Solutions of globular proteins and monoclonal antibodies (mAbs) possess complex pH and concentration dependence of viscosity (h) and diffusivity (D). Despite these complexities, dilute solution properties like diffusion interaction parameter (k_D) and second virial coefficient (B₂₂) have recently been proposed to predict colloidal stability and at high protein concentrations. In this two-part talk, I will first discuss recent studies that illustrate the hazards of extrapolating dilute solution properties to concentrated solutions. We have investigated buffered Bovine Serum Albumin (BSA) solutions of 2 mg/mL < [BSA] < 500 mg/mL and 3.0 < pH < 7.4, respectively and found clear evidence for pH and concentration dependencies of conformation from our small-angle neutron scattering (SANS) data. Previous small-angle scattering data only reached a maximum [BSA] of 50 mg/mL¹, but clearly showed a coil to molten globule transition for [BSA] > 50 mg/mL. Our SANS data revealed that the wavevector (q)-dependent form factor, P(q), which describes scattering from a single chain and hence the conformation, clearly changes with concentration. P(q), measured by definition in dilute solution, changes at high [BSA]. Conformation changes with concentration and pH significantly impact the intermolecular interaction potential: B₂₂ does not predict high concentration solution h of a “simple” globular protein like BSA, at pH 3.0 and 5.0. This complexity was further accentuated in our static light scattering and dynamic light scattering (DLS), SANS, and rheology data on a buffered IgG1 solution over a wide pH (3.0 < pH <11.0) and concentration (2 mg/mL <[IgG 1] < 500 mg/mL) range. Similar to BSA, this IgG1 solution also showed weak correlation of B₂₂ and k_D with η, and, its pH- and [IgG 1]-dependent conformation could not be modeled with a P(q) invariant with [IgG 1] and pH. Clearly, there are severe limitations of correlating dilute solution metrics with high concentration protein formulation η.

Next, I will discuss our attempts to understand the stabilization and h modulation mechanism of Arginine Hydrochloride (Arg.HCl). Since the k_D did not correlate with η , we performed SANS on undiluted IgG 1 formulations to quantify the intermolecular interaction potential and discern molecular conformation. When 300 mM Arg.HCl was added to the 120 mg/mL IgG 1formulation, a 1.7X η decrease was seen but a 1 M Arg.HCl increased h 1.4X, relative to the formulation with 300 mM Arg.HCl). SANS data suggested that Arg.HCl imparts colloidal stability to the mAb solution via hydration forces that are activated by concentration-dependent Arg.HCl-Arg.HCl and Arg.HCl-mAb interactions. Our results re-emphasize the critical and classical, yet overlooked, role of chain conformation and protein hydration in solution rheology and thermodynamics. We are currently characterizing hydration layer effects on viscosity using inelastic neutron scattering.

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