Probing Shear Thinning Effects on IgG Molecules at the Air-water Interface via Viscosity and Rheological Measurements

Abstract

Shear thinning behavior, observed in shear viscosity tests of IgG therapeutic molecules, may lead to inaccurate reporting of apparent viscosity, depending on the severity of the shear thinning behavior. To determine whether shear thinning is an intrinsic bulk property or an interfacial phenomenon, shear thinning behavior was tested as a function of bulk concentration with IgG1 and IgG2 molecules. Indeed if shear thinning is a bulk property, then the higher the concentration, the greater the shear thinning effect. The 70 mg/ml and 0.007 mg/ml samples showed the least shear thinning in comparison to 0.7 mg/ml concentrations of both the IgG1 and IgG2 molecules. This suggests that high bulk concentration did not produce the greatest shear thinning effects; therefore bulk properties do not contribute to the shear thinning effect. To test the sensitivity of the IgG molecules at the air-water interface, the surface area to volume ratio (SA:V) of samples exposed to the air-water interface were varied by the measuring system. The measuring systems used were: Cone and Plate 25 mm (CP 25), Cone and Plate 50 mm (CP 50) and Double Gap (DG). Their respective SA:V exposed to air were: 155 m-1, 108 m-1, and 33 m-1 with an Anton Paar MCR 302 rheometer. For both IgG1 and IgG2 molecules, the measuring systems with the highest SA:V ratios produced the most dramatic shear thinning effects. IgG1 molecules were more sensitive to changes in SA:V but IgG2 molecules showed higher magnitude shear thinning effects. To further probe the behavior of the IgG molecules at the air-water interface, interfacial oscillatory rheology with a Bicone (BC) was performed at a constant stress and strain (1 Hz, 1 %). The IgG molecules showed solid behavior (G'i) at 0.7 mg/ml over a 22 hour period. At 70 mg/ml and 0.007 mg/ml, liquid behavior (G''i) was dominant for both molecules. In fact, at 70 mg/ml G'i was not detected by the rheometer. Furthermore, the addition of polysorbate 20 (PS20) minimized shear thinning behavior as well as G'i. IgG1 had a G'i/G''i crossover point over half an hour after testing while IgG2 had a crossover point seconds into the test. This suggests different film formation behavior for IgG1 and IgG2. The slow continuous growth of G'i over 22 hours suggests the formation of multi-layer's of mAbs beneath the air-water interfacial monolayer.[4] To orthogonally verify the presence of IgG molecules at the air-water interface, interfacial tension (IFT) was also measured at the 0.7 mg/ml IgG molecule concentrations and in the respective IgG buffers. For both IgG molecules, IFT was reduced relative to the buffers.