Biology subjects

Fodera, V.

Publications and source records attributed to Fodera, V..

10 recordsLinked to original sources

bioRxiv2025

Rule-based mitigation of charge asymmetry-triggered monoclonal antibody self-assembly

In a pharmaceutical setting, understanding the factors governing monoclonal antibody (mAb) attractive interactions in formulations is highly warranted as many solution phenomena such as liquid-liquid phase separation (LLPS) result from their preferential self-interaction. While the effect of locally accumulated charge in the variable region has been recognized as an important factor in mediating non-specific mAb self-assembly, the effect of charge asymmetry, i.e. the distribution of oppositely charges residues, has been much less studied experimentally. Moreover, most studies restrict such analyses to the variable region of mAbs, leaving out possible contributions from the constant region of the molecule to the observed sticky behavior. Hence, the aim of this work is to correlate the charge asymmetry over the entire mAb surface to the extent of attractive self-interaction. To do so, we selected three mAbs with distinct solvent exposed surface distribution of charged residues, for which we computationally assessed the charge asymmetry and defined an apparent molecular stickiness ranking. We then tested this ranking experimentally by evaluating their ability to engage in attractive self-interaction as a function of mAb concentration and ionic strength. Experimental data included a combination of small-angle X-ray scattering, dynamic light scattering and micro-flow imaging. We show that the mAbs with oppositely charged Fab and Fc domains are characterized by overall attractive protein-protein interactions in solution amounting to diverse sub-visible morphologies, which vary non-linearly with mAb concentration and ionic strength. As a proof of concept, we also report the absence of any of such assemblies for the mAb with like-charged Fab and Fc domains, resulting in an overall repulsive behavior in solution. Altogether, we show how to utilize charge distribution analyses of full-length mAbs to rationally develop formulations that prevent problematic self-assembly. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/686261v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1c29a4dorg.highwire.dtl.DTLVardef@120644borg.highwire.dtl.DTLVardef@1989b61org.highwire.dtl.DTLVardef@12720_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Decoupling liquid-liquid phase separation and opalescence from stress-induced aggregation in therapeutic mAb formulations

Liquid-liquid phase separation (LLPS) and high opalescence are two self-association phenomena commonly encountered in monoclonal antibody (mAb) formulations. Because of their impact on colloidal stability, they are commonly avoided, due to a suspected link with aggregation and reduced product shelf-life. However, the molecular underpinnings and interrelation between these phenomena remain unclear, complicating predictions of their occurrence. By combining light and X-ray scattering techniques with microscopy and advanced microfluidic setups, we here report the delicate phase behavior of a model mAb, named mAb1. This is characterized by rapid clustering and LLPS in a narrow NaCl range, above which it transitions into an opalescent state devoid of micron-sized assemblies, yet retaining a similar interaction fingerprint. Using Monte Carlo simulations, we report that the macroscopic solution state of mAb1 is controlled by a positive patch, whose degree of charge screening determines whether LLPS or opalescence will take place. Specifically, neutralization of this patch via counterion interactions diminishes intermolecular repulsion and favors the concerted action of weaker dipole-dipole/hydrophobic interactions, amounting to the creation of a new solution phase, via LLPS. Further NaCl addition distributes ions more uniformly across the surface, attenuating these attractive interactions, leading to the dismantling of droplets while preserving solution opalescence. Finally, we show that LLPS and opalescence are decoupled from stirring-induced aggregation, challenging an unequivocal relationship between these phenomena. Significance StatementTailoring formulations to maximize the stability of therapeutic antibodies is crucial for their development. This is complicated by their tendency for self-association at high concentrations, where increased opalescence and phase separation, that are thought to precede irreversible aggregation, are routinely observed. Here, we studied the molecular underpinnings of mAb opalescence versus liquid-liquid phase separation. We report the mechanisms determining the two phenomena and provide a foundation for their prediction, which may guide the rational development of mAb formulations. We further show that LLPS and opalescence can be decoupled from stress-induced aggregation. We hypothesize that excluding mAbs from the bulk solvent via LLPS may even be harnessed to enhance drug product stability.

biophysics↗

bioRxiv2025

A low-complexity linker as a driver of intra- and intermolecular interactions in DNAJB chaperones

J-domain proteins (JDPs) act as major regulators of the proteostasis network by driving the specificity of the Hsp70 machine. Their important functions are mediated by a low-complexity glycine-/phenylalanine-rich region (GF-linker) that links the folded J-domain with the substrate binding domain. Recently, we and others have shown that in an autoinhibited JDP state, an -helix formed within GF blocks the Hsp70 binding site on the J-domain. However, the role of the disordered GF-linker in autoinhibition and how the latter is released, are still not understood. Here, using autoinhibited DNAJB1 and DNAJB6 constructs, we show that in combination with the J-domain, the GF-linker creates a hydrophobic, partially collapsed cluster that shows a remarkable degree of allosteric communication, disruption of which can lead to destabilisation of autoinhibition. Apart from this crucial intramolecular role, we reveal that the GF-linker can also be recognised by the substrate-binding domain of Hsc70 and dictate the lifetime of the entire JDP-Hsc70 complex. Importantly, both the intra- and intermolecular GF-linker functions are DNAJB-class member-specific.

