Water Proton Transverse Relaxation Research Articles

Low-field NMR Works as a Rapid, Automatic, Non-Invasive and Wide-Scale Coverage Technique for Aggregates Indication in Biomacromolecule Development

Protein aggregation is challenging for biopharmaceutical drug, because it affects the stability of protein formulations in real-time. However, current techniques for protein aggregate indication meet a number of limitations including limited aggregate size range, complex pre-treatments and lack of chromatographic approaches. Herein, a rapid, automatic, non-invasive and wide-scale coverage technique for aggregates indication is developed to overcome these challenges. Firstly, the response of low-field nuclear magnetic resonance (LF-NMR) to the aggregates is explored by making a comparison with certain established techniques. LF-NMR achieves a high sensitivity of water proton transverse relaxation rate (R2 of H2O, hereinafter referred as R2(H2O)) to protein aggregates from nanometer to micrometer. Then, the quantitative relationship between R2(H2O) and aggregates is investigated furtherly. R2(H2O) could serve as an all-size coverage protein aggregates indicator during development. As a non-invasive method, LF-NMR does not need any sample handling. It takes only 44 s for one test, and saves a lot of manpower, materials and costs. Compared with other established analytical techniques, the technique developed here could be a powerful tool for a rapid, automatic, non-invasive and wide-scale coverage technique for aggregates indication in biomacromolecule development.

Relationship of Iron Intake, Ferritin, and Hepcidin with the Transverse Relaxation Rate of Water Protons in the Pancreas.

(1) Background: There is a paucity of markers of iron metabolism in health and disease. The aim was to investigate the associations of iron metabolism with pancreas transverse water proton relaxation rate (R2water) in healthy individuals and people after an attack of pancreatitis. (2) Methods: All participants underwent a 3.0 T magnetic resonance imaging of the abdomen on the same scanner. High-speed T2-corrected multi-echo (HISTO) acquisition at single-voxel magnetic resonance spectroscopy and inline processing were used to quantify pancreas R2water. Habitual dietary intake of iron was determined using the EPIC-Norfolk food frequency questionnaire. Circulating levels of ferritin and hepcidin were measured. Generalised additive models were used, adjusting for age, sex, body mass index, and haemoglobin A1c. (3) Results: A total of 139 individuals (47 healthy individuals, 54 individuals after acute pancreatitis, and 38 individuals after chronic pancreatitis) were included. Total dietary intake of iron was significantly associated with pancreas R2water, consistently in healthy individuals (p < 0.001), individuals after acute pancreatitis (p < 0.001), and individuals after chronic pancreatitis (p < 0.001) across all the statistical models. Ferritin was significantly associated with pancreas R2water, consistently in healthy individuals (p < 0.001), individuals after acute pancreatitis (p < 0.001), and individuals after chronic pancreatitis (p = 0.01) across all adjusted models. Hepcidin was significantly associated with pancreas R2water in individuals after acute pancreatitis (p < 0.001) and individuals after chronic pancreatitis (p = 0.04) in the most adjusted model. (4) Conclusions: Pancreas R2water, corrected for T2, is related to iron metabolism in both health and pancreatitis. This non-invasive marker could be used for automated in vivo identification of intra-pancreatic iron deposition.

Orientation dependent proton transverse relaxation in human brain white matter: The magic angle effect on a cylindrical helix

