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.

Droplet-Based Microfluidic Temperature-Jump Platform for the Rapid Assessment of Biomolecular Kinetics.

Protein folding, unfolding, and aggregation are important in a variety of biological processes and intimately linked to "protein misfolding diseases". The ability to perform experiments at different temperatures allows the extraction of important information regarding the kinetics and thermodynamics of such processes. Unfortunately, conventional stopped-flow methods are difficult to implement, generate limited information, and involve complex sample handling. To address this issue, we present a temperature-controlled droplet-based microfluidic platform that allows measurement of reaction kinetics on millisecond to second timescales and at temperatures between ambient and 90 °C. The utility of the microfluidic platform for measuring fast biomolecular kinetics at high temperatures is showcased through the investigation of the unfolding kinetics of haloalkane dehalogenases and the elongation of fibrils composed of the amyloid ß peptide associated with Alzheimer's disease. In addition, a deep-ultraviolet (UV) fluorescence microscope was developed for the on-chip recording of protein intrinsic fluorescence spectrum originating from aromatic amino acid residues. We envision that the developed optofluidic platform will find wide applicability in the analysis of biological processes, such as protein refolding and phase separation.

Dynamic arrest and aging of biomolecular condensates are modulated by low-complexity domains, RNA and biochemical activity.

Biomolecular condensates require suitable control of material properties for their function. Here we apply Differential Dynamic Microscopy (DDM) to probe the material properties of an in vitro model of processing bodies consisting of out-of-equilibrium condensates formed by the DEAD-box ATPase Dhh1 in the presence of ATP and RNA. By applying this single-droplet technique we show that condensates within the same population exhibit a distribution of material properties, which are regulated on several levels. Removal of the low-complexity domains (LCDs) of the protein decreases the fluidity of the condensates. Structured RNA leads to a larger fraction of dynamically arrested condensates with respect to unstructured polyuridylic acid (polyU). Promotion of the enzymatic ATPase activity of Dhh1 reduces aging of the condensates and the formation of arrested structures, indicating that biochemical activity and material turnover can maintain fluid-like properties over time.

Sequestration within biomolecular condensates inhibits Aß-42 amyloid formation.

Biomolecular condensates are emerging as an efficient strategy developed by cells to control biochemical reactions in space and time by locally modifying composition and environment. Yet, local increase in protein concentration within these compartments could promote aberrant aggregation events, including the nucleation and growth of amyloid fibrils. Understanding protein stability within the crowded and heterogeneous environment of biological condensates is therefore crucial, not only when the aggregation-prone protein is the scaffold element of the condensates but also when proteins are recruited as client molecules within the compartments. Here, we investigate the partitioning and aggregation kinetics of the amyloidogenic peptide Abeta42 (Aß-42), the peptide strongly associated with Alzheimer's disease, recruited into condensates based on low complexity domains (LCDs) derived from the DEAD-box proteins Laf1, Dbp1 and Ddx4, which are associated with biological membraneless organelles. We show that interactions between Aß-42 and the scaffold proteins promote sequestration and local increase of the peptide concentration within the condensates. Yet, heterotypic interactions within the condensates inhibit the formation of amyloid fibrils. These results demonstrate that biomolecular condensates could sequester aggregation-prone proteins and prevent aberrant aggregation events, despite the local increase in their concentration. Biomolecular condensates could therefore work not only as hot-spots of protein aggregation but also as protective reservoirs, since the heterogenous composition of the condensates could prevent the formation of ordered fibrillar aggregates.

The role of surfaces on amyloid formation.

Interfaces can strongly accelerate or inhibit protein aggregation, destabilizing proteins that are stable in solution or, conversely, stabilizing proteins that are aggregation-prone. Although this behaviour is well-known, our understanding of the molecular mechanisms underlying surface-induced protein aggregation is still largely incomplete. A major challenge is represented by the high number of physico-chemical parameters involved, which are highly specific to the considered combination of protein, surface properties, and solution conditions. The key aspect determining the role of interfaces is the relative propensity of the protein to aggregate at the surface with respect to bulk. In this review, we discuss the multiple molecular determinants that regulate this balance. We summarize current experimental techniques aimed at characterizing protein aggregation at interfaces, and highlight the need to complement experimental analysis with theoretical modelling. In particular, we illustrate how chemical kinetic analysis can be combined with experimental methods to provide insights into the molecular mechanisms underlying surface-induced protein aggregation, under both stagnant and agitation conditions. We summarize recent progress in the study of important amyloids systems, focusing on selected relevant interfaces.

