Real-Time Observation of Protein Aggregation at Liquid-Liquid Interfaces in a Microfluidic Device

A droplet microfluidic device to capture in real-time protein aggregation at liquid-liquid interfaces is described. In contrast to conventional methods, typically characterized by a lag time between the application of interfacial stress and the measurement of protein aggregation, here protein adsorption, the formation of a viscoelastic protein layer, aggregation, and shedding of protein particles into solution is simultaneously monitored. The device is applied to analyze the stability of antibody formulations over a wide range of concentrations (1-180 mg mL^-1) at the silicone oil (SO)-water interface under controlled mechanical deformation. The adsorption onto oil droplets induces the formation of a viscoelastic protein layer on a subsecond timescale, which progressively restricts the relaxation of the droplets within the chip. Upon mechanical rupture, the protein layer releases particles in solution. The rate of particle formation increases strongly with concentration, similar to the bulk viscosity. Concentrations above 120 mg mL^-1 lead to aggregation in seconds and drastically decrease the mechanical perturbations required to shed protein particles in solution. These results are important for the development of formulations at high-protein concentrations (>100 mg mL^-1) and indicate that particular attention should be given to interface-induced particle formation in this concentration range. In this context, low-volume microfluidic platforms allow the assessment of protein physical instabilities early in development and represent attractive tools to optimize antibody stability and formulation design consuming limited amounts of material.