Quantifying the Risks of Asparagine Deamidation and Aspartate Isomerization in Biopharmaceuticals by Computing Reaction Free-Energy Surfaces

Early identification of asparagine deamidation and aspartate isomerization degradation sites can facilitate the successful development of biopharmaceuticals. Several knowledge-based models have been proposed to assess these degradation risks. In this study, we propose a physics-based approach to identify the degradation sites on the basis of the free-energy barriers along the prechemical conformational step and the chemical reaction pathway. These contributions are estimated from classical and quantum mechanics/molecular mechanics molecular dynamics simulations. The computed barriers are compared to those for reference reactions in water within GNG and GDG sequence motifs in peptides (which demonstrate the highest degradation rates). Two major factors decreasing the degradation rates relative to the reference reactions are steric hindrance toward accessing reactive conformations and replacement of water by less polar side chains in the solvation shell of transition states. Among the potential degradation sites in the complementarity-determining region of trastuzumab and between two DK sites in glial cell-derived neurotropic factor, this method identified N³⁰T, N⁵⁵G, D¹⁰²G, and D⁹⁵K, respectively, in agreement with experiments. This approach can be incorporated in early computational screening of chemical degradation sites in biopharmaceuticals

Elucidation of lipid nanoparticle surface structure in mRNA vaccines

Abstract Lipid nanoparticles (LNPs) have been used as a carrier for messenger RNA (mRNA) vaccines. Surface properties of LNPs are important to the stability and function of mRNA vaccines. Polyethylene-glycol (PEG) is a functional lipid at the surface of LNPs that improves colloidal stability, increases circulation time, and impacts cellular uptake. In this study, we explore in-depth lipid composition at the surface of mRNA-LNPs using high-field nuclear magnetic resonance (NMR) spectroscopy. Our results provide a unique surface lipid profile of intact LNPs identifying PEG chains and partial ionizable lipids are present with quantification capability. The surface PEG density is determined to reveal the brush-like conformation on the surface of mRNA-LNPs. Furthermore, we implement a diffusion NMR strategy for routine testing of formulated drug products during drug development. Comparative NMR analysis of different vaccine preparations and stability samples provides a global view of the mRNA-LNP surface structure for enhanced product knowledge

Top-Down Structural Analysis of an Intact Monoclonal Antibody by Electron Capture Dissociation-Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry

Top-down electron capture dissociation (ECD) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was performed for structural analysis of an intact monoclonal antibody (IgG1kappa (κ) isotype, ∼148 kDa). Simultaneous ECD for all charge states (42+ to 58+) generates more extensive cleavages than ECD for an isolated single charge state. The cleavages are mainly localized in the variable domains of both heavy and light chains, the respective regions between the variable and constant domains in both chains, the region between heavy-chain constant domains C_H2 and C_H3, and the disulfide bond (S–S)-linked heavy-chain constant domain C_H3. The light chain yields mainly N-terminal fragment ions due to the protection of the interchain disulfide bond between light and heavy chain, and limited cleavage sites are observed in the variable domains for each chain, where the S–S spans the polypeptide backbone. Only a few cleavages in the S–S-linked light-chain constant domain, hinge region, and heavy-chain constant domains C_H1 and C_H2 are observed, leaving glycosylation uncharacterized. Top-down ECD with a custom-built 9.4 T FTICR mass spectrometer provides more extensive sequence coverage for structural characterization of IgG1κ than does top-down collision-induced dissociation (CID) and electron transfer dissociation (ETD) with hybrid quadrupole time-of-flight instruments and comparable sequence coverage for top-down ETD with orbitrap mass analyzers

Top-Down Structural Analysis of an Intact Monoclonal Antibody by Electron Capture Dissociation-Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry

Top-down electron capture dissociation (ECD) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was performed for structural analysis of an intact monoclonal antibody (IgG1kappa (κ) isotype, ∼148 kDa). Simultaneous ECD for all charge states (42+ to 58+) generates more extensive cleavages than ECD for an isolated single charge state. The cleavages are mainly localized in the variable domains of both heavy and light chains, the respective regions between the variable and constant domains in both chains, the region between heavy-chain constant domains C_H2 and C_H3, and the disulfide bond (S–S)-linked heavy-chain constant domain C_H3. The light chain yields mainly N-terminal fragment ions due to the protection of the interchain disulfide bond between light and heavy chain, and limited cleavage sites are observed in the variable domains for each chain, where the S–S spans the polypeptide backbone. Only a few cleavages in the S–S-linked light-chain constant domain, hinge region, and heavy-chain constant domains C_H1 and C_H2 are observed, leaving glycosylation uncharacterized. Top-down ECD with a custom-built 9.4 T FTICR mass spectrometer provides more extensive sequence coverage for structural characterization of IgG1κ than does top-down collision-induced dissociation (CID) and electron transfer dissociation (ETD) with hybrid quadrupole time-of-flight instruments and comparable sequence coverage for top-down ETD with orbitrap mass analyzers

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

ABSTRACTThe 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

Oligonucleotide mapping via mass spectrometry to enable comprehensive primary structure characterization of an mRNA vaccine against SARS-CoV-2

Abstract Oligonucleotide mapping via liquid chromatography with UV detection coupled to tandem mass spectrometry (LC-UV-MS/MS) was recently developed to support development of Comirnaty, the world’s first commercial mRNA vaccine which immunizes against the SARS-CoV-2 virus. Analogous to peptide mapping of therapeutic protein modalities, oligonucleotide mapping described here provides direct primary structure characterization of mRNA, through enzymatic digestion, accurate mass determinations, and optimized collisionally-induced fragmentation. Sample preparation for oligonucleotide mapping is a rapid, one-pot, one-enzyme digestion. The digest is analyzed via LC-MS/MS with an extended gradient and resulting data analysis employs semi-automated software. In a single method, oligonucleotide mapping readouts include a highly reproducible and completely annotated UV chromatogram with 100% maximum sequence coverage, and a microheterogeneity assessment of 5′ terminus capping and 3′ terminus poly(A)-tail length. Oligonucleotide mapping was pivotal to ensure the quality, safety, and efficacy of mRNA vaccines by providing: confirmation of construct identity and primary structure and assessment of product comparability following manufacturing process changes. More broadly, this technique may be used to directly interrogate the primary structure of RNA molecules in general