title: Profiling formulated monoclonal antibodies by (1)H NMR spectroscopy.
abstract: Nuclear magnetic resonance (NMR) is arguably the most direct methodology for characterizing the higher-order structure of proteins in solution. Structural characterization of proteins by NMR typically utilizes heteronuclear experiments. However, for formulated monoclonal antibody (mAb) therapeutics, the use of these approaches is not currently tenable due to the requirements of isotope labeling, the large size of the proteins, and the restraints imposed by various formulations. Here, we present a new strategy to characterize formulated mAbs using (1)H NMR. This method, based on the pulsed field gradient stimulated echo (PGSTE) experiment, facilitates the use of (1)H NMR to generate highly resolved spectra of intact mAbs in their formulation buffers. This method of data acquisition, along with postacquisition signal processing, allows the generation of structural and hydrodynamic profiles of antibodies. We demonstrate how variation of the PGSTE pulse sequence parameters allows proton relaxation rates and relative diffusion coefficients to be obtained in a simple fashion. This new methodology can be used as a robust way to compare and characterize mAb therapeutics.
authors.affiliation: Molecular Structure and Characterization and ‡Process and Product Development, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.
authors.forename: Leszek
authors.initials: L
authors.lastname: Poppe
authors.forename: John B
authors.initials: JB
authors.lastname: Jordan
authors.forename: Ken
authors.initials: K
authors.lastname: Lawson
authors.forename: Matthew
authors.initials: M
authors.lastname: Jerums
authors.forename: Izydor
authors.initials: I
authors.lastname: Apostol
authors.forename: Paul D
authors.initials: PD
authors.lastname: Schnier
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D011487:Protein Conformation
chemical_list: D000911:Antibodies, Monoclonal