Best Practices in Utilization of 2D-NMR Spectral Data as the Input for Chemometric Analysis in Biopharmaceutical Applications

Abstract

Quality attributes (QAs) are measureable parameters of a biologic that impact product safety and efficacy and are essential characteristics that are linked to positive patient health outcomes. One QA, higher order structure (HOS), is directly coupled to the function of protein biologics, and deviations in this QA may cause adverse effects. To address the critical need for HOS assessment, methods for analyzing structural fingerprints from 2D nuclear magnetic resonance spectroscopy (2D-NMR) spectra have been established for drug substances as large as monoclonal antibody therapeutics. Here, chemometric analyses have been applied to 2D 1 H, 13 C-methyl NMR correlation spectra of the IgG1κ NIST monoclonal antibody (NISTmAb), recorded at natural isotopic abundance, to benchmark the performance and robustness of the methods. In particular, a variety of possible spectral input schemes (e.g., chemical shift, peak intensity, and total spectral matrix) into chemometric algorithms are examined using two case studies: (1) a large global 2D-NMR interlaboratory study and (2) a blended series of enzymatically glycan-remodeled NISTmAb isoforms. These case studies demonstrate that the performance of chemometric algorithms using either peak positions or total spectral matrix as the input will depend on the study design and likely be product-specific. In general, peak positions are found to be a more robust spectral parameter for input into chemometric algorithms, whereas the total spectral matrix approach lends itself to easier automation and requires less user intervention. Analysis with different input data also shows differences in sensitivity to certain changes in HOS, highlighting that product knowledge will further guide appropriate method selection based on the fit-for-purpose application in the context of biopharmaceutical development, production, and quality control.

Interlaboratory Studies Using the NISTmAb to Advance Biopharmaceutical Structural Analytics

“…As such, the PCA on peak position confirms the reproducibility of the measurement and shows the applicability of the method even at 500 MHz. Validation of a cluster assignment for a spectrum could be evaluated using the Dunn index or silhouette values ( Brinson et al, 2020 ).…”

“…To fully harmonize the experimental methods and the analysis components of the 2D-NMR method, a global interlaboratory study was conducted with equal representation from academia, government, and industry, involving 26 laboratories from 9 countries, using the NIST-Fab, the Fab fragment derived from papain cleavage of the NISTmAb as the model therapeutic protein for the IgG1 molecular class ( Brinson et al, 2019 ). In addition, two subsequent studies ( Brinson et al, 2020 ; Sheen et al, 2020 ) followed, which used the interlaboratory data to further develop NMR processing and analysis tools well suited for biopharmaceutical applications.…”

Quantification of natural abundance NMR data differentiates the solution behavior of monoclonal antibodies and their fragments

“… 51 This technique can also be used to evaluate the behavior of one molecule that is subjected to different solution or instrument conditions. 38 , 40 One criterion for PCA is that the input data array, across all samples, must have an equivalent number of values. Therefore, the direct application of PCA to 2D NMR, of different Ab(L/F)s, is not straightforward because the structural scaffolds, size and behavior of samples will generate a different number of resonance peaks within each spectrum.…”

“… 31 , 36 , 37 Natural abundance 2D NMR applied to mAbs and Fabs has largely been championed by the Marino group, where they demonstrated multiple applications including detected differences in mAb glycosylation, reported a statistical approach to assess large NMR signal differences between a set of biologics samples, and established the robustness and variability of 2D [ 1 H, 13 C]-HSQC fingerprinting for biosimilar comparison. 30 , 36 , 38–41 However, extracting and comparing quantitative aspects between different types of Ab(L/F)s still remains a challenge.…”

Role of NMR in High Ordered Structure Characterization of Monoclonal Antibodies

“…It was reported that Fc and Fab fragments yielded methyl fingerprint spectra in a much shorter time in approximately 30 min [52]. It should be noted that an international effort of standardization to make the 2D NMR method a highly confident routine way to assess HOS of mAb is underway [63][64][65][66].…”