NMR Spectroscopy for Protein Higher Order Structure Similarity Assessment in Formulated Drug Products.

Deyun Wang,You Zhuo,Kang Chen,Cameron J. Smith,Sharadrao M. Patil,David A. Keire,Mike Karfunkle

doi:10.3390/molecules26144251

Abstract

Peptide and protein drug molecules fold into higher order structures (HOS) in formulation and these folded structures are often critical for drug efficacy and safety. Generic or biosimilar drug products (DPs) need to show similar HOS to the reference product. The solution NMR spectroscopy is a non-invasive, chemically and structurally specific analytical method that is ideal for characterizing protein therapeutics in formulation. However, only limited NMR studies have been performed directly on marketed DPs and questions remain on how to quantitively define similarity. Here, NMR spectra were collected on marketed peptide and protein DPs, including calcitonin-salmon, liraglutide, teriparatide, exenatide, insulin glargine and rituximab. The 1D 1H spectral pattern readily revealed protein HOS heterogeneity, exchange and oligomerization in the different formulations. Principal component analysis (PCA) applied to two rituximab DPs showed consistent results with the previously demonstrated similarity metrics of Mahalanobis distance (DM) of 3.3. The 2D 1H-13C HSQC spectral comparison of insulin glargine DPs provided similarity metrics for chemical shift difference (Δδ) and methyl peak profile, i.e., 4 ppb for 1H, 15 ppb for 13C and 98% peaks with equivalent peak height. Finally, 2D 1H-15N sofast HMQC was demonstrated as a sensitive method for comparison of small protein HOS. The application of NMR procedures and chemometric analysis on therapeutic proteins offer quantitative similarity assessments of DPs with practically achievable similarity metrics.

Highlights

Complex generic and biosimilar drug products (DPs) are increasingly developed and comprehensive analysis of these DPs is the foundation for their regulatory approval [1,2,3,4]
The peptide and protein drug products (DPs) listed in Table 1 were sourced from the US market except Reditux®, which was sourced from India
All the drug products (DP) listed in Table 1 were sourced from the US market except Reditux®, which was sourced from the India market

Summary

Introduction

Complex generic and biosimilar drug products (DPs) are increasingly developed and comprehensive analysis of these DPs is the foundation for their regulatory approval [1,2,3,4]. Proteins can be chemically modified either purposely, e.g., pegylation, or unintentionally, e.g., oxidation, which could introduce variability to protein HOS [10,11,12]. All these factors and the accompanying sensitivity to solution conditions necessitate characterizing the protein chemistry and HOS with minimal perturbation to the formulation by ideally using DPs [13].

Methods

Results

Conclusion