Combining Unfolding Reversibility Studies and Molecular Dynamics Simulations to Select Aggregation-Resistant Antibodies

Abstract

The efficient development of new therapeutic antibodies relies on developability assessment with biophysical and computational methods to find molecules with drug-like properties such as resistance to aggregation. Despite the many novel approaches to select well-behaved proteins, antibody aggregation during storage is still challenging to predict. For this reason, there is a high demand for methods that can identify aggregation-resistant antibodies. Here, we show that three straightforward techniques can select the aggregation-resistant antibodies from a dataset with 13 molecules. The ReFOLD assay provided information about the ability of the antibodies to refold to monomers after unfolding with chemical denaturants. Modulated scanning fluorimetry (MSF) yielded the temperatures that start causing irreversible unfolding of the proteins. Aggregation was the main reason for poor unfolding reversibility in both ReFOLD and MSF experiments. We therefore performed temperature ramps in molecular dynamics (MD) simulations to obtain partially unfolded antibody domains in silico and used CamSol to assess their aggregation potential. We compared the information from ReFOLD, MSF, and MD to size-exclusion chromatography (SEC) data that shows whether the antibodies aggregated during storage at 4, 25, and 40 °C. Contrary to the aggregation-prone molecules, the antibodies that were resistant to aggregation during storage at 40 °C shared three common features: (i) higher tendency to refold to monomers after unfolding with chemical denaturants, (ii) higher onset temperature of nonreversible unfolding, and (iii) unfolding of regions containing aggregation-prone sequences at higher temperatures in MD simulations.

In Situ Monitoring of Protein Unfolding/Structural States under Cold High-Pressure Stress

“…Cold temperature chains (refrigerated and subzero regimes) are currently the preferred best option to prolong the shelf-life of therapeutic protein solutions, as well as to assure protein stability during the transfer of bulk drug substance to drug product facilities. , That notwithstanding, cold-induced denaturation is expected to occur at a sufficiently low temperature ( T ) and thereby accelerate unwanted protein–protein association events over a long-term storage period. , To this end, understanding protein structural changes under different stresses can be used as an indicator of protein conformational stability and aggregation propensity during formulation development stages. − …”

Approaches to expand the conventional toolbox for discovery and selection of antibodies with drug-like physicochemical properties

“…These measurements can be performed at any temperature of interest and the data is complementary to the thermal denaturation data to estimate the non-native aggregation propensity of antibody candidates. 110 …”

“… 31 Several strategies exist to screen for candidates with low tendency to aggregate based on the analysis of protein-protein, protein-interface interactions or refoldability. 27 , 28 , 93 , 110 However, it is currently close to impossible to predict the tendency of candidates to form larger aggregates, i.e., subvisible and visible protein particles, during production, storage, and post-production handling.…”

It is Never Too Late for a Cocktail - Development and Analytical Characterization of Fixed-dose Antibody Combinations

“…There is substantial knowledge about how to select and develop conventional antibodies with desired physicochemical behaviour. [28][29][30][31] Third, a bispecific antibody will be produced as one DS, while the mAbs in FDCs could be produced as different DSs.…”