Influence of Tableting on the Conformation and Thermal Stability of Trypsin as a Model Protein

The objective of this study was to investigate the influence of compaction on the conformation of trypsin, its transition temperature (Tm ) of unfolding, and its folding reversibility after thermal denaturation. Plain trypsin was compacted at 40-382 MPa. Pressure-induced changes in the trypsin conformation and the extent of their reversibility were determined using solid- and liquid-state IR spectroscopy together with principal component analysis and an area overlap approach. Trypsin enzymatic activity was determined by a photometric assay. Liquid-state differential scanning calorimetry was performed to determine the Tm as well as the folding reversibility after thermal denaturation of the reconstituted samples. It was found that compacted samples showed reduced activity accompanied by an altered secondary structure. Conformational changes that occur in the solid state were partially reversible upon tablet reconstitution. Aqueous-state IR spectroscopy combined with partial least squares was shown to be a powerful tool to follow irreversible structural changes and evaluate sample bioactivity. Besides its conformation, the thermal stability of trypsin was altered as a result of the applied compaction pressure, indicated by a reduced folding reversibility. In conclusion, this study reveals that tableting can have a negative impact on the biological quality of protein APIs.