biophysics↗

bioRxiv2024

Amyloid Morphology is Encoded in H-bonds and Electrostatics Interactions Ruling Protein Phase Separation

Ion-protein interactions regulate biological processes and are the basis of key strategies of modulating protein phase diagrams and stability in drug development. Here, we report the mechanisms by which H-bonds and electrostatic interactions in ion-protein systems determine phase separation and amyloid formation. Using microscopy, small-angle X-ray scattering, circular dichroism and atomistic molecular dynamics (MD) simulations, we found that anions specifically interacting with insulin induced phase separation by neutralising the protein charge and forming H-bond bridges between insulin molecules. The same interaction was responsible for an enhanced insulin conformational stability and resistance to oligomerisation. Under aggregation conditions, the anion-protein interaction translated into the activation of a coalescence process, leading to amyloid-like microparticles. This reaction is alternative to conformationally-driven pathways, giving rise to elongated amyloid-like fibrils and occurs in the absence of preferential ion-protein binding. Our findings depict a unifying scenario in which common interactions dictated both phase separation at low temperatures and the occurrence of pronounced heterogeneity in the amyloid morphology at high temperatures, similar to what has previously been reported for protein crystal growth.

biophysics↗

bioRxiv2022

Heterogeneous and Surface-Catalyzed Amyloid Aggregation Monitored by Spatially Resolved Fluorescence and Single Molecule Microscopy

Amyloid aggregation is associated with many diseases and may also occur in therapeutic protein formulations. Addition of co-solutes is a key strategy to modulate the stability of proteins in pharmaceutical formulations and select inhibitors for drug design in the context of diseases. However, the heterogeneous nature of this multi-component system in terms of structures and mechanisms poses a number of challenges for the analysis of the chemical reaction. Combining a spatially resolved fluorescence approach with single molecule microscopy and machine learning approaches, we disentangle the different contributions from multiple species within a single aggregation experiment. Moreover, we link the presence of interfaces to the degree of heterogeneity of the aggregation kinetics and retrieve the rate constants and underlying mechanisms for single aggregation events, providing a general tool for a comprehensive analysis of self-assembly reactions. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/510935v2_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@c678eaorg.highwire.dtl.DTLVardef@2c6decorg.highwire.dtl.DTLVardef@e4c2e4org.highwire.dtl.DTLVardef@b741aa_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

In vitro and in vivo immunogenicity assessment of protein aggregate characteristics

The immunogenicity risk of therapeutic protein aggregates has been extensively investigated over the past decades. While it is established that not all aggregates are equally immunogenic, the specific aggregate characteristics which are most likely to induce an immune response, remain ambiguous. The aim of this study was to perform comprehensive in vitro and in vivo immunogenicity assessment of human insulin aggregates varying in size, structure and chemical modifications, while keeping other morphological characteristics constant. We found that flexible aggregates with highly altered secondary structure were most immunogenic in all setups, while compact aggregates with native-like structure were found to be immunogenic primarily in vivo. Moreover, sub-visible (1-100 {micro}m) aggregates were found to be more immunogenic than sub-micron (0.1-1 {micro}m) aggregates, while chemical modifications (deamidation, ethylation and covalent dimers) were not found to have any measurable impact on immunogenicity. The findings highlight the importance of utilizing aggregates varying in few characteristics for assessment of immunogenicity risk of specific morphological features and provides a universal workflow for reliable particle analysis in biotherapeutics.

immunology↗

bioRxiv2022

Morphological integrity of insulin amyloid-like aggregates depends on preparation methods and post-production treatments

Protein aggregates are often varying extensively in their morphological characteristics, which may lead to various biological outcomes related to e.g., immunogenicity risk. However, isolation of aggregates with a specific morphology within an ensemble is often challenging. To gain vital knowledge on the effects of aggregate characteristics, samples containing a single morphology must be produced by direct control of the aggregation process. Moreover, the formed aggregates need to be in a solvent suitable for biological assays, while keeping their morphology intact. Here we evaluated the dependence of morphology and integrity of amyloid-like fibrils and spherulites on preparation conditions and post-treatment methods. Samples containing either amyloid-like fibrils or spherulites produced from human insulin in acetic acid solutions are dependent on the presence of salt (NaCl). Moreover, mechanical shaking (600 rpm) inhibits spherulite formation, while only affecting the length of the formed fibrils compared to quiescent conditions. Besides shaking, the initial protein concentration in the formulation was found to control fibril length. Surprisingly, exchanging the solvent used for aggregate formation to a physiologically relevant buffer, had a striking effect on the morphological integrity of the fibril and spherulite samples. Especially the secondary structure of one of our spherulite samples presented dramatic changes of the aggregated {beta}-sheet content after solvent exchange, emphasizing the importance of the aggregate stability. These results and considerations have profound implications on the data interpretation and should be implemented in the workflow for both fundamental characterization of aggregates as well as assays for evaluation of their corresponding biological effects.