PurposeTo overcome some limitations of previous proton orientation-dependent transverse relaxation formalisms in the human brain white matter (WM) by a generalized magic angle effect function. MethodsA cylindrical helix model was developed embracing anisotropic rotational and translational diffusion of restricted molecules in WM, with the former characterized by an axially symmetric system. Transverse relaxation rates R2 and R2∗ were divided into isotropic R2i and anisotropic parts, R2a ∗ f(α,Φ − ε0), with α denoting an open angle and ε0 an orientation (Φ) offset from DTI-derived primary diffusivity direction. The proposed framework (Fit A) was compared to prior models without ε0 on previously published water and methylene proton transverse relaxation rates from developing, healthy, and pathological WM at 3 T. Goodness of fit was represented by root-mean-square error (RMSE). F-test and linear correlation were used with statistical significance set to P ≤ 0.05. ResultsFit A significantly (P < 0.01) outperformed prior models as demonstrated by reduced RMSEs, e.g., 0.349 vs. 0.724 in myelin water. Fitted ε0 was in good agreement with calculated ε0 from directional diffusivities. Compared with those from healthy adult, the fitted R2i, R2a, and α from neonates were substantially reduced but ε0 increased, consistent with developing myelination. Significant positive (R2i) and negative (α and R2a) correlations were found with aging (demyelination) in elderly. ConclusionThe developed framework can better characterize orientation dependences from a wide range of proton transverse relaxation measurements in the human brain WM, thus shedding new light on myelin microstructural alterations at the molecular level.

Effect of ultrasound on mill starch and protein in ultrasound-assisted laboratory-scale corn wet-milling

Ultrasound was applied to facilitate starch-protein separation during ultrasound-assisted laboratory-scale corn wet-milling. The effects of ultrasound on the properties of mill starch and protein were investigated to explore the possible mechanisms in this study. A higher starch yield was obtained with sonication treatment in starch-protein separation (15 min, 200 W) and the results showed that ultrasound did not induce damage to the product starch. The differences in the distribution of water proton transverse relaxation time for mill starch suggested that the interaction between starch granules and protein in mill starch was destroyed. The free sulfhydryl content increased by 37.21% and the disulfide bond content decreased by 43.66% of the separated proteins indicated that ultrasound destroyed the disulfide bonds effectively (p < 0.05). Sonication treatment for longer time or at higher power decreased α-helix and β-turn while increased β-sheet and random coil (p < 0.05). Ultrasound increased surface hydrophobicity and decreased the thermal stability of the protein (p < 0.05). The higher intensity of ultraviolet–visible spectroscopy and ultraviolet-difference absorption spectroscopy indicated the unfolding of protein by sonication treatments. Ultrasound may break down the disulfide bonds and hydrogen bonds of protein molecules, effectively releasing starch from the disintegrated protein agglomerates.

Monitoring of the sedimentation kinetics of vaccine adjuvants using water proton NMR relaxation.

Suspensions of solid particles find applications in many areas-mining, waste treatment, and in pharmaceutical formulations. Pharmaceutical suspensions include aluminum-adjuvanted vaccines are widely administered to millions of people worldwide annually. Hence, the stability parameters of such suspensions, for example, sedimentation rate and the compactness of the formed sediments, are of great interest to achieve the most optimal and stable formulations. Unlike currently used analytical techniques involving visual observations and/or monitoring of several optical properties using specialized glassware, water proton nuclear magnetic resonance (wNMR) used in this work allows one to analyze samples in their original sealed container regardless of its opacity and/or labeling. It was demonstrated that the water proton transverse relaxation rate could be used to monitor in real time the sedimentation process of two widely used aluminum adjuvants-Alhydrogel® and Adju-Phos®. Using wNMR, we obtained valuable information on the sedimentation rate, dynamics of the supernatant and sediment formation, and the sedimentation volume ratio (SVR) reflecting the compactness of the formed sediment. Results on SVR from wNMR were verified by caliper measurements. Verification of the sedimentation rate results from wNMR by other analytical techniques is challenging due to differences in the measured attributes and even units of the reported rate. Nonetheless, our results demonstrate the practical applicability of wNMR as an analytical tool to study pharmaceutical suspensions, for example, aluminum-adjuvanted vaccines, to provide higher quality and more efficient vaccines. Such analyses could be carried out in the original container of a suspension drug product to study its colloidal stability and to monitor its quality over time without compromising product integrity.

