Biosimilar structural comparability assessment by NMR: from small proteins to monoclonal antibodies

Abstract

Biosimilar drug products must have a demonstrated similarity with respect to the reference product’s molecules in order to ensure both the effectiveness of the drug and the patients’ safety. In this paper the fusion framework of a highly sensitive NMR fingerprinting approach for conformational changes and mathematically-based biosimilarity metrics is introduced. The final goal is to translate the complex spectral information into biosimilarity scores, which are then used to estimate the degree of similarity between the biosimilar and the reference product. The proposed method was successfully applied to a small protein, i.e., filgrastim (neutropenia treatment), which is the first biosimilar approved in the United States, and a relatively large protein, i.e., monoclonal antibody rituximab (lymphoma treatment). This innovative approach introduces a new level of sensitivity to structural changes that are induced by, e.g., a small pH shift or other changes in the protein formulation.

Effect of Polysorbate 20 and Polysorbate 80 on the Higher-Order Structure of a Monoclonal Antibody and Its Fab and Fc Fragments Probed Using 2D Nuclear Magnetic Resonance Spectroscopy

“…Published accounts of the application of 2D NMR techniques to study antibodies are only two years old 33,67,72 , and this study for the first time shows the power of simple 2D NMR techniques in probing how excipients in therapeutic formulations interact with mAbs. Future availability of NMR peak assignments for mAbs, similar to the recently solved backbone assignments for Fc 73 , will further help in pinpointing the observed structural and dynamics changes to specific local regions in mAbs.…”

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

“…1–6 Indeed, we have recently demonstrated the feasibility of recording two-dimensional (2D) heteronuclear 1 H- 15 N and 1 H- 13 C correlation spectra on mAbs, 7–10 however, questions remain as to best practices for spectral analysis in connection with determination of structural comparability of HOS. 5 …”

Platform development for expression and purification of stable isotope labeled monoclonal antibodies inEscherichia coli

“…36,47–49 More recently, the 2D-NMR method is becoming more established as a tool for assessing mAb HOS. 33,35,50,51 However, in the absence of resonance assignments 2D-NMR spectra can only be used as a composite high resolution structural fingerprint. The E. coli expression described herein provides an assessable platform to label a mAb with stable isotopes.…”