Quantifying the Risks of Asparagine Deamidation and Aspartate Isomerization in Biopharmaceuticals by Computing Reaction Free-Energy Surfaces

Early identification of asparagine deamidation and aspartate isomerization degradation sites can facilitate the successful development of biopharmaceuticals. Several knowledge-based models have been proposed to assess these degradation risks. In this study, we propose a physics-based approach to identify the degradation sites on the basis of the free-energy barriers along the prechemical conformational step and the chemical reaction pathway. These contributions are estimated from classical and quantum mechanics/molecular mechanics molecular dynamics simulations. The computed barriers are compared to those for reference reactions in water within GNG and GDG sequence motifs in peptides (which demonstrate the highest degradation rates). Two major factors decreasing the degradation rates relative to the reference reactions are steric hindrance toward accessing reactive conformations and replacement of water by less polar side chains in the solvation shell of transition states. Among the potential degradation sites in the complementarity-determining region of trastuzumab and between two DK sites in glial cell-derived neurotropic factor, this method identified N<sup>30</sup>T, N<sup>55</sup>G, D<sup>102</sup>G, and D<sup>95</sup>K, respectively, in agreement with experiments. This approach can be incorporated in early computational screening of chemical degradation sites in biopharmaceuticals.