11 research outputs found
Temporal and spatial characterisation of protein liquid-liquid phase separation using NMR spectroscopy
Controlled release and characterisation of photocaged molecules using in situ LED illumination in solution NMR spectroscopy
Assessing Photostability of mAb Formulations In Situ Using Light-Coupled NMR Spectroscopy
Comprehensive Assessment of Protein and Excipient Stability in Biopharmaceutical Formulations Using H-1 NMR Spectroscopy
[Image: see text] Biopharmaceutical proteins are important drug therapies in the treatment of a range of diseases. Proteins, such as antibodies (Abs) and peptides, are prone to chemical and physical degradation, particularly at the high concentrations currently sought for subcutaneous injections, and so formulation conditions, including buffers and excipients, must be optimized to minimize such instabilities. Therefore, both the protein and small molecule content of biopharmaceutical formulations and their stability are critical to a treatment’s success. However, assessing all aspects of protein and small molecule stability currently requires a large number of analytical techniques, most of which involve sample dilution or other manipulations which may themselves distort sample behavior. Here, we demonstrate the application of (1)H nuclear magnetic resonance (NMR) spectroscopy to study both protein and small molecule content and stability in situ in high-concentration (100 mg/mL) Ab formulations. We show that protein degradation (aggregation or fragmentation) can be detected as changes in 1D (1)H NMR signal intensity, while apparent relaxation rates are specifically sensitive to Ab fragmentation. Simultaneously, relaxation-filtered spectra reveal the presence and degradation of small molecule components such as excipients, as well as changes in general solution properties, such as pH. (1)H NMR spectroscopy can thus provide a holistic overview of biopharmaceutical formulation content and stability, providing a preliminary characterization of degradation and acting as a triaging step to guide further analytical techniques
19F Dark-State Exchange Saturation Transfer NMR Reveals Reversible Formation of Protein-Specific Large Clusters in High Concentration Protein Mixtures
Proteins
frequently exist as high-concentration mixtures, both
in biological environments and increasingly in biopharmaceutical co-formulations.
Such crowded conditions promote protein–protein interactions,
potentially leading to formation of protein clusters, aggregation,
and phase separation. Characterizing these interactions and processes in situ in high-concentration mixtures is challenging due
to the complexity and heterogeneity of such systems. Here we demonstrate
the application of the dark-state exchange saturation transfer (DEST)
NMR technique to a mixture of two differentially 19F-labeled
145 kDa monoclonal antibodies (mAbs) to assess reversible temperature-dependent
formation of small and large protein-specific clusters at concentrations
up to 400 mg/mL. 19F DEST allowed quantitative protein-specific
characterization of the cluster populations and sizes for both mAbs
in the mixture under a range of conditions. Additives such as arginine
glutamate and NaCl also had protein-specific effects on the dark-state
populations and cluster characteristics. Notably, both mAbs appear
to largely exist as separate self-associated clusters, which mechanistically
respond differently to changes in solution conditions. We show that
for mixtures of differentially 19F-labeled proteins DEST
NMR can characterize clustering in a protein-specific manner, offering
unique tracking of clustering pathways and a means to understand and
control them
Research data for "Simple and effective in-situ sample illumination for Electron Paramagnetic Resonance"
Raw Electron Paramagnetic Resonance (EPR) data used in the analysis of EPRTorch performance.Accepted in ChemComm. DOI pending, will be added once available
Investigating Liquid–Liquid Phase Separation of a Monoclonal Antibody Using Solution-State NMR Spectroscopy: Effect of Arg·Glu and Arg·HCl
Liquid–liquid
phase separation (LLPS) of monoclonal antibody
(mAb) formulations involves spontaneous separation into dense (protein-rich)
and diluted (protein-lean) phases and should be avoided in the final
drug product. Understanding the factors leading to LLPS and ways to
predict and prevent it would therefore be highly beneficial. Here
we describe the link between LLPS behavior of an IgG1 mAb (mAb5),
its solubility, and parameters extracted using <sup>1</sup>H NMR spectroscopy,
for various formulations. We show that the formulations demonstrating
least LLPS lead to the largest mAb5 NMR signal intensities. In the
formulations exhibiting the highest propensity to phase-separate the
mAb NMR signal intensities are the lowest, even at higher temperatures
without visible phase separation, suggesting a high degree of self-association
prior to distinct phase separation. Addition of arginine glutamate
prevented LLPS and led to a significant increase in the observed mAb
signal intensity, whereas the effect of arginine hydrochloride was
only marginal. Solution NMR spectroscopy was further used to characterize
the protein-lean and protein-rich phases separately and demonstrated
that protein self-association in the protein-rich phase can be significantly
reduced by arginine glutamate. Solution NMR spectroscopy may be useful
as a tool to assess the propensity of mAb solutions to phase-separate
Light‑FESTA: enhancing characteristic 1H signal patterns of fluorinated molecules
This folder contains all NMR raw data for the publication entitled "Lighting up spin systems: enhancing characteristic 1H signal patterns of fluorinated molecules", as well as relevant pulse programs and processing macros for Bruker spectrometers