The purpose of this study was to assess the stability of protein formulations using a device designed to generate defined, quantifiable levels of shear in the presence of a solid-liquid interface. The device, based on a rotating disk, produced shear strain rates of up to 3.4 x 10(4) s(-1) (at 250 rps) and was designed to exclude air-liquid interfaces and enable temperature to be controlled. Computational fluid dynamics (CFD) was used to study the fluid flow patterns within the device and to determine the shear strain rate (s(-1)) at a range of disk speeds. The device was then used to study the effect on a monoclonal IgG4 of high levels of shear at the solid-liquid interface. Monomeric antibody concentration and aggregation of the protein in solution were monitored by gel permeation HPLC and turbidity at 350 nm. High shear strain rates were found to cause significant levels of protein aggregation and precipitation with reduction of protein monomer following first-order kinetics. Monomer reduction rate was determined for a range of disk speeds and found to have a nonlinear relationship with shear strain rate, indicating the importance of identifying and minimizing such environments during processing.

A rotating disk shear device was used to study the effect of interfacial shear on the structural integrity of human monoclonal antibodies of IgG4 isotype. Factors associated with the solution conditions (pH, ionic strength, surfactant concentration, temperature) and the interface (surface roughness) were studied for their effect on the rate of IgG4 monomer loss under high shear conditions. The structural integrity of the IgG4 was probed after exposure to interfacial shear effects by SDS-PAGE, IEF, dynamic light scattering, and peptide mapping by LC-MS. This analysis revealed that the main denaturation pathway of IgG4 exposed to these effects was the formation of large insoluble aggregates. Soluble aggregation, breakdown in primary structure, and chemical modifications were not detected. The dominant factors found to affect the rate of IgG4 monomer loss under interfacial shear conditions were found to be pH and the nanometer-scale surface roughness associated with the solid-liquid interface. Interestingly, temperature was not found to be a significant factor in the range tested (15-45 degrees C). The addition of surfactant was found to have a significant stabilizing effect at concentrations up to 0.02% (w/v). Implications of these findings for the bioprocessing of this class of therapeutic protein are briefly discussed.

No abstract

Lyophilized protein formulations must be reconstituted back into solution prior to patient administration and in this regard long reconstitution times are not ideal. The factors that govern reconstitution time remain poorly understood. The aim of this research was to understand the influence of the lyophilization cooling profile (including annealing) on the resulting cake structure and reconstitution time. Three protein formulations (BSA 50mg/ml, BSA 200mg/ml and IgG1 40mg/ml, all in 7% w/v sucrose) were investigated after cooling at either 0.5°C/min, or quench cooling with liquid nitrogen with/without annealing. Significantly longer reconstitution times were observed for the lower protein concentration formulations following quench cool. Porosity measurements found concomitant increases in the surface area of the porous cake structure but a reduction in total pore volume. We propose that slow reconstitution results from either closed pores or small pores impeding the penetration of water into the lyophilized cake.