11 research outputs found
Proceedings of Patient Reported Outcome Measure’s (PROMs) Conference Oxford 2017: Advances in Patient Reported Outcomes Research
A33-Effects of Out-of-Pocket (OOP) Payments and Financial Distress on Quality of Life (QoL) of People with Parkinson’s (PwP) and their Carer
Comprehensive assessment of protein and excipient stability in bio-pharmaceutical formulations using 1H NMR spectroscopy
Stability of a high-concentration monoclonal antibody solution produced by liquid-liquid phase separation
Subcutaneous injection of a low volume (100 mg/mL) formulation is an attractive administration strategy for monoclonal antibodies (mAbs) and other biopharmaceutical proteins. Using concentrated solutions may also be beneficial at various stages of bioprocessing. However, concentrating proteins by conventional techniques, such as ultrafiltration, can be time consuming and challenging. Isolation of the dense fraction produced by macroscopic liquid–liquid phase separation (LLPS) has been suggested as a means to produce high-concentration solutions, but practicality of this method, and the stability of the resulting protein solution have not previously been demonstrated. In this proof-of-concept study, we demonstrate that LLPS can be used to concentrate a mAb solution to >170 mg/mL. We show that the structure of the mAb is not altered by LLPS, and unperturbed mAb is recoverable following dilution of the dense fraction, as judged by (1)H nuclear magnetic resonance spectroscopy. Finally, we show that the physical properties and stability of a model high concentration protein formulation obtained from the dense fraction can be improved, for example through the addition of the excipient arginine·glutamate. This results in a stable high-concentration protein formulation with reduced viscosity and no further macroscopic LLPS. Concentrating mAb solutions by LLPS represents a simple and effective technique to progress toward producing high-concentration protein formulations for bioprocessing or administration. Abbreviations Arginine·glutamate (Arg·Glu), Carr-Purcell-Meiboom-Gill (CPMG), critical temperature (T(C)), high-performance size-exclusion chromatography (HPSEC), liquid–liquid phase separation (LLPS), monoclonal antibody (mAb), nuclear magnetic resonance (NMR), transverse relaxation rate (R(2)
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
Control of Peptide Aggregation and Fibrillation by Physical PEGylation
Peptide therapeutics have the potential to self-associate, leading to aggregation and fibrillation. Noncovalent PEGylation offers a strategy to improve their physical stability; an understanding of the behavior of the resulting polymer/ peptide complexes is, however, required. In this study, we have performed a set of experiments with additional mechanistic insight provided by in silico simulations to characterize the molecular organization of these complexes. We used palmitoylated vasoactive intestinal peptide (VIP-palm) stabilized by methoxy-poly(ethylene glycol)(skDa)-cholane (PEG-cholane) as our model system. Homogeneous supramolecular assemblies were found only when complexes of PEG-cholane/VIP-palm exceeded a molar ratio of 2:1; at and above this ratio, the simulations showed minimal exposure of VIP-palm to the solvent. Supramolecular assemblies formed, composed of, on average, 9-11 PEG-cholane/VIP-palm complexes with 2:1 stoichiometry. Our in silico results showed the structural content of the helical conformation in VIP-palm increases when it is complexed with the PEG-cholane molecule; this behavior becomes yet more pronounced when these complexes assemble into larger supramolecular assemblies. Our experimental results support this: the extent to which VIP-palm loses helical structure as a result of thermal denaturation was inversely related to the PEG-cholane:VIP-palm molar ratio. The addition of divalent buffer species and increasing the ionic strength of the solution both accelerate the formation of VIP-palm fibrils, which was partially and fully suppressed by 2 and >4 mol equivalents of PEG-cholane, respectively. We conclude that the relative freedom of the VIP-palm backbone to adopt nonhelical conformations is a key step in the aggregation pathway.Peer reviewe
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Research data supporting "Selective, non-covalent conjugation of synthetic peptides with recombinant proteins mediated by host–guest chemistry"
Raw data to support experiments reported in the main manuscript and ESI of the publicationThis research data supports “Selective, non-covalent conjugation of synthetic peptides with recombinant proteins mediated by host–guest chemistry” which has been published in “Chemical Communications".This work was supported by the ERC [grant number ASPiRe No. 240629]
Impact of a Heat Shock Protein Impurity on the Immunogenicity of Biotherapeutic Monoclonal Antibodies
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
Intense Pulse Light (IPL)
Intense Pulsed Light (IPL) is a light-emitting system that is capable of emitting filtered polychromatic broad bandwidth wavelengths between 515 and 1,200 nm.
The emission of wavelengths is controlled by both an internal filter that blocks undesired wavelengths from being emitted and a “heat-sink” effect that allows the controlled transfer of thermal energy from an object at high temperature to an object at lower temperature