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 linewidths. 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.

Platform development for expression and purification of stable isotope labeled monoclonal antibodies inEscherichia coli

“…To determine if glycosylation is responsible for the slower migration of the NISTmAb heavy chain, an aglycosylated form of the NISTmAb was generated using PNGase F as previously described. 21 As shown in Figure 2, the migration of the eNISTmAb heavy chain is similar to the aglycosylated form of the NISTmAb heavy chain (Figure 2, compare lane 3 to lane 2), suggesting that the faster migration of the eNISTmAb heavy chain is due to the lack of glycosylation. Subsequent mass spectral analysis confirmed that the intact masses of the heavy and light chains matched the theoretical masses.…”

“…21,29–36 The appropriate 2D-NMR method was applied to each eNISTmAb sample, e.g ., 1 H, 13 C constant time (CT) heteronuclear single quantum coherence spectroscopy (HSQC) for the U- 13 C sample; 1 H, 15 N gradient-selected (g)HSQC for the U- 15 N sample, and both methods for the triply-labeled 2 H, 13 C, 15 N sample (Figure 6, Fig. S6).…”

“…In total, the observed spectral shifts in the 1 H, 13 C eNISTmAb spectra can be tentatively assigned to the lack of glycosylation of the eNISTmAb and the resulting effects on Fc structure from the lack of glycosylation. 21 …”

Diffusion Profiling of Therapeutic Proteins by Using Solution NMR Spectroscopy

“…Together 1D and 2D NMR profiling methods offer a way to quantify spectral/structural differences between similar proteins, but more information is often needed to capture complicated solution behavior. One example, highlighted in Figure , is for a fast‐acting insulin analogue.…”

“…Ongoing efforts are focused on validating the diffusion profile interpretation of protein behavior by using orthogonal biophysical methods and combining the diffusion profile with NMR‐relaxation measurements to better relate the observed behavior to changes in both structure and dynamics. The diffusion profile shows promise as another 1 H NMR‐based profiling method that can easily be applied to study interactions and higher‐order structure and as a useful addition to the 1D PROFILE and 2D fingerprint methods, as a way to interpret observed changes in NMR spectra in terms of the solution behavior of the protein. All of these methods fit together in various aspects of discovery and development: they can be used to investigate the intrinsic behavior of the protein itself as well as subtle differences or changes in its behavior in formulation.…”