Mass Spectrometric ITEM-ONE and ITEM-TWO Analyses Confirm and Refine an Assembled Epitope of an Anti-Pertuzumab Affimer.

Intact Transition Epitope Mapping-One-step Non-covalent force Exploitation (ITEM-ONE) analysis reveals an assembled epitope on the surface of Pertuzumab, which is recognized by the anti-Pertuzumab affimer 00557_709097. It encompasses amino acid residues NSGGSIYNQRFKGR, which are part of CDR2, as well as residues FTLSVDR, which are located on the variable region of Pertuzumab's heavy chain and together form a surface area of 1381.46 Å 2 . Despite not being part of Pertuzumab's CDR2, the partial sequence FTLSVDR marks a unique proteotypic Pertuzumab peptide. Binding between intact Pertuzumab and the anti-Pertuzumab affimer was further investigated using the Intact Transition Epitope Mapping-Thermodynamic Weak-force Order (ITEM-TWO) approach. Quantitative analysis of the complex dissociation reaction in the gas phase afforded a quasi-equilibrium constant (KD m0g#) of 3.07 × 10 -12 . The experimentally determined apparent enthalpy (ΔHm0g#) and apparent free energy (ΔGm0g#) of the complex dissociation reaction indicate that the opposite reaction-complex formation-is spontaneous at room temperature. Due to strong binding to Pertuzumab and because of recognizing Pertuzumab's unique partial amino acid sequences, the anti-Pertuzumab affimer 00557_709097 is considered excellently suitable for implementation in Pertuzumab quantitation assays as well as for the accurate therapeutic drug monitoring of Pertuzumab in biological fluids.