Precision and robustness of 2D-NMR for structure assessment of filgrastim biosimilars

Abstract

With the advent of biosimilar version of brand biologics, regulatory authorities in all major jurisdictions throughout the world have developed guidance documents to facilitate their approval [1][2][3][4] . The common theme in the new guidance documents is the stipulation that sponsors must demonstrate biosimilarity between their proposed product and an approved reference product by using state-of-the-art analytical technologies to reduce the size of clinical studies. In the US guidance documents a "totality of evidence" approach is described that can be used to establish the degree of similarity and guide regulatory decision making 3, 4 .Higher order structure is an important quality attribute of biosimilars that must be assessed in a thorough comparability exercise. To date, the higher order structure has been evaluated with low-resolution techniques, such as circular dichroism, Fourier transform infrared and Raman spectroscopies, and indirectly with several biological and stability assays 5 . In 2008, a two dimensional nuclear magnetic resonance (2D-NMR) spectroscopy approach was first applied to the high-resolution assessment of the higher order structure of a native Correspondence should be addressed to

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.…”

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.…”

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

“…Four of the samples were from a previous study and will be referred to as lot 1 [10]. Four new samples, herein referred to as lot 2, were purchased for this follow-up study: Amgen (Neupogen ® , Lot: 1036977, Expiry: April 2015), Biocon (NUFIL Safe ™ , Batch number: N010069, Exp: Aug. 2014), INTAS (Neukine ® , B.…”

“…Recently, Ghasriani et al reported a study that evaluated the inter-laboratory precision and robustness of the 2D-NMR method using NMR systems equipped with highly sensitive NMR cold probes [10]. One innovator filgrastim product (Neupogen ® ) and three Indian-sourced products were evaluated in their fully formulated state.…”

“…Filgrastim’s primary structure consists of 175 amino acid residues, including one extra methionine at the N-terminus of the native 174-residue protein. Through the inter-laboratory comparison, Ghasriani et al found that 2D-NMR provides a highly robust and reproducible measurement, and established an inter-laboratory measurement precision of 8 parts per billion (ppb) for the method [10]. …”