Aggregation in concentrated protein solutions: insights from rheology and small-angle neutron scattering

Maria Monica Castellanos (Pennsylvania State University)

Aggregation of bio-therapeutic proteins is currently one of the major challenges in the pharmaceutical industry, because aggregates could induce immunogenic responses and compromise the quality of the product. The first part of this talk will focus on the rheology of protein solutions, and how aggregation affects the bulk and interfacial viscosity of proteins. Solutions of Bovine Serum Albumin (BSA, isoelectric point pI=4.95) in surfactant-free Phosphate Buffered Saline (pH = 7.4), and a monoclonal antibody (mAb, IgG1, pI = 8.6, 145 kDa) in its formulation buffer (20 mM Histidine/Histidine Hydrochloride at pH = 6.0, 60 mg/mL of trehalose and 0.2 mg/mL polysorbate 80) were studied. The mAb solution was incubated at 40 oC to promote aggregation. Both protein solutions show a signature of shear yielding, leading to an increase in the low shear viscosity. However, bulk and interfacial rheology measurements proved that this increase in viscosity is mostly due to protein adsorption at the air/water interface in surfactant-free BSA solutions, whereas aggregates are responsible for yielding in surfactant- laden mAb solutions. The fresh surfactant-laden mAb solution behaves as a simple Newtonian fluid. But after incubation at 40 oC for about a month, the mAb solution aggregates and shows an increase in the low shear viscosity. The aggregated mAb solution becomes Newtonian after passing the solution through a 0.2 µm filter. In the second part of the talk, small-angle neutron scattering (SANS) experiments will be discussed. SANS was used to characterize the mAb aggregates responsible for the non-Newtonian rheology observed. SANS experiments were performed at the NIST Center for Neutron Research with three configurations in order to access wavevectors q in the range 0.001 1/Å < q < 0.6 1/Å. SANS suggests the presence of a weak repulsive barrier before proteins fall into their primary minimum and aggregate reversibly, unless a favorable contact with high binding energy occurs. After incubation at 40 oC, two types of mAb aggregates were identified: oligomers with average radius of gyration of ~10 nm and fractal aggregates (self-similar structures) in the submicron scale formed by a slow reaction-limited aggregation process. The latter aggregates are responsible for the low shear viscosity increase observed in the rheological measurements. Biophysical characterization techniques commonly used in the bio- pharmaceutical industry support the conclusions obtained from rheology and SANS.

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