Quality assurance at the point-of-care: Noninvasively detecting vaccine freezing variability using water proton NMR

Aluminum-adjuvanted vaccines are freeze-sensitive products that require attentive cold chain adherence. Freeze/thaw events can be tested using “The World Health Organization Shake Test”, a qualitative test whereby a vial from the batch suspected to have been frozen is checked to infer whether the whole batch has been frozen. In this paper, we present a noninvasive and quantitative method to detect whether a vial of liquid vaccine has experienced freeze/thaw using the water proton transverse relaxation rate by Nuclear Magnetic Resonance relaxometry (wNMR relaxometry). Importantly, wNMR relaxometry does not compromise the vial’s integrity so the analyzed vial can be used for vaccination if it meets the quality specifications. Vial-to-vial variability in freezing susceptibility within a single carton of vaccine vials was also detected, both by visual observation and concurrently by wNMR relaxometry. This variability brings into question the practice of using one or a few vials in a batch of vaccines to infer about the quality of the whole batch.

Freeze-thaw Properties of β-glucan Gels

The network structure of β-glucan polymers and the presence of water have significant effects on the properties of β-glucan gels produced by freeze-thaw cycles. The properties of the gel are influenced by its fine structure, molecular size, mass fraction, number of freeze-thaw cycles, and temperature of the β-glucan. To characterize β-glucan freeze-thaw gels, we used low-field nuclear magnetic resonance (LF-NMR) to measure water proton transverse relaxation in aqueous β-glucan solutions during storage. The results indicated that microphase separation occurred during cryogelation, and three water components were identified in the cryostructure. The spin-spin relaxation time was analyzed on the basis of chemical exchange and diffusion exchange theory. The location of each water component was identified in the porous microstructure of the cryogel. The pore size measured from scanning electron microscopy (SEM) images agreed with the pore size estimated from relaxation time. The formation of cryogel was confirmed by rheological. The results suggested that LF-NMR can monitor the polysaccharide cryogelation process by the evolution of spin-spin relaxation characteristics.

Magnetic Resonance Relaxometry for Determination of Protein Concentration and Aggregation.

The water-proton signal, overwhelmingly considered a nuisance in nuclear magnetic resonance spectroscopy, is advantageously used as a tool to assess protein concentration and to detect protein aggregates in aqueous solutions. The protocols in this article describe use of the water-proton transverse relaxation rate to determine concentration and aggregate content in protein solutions. Detailed recommendations and description of the parameter settings and data processing ensure successful implementation of this technique, even by a user with limited experience in magnetic resonance relaxometry. All measurements are done noninvasively, in a sealed container, without sampling or otherwise aliquoting the solution. The magnetic resonance relaxometry approach offered in this article could be advantageous for analysis of biologics formulations or when use of conventional analytical techniques is not possible. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Nuclear magnetic resonance (NMR) relaxometry to measure protein concentration Basic Protocol 2: NMR relaxometry to measure protein aggregation.

Flow Water Proton NMR: In-Line Process Analytical Technology for Continuous Biomanufacturing.

Continuous manufacturing of biologics is one of the priorities of the biopharmaceutical industry. However, its widespread implementation is hampered by a lack of noninvasive/nondestructive process analytical technology (PAT) systems capable of real-time in-line monitoring of product flow parameters, such as concentration and/or aggregate content. We have previously demonstrated that, under nonflow conditions, the water proton transverse relaxation rate, R2(1H2O), is sensitive to protein concentration and aggregate content in biopharmaceutical formulations. In the present work, we explored the potential of water proton NMR under flow conditions (flow-wNMR) to use R2(1H2O) as a quantitative indicator of protein concentration variations and aggregate levels in the process flow. We show that, under flow conditions, R2(1H2O) is sensitive to rather small changes in protein concentration (<1 mg/mL) and is capable to detect variations in the aggregate content of <1%. Our findings suggest that flow-wNMR could be advantageously used as a real-time in-line noninvasive PAT for continuous biomanufacturing.

