at 500 and 1000 s –1 to verify the Newtonian plateau using the C-chip following a previously reported procedure. , For selected systems, η was also measured at up to 60,000 s –1 using the E-chip to detect the presence of shear thinning, with further discussion of the procedure in Section S1.1 of the Electronic Supporting Information (ESI). To reduce changes from small variation in concentration, the inherent η is also reported η normali normaln normalh = ln ( η / η 0 ) C normalp where η 0 is the solvent η and C p is the protein concentration.…”

“…SAXS at 130 mg/mL was measured on a custom setup from Xenocs (Holyoke MA) equipped with two scatterless square slits, a Xenocs Genix 3D microfocused X-ray source and a PILATUS 300 detector as described previously. , Briefly, the samples were sealed in quartz capillary tubes and exposed for ∼1 h in the “MAXS” mode (0.01 to 0.5 Å –1 ) to ensure a good signal-to-noise ratio. SAXS for protein concentrations <30 mg/mL was measured at beamline 7a of the Cornell High Energy Synchrotron Source (CHESS) using a PILATUS 300 detector as described previously . Briefly, multiple 1s exposures were collected and averaged to ensure low noise while verifying that no radiation damage was present .…”

Using Cluster Theory to Calculate the Experimental Structure Factors of Antibody Solutions

“…However, instead of plotting the intensity, we calculate an effective measured solution structure factor S ef f (q) where we divide the total normalized intensity with the monomer form factor, i.e. (25) where ⟨S c (q)⟩ w is the weight-average internal cluster structure factor. We thus compare the measured S ef f (q) with the calculated quantity ⟨S c (q)⟩ w S c ef f (q).…”

“… 15 Specifically to mAbs, there are several studies showing that the increased viscosity in concentrated solutions of mAbs is linked to cluster formation. 13 , 14 , 16 − 25 Many of these works have made attempts to characterize cluster formation in mAb solutions, and to interpret antibody solution properties through analogies with colloids or polymers. In particular, experimental scattering techniques were used to investigate protein interactions and self-association.…”

“…The presence of such clusters strongly influences the zero shear viscosity η 0 of concentrated solutions, resulting in an arrest transition at lower concentrations when compared to a purely monomeric solution . Specifically to mAbs, there are several studies showing that the increased viscosity in concentrated solutions of mAbs is linked to cluster formation. ,,− Many of these works have made attempts to characterize cluster formation in mAb solutions, and to interpret antibody solution properties through analogies with colloids or polymers. In particular, experimental scattering techniques were used to investigate protein interactions and self-association. ,,− Numerically, the first coarse-grained model for the study of antibody self-association dates back to 2012, where Chaudhri and co-workers proposed 12 and 26 bead-based models arranged in a Y-shape and demonstrated the formation of clusters for two model antibodies .…”