Measuring Protein Aggregation and Stability Using High-Throughput Biophysical Approaches

Abstract

Structure-function relationships of biological macromolecules, in particular proteins, provide crucial insights for fundamental biochemistry, medical research and early drug discovery. However, production of recombinant proteins, either for structure determination, functional studies, or to be used as biopharmaceutical products, is often hampered by their instability and propensity to aggregate in solution in vitro. Protein samples of poor quality are often associated with reduced reproducibility as well as high research and production expenses. Several biophysical methods are available for measuring protein aggregation and stability. Yet, discovering and developing means to improve protein behaviour and structure-function integrity remains a demanding task. Here, we discuss workflows that are made possible by adapting established biophysical methods to high-throughput screening approaches. Rapid identification and optimisation of conditions that promote protein stability and reduce aggregation will support researchers and industry to maximise sample quality, stability and reproducibility, thereby reducing research and development time and costs.

Structural, kinetic, and thermodynamic aspects of insulin aggregation

“…Many biochemical and spectroscopic methods, as detailed in Table 1, have been explored to measure the monomeric depletion rate and the rate of formation of oligomeric species. 40,[43][44][45][46][47][48][49] Several other advanced graph fitting and theoretical attempts have also been made to derive a standard set of equations for therapeutic protein fibrillation, taking the secondary nucleation, heterogeneity, and environmental conditions into consideration. 42,[50][51][52][53] The structural aspects of insulin aggregation have been discussed in the next section.…”

Effect of differences preheating temperatures on the functional and physical properties of soy protein concentrate

“…The decrease in solubility in the preheating treatment indicated the occurrence of denaturation and the formation of insoluble aggregates. The formation of aggregates can increase protein stability, thereby preventing further aggregation [28]. Therefore, proteins with low solubility can be utilized to minimize excessive aggregation when proteins are heated during the cooking process, which might result in hardness in foods.…”

“…Exposure to hydrophobic bonds increases the hydrophobicity of the protein surface, which results in decreased protein stability [33]. Proteins with low stability will more easily interact, form bonds with other proteins, and form protein aggregates, which are generally irreversible and very stable [28]. This results in reduced interactions between proteins in solution and a decrease in viscosity.…”

“…The decrease in protein digestibility in the preheating treatment compared to the control treatment can also be caused by the denaturation process and the formation of protein aggregates during the preheating. Protein aggregates are generally irreversible and very stable [28]. Thus, the formation of aggregates inhibits the binding of enzymes to proteins, so that the digestibility of proteins decreases [25].…”

Overcoming aggregation with laser heated nanoelectrospray mass spectrometry: thermal stability and pathways for loss of bicarbonate from carbonic anhydrase II

“…15,17 Fluorescence-based and calorimetric assays are routinely used to study the aggregation temperature of monoclonal antibodies and other biotherapeutic complexes from different buffer conditions. [21][22][23][24] However, all of these methods generally require purified samples and do not provide the type of detailed information that mass spectrometry (MS) can provide about solution conformers, unfolding intermediates, or ligand-binding.…”