biophysics↗

bioRxiv2021

Direct Observation of Anisotropy-Driven Formation of Amyloid Protein Core-Shell Structures in Real-time by Super-resolution Microscopy

The misfolding of proteins and their aggregation in the form of fibrils or amyloid-like spherulites are involved in a spectrum of pathological abnormalities. Our current understanding of protein amyloid aggregation mechanisms has primarily relied on the use of spectrometric methods to determine the average growth rates and diffraction limited microscopes with low temporal resolution to observe the large-scale morphologies of intermediates. We developed a REal-time kinetics via binding and Photobleaching LOcalisation Microscopy (REPLOM) super-resolution method to directly observe and quantify the existence and abundance of diverse aggregate morphologies below the diffraction limit and extract their heterogeneous growth kinetics. Our results revealed that even the growth of a microscopically identical aggregates, e.g. amyloid spherulites, may follow distinct pathways. Specifically, spherulites do not exclusively grow isotropically but, surprisingly, may also grow anisotropically, following similar pathways as reported for minerals and polymers. Combining our technique with machine learning approaches, we associated growth rates to specific morphological transitions and provided energy barriers and the energy landscape at the level of single aggregate morphology. Our unifying framework for the detection and analysis of spherulite growth can be extended to other self-assembled systems characterized by a high degree of heterogeneity, disentangling the broad spectrum of diverse morphologies at the single-molecule level.

biophysics↗

bioRxiv2021

Polysorbate 80 Controls Morphology, Structure and Stability of Human Insulin Amyloid-Like Spherulites

Amyloid protein aggregates are not only associated with neurodegenerative diseases and may also occur as unwanted by-products in protein-based therapeutics. Surfactants are often employed to stabilize protein formulations and reduce the risk of aggregation. However, surfactants alter protein-protein interactions and may thus modulate the physicochemical characteristics of any aggregates formed. Human insulin aggregation was induced at low pH in the presence of varying concentrations of the surfactant polysorbate 80. Various spectroscopic and imaging methods were used to study the aggregation kinetics, as well as structure and morphology of the formed aggregates. Molecular dynamics simulations were employed to investigate the initial interaction between the surfactant and insulin. Addition of polysorbate 80 slowed down, but did not prevent, aggregation of insulin. Amyloid spherulites formed under all conditions, with a higher content of intermolecular beta-sheets in the presence of the surfactant above its critical micelle concentration. In addition, a denser packing was observed, leading to a more stable aggregate. Molecular dynamics simulations suggested a tendency for insulin to form dimers in the presence of the surfactant, indicating a change in protein-protein interactions. It is thus shown that surfactants not only alter aggregation kinetics, but also affect physicochemical properties of any aggregates formed.

biophysics↗

bioRxiv2020

Zinc Determines Dynamical Properties and Aggregation Kinetics of Human Insulin

Protein aggregation is a widespread process leading to deleterious consequences in the organism, with amyloid aggregates being important not only in biology but also for drug design and biomaterial production. Insulin is a protein largely used in diabetes treatment and its amyloid aggregation is at the basis of the so-called insulin-derived amyloidosis. Here we uncover the major role of zinc in both insulin dynamics and aggregation kinetics at low pH, where the formation of different amyloid superstructures (fibrils and spherulites) can be thermally induced. Amyloid aggregation is accompanied by zinc release and the suppression of water-sustained insulin dynamics, as shown by particle-induced X-ray emission and X-ray absorption spectroscopy and by neutron spectroscopy, respectively. Our study shows that zinc binding stabilizes the native form of insulin by facilitating hydration of this hydrophobic protein and suggests that introducing new binding sites for zinc can improve insulin stability and tune its aggregation propensity.Statement of Significance Localized amyloidosis occurs at insulin injection sites for diabetes treatment, leading to deleterious inflammations known as insulin-derived amyloidosis. Amyloid superstructures are also promising candidates in the field of biomaterials. Here we revealed that zinc, coordinated to insulin in the native form, is released upon amyloid aggregation, when insulin forms superstructures known as fibrils and spherulites. Zinc release leads to a full suppression of functionally essential protein dynamics through a modification of the protein’s hydration properties and completely modifies insulin amyloid kinetics. The results suggest that changes in protein hydration upon zinc binding/release modifies both stability and dynamics of insulin and might then be a general strategy to control protein stability and tune protein aggregation into amorphous and ordered superstructures.View Full Text

biophysics↗