Understanding the interactions between biological macromolecules and material surfaces is crucial for various biotechnological applications. Techniques like QCM-D offer real-time insight into interfacial processes such as protein adsorption and cell fouling, aiding in the design of surfaces for medical devices and beyond. Its versatility makes it a standout tool in this field. In this blog post, we present an example where QCM-D was used to study antibody fouling on steel surfaces,1 including the study motivation, measurement concepts, data analysis, and future possibilities that will be helpful for novice and advanced users alike.
Therapeutic proteins such as monoclonal antibodies are an emerging class of medicine and quality control of the manufacturing and storage processes is critical to developing safe, well-characterized products. When antibodies are produced, they come into contact with various material surfaces such as steel and glass and these protein-material interactions can induce protein aggregation in some cases. The aggregates can be smaller than filtration pore sizes and thus remain in the final product, so it is important to characterize potential aggregate formation on different material surfaces and to characterize the material properties of adsorbed antibodies at the solid-liquid interface. In this case study, QCM-D technology is shown to detect antibody fouling on steel surfaces and demonstrates the ability to characterize complex (two-layer) adsorbate formation using physics-based modeling approaches.
The adsorption kinetics of a monoclonal antibody onto stainless steel-coated surfaces was monitored as a function of antibody concentration by the QCM-D technique.1 The protocol involved a buffer baseline signal, injection of antibody solution at a relatively low flow rate, and then a buffer rinse step at a relatively high flow rate. The data were collected at multiple overtones in order to analyze the adsorbate properties with an appropriate model. During antibody adsorption, the measurement responses were complex, and a two-layer Voigt model was applied to analyze the thickness, shear modulus, and interfacial viscosity of each layer. On the other hand, after buffer rinsing, it was possible to apply the simpler Sauerbrey model that assumes a single, rigid adlayer and can directly convert the resonance frequency shift into the adsorbed mass.
The results showed that antibody adsorption onto steel surfaces occurred in a concentration-dependent manner (Fig. 1). At low antibody concentration, a rigid antibody layer of ~5 nm thickness adhered at the solid-liquid interface. At higher concentrations, there was a two-stage adsorption process consisting of a dense, rigid layer of ~7 nm thickness on the steel surface and a much thicker but less dense upper layer of up to ~250 nm thickness. The two-stage adsorption process was directly detected from the time-resolved QCM-D measurement signals while the complex film properties were obtained from subsequent modeling. After buffer rinsing, only rigidly attached antibody molecules remained on the steel surface so it was possible to use the Sauerbrey model for quantitative analysis and there was more remaining adsorbate in high antibody concentration cases than in low concentration cases.
Figure 1. Monoclonal antibody adsorption and aggregation on steel surfaces. Representative QCM-D resonance frequency (Δf) and energy dissipation (ΔD) signals as a function of time at multiple overtones for low and high concentration antibody adsorption. Corresponding layer thicknesses obtained from different QCM-D modeling approaches.
The QCM-D technique is highly sensitive to the viscoelastic properties of adsorbed biomacromolecules, which enables various modeling options to analyze one- and two-layer films. If the film is laterally homogenous, it is possible to extract quantitative information about film properties such as thickness, shear modulus, and viscosity. This case study shows the potential of rationally applying different modeling approaches in distinct application scenarios while such efforts can also be integrated with changing experimental parameters such as antibody type, solution conditions, and flow conditions. Interestingly, the QCM-D technique was able to detect the presence of the liquid-like upper layer at high antibody concentrations, whereas it was not possible to detect this second layer with neutron reflectivity due to weak contrast.
Various industrial processes involve biomacromolecules such as proteins contacting material surfaces and the QCM-D technique is adept at characterizing the mass and viscoelastic properties of resulting adsorbates due to protein-material and protein-protein interactions. The capability to probe interfacial rheology can also be useful for studying thin polymer films and polyelectrolyte complexes, including stimuli-responsive systems that can exhibit softer or more rigid properties depending on the environmental conditions.
Download the overview to learn more about how QCM-D can be used to measure molecular interaction at surfaces and interfaces in biotechnology and biophysics.
Malin graduated in engineering physics in 2006, where her research focused on the QCM-D technology. Since then, she has been scrutinizing the how’s and why’s of the world in general, and the world of QCM-D in particular.
