Microfluidic Approaches for Investigating Aggregated Forms of Disease-Related Proteins

The self-assembly of soluble proteins is a common phenomenon observed in nature. It ranges from the formation of functional biomaterials, such as actin filaments and tubulin microtubules that form the cytoeskeleton, to the abnormal deposition of aberrant aggregates that lead to disease. There are nearly 50 diseases currently associated with protein misfolding and amyloid formation, among them several neurodegenerative disorders such as Alzheimer's, Parkinson's and prion diseases. Recent evidence indicates that the most toxic species are the low molecular weight oligomers formed during the onset of the aggregation process. These oligomeric species are commonly difficult to analyse due to their low abundance, transient nature and heterogeneity in their structure. In fact, prions, known to be associated with transmissible spongiform encephalopathies, have not yet been fully characterised as parts of the prion composition seem to lose infectivity through various \textit{in vitro} purification and isolation procedures. In nature, non-homogeneous solution conditions and molecular crowding are key factors as there might be multiple species associated with the disease. However, traditional biophysical techniques allow the study of biomolecules under ideal conditions, such as high concentrations and monodisperse size distribution. Furthermore, among the limitations of the traditional methods are the sensitivity as well as the sample preparation, which in some cases may require to chemically modify the molecule. In this thesis, I describe the development of a sensitive label-free method to enable the study of protein complexes and proteins under biologically relevant environment, such as brain homogenates, by combining immunochemistry with microfluidic strategies. In this thesis I describe the development of Immuno Diffusional Sizing (IDS), a method that combines diffusional sizing with the sensitivity and specificity of immunoassay detection. In Chapter 3, I explain the optimisation of the devices developed to size a range of particle sizes in heterogeneous mixtures, from small protein monomers to large aggregates. IDS is a label-free method capable of sizing proteins down to the nano- and picomolar concentrations, in complex solutions. The applications of this method to monomeric and aggregated forms of disease-related proteins are described in Chapter 4. IDS has been applied to study the Prion Protein (PrP) in its native and infectious forms. Furthermore, I studied of the proteinase resistant form (PrP\textsuperscript{res}) extracted from the brains of infected mice specimens. IDS was applied for studying disease-relevant species, such as intrinsically disordered proteins, e.g. $\alpha$-Synuclein (aSyn) and A$\beta$40, and their respective oligomers. Another advantage of this method is its applicability for studies of brain aSyn from transgenic overexpressing mice (OVX). Furthermore, I explore the potential of IDS for the study of post-translational modifications (PTMs) of specific proteins, such as the ubiquitination of aSyn \textit{in vitro}. The efforts performed during this work to achieve TR-FRET detection on-chip are described in Chapter 5. A comparison of two commercial immunoassays was performed in order to determine the suitability for its adaptation from a platereader to a microfluidic device. The design and adaptation of an optics platform for FRET detection on-chip is also discussed. My research on the mechanisms of the molecular chaperone $\alpha$B-Crystallin (aBC) is described in Chapter 6. I investigated the aggregation process of insulin in the presence of aBC. I have used a combination of bulk techniques to study the kinetics of the systems, as well as microfluidics to generate microdroplets that allow the direct observation of rare nucleation events leading to protein aggregation. Overall, in this work I describe the development of a novel microfluidic technique, its optimisation and application to multiple systems related to protein misfolding diseases, including prions and intrinsically disordered proteins. Purification of the sample is not necessary to study the target protein because the detection of the target protein after diffusional separation is made with a sandwich immunoassay, which allows to study disease-related proteins under native conditions in a label-free manner, which is usually difficult with traditional sizing techniques. The applications of this technology could be expanded to characterise other antigens in body fluids, like cerebrospinal fluid (CSF) of blood serum.CONACYT (Mexican National Council on Science and Technology) Cambridge Trus

Resolving protein mixtures using microfluidic diffusional sizing combined with synchrotron radiation circular dichroism

Circular dichroism spectroscopy has become a powerful tool to characterise proteins and other biomolecules. For heterogeneous samples such as those present for interacting proteins, typically only average spectroscopic features can be resolved. Here we overcome this limitation by using free-flow microfluidic size separation in-line with synchrotron radiation circular dichroism to resolve the secondary structure of each component of a model protein mixture containing monomers and fibrils. To enable this objective, we have integrated far-UV compatible measurement chambers into PDMS-based microfluidic devices. Two architectures are proposed so as to accommodate for a wide range of concentrations. The approach, which can be used in combination with other bulk measurement techniques, paves the way to the study of complex mixtures such as the ones associated with protein misfolding and aggregation diseases including Alzheimer’s and Parkinson’s diseases

Scaling analysis reveals the mechanism and rates of prion replication in vivo.