Establishment of a scalable microfluidic assay for characterization of population-based neutrophil chemotaxis.

BACKGROUND: Regulation of neutrophil chemotaxis and activation plays crucial roles in immunity, and dysregulated neutrophil responses can lead to pathology as seen in neutrophilic asthma. Neutrophil recruitment is key for initiating immune defense and inflammation, and its modulation is a promising therapeutic target. Microfluidic technology is an attractive tool for characterization of neutrophil migration. Compared to transwell assays, microfluidic approaches could offer several advantages, including precis e control of defined chemokine gradients in space and time, automated quantitative analysis of chemotaxis, and high throughput. METHODS: We established a microfluidic device for fully automated, quantitative assessment of neutrophil chemotaxis. Freshly isolated mouse neutrophils from bone marrow or human neutrophils from peripheral blood were assessed in real time using an epifluorescence microscope for their migration toward the potent chemoattractants C-X-C-motif ligand 2 (CXCL2) and CXCL8, without or with interleukin-4 (IL-4) pre-incubation. RESULTS: Our microfluidic device allowed the precise and reproducible determination of the optimal CXCL2 and CXCL8 concentrations for mouse and human neutrophil chemotaxis, respectively. Furthermore, our microfluidic assay was able to measure the equilibrium and real-time dynamic effects of specific modulators of neutrophil chemotaxis. We demonstrated this concept by showing that IL-4 receptor signaling in mouse and human neutrophils inhibited their migration toward CXCL2 and CXCL8, respectively, and this inhibition was time-dependent. CONCLUSION: Collectively, our microfluidic device shows several advantages over traditional transwell migration assays and its design is amenable to future integration into multiplexed high-throughput platforms for screening of molecules that modulate the chemotaxis of different immune cells.

Synergistic effects of flow and interfaces on antibody aggregation.

During the manufacturing process, solutions of protein-based drugs are exposed to hydrodynamic forces, which can potentially affect protein stability and aggregation. Despite being an area of extensive investigation, the effect of hydrodynamic flow on protein aggregation is still controversial. In this study, we designed an experimental setup that allowed us to investigate flow- and interface-induced protein aggregation of two model immunoglobulins in the presence of well-defined flow stresses and solid-liquid interfaces. Within the range of shear rates typically encountered in bioprocessing ( γ̇=10-103 s-1 ), we observed that increasing the shear rate by three orders of magnitude had a negligible effect on protein aggregation. By contrast, changes in the materials of the syringe barrels had a dramatic effect on the monomer loss, demonstrating the key role of solid-liquid interfaces in flow-induced aggregation. This finding was confirmed by the observed inverse dependence of the aggregation rate on the initial protein concentration, which is inconsistent with mechanisms of protein aggregation in bulk solution. Overall, our results reveal the presence of a synergistic effect of interfaces and hydrodynamic flow in flow-induced protein aggregation, which arises from the formation of protein particles or films on interfaces followed by displacement by flow or mechanical scraping.

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.

Sensitivity analysis of the variability of amyloid aggregation profiles.

In vitro kinetic assays of amyloid formation represent a central tool in many areas of biotechnological and biomedical sciences, including drug discovery against a variety of neurodegenerative disorders. The conversion of soluble peptides and proteins into insoluble amyloid aggregates follows macroscopic kinetic profiles that are commonly characterized by a certain degree of variability. This variability can challenge the investigation of the molecular determinants of the aggregation process, and its molecular origin remains largely elusive. Here, we analyze the aggregation profiles of four different amyloidogenic proteins that follow distinct microscopic mechanisms. We show that the variability of the kinetic traces, described by the standard deviation of the aggregation lag-phase, is linearly proportional to the duration of the aggregation process. By applying a sensitivity analysis we demonstrate that this behaviour arises from an initial fixed perturbation of one or more of the kinetic parameters of the aggregation network, and does not involve any amplification of the perturbation during the aggregation process. Moreover, our parametric sensitivity analysis shows that the experimentally measured variability is compatible with variations in the initial monomer concentration that are consistently smaller compared to all the other kinetic parameters, denoting a higher sensitivity of the amyloid aggregation process with respect to this parameter. Overall, these results show that the variability of aggregation profiles in large volumes (µl) depends on the initial conditions and not on intrinsic stochasticity. The accurate control of the initial conditions is therefore crucial to decrease this variability.

