Profiling Formulated Monoclonal Antibodies by ¹H 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.

On the Analytical Superiority of 1D NMR for Fingerprinting the Higher Order Structure of Protein Therapeutics Compared to Multidimensional NMR Methods

“…The NMR measurements were performed on either Bruker 800 or 600 MHz spectrometers. PROFILE spectra data processing was performed with Topspin 3.0 and in-house developed scripts in Matlab, as described in ref 9. The similarity scores were finally compared by the ANOVA statistical analysis.…”

“…We recently proposed the protein fingerprint by line shape enhancement (PROFILE) methodology as an alternative approach to two-dimensional NMR fingerprinting methods. 9 This method exploits differences in the diffusion properties of large antibodies and formulation components to generate a highly resolved one-dimensional 1 H spectrum of the entire mAb, devoid of solvent and excipients. We demonstrated that, for isotopically labeled protein samples, the PROFILE similarity is commensurable and more sensitive to structural changes than spectral similarity as measured from 2D spectra acquired for the same mAbs.…”

Multivariate Analysis of Two-Dimensional 1H, 13C Methyl NMR Spectra of Monoclonal Antibody Therapeutics To Facilitate Assessment of Higher Order Structure

“…3,13 In addition to producing scoring plots to provide statistical differentiation of HOS variation, PCA also produces spectral loading plots, a subset of the full data-matrix describing the sources of variability along each principal component. These results can be visualized as spectra and thus can reveal which particular regions of the spectrum are undergoing variation.…”

High-throughput identification of monoclonal antibodies after compounding by UV spectroscopy coupled to chemometrics analysis

“…However, infrared, Raman, Fourier transform infrared (FTIR), or nuclear magnetic resonance (NMR) present some technical limitations for a fast quality control in a hospital environment that can arise from excipients and water interferences, long sample preparation, the requirement of sample pretreatment, or time-consuming experiments. Recently, NMR spectroscopy has been used to provide a spectral fingerprint of therapeutic mAbs [12]. However, important handling and sample preparation, not compatible with a QC online to patient administration, is required.…”