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

The higher order structure (HOS) of monoclonal antibodies (mAbs) is an important quality attribute with strong contribution to clinically relevant biological functions and drug safety. Due to the multi-faceted nature of HOS, the synergy of multiple complementary analytical approaches can substantially improve the understanding, accuracy, and resolution of HOS characterization. In this study, we applied one- and two-dimensional (1D and 2D) nuclear magnetic resonance (NMR) spectroscopy coupled with chemometric analysis, as well as circular dichroism (CD), differential scanning calorimetry (DSC), and fluorescence spectroscopy as orthogonal methods, to characterize the impact of methionine (Met) oxidation on the HOS of an IgG1 mAb. We used a forced degradation method involving concentration-dependent oxidation by peracetic acid, in which Met oxidation is site-specifically quantified by liquid chromatography-mass spectrometry. Conventional biophysical techniques report nuanced results, in which CD detects no change to the secondary structure and little change in the tertiary structure. Yet, DSC measurements show the destabilization of Fab and Fc domains due to Met oxidation. More importantly, our study demonstrates that 1D and 2D NMR and chemometric analysis can provide semi-quantitative analysis of chemical modifications and resolve localized conformational changes with high sensitivity. Furthermore, we leveraged a novel 15N-Met labeling technique of the antibody to directly observe structural perturbations at the oxidation sites. The NMR methods described here to probe HOS changes are highly reliable and practical in biopharmaceutical characterization.

Characterization of Higher Order Structural Changes of a Thermally Stressed Monoclonal Antibody via Mass Spectrometry Footprinting and Other Biophysical Approaches.

Characterizing changes in the higher order structure (HOS) of monoclonal antibodies upon stressed conditions is critical to gaining a better understanding of the product and process. One single biophysical approach may not be best suited to assess HOS comprehensively; thus, the synergy from multiple, complementary approaches improves characterization accuracy and resolution. In this study, we employed two mass spectrometry (MS )-based footprinting techniques, namely, fast photochemical oxidation of proteins (FPOP)-MS and hydrogen-deuterium exchange (HDX)-MS, supported by dynamic light scattering (DLS), differential scanning calorimetry (DSC), circular dichroism (CD), and nuclear magnetic resonance (NMR) to study changes to the HOS of a mAb upon thermal stress. The biophysical techniques report a nuanced characterization of the HOS in which CD detects no changes to the secondary or tertiary structure, yet DLS measurements show an increase in the hydrodynamic radius. DSC indicates that the stability decreases, and chemical or conformational changes accumulate with incubation time according to NMR. Furthermore, whereas HDX-MS does not indicate HOS changes, FPOP-MS footprinting reveals conformational changes at residue resolution for some amino acids. The local phenomena observed with FPOP-MS indicate that several residues show various patterns of degradation during thermal stress: no change, an increase in solvent exposure, and a biphasic response to solvent exposure. All evidences show that FPOP-MS efficiently resolves subtle structural changes and novel degradation pathways upon thermal stress treatment at residue-level resolution.

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

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.

Allosteric Impact of the Variable Insert Loop in Vaccinia H1-Related (VHR) Phosphatase.

Dynamics and conformational motions are important to the activity of enzymes, including protein tyrosine phosphatases. These motions often extend to regions outside the active site, called allosteric regions. In the tyrosine phosphatase Vaccinia H1-related (VHR) enzyme, we demonstrate the importance of the allosteric interaction between the variable insert region and the active-site loops in VHR. These studies include solution nuclear magnetic resonance, computation, steady-state, and rapid kinetic measurements. Overall, the data indicate concerted millisecond motions exist between the variable insert and the catalytic acid loop in wild-type (WT) VHR. The 150 ns computation studies show a flexible acid loop in WT VHR that opens during the simulation from its initial closed structure. Mutation of the variable insert residue, asparagine 74, to alanine results in a rigidification of the acid loop as observed by molecular dynamics simulations and a disruption of crucial active-site hydrogen bonds. Moreover, enzyme kinetic analysis shows a weakening of substrate affinity in the N74A mutant and a >2-fold decrease in substrate cleavage and hydrolysis rates. These data show that despite being nearly 20 Å from the active site, the variable insert region is linked to the acid loop by coupled millisecond motions, and that disruption of the communication between the variable insert and active site alters the normal catalytic function of VHR and perturbs the active-site environment.