Biology subjects

Lenton, S.

Publications and source records attributed to Lenton, S..

4 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

Lipopolysaccharide lateral mobility in the Gram-negative bacterial outer membrane is confined and governed by interactions within the conserved Lipid A anchor

The Gram-negative bacterial cell envelope is defined by an asymmetric outer membrane where the outer leaflet adopts a highly ordered structure composed principally of lipopolysaccharide molecules. The organisation and dynamics of these glycolipids are key to the ability of the outer membrane to act as an innate barrier against chemical and antibiotic challenges, and as a load bearing element for the cell. Strong intermolecular forces are thought to govern the lateral diffusion of lipopolysaccharide in the outer membrane, but the extent and molecular basis of this diffusion has remained a controversial topic for over 50 years. Here we use a bio-orthogonal labelling strategy and in vivo fluorescence microscopy to unequivocally demonstrate extreme lateral confinement of lipopolysaccharide in the outer membrane of Escherichia coli, regardless of carbohydrate domain size and structure. We specifically identify magnesium cation-mediated interactions at the base of the carbohydrate and hydrophobic interactions within the lipid milieu as critical for lipopolysaccharide confinement. Importantly, these traits are conserved across multiple pathogenic species irrespective of O-antigen and capsular serotype. Together, these findings establish lipopolysaccharide endotoxin lateral confinement as a ubiquitous feature of the outer membrane and highlight potential universal vulnerabilities of the bacterial cell envelope.

microbiology↗

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↗