Prions consist of pathological aggregates of cellular prion protein and have the ability to replicate, causing neurodegenerative diseases, a phenomenon mirrored in many other diseases connected to protein aggregation, including Alzheimer's and Parkinson's diseases. However, despite their key importance in disease, the individual processes governing this formation of pathogenic aggregates, as well as their rates, have remained challenging to elucidate in vivo. Here we bring together a mathematical framework with kinetics of the accumulation of prions in mice and microfluidic measurements of aggregate size to dissect the overall aggregation reaction into its constituent processes and quantify the reaction rates in mice. Taken together, the data show that multiplication of prions in vivo is slower than in in vitro experiments, but efficient when compared with other amyloid systems, and displays scaling behavior characteristic of aggregate fragmentation. These results provide a framework for the determination of the mechanisms of disease-associated aggregation processes within living organisms

Microfluidic characterisation reveals broad range of SARS-CoV-2 antibody affinity in human plasma.

Funder: Herchel Smith FundFunder: St John’s College CambridgeFunder: Centre for Misfolding Diseases, CambridgeFunder: Swiss FCS and the Forschungskredit of the University of ZurichFunder: Frances and Augustus Newman FoundationFunder: BBRSCFunder: NOMIS FoundationThe clinical outcome of SARS-CoV-2 infections, which can range from asymptomatic to lethal, is crucially shaped by the concentration of antiviral antibodies and by their affinity to their targets. However, the affinity of polyclonal antibody responses in plasma is difficult to measure. Here we used microfluidic antibody affinity profiling (MAAP) to determine the aggregate affinities and concentrations of anti-SARS-CoV-2 antibodies in plasma samples of 42 seropositive individuals, 19 of which were healthy donors, 20 displayed mild symptoms, and 3 were critically ill. We found that dissociation constants, K d, of anti-receptor-binding domain antibodies spanned 2.5 orders of magnitude from sub-nanomolar to 43 nM. Using MAAP we found that antibodies of seropositive individuals induced the dissociation of pre-formed spike-ACE2 receptor complexes, which indicates that MAAP can be adapted as a complementary receptor competition assay. By comparison with cytopathic effect-based neutralisation assays, we show that MAAP can reliably predict the cellular neutralisation ability of sera, which may be an important consideration when selecting the most effective samples for therapeutic plasmapheresis and tracking the success of vaccinations

Continuous population-level monitoring of SARS-CoV-2 seroprevalence in a large European metropolitan region

Effective public health measures against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against three SARS-CoV-2 proteins. We used TRABI for continuous seromonitoring of hospital patients and blood donors (n = 72'250) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period). We found that antibodies waned with a half-life of 75 days, whereas the cumulative incidence rose from 2.3% in June 2020 to 12.2% in mid-December 2020. A follow-up health survey indicated that about 10% of patients infected with wildtype SARS-CoV-2 sustained some symptoms at least twelve months post COVID-19. Crucially, we found no evidence of a difference in long-term complications between those whose infection was symptomatic and those with asymptomatic acute infection. The cohort of asymptomatic SARS-CoV-2-infected subjects represents a resource for the study of chronic and possibly unexpected sequelae

Design of Biopharmaceutical Formulations Accelerated by Machine Learning

In addition to activity, successful biological drugs must exhibit a series of suitable developability properties, which depend on both protein sequence and buffer composition. In the context of this high-dimensional optimization problem, advanced algorithms from the domain of machine learning are highly beneficial in complementing analytical screening and rational design. Here, we propose a Bayesian optimization algorithm to accelerate the design of biopharmaceutical formulations. We demonstrate the power of this approach by identifying the formulation that optimizes the thermal stability of three tandem single-chain Fv variants within 25 experiments, a number which is less than one-third of the experiments that would be required by a classical DoE method and several orders of magnitude smaller compared to detailed experimental analysis of full combinatorial space. We further show the advantage of this method over conventional approaches to efficiently transfer historical information as prior knowledge for the development of new biologics or when new buffer agents are available. Moreover, we highlight the benefit of our technique in engineering multiple biophysical properties by simultaneously optimizing both thermal and interface stabilities. This optimization minimizes the amount of surfactant in the formulation, which is important to decrease the risks associated with corresponding degradation processes. Overall, this method can provide high speed of converging to optimal conditions, the ability to transfer prior knowledge, and the identification of new nonlinear combinations of excipients. We envision that these features can lead to a considerable acceleration in formulation design and to parallelization of operations during drug development.ISSN:1543-8384ISSN:1543-839

A Relationship between the Structures and Neurotoxic Effects of Aβ Oligomers Stabilized by Different Metal Ions.

