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

Abstract

An important aspect in the analytical characterization of protein therapeutics is the comprehensive characterization of higher order structure (HOS). Nuclear magnetic resonance (NMR) is arguably the most sensitive method for fingerprinting HOS of a protein in solution. Traditionally, (1)H-(15)N or (1)H-(13)C correlation spectra are used as a "structural fingerprint" of HOS. Here, we demonstrate that protein fingerprint by line shape enhancement (PROFILE), a 1D (1)H NMR spectroscopy fingerprinting approach, is superior to traditional two-dimensional methods using monoclonal antibody samples and a heavily glycosylated protein therapeutic (Epoetin Alfa). PROFILE generates a high resolution structural fingerprint of a therapeutic protein in a fraction of the time required for a 2D NMR experiment. The cross-correlation analysis of PROFILE spectra allows one to distinguish contributions from HOS vs protein heterogeneity, which is difficult to accomplish by 2D NMR. We demonstrate that the major analytical limitation of two-dimensional methods is poor selectivity, which renders these approaches problematic for the purpose of fingerprinting large biological macromolecules.

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.…”

Spin Diffusion Editing for Structural Fingerprints of Therapeutic Antibodies

“…Comparisons of biosimilars to innovator products should also be possible. Similarity measures, based on correlation coefficients between 1D spectral vectors of a reference sample and a test sample as applied to data in Figures 5 and 6, could provide a means of quantitating these differences 7 .…”

“…However, there are special cases, such as the observation of 13 C- 1 H methyl correlations, where observations on whole antibodies have been achieved 6 . The length of acquisition is still long, and a recent analysis has suggested that, for applications to large numbers of samples, alternate methods that depend on one dimensional (1D) proton NMR should be considered 7 .…”

Simple NMR methods for evaluating higher order structures of monoclonal antibody therapeutics with quinary structure

“…Recently, several laboratories have applied 1D 1 H and 2D heteronuclear NMR profiling protocols for structure evaluation of protein therapeutics [10,13,14,24-29], including the proposed biosimilar rituximab at 22 mg/mL [15] and the NIST mAb at 40 mg/mL [12]. Previously Poppe et al [9] developed a 1D- 1 H diffusion filtering method, which reduced the sucrose peaks in the spectrum and was applied on Amgen’s proprietary mAb samples concentrated at 30 mg/mL in 9% sucrose or phosphate buffer [30]. Most recently Franks et al [14] added a saturation transfer pulse segment in front of the diffusion filtering, which appears to suppress the trehalose resonances more efficiently with selective spin saturation at non-excipient chemical shift ranges.…”

“…The method was demonstrated on Pfizer’s proprietary mAb at 11 or 34 mg/mL in a trehalose formulation reconstituted in nearly neat D 2 O [14]. For molecules as large as a 150 kDa mAb, both the 2D heteronuclear NMR and most of the 1D diffusion-filtered 1 H NMR have employed sophisticated data acquisition and processing, high protein concentrations and high temperatures (40–50 °C) [9,12,30], aiming to compensate for the lower S/N caused by short spin-spin relaxation times (T 2 ).…”