Antibodies Adsorbed to the Air–Water Interface Form Soft Glasses

Abstract

When monoclonal antibodies are exposed to an air−water interface, they form aggregates, which negatively impacts their performance. Until now, the detection and characterization of interfacial aggregation have been difficult. Here, we exploit the mechanical response imparted by interfacial adsorption by measuring the interfacial shear rheology of a model antibody, anti-streptavidin immunoglobulin-1 (AS-IgG1), at the air−water interface. Strong viscoelastic layers of AS-IgG1 form when the protein is adsorbed from the bulk solution. Creep experiments correlate the compliance of the interfacial protein layer with the subphase solution pH and bulk concentration. These, along with oscillatory strain amplitude and frequency sweeps, show that the viscoelastic behavior of the adsorbed layers is that of a soft glass with interfacial shear moduli on the order of 10 −3 Pa m. Shifting the creep compliance curves under different applied stresses forms master curves consistent with stress−time superposition of soft interfacial glasses. The interfacial rheology results are discussed in the context of the interface-mediated aggregation of AS-IgG1.

Interfacial Pressure and Viscoelasticity of Antibodies and Their Correlation to Long-Term Stability in Formulation

“…An exponential dependence of the elastic modulus is observed in concentrated colloidal gels and glasses as described elsewhere. 34,36 Naturally then, the observation of visible particle counts also correlates with the protein concentration in this hydrophobic region at the interface, showing a linear correlation with an R 2 of 0.96 as shown in Figure 7B. The total amount of absorbed protein (i.e., the total volume fraction of mAb at the interface) also correlates with the interfacial elastic modulus and visible particle counts, as shown in Figure S8 (Supporting Information).…”

“…At hydrophobic interfaces such as the air–water interface, proteins are believed to undergo conformational change to expose hydrophobic residues to the hydrophobic phase (i.e., air in this system) and lower overall free energy. , This structural rearrangement leads to enhancement in hydrophobic interactions with the tightly packed, neighboring molecules, giving rise to the β-sheet formation, as has been confirmed for some hydrophobins and monoclonal antibodies. , As discussed, the interfacial elastic (or storage) modulus is a metric to quantify the interprotein interaction at the air–water interface, which could reflect the long-term instability (or particulate formation propensity) of antibodies in solution . Thus, it is reasonable to interpret the enhancement in interprotein interactions at the interface as probed by interfacial rheology as being due to strong interprotein hydrophobic attractions, such as β-sheet formation, as indicated by significant enhancement in protein concentration in the hydrophobic region at the air interface, as shown by the strong peak in the XRR measurements.…”

“…The interfacial elastic modulus, G s ′, is approximately an order of magnitude greater than the interfacial viscous modulus, G s ″, and over the measured frequency range, the moduli are only weakly dependent on frequency, typical of soft glassy materials. 36 Moreover, the strain amplitude sweep in Figure S1 (Supporting Information) does not show a maximum in G s ″ immediately beyond the yield, 37 confirming that the adsorbed mAb layer at the air− water interface exhibits soft glassy rheological properties. 38,39 The soft glassy characteristics are consistent with a densely adsorbed layer of mAbs at the air−water interface.…”

“…14,50 As discussed, the interfacial elastic (or storage) modulus is a metric to quantify the interprotein interaction at the air−water interface, which could reflect the long-term instability (or particulate formation propensity) of antibodies in solution. 36 Thus, it is reasonable to interpret the enhancement in interprotein interactions at the interface as probed by interfacial rheology as being due to strong interprotein hydrophobic attractions, such as β-sheet formation, as indicated by significant enhancement in protein concentration in the hydrophobic region at the air interface, as shown by the strong peak in the XRR measurements. Indeed, the interfacial elastic modulus G s ′ follows a strong exponential scaling with an exponent of 10.2 ± 1.8 with the volume fraction of proteins in the hydrophobic region at the interface as demonstrated in Figure 7A.…”

Using Passive Microrheology to Measure the Evolution of the Rheological Properties of NIST mAb Formulations during Adsorption to the Air–Water Interface

“…Recently, Wood et al. studied the response to the application of shear stress to a mAb film formed at the air–water interface and showed that the protein film formed a soft glass at the air–water interface . More recently, Pham et al also correlated the rheology of industrially relevant mAbs to their long-term stability under storage conditions .…”