D‐Glucuronic Acid‐Coated Ultrasmall Paramagnetic Ln2O3 (Ln = Tb, Dy, and Ho) Nanoparticles: Magnetic Properties, Water Proton Relaxivities, and Fluorescence Properties

Many of the lanthanide‐based nanoparticles are valuable in magnetic resonance imaging (MRI) because they have excellent magnetic properties that make them suitable for MRI contrast agents, while some of them have additional optical properties and thus can serve as fluorescence imaging (FI) agents as well. Herein, we synthesized d‐glucuronic acid‐coated ultrasmall Ln2O3 (Ln = Tb, Dy, and Ho) nanoparticles with average diameters of ∼ 2 nm. All the nanoparticles showed unsaturated but appreciable magnetization of ∼ 4 emu/g at an applied field of 1.8 T at room temperature, and consequently, high transverse water proton relaxivities (r2) of ∼ 7 s–1 mm–1 at 1.5 T, ∼ 35 s–1 mm–1 at 3.0 T, and ∼ 55 s–1 mm–1 at 9.4 T with negligible longitudinal water proton relaxivities (r1), thus being eligible for use as T2 MRI contrast agents at high MR fields. In addition, d‐glucuronic acid‐coated ultrasmall Tb2O3 nanoparticles exhibited fluorescence in the green region, useful for dual‐modal imaging.

Nondestructive Quantitative Inspection of Drug Products Using Benchtop NMR Relaxometry-the Case of NovoMix® 30.

Batch-level inference-based quality control is the standard practice for drug products. However, rare drug product defects may be missed by batch-level statistical sampling, where a subset of vials in a batch is tested quantitatively but destructively. In 2013, a suspension insulin product, NovoLog® Mix 70/30 was recalled due to a manufacturing error, which resulted in insulin strength deviations up to 50% from the labeled value. This study analyzed currently marketed FlexPen® devices by the water proton transverse relaxation rate using a benchtop nuclear magnetic resonance relaxometer. The water proton transverse relaxation rate was found to be sensitive to detecting concentration changes of the FlexPen® product. These findings support the development of vial-level verification-based quality control for drug products where every vial in a batch is inspected quantitatively but nondestructively.

Use of Water Proton NMR to Characterize Protein Aggregates: Gauging the Response and Sensitivity.

Water proton transverse relaxation rate R2(1H2O) measurements by NMR stand out as a powerful noninvasive tool to detect protein aggregates, including subvisible particles in biopharmaceutical formulations. To understand the applicability of water proton NMR ( wNMR), we studied the response and sensitivity of wNMR to the aggregates of a monoclonal antibody (mAb) within a wide size range at different aggregate levels, for three different physical stresses: freeze-thaw cycling, heating, and agitation. We compared the sensitivity and response of wNMR with those observed by conventional techniques of size exclusion chromatography (SEC), microflow imaging (MFI), and dynamic light scattering (DLS). Our findings showed that wNMR detects mAb aggregates within wide aggregate levels and in a wide range of aggregate sizes. wNMR was sensitive to an increase in soluble protein aggregates in the range of <1.0%. In most cases, wNMR demonstrated linear response toward the aggregate fraction. Nonlinearity of such response potentially points to the presence of larger size aggregates that possibly rearrange and/or dissociate upon dilution. The results demonstrate the potential of wNMR as a quantitative and noninvasive analytical tool for characterizing protein aggregates in biopharmaceutical formulations.

Low-Field NMR in Nondestructive Quantitative Inspection of Drug Products

Batch-to-batch inference-based quality control of drug products has historically been very effective. However, infrequent incidences of drug defects may be missed by traditional batch-to-batch testing, especially for complex drugs, e.g. biologics. Compared to small molecule drugs, complex drugs have many more factors involved their production and manufacturing, and are more prone to degrade with temperature stresses or agitation during transportation and storage. Relying on batch testing, the sole inspection method of released drug products is human visual inspection. Here we investigate the use of low-field NMR (23 MHz) to inspect four types of drug products, each addressing a different potential quality control problem: 1. Novolog FlexPen® (wrong dosage), 2. Ferrlecit® vs. generic (false equivalence), 3. Alhydrogel® (uneven filling of alum adjuvant), 4. Daptacel®, Engerix-B®, and Vaqta® vaccines (freeze/thaw stress). In every case, during our experiments the product (vial or pen) remained sealed without any tampering; low-field NMR as a method for drug product inspection is non-destructive. The water proton transverse relaxation rate, R2(1H2O), was found to be sensitive to concentration changes in aluminum hydroxide adjuvants, and insulin protein. Furthermore, differences in brand name and generic drug products were detected by the R2(1H2O), where properties measured by other analytical methods failed to detect differences. These findings suggest that low-field 1H2O NMR could serve as a non-destructive inspection method for drug products, such as insulin pens and vaccines.

