A method for determining the amount of a damaged form of a protein

Abstract

The procedure for determining damaged forms of proteins using adsorption on hydrophobic microparticles represents a methodology for the qualitative and quantitative determination of damaged therapeutic proteins, with a subsequent impact on the development and validation of protein drugs. The methodology enables the quantitative determination of the amount of damaged / inactive forms of proteins in the presence of active / functional forms. In order to determine the damaged forms of the protein, about 10-6 grams of the sample is needed, which makes it possible to analyze a number of different external conditions suitable for the storage of protein drugs. The system includes uncharged hydrophobic microparticles to which proteins are bound by passive adsorption depending on their conformation (active and damaged forms). The adsorption mechanism is based primarily on hydrophobic interactions between the hydrophobic part of the adsorbed protein and the hydrophobic surface of the microparticles. By damaging the native form of the protein, the hydrophobic parts of the protein are exposed to the surface. Such a damaged form binds to microparticles to an increased extent compared to the native protein, and based on this difference, it is possible to determine the concentration of both forms side by side.