Publication status: PublishedOligomeric assemblies of the amyloid β peptide (Aβ) have been investigated for over two decades as possible neurotoxic agents in Alzheimer's disease. However, due to their heterogeneous and transient nature, it is not yet fully established which of the structural features of these oligomers may generate cellular damage. Here, we study distinct oligomer species formed by Aβ40 (the 40-residue form of Aβ) in the presence of four different metal ions (Al3+, Cu2+, Fe2+, and Zn2+) and show that they differ in their structure and toxicity in human neuroblastoma cells. We then describe a correlation between the size of the oligomers and their neurotoxic activity, which provides a type of structure-toxicity relationship for these Aβ40 oligomer species. These results provide insight into the possible role of metal ions in Alzheimer's disease by the stabilization of Aβ oligomers.UKRI 10059436 and 1006110

A Relationship between the Structures and Neurotoxic Effects of Aβ Oligomers Stabilized by Different Metal Ions.

Oligomeric assemblies of the amyloid β peptide (Aβ) have been investigated for over two decades as possible neurotoxic agents in Alzheimer's disease. However, due to their heterogeneous and transient nature, it is not yet fully established which of the structural features of these oligomers may generate cellular damage. Here, we study distinct oligomer species formed by Aβ40 (the 40-residue form of Aβ) in the presence of four different metal ions (Al3+, Cu2+, Fe2+, and Zn2+) and show that they differ in their structure and toxicity in human neuroblastoma cells. We then describe a correlation between the size of the oligomers and their neurotoxic activity, which provides a type of structure-toxicity relationship for these Aβ40 oligomer species. These results provide insight into the possible role of metal ions in Alzheimer's disease by the stabilization of Aβ oligomers.UKRI 10059436 and 1006110

Microfluidic characterisation reveals broad range of SARS-CoV-2 antibody affinity in human plasma

The clinical outcome of SARS-CoV-2 infections, which can range from asymptomatic to lethal, is crucially shaped by the concentration of antiviral antibodies and by their affinity to their targets. However, the affinity of polyclonal antibody responses in plasma is difficult to measure. Here we used microfluidic antibody affinity profiling (MAAP) to determine the aggregate affinities and concentrations of anti–SARS-CoV-2 antibodies in plasma samples of 42 seropositive individuals, 19 of which were healthy donors, 20 displayed mild symptoms, and 3 were critically ill. We found that dissociation constants, Kd, of anti–receptor-binding domain antibodies spanned 2.5 orders of magnitude from sub-nanomolar to 43 nM. Using MAAP we found that antibodies of seropositive individuals induced the dissociation of pre-formed spike-ACE2 receptor complexes, which indicates that MAAP can be adapted as a complementary receptor competition assay. By comparison with cytopathic effect–based neutralisation assays, we show that MAAP can reliably predict the cellular neutralisation ability of sera, which may be an important consideration when selecting the most effective samples for therapeutic plasmapheresis and tracking the success of vaccinations

Scaling analysis reveals the mechanism and rates of prion replication in vivo

Prions consist of pathological aggregates of cellular prion protein and have the ability to replicate, causing neurodegenerative diseases, a phenomenon mirrored in many other diseases connected to protein aggregation, including Alzheimer’s and Parkinson’s diseases. However, despite their key importance in disease, the individual processes governing this formation of pathogenic aggregates, as well as their rates, have remained challenging to elucidate in vivo. Here we bring together a mathematical framework with kinetics of the accumulation of prions in mice and microfluidic measurements of aggregate size to dissect the overall aggregation reaction into its constituent processes and quantify the reaction rates in mice. Taken together, the data show that multiplication of prions in vivo is slower than in in vitro experiments, but efficient when compared with other amyloid systems, and displays scaling behavior characteristic of aggregate fragmentation. These results provide a framework for the determination of the mechanisms of disease-associated aggregation processes within living organisms