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

Abstract

Two-dimensional (2D) ¹H-¹³C methyl NMR provides a powerful tool to probe the higher order structure (HOS) of monoclonal antibodies (mAbs), since spectra can readily be acquired on intact mAbs at natural isotopic abundance, and small changes in chemical environment and structure give rise to observable changes in corresponding spectra, which can be interpreted at atomic resolution. This makes it possible to apply 2D NMR spectral fingerprinting approaches directly to drug products in order to systematically characterize structure and excipient effects. Systematic collections of NMR spectra are often analyzed in terms of the changes in specifically identified peak positions, as well as changes in peak height and line widths. A complementary approach is to apply principal component analysis (PCA) directly to the matrix of spectral data, correlating spectra according to similarities and differences in their overall shapes, rather than according to parameters of individually identified peaks. This is particularly well-suited for spectra of mAbs, where some of the individual peaks might not be well resolved. Here we demonstrate the performance of the PCA method for discriminating structural variation among systematic sets of 2D NMR fingerprint spectra using the NISTmAb and illustrate how spectral variability identified by PCA may be correlated to structure.

Figure 1

Comparison of ¹H-¹³C methyl spectra of the NISTmAb with the DeGly NISTmAb by spectral overlay (A) and linear correlation analysis (B) as well as the native NISTmAb with the ExoGal NISTmAb by spectral overlay (C) and linear correlation analysis (D). Reported correlation coefficients, R_N, are normalized to the expectation value based on the average S/N of the datasets.

Figure 2

PCA Scatterplot from 20 ¹H-¹³C-methyl spectra of NISTmAb isoforms collected at 900 MHz and 50 °C. Points in the scatterplot correspond to spectra listed in Table S2. Dashed lines correspond to the 95 % confidence interval of the spread for each cluster.

Figure 3

Spectral loading plots of the ExoGal- (A) and DeGly- (B) NISTmAb, showing the variance in the 2nd principal component (red and blue contours) of respective 900 MHz ¹H-¹³C methyl spectral series overlaid on the average series spectrum (grey contours). Red/blue contours from the 2^nd PC loading plot show areas of greater/lesser intensity in the ExoGal and DeGly isoform spectra relative to the average spectrum of the series.

Figure 4

PCA Scatterplot from 26 ¹H-¹³C-methyl spectra of blended ExoGal/native NISTmAb collected at 900 MHz and 50 °C. Dashed grey lines correspond to the 95 % confidence interval of the spread for each cluster. Dashed red lines on the 15 %, 7.5 % and native clusters correspond to the 2 σ interval of the spread in the respective clusters.