Effect of Hierarchical Cluster Formation on the Viscosity of Concentrated Monoclonal Antibody Formulations Studied by Neutron Scattering

Abstract

Recently, reversible cluster formation was identified as an underlying cause of anomalously large solution viscosities observed in some concentrated monoclonal antibody (mAb) formulations, which poses a major challenge to the use of subcutaneous injection for some mAbs. A fundamental understanding of the structural and dynamic origins of high viscosities in concentrated mAb solutions is thus of significant relevance to mAb applications in human health care, as well as being of scientific interest. Herein, we present a detailed investigation of an IgG1-based mAb to relate the short-time dynamics and microstructure to significant viscosity changes over a range of pharmaceutically relevant physiochemical conditions. The combination of light scattering, small-angle neutron scattering, and neutron spin echo measurement techniques conclusively demonstrates that, upon addition of Na2SO4, these antibodies form strongly bound reversible dimers at dilute concentrations that interact with each other to form large, loosely bound, transient clusters when concentrated. This hierarchical structure formation in solution causes a significant increase in the solution viscosity.

Biophysical characterization and molecular simulation of electrostatically driven self‐association of a single‐chain antibody

“…PPI have been related to the rates and mechanisms of protein aggregation, as well as the morphology of resulting aggregates . PPI may also influence protein solubility and the viscosity of protein solutions . Recent work has focused on optimization of protein solution properties using formulation and/or protein engineering approaches that target protein–protein interactions …”

“…[13][14][15] PPI may also influence protein solubility and the viscosity of protein solutions. [6][7][8]16 Recent work has focused on optimization of protein solution properties using formulation and/or protein engineering approaches that target protein-protein interactions. 4,8,17,18 Experimental characterization of electrostatic protein-protein interactions often relies on measuring bulk solution properties as a function of protein concentration across a range of pH and ionic strength conditions.…”

A Colloid Approach to Self-Assembling Antibodies

“…Despite the extensive experimental and theoretical work devoted to protein crowding and its effects on the resulting stability and flow properties at high protein concentration, our ability to predict for example the concentration dependence of the zero shear viscosity η 0 and the location of an arrest or glass transition is still limited [5][6][7][8][9][10][11][12][13][14]. For antibody solutions this is a particularly difficult problem as attractive interactions often lead to reversible self-association between the antibody molecules [7,9,[15][16][17], making the change in solution flow properties highly sensitive to the protein concentration [18][19][20][21].A number of studies have made attempts to characterize cluster formation in mAb solutions, and to interpret antibody solution properties through analogies with colloids or polymers. In particular, scattering techniques were used to investigate protein interactions and selfassociation in antibody formulations [7,9,[22][23][24][25][26][27].…”

“…For antibody solutions this is a particularly difficult problem as attractive interactions often lead to reversible self-association between the antibody molecules [7,9,[15][16][17], making the change in solution flow properties highly sensitive to the protein concentration [18][19][20][21].A number of studies have made attempts to characterize cluster formation in mAb solutions, and to interpret antibody solution properties through analogies with colloids or polymers. In particular, scattering techniques were used to investigate protein interactions and selfassociation in antibody formulations [7,9,[22][23][24][25][26][27]. While investigations of the self-association behavior of various mAb formulations have frequently addressed mAb selfassociation and its effect on flow properties, we are far from having any predictive understanding and a generally accepted methodology and/or theoretical framework to detect antibody association and model mAb interactions quantitatively.…”

Fluids with competing interactions. II. Validating a free energy model for equilibrium cluster size

“…This class of interactions can drive the reversible formation of equilibrium cluster phases composed of self-terminating aggregates (droplets) of monomers. Such cluster phases have been the focus of much recent work, ranging from theoretical and computational studies of idealized colloidal or nanoparticle suspensions [7][8][9][10][11][12][13][14][15][16][17][18][19] to experimental demonstrations for both archetypal colloidal particles [20][21][22][23] and heterogeneous monomers with anisotropic interactions like proteins [24][25][26][27][28][29][30][31] . Despite the range of materials and lengthscales, a) Electronic mail: truskett@che.utexas.edu the broad underlying formulation principles appear universal: induce (or allow) depletion (dispersion) attractions between monomers to drive aggregation while simultaneously controlling electrostatic repulsions between the ionic doublelayers of monomers such that they collectively build up to attenuate growth.…”