During manufacture of antibody molecules, they are subjected to mechanical stress generated by processes such as pumping and filtration. This causes denaturation and aggregation due to exposure of the protein to air-liquid interfaces and shear forces, resulting in the ultimate loss in bioactivity. Hence during candidate selection, data regarding the aggregation propensity of antibody molecules in various buffer conditions aid in the prediction of problems during manufacturing and shelf-life stability. Shaking stress is a convenient method to screen the robustness of antibodies at air-liquid interfaces.
The fluorophore Thioflavin T (ThT) increases its fluorescence when binding to β-sheet structures that form during antibody aggregation. A BMG LABTECH microplate reader detected ThT fluorescence indicative of antibody aggregation and exposed the samples to mechanical stress by constant shaking of the microplate. Differences in aggregation-propensities of antibodies were identified using this method.