Biology subjects

Groenning, M.

Publications and source records attributed to Groenning, M..

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↗

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↗