Controlling the viscosities of antibody solutions through control of their binding sites

Abstract

For biotechnological drugs, it is desirable to formulate antibody solutions with low viscosities. We go beyond previous colloid theories in treating protein–protein self–association of molecules that are antibody–shaped and flexible and have spatially specific binding sites. We consider interactions either through fragment antigen (Fab–Fab) or fragment crystalizable (Fab–Fc) binding. Wertheim's theory is adapted to compute the cluster–size distributions, viscosities, second virial coefficients, and Huggins coefficients, as functions of antibody concentration. We find that the aggregation properties of concentrated solutions can be anticipated from simpler–to–measure dilute solutions. A principal finding is that aggregation is controllable, in principle, through modifying the antibody itself, and not just the solution it is dissolved in. In particular: (i) monospecific antibodies having two identical Fab arms can form linear chains with intermediate viscosities. (ii) Bispecific antibodies having different Fab arms can, in some cases, only dimerize, having low viscosities. (iii) Arm–to–Fc binding allows for three binding partners, leading to networks and high viscosities.

Theory for the Liquid–Liquid Phase Separation in Aqueous Antibody Solutions

“…To construct the Y-shape molecule (see Figure 1), we use seven hard spheres of diameter σ , having attractive short-range sites located on the surface. 33 The sites named A are shown in green, sites named B in blue, C in red, D in orange, and sites E are shown in black; only the sites of the same color are allowed to make bonds. Sphere number 1 is at the center of the molecule and has 3 D sites, contacting spheres 2, 3, and 4.…”

“…In this study we model the mAbs molecule as an assembly of seven hardspheres. 33 The model molecule has a shape of the letter Y, two upper terminals are named fragment antigen-binding domains Fab and Fab′ (in the case of unequal Fab domains), and the bottom one is called the fragment crystallizable, Fc, terminal (see Figure 1). For simplicity we denote Fab by the letter A, Fab′ by B, and the Fc domain by C. Such Y-shaped particles may then interact via the attractive sites A, B, and C to form aggregates.…”

“…In the preceding study 33 we analyzed experimental viscosities and we did not examine possible phase separations. To fill this gap, our present work explores the liquid–liquid phase separation (LLPS) in aqueous monoclonal antibody solutions.…”

Potential and limits of a colloid approach to protein solutions

“…There have again been numerous studies where researchers tried to determine the molecular origins of self-association, and to predict enhanced self-association and viscosity based on simple low concentration parameters such as B 2 * and k D , albeit mostly with limited success. 130,134,[166][167][168][169][170][171][172] Given the difficulties in trying to reproduce D s c (q*)/D 0 or the location of the arrest line for globular proteins, it seems out of question to arrive at some prediction of the location of an arrest line or the concentration dependence of the relative viscosity Z r for mAbs that exhibit a concentration-dependent self-association and a corresponding strong increase of Z r at higher concentrations. However, the observation previously made for lysozyme cluster fluids, where Z r was found to follow the Quemada relation, 61 clearly indicates that there is hope!…”

Toward Drug-Like Multispecific Antibodies by Design

“…To better understand the molecular origins of viscous antibody formulations and particularly for bispecific antibodies, a previous theoretical study used covalently-linked hard spheres with different attractive interactions to model antibodies as Y-shaped molecules [ 172 ]. Interestingly, such models show that bispecific antibodies with two different binding arms tend to be less viscous than monospecific IgG, as linkages through a single Fab arm form dimers instead of the higher-order networks formed by bivalent monospecific antibodies with similar Fab arms.…”