A hydrophobic low-complexity region regulates aggregation of the yeast pyruvate kinase Cdc19 into amyloid-like aggregates in vitro.

Cells form stress granules (SGs) upon stress stimuli to protect sensitive proteins and RNA from degradation. In the yeast Saccharomyces cerevisiae, specific stresses such as nutrient starvation and heat-shock trigger recruitment of the yeast pyruvate kinase Cdc19 into SGs. This RNA-binding protein was shown to form amyloid-like aggregates that are physiologically reversible and essential for cell cycle restart after stress. Cellular Cdc19 exists in an equilibrium between a homotetramer and monomer state. Here, we show that Cdc19 aggregation in vitro is governed by protein quaternary structure, and we investigate the physical-chemical basis of Cdc19's assembly properties. Equilibrium shift toward the monomer state exposes a hydrophobic low-complexity region (LCR), which is prone to induce intermolecular interactions with surrounding proteins. We further demonstrate that hydrophobic/hydrophilic interfaces can trigger Cdc19 aggregation in vitro Moreover, we performed in vitro biophysical analyses to compare Cdc19 aggregates with fibrils produced by two known dysfunctional amyloidogenic peptides. We show that the Cdc19 aggregates share several structural features with pathological amyloids formed by human insulin and the Alzheimer's disease-associated Aß42 peptide, particularly secondary ß-sheet structure, thermodynamic stability, and staining by the thioflavin T dye. However, Cdc19 aggregates could not seed aggregation. These results indicate that Cdc19 adopts an amyloid-like structure in vitro that is regulated by the exposure of a hydrophobic LCR in its monomeric form. Together, our results highlight striking structural similarities between functional and dysfunctional amyloids and reveal the crucial role of hydrophobic/hydrophilic interfaces in regulating Cdc19 aggregation.

Mechanistic Origin of the Combined Effect of Surfaces and Mechanical Agitation on Amyloid Formation.

Interactions between proteins and surfaces in combination with hydrodynamic flow and mechanical agitation can often trigger the conversion of soluble peptides and proteins into aggregates, including amyloid fibrils. Despite the extensive literature on the empirical effects of surfaces and mechanical forces on the formation of amyloids, the molecular details of the mechanisms underlying this behavior are still elusive. This limitation is, in part, due to the complex reaction network underlying the formation of amyloids, where several microscopic reactions of nucleation and growth can occur both at the interfaces and in bulk. In this work, we design a high-throughput assay based on nanoparticles and we apply a chemical kinetic platform to analyze the mechanisms underlying the effect of surfaces and mechanical forces on the formation of amyloid fibrils from human insulin under physiological conditions. By considering a variety of polymeric nanoparticles with different surface properties we explore a broad range of repulsive and attractive interactions between insulin and surfaces. Our analysis shows that hydrophobic interfaces induce the formation of amyloid fibrils by specifically promoting the primary heterogeneous nucleation rate. In contrast, mechanical forces accelerate the formation of amyloid fibrils by favoring mass transport and further amplify the number of fibrils by promoting fragmentation events. Thus, surfaces and agitation have a combined effect on the kinetics of protein aggregation observed at the macroscopic level but, individually, they each affect distinct microscopic reaction steps: the presence of interfaces generates primary nucleation events of fibril formation, which is then amplified by mechanical forces. These results suggest that the inhibition of surface-induced heterogeneous nucleation should be considered a primary target to suppress aggregation and explain why in many systems the simultaneous presence of surfaces and hydrodynamic flow enhances protein aggregation.