Application of 2D-NMR with room temperature NMR probes for the assessment of the higher order structure of filgrastim

Abstract

The higher order structure (HOS) of biotherapeutics is a critical quality attribute that can be evaluated by nuclear magnetic resonance (NMR) spectroscopy at atomic resolution. NMR spectral mapping of HOS can be used to establish HOS consistency of a biologic across manufacturing changes or to compare a biosimilar to an innovator reference product. A previous inter-laboratory study performed using filgrastim drug products demonstrated that two-dimensional (2D)-NMR 1HN-15NH heteronuclear correlation spectroscopy is a highly robust and precise method for mapping the HOS of biologic drugs at natural abundance using high sensitivity NMR ‘cold probes.’ Here, the applicability of the 2D-NMR method to fingerprint the HOS of filgrastim products is demonstrated using lower sensitivity, room temperature NMR probes. Combined chemical shift deviation and principal component analysis are used to illustrate the performance and inter-laboratory precision of the 2D-NMR method when implemented on room temperature probes.

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

“…Indeed, a previous NMR interlaboratory study on the small protein therapeutic filgrastim, displayed a slight clustering of spectra by instrument, although, experimental parameters and instrument performance were not carefully matched in this study. 18,19 Sample conditions must also be carefully considered. Because signals from formulation components can be orders of magnitude greater than those from the protein, even minor differences in formulation conditions may lead to bias in the PCA results.…”

“…Indeed a number of 1D 1 H NMR based methods have been recently proposed for chemometric HOS analysis of mAbs. ,,− The application of chemometric approaches to the problem of 2D spectral analysis of protein therapeutics is less well established but is actively being explored. Proposed approaches include direct point-to-point linear correlation analysis of the processed data matrix, spectral covariance analysis, multivariate principal component analysis both on the processed data matrix , and on tables of peak positions and intensities, spectral distance metrics such as nearest neighbor approaches on peak tables, and total spectral Euclidian deviation …”

Integrative Approaches in Structural Biology: A More Complete Picture from the Combination of Individual Techniques

“…However, PEGylation is a challenge for structural biologist: (1) It inhibits the crystallization of the protein [30], (2) causes serious problems to the standard structural biology techniques [31,32,33,34,35], and (3) limits the use of solution NMR to the small proteins because it significantly increases the hydrodynamic volume of the protein itself [30,36,37,38,39]. Therefore, it is not possible to obtain information at the atomic level to drive any possible structure-based optimization processes using only a single biophysical method.…”

Native peptide mapping – A simple method to routinely monitor higher order structure changes and relation to functional activity

“…[1][2][3] Therefore, the process of defining and controlling product critical quality attributes (CQA), which are defined by the quality by design approach (QbD), [4][5][6] is crucial, and ensures that the safety and efficacy of the product are maintained. 7 The higher order structure (HOS) of a product, which may be considered a CQA, can be studied by a wide range of techniques, such as fluorescence detection, 8 circular dichroism, [9][10][11][12][13] X-ray crystallography, [14][15][16] nuclear magnetic resonance (NMR), [17][18][19] epitope detection and Fourier-transform infrared spectroscopy. [20][21][22][23] This characterization approach is in line with the U.S. Food and Drug Administration's Quality by design (QbD) initiative 24 linking a desired product quality with physico-chemical or biological properties/characteristics.…”

“…The study allowed differentiation between different products. 19 A significant disadvantage of existing HOS analysis techniques is their lack of applicability within a routine quality control (QC) environment. The techniques require expensive equipment, highly trained operators, complex post-acquisition treatment of data and software that would comply with the data integrity requirements prescribed in cGMP.…”