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.

Microfluidic Stress Device to Decouple the Synergistic Effect of Shear and Interfaces on Antibody Aggregation.

Protein denaturation and aggregation resulting from the effects of interfacial stress, often enhanced by flow and shear stress, pose significant challenges in the production of therapeutic proteins and monoclonal antibodies. The influence of flow on protein stability is closely intertwined with interfacial effects. In this study, we have developed a microfluidic device capable of exposing low volume (< 320 µL) protein solutions to highly uniform shear. To disentangle the synergistic impact of flow and interfaces on protein aggregation, we fabricated two devices composed of different materials, namely poly(methyl methacrylate) (PMMA) and stainless steel. Upon application of shear, we observed formation of protein particles in the micron-size range. Notably, The number of particles generated in the steel devices was ∼ 3.5 fold lower than in the PMMA device, hinting at an interface-mediated effect. With increasing the protein concentration from 1 to 50 mg/mL we observed a saturation in the amount of aggregates, further confirming the key role of solid-liquid interfaces in inducing particle formation. Introduction of non-ionic surfactants prevented protein aggregation, even at the highest tested protein concentration and low surfactant concentrations of 0.05 mg/mL. Overall, our findings corroborate the synergistic impact of shear and interface effects on protein aggregation. The device developed in this study offers a small-scale platform for assessing the stability of antibody formulations throughout various stages of the development and manufacturing process.

Comparison of the Protective Effect of Polysorbates, Poloxamer and Brij on Antibody Stability Against Different Interfaces.

Therapeutic proteins and antibodies are exposed to a variety of interfaces during their lifecycle, which can compromise their stability. Formulations, including surfactants, must be carefully optimized to improve interfacial stability against all types of surfaces. Here we apply a nanoparticle-based approach to evaluate the instability of four antibody drugs against different solid-liquid interfaces characterized by different degrees of hydrophobicity. We considered a model hydrophobic material as well as cycloolefin-copolymer (COC) and cellulose, which represent some of the common solid-liquid interfaces encountered during drug production, storage, and delivery. We assess the protective effect of polysorbate 20, polysorbate 80, Poloxamer 188 and Brij 35 in our assay and in a traditional agitation study. While all nonionic surfactants stabilize antibodies against the air-water interface, none of them can protect against hydrophilic charged cellulose. Polysorbates and Brij increase antibody stability in the presence of COC and the model hydrophobic interface, although to a lesser extent compared to the air-water interface, while Poloxamer 188 has a negligible stabilizing effect against these interfaces. These results highlight the challenge of fully protecting antibodies against all types of solid-liquid interfaces with traditional surfactants. In this context, our high-throughput nanoparticle-based approach can complement traditional shaking assays and assist in formulation design to ensure protein stability not only at air-water interfaces, but also at relevant solid-liquid interfaces encountered during the product lifecycle.

Surface-Induced Protein Aggregation and Particle Formation in Biologics: Current Understanding of Mechanisms, Detection and Mitigation Strategies.

Protein stability against aggregation is a major quality concern for the production of safe and effective biopharmaceuticals. Amongst the different drivers of protein aggregation, increasing evidence indicates that interactions between proteins and interfaces represent a major risk factor for the formation of protein aggregates in aqueous solutions. Potentially harmful surfaces relevant to biologics manufacturing and storage include air-water and silicone oil-water interfaces as well as materials from different processing units, storage containers, and delivery devices. The impact of some of these surfaces, for instance originating from impurities, can be difficult to predict and control. Moreover, aggregate formation may additionally be complicated by the simultaneous presence of interfacial, hydrodynamic and mechanical stresses, whose contributions may be difficult to deconvolute. As a consequence, it remains difficult to identify the key chemical and physical determinants and define appropriate analytical methods to monitor and predict protein instability at these interfaces. In this review, we first discuss the main mechanisms of surface-induced protein aggregation. We then review the types of contact materials identified as potentially harmful or detected as potential triggers of proteinaceous particle formation in formulations and discuss proposed mitigation strategies. Finally, we present current methods to probe surface-induced instabilities, which represent a starting point towards assays that can be implemented in early-stage screening and formulation development of biologics.

Left ventricular twist predicts mortality in severe aortic stenosis.

OBJECTIVE: Left ventricular (LV) twist is a major component of ventricular mechanics reflecting the helical orientation of cardiac fibres and compensating for afterload mismatch. However, it is not known whether it determines outcome after transcatheter aortic valve implantation (TAVI). This study sought to investigate TAVI-induced short-term changes of LV twist and to define its role in outcome prediction. METHODS: A total of 146 patients (median age 81.78 years, 50.7% male) undergoing TAVI for severe aortic stenosis were included. LV rotation and twist were determined by speckle tracking echocardiography within 3 months before and 2 weeks after TAVI. All-cause mortality at 2 years was defined as primary end point. RESULTS: Patients who survived exhibited a higher apical peak systolic rotation (APSR) (p<0.001), twist (p=0.003) and torsion (p=0.019) pre-TAVI compared with those who died (n=22). Within 2 weeks after TAVI, APSR, twist and torsion decreased in patients who survived (all p<0.001), while no change occurred in those who died. Cox regression analysis showed an association of pre-TAVI APSR (HR 0.92, p=0.010), twist (HR 0.93, p=0.018) and torsion (HR 0.68, p=0.040) with all-cause mortality and an even stronger association of the respective changes after TAVI (∆APSR: HR 1.15, p<0.001; ∆twist: HR 1.14, p<0.001; ∆torsion: HR 2.53, p<0.001). All the parameters determined outcome independently of global longitudinal strain (GLS) and LV ejection fraction (LVEF). CONCLUSION: APSR, twist and torsion pre-TAVI as well as their change within 2 weeks after TAVI predict 2-year all-cause mortality after TAVI, adding incremental prognostic value to LVEF and GLS.

Dynamics of Synthetic Membraneless Organelles in Microfluidic Droplets.

Cells can form membraneless organelles by liquid-liquid phase separation. As these organelles are highly dynamic, it is crucial to understand the kinetics of these phase transitions. Here, we use droplet-based microfluidics to mix reagents by chaotic advection and observe nucleation, growth, and coarsening in volumes comparable to cells (pL) and on timescales of seconds. We apply this platform to analyze the dynamics of synthetic organelles formed by the DEAD-box ATPase Dhh1 and RNA, which are associated with the formation of processing bodies in yeast. We show that the timescale of phase separation decreases linearly as the volume of the compartment increases. Moreover, the synthetic organelles coarsen into one single droplet via gravity-induced coalescence, which can be arrested by introducing a hydrogel matrix that mimics the cytoskeleton. This approach is an attractive platform to investigate the dynamics of compartmentalization in artificial cells.