Structural Characterization of Modification on the Interface between a Ligand and its Receptor for Biopharmaceuticals

The binding of a protein to its target is a major mode of action for most biopharmaceutical therapies with over 70% of biopharmaceuticals involved in binding between the protein and its target. The interfaces between a biopharmaceutical and its target are key regions for its efficacy. Any modifications to the amino acids at the interfaces invariably affect interactions between the biopharmaceutical and its receptor and may result in lowering therapeutic efficacy. Degradations of biopharmaceuticals by asparagine (Asn) deamidation and/or aspartate (Asp) isomerization have been well characterized and those modifications at the interfaces have resulted in a loss of activity. To characterize modification hot-spots on the interfaces, it is necessary to identify the amino acid residues on the interfaces. We recently addressed a visualization tool for amino acids on the interfaces between a protein ligand and its receptor. This tool was applied to visualize ligand protein-receptor interaction and antigen-antibody interaction. As a model system for ligand protein-receptor interaction, erythropoietin (EPO) and its receptor were selected and amino acids on the interfaces were identified. Modifications on the interfaces were then investigated. Deamidation of Asn was identified at two amino acid residues, Asn47 and Asp147, on Interface 1 of EPO. The relative contents of deamidated residues on the interface of EPO were in the range of 3-5% of the total. As a model system for antigen-antibody interaction, Herceptin and its receptor, HER2, were chosen and amino acids on the interfaces were identified. Then modifications on the interfaces were assessed. Deamidation of the light chain Asn30 and heavy chain Asn55 were identified. The relative contents of the deamidated residues on the interfaces were in the range of 8-9% of the total. Along with deamidation, another modification, isomerization, was identified at the amino acid residue Asp102 of the heavy chain, and the level of oxidation was 13.5% of the total. Our studies provide a targeted method focusing on the interface between a protein and its target that can be coupled with other applications, for example, identification of modified amino acids on the interfaces.