Biology subjects

Brakti, I.

Publications and source records attributed to Brakti, I..

3 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↗

bioRxiv2024

The importance of stereochemistry in the disorder-order continuum of protein-protein interactions

Intrinsically disordered proteins can bind via the formation of highly disordered protein complexes without the formation of 3D-structure. Most naturally occurring proteins are "left-handed" or levorotatory (L), made up only of L-amino acids, imprinting molecular structure and communication with stereochemistry. In contrast, their mirror image "right-handed" or dextrorotatory (D) amino acids are rare in Nature. Whether disordered protein complexes are truly independent of 3D-topology and thus of chiral constraints is not clear. To test the chiral constraints of disordered protein-protein interactions, a set of interacting protein pairs covering the disorder-order continuum was chosen as representative examples. By observing both the natural ligands and their stereochemical mirror images in free and bound states, we discovered that chirality was inconsequential in a fully disordered complex. However, if the interaction relied on the ligand undergoing coupled folding and binding, correct stereochemistry was essential. Between these extremes, binding could be observed for the D-ligand with a strength that correlated with the amount of disorder in the final complex. These findings have important implications for our understanding of protein-protein interactions, the molecular processes leading to complex formation, the use of D-peptides in drug discovery, and the chemistry of protein evolution of the first living entities on Earth.

biophysics↗