Application of NMR and Chemometric Analyses to Better Understand the Quality Attributes in pH and Thermally Degraded Monoclonal Antibodies

Abstract

Purpose Nuclear magnetic resonance (NMR) spectroscopy provides the sensitivity and specificity to probe the higher order structure (HOS) of monoclonal antibodies (mAbs) for potential changes. This study demonstrates an application of chemometric tools to measure differences in the NMR spectra of mAbs after forced degradation relative to the respective unstressed starting materials. Methods Samples of adalimumab (Humira, ADL-REF) and trastuzumab (Herceptin, TRA-REF) were incubated in three buffer-pH conditions at 40°C for 4 weeks to compare to a control sample that was left unstressed. Replicate 1D 1H and 2D 1H-13C HMQC NMR spectra were collected on all samples. Chemometric analyses such as Easy Comparability of HOS (ECHOS), PROtein FIngerprinting by Lineshape Enhancement (PROFILE), and Principal Component Analysis (PCA) were applied to capture and quantitate differences between the spectra. Results Visual and statistical inspection of the 2D 1H-13C HMQC spectra of adalimumab and trastuzumab after forced degradation conditions shows no changes in the spectra relative to the unstressed material. Chemometric analysis of the 1D 1H NMR spectra shows only minor changes in the spectra of adalimumab after forced degradation, but significant differences in trastuzumab. Conclusion The chemometric analyses support the lack of statistical differences in the structure of pH-thermal stressed adalimumab, however, it reveals conformational changes or chemical modifications in trastuzumab after forced degradation. Application of chemometrics in comparative NMR studies enables HOS characterization and showcases the sensitivity and specificity in detecting differences in the spectra of mAbs after pH-thermal forced degradation with respect to local and global protein structure.

Comprehensive characterization of higher order structure changes in methionine oxidized monoclonal antibodies via NMR chemometric analysis and biophysical approaches

“… 43 , 44 The similarity score provides robust analysis for intergroup comparison between stressed samples and the control given that the spectral quality and processing are consistent. 45 FDA-based researchers successfully leveraged PCA and Mahalanobis distance (D M ) measurements of 1D 1 H spectra to assess HOS similarity of marketed insulin biosimilars. 36 Additionally, a study led by researchers at the National Institute of Standards and Technology (NIST) demonstrated the robustness of PCA in examining the HOS of NISTmAb via the 2D methyl 1 H- 13 C NMR spectra collected across different academic, industrial, and research laboratories from different countries.…”