Water Proton Flow-NMR—A Novel Tool for Real-Time In-Line Process Monitoring in Biomanufacturing

Due to serious limitations, such as experiment duration, relaxation parameters, flow-NMR has limited applications to study structure and properties of a solute, especially, in the case of biomacromolecules, or in the manufacturing of biologics. Unlike conventional structural applications of NMR spectroscopy targeted towards complex solute biomacromolecules, its approaches directed to study the interactions between solute and solvent are less explored. However, in aqueous solutions, water molecules bear useful information about the state of solute molecule, in particular, biomacromolecules, e.g., proteins, actively interacting with water molecules. We have already demonstrated that water proton transverse relaxation rate, R2(1H2O), is a powerful indicator of protein aggregation sensitive to a wide range of aggregate sizes. Less is known about flow-NMR potential to explore water-proton interactions. Meanwhile, due to generally high water concentration in aqueous solutions of biomacromolecules and long relaxation times of water, many existing limitations of flow-NMR are lifted when it comes to study water-protein interactions mediated by relaxation. We report here the results obtained by a newly designed low-field benchtop flow-NMR instrument, which can monitor R2(1H2O) within the flow rates ranging from 0 to 50 mL/min. In our experiments, we demonstrated that R2(1H2O) in a flow depends on a flow rate in accordance with theoretically predicted residence time component. We also showed the sensitivity of water flow-NMR to the pH and ionic strength/conductivity of buffered aqueous solutions. R2(1H2O) was also found sensitive to the concentration of proteins in solution in the range from 0 to 50 mg/mL and flow rates from 0 to 50 mL/min. Our results pave the way for the application of water-proton flow-NMR as a noninvasive process analytical technology in continuous biomanufacturing. In a product flow, this unique technique could detect in-line concentration changes and protein aggregates.

Facile synthesis of stable colloidal suspension of amorphous carbon nanoparticles in aqueous medium and their characterization

In this study, amorphous carbon nanoparticles (CNPs) with an average diameter of 2.2 nm were synthesized by reducing dextrose (C6H12O6) with sodium hydroxide in an aqueous medium. The amorphous CNPs formed stable colloidal suspensions in water owing to the presence of hydrophilic functional groups on the nanoparticle surfaces. The amorphous CNPs exhibited ultraviolet (UV)-visible absorption (λabs) at 267 nm and emission (λem) at 453 nm under UV irradiation; thus, the colloidal suspension appeared sky-blue in color under UV irradiation. The amorphous CNPs were paramagnetic with weak magnetization at room temperature and exhibited small longitudinal (r1) and transverse (r2) water proton relaxivities of 0.036 and 0.068 s−1mM−1, respectively. The amorphous CNPs exhibited no cellular toxicity up to the measured carbon concentration of 500 μM and presented fluorescence microscopy images on a micrometer scale, thus demonstrating their utility in fluorescence bio-imaging applications.

Water Proton NMR: A Tool for Protein Aggregation Characterization.

Formulation stability is a critical attribute of any protein-based biopharmaceutical drug due to a protein's inherent tendency to aggregate. Advanced analytical techniques currently used for characterization of protein aggregates are prone to a number of limitations and usually require additional manipulations with the sample, such as dilution, separation, labeling, and use of special cuvettes. In the present work, we compared conventional techniques for the analysis of protein aggregates with a novel approach that employs the water proton transverse relaxation rate R2(1H2O). We explored differences in the sensitivity of conventional techniques, size-exclusion chromatography (SEC), microflow imaging (MFI), and dynamic light scattering (DLS), and water NMR (wNMR) toward the presence of monoclonal antibody aggregates generated by different stresses. We demonstrate that wNMR outperformed SEC, DLS, and MFI in that it was most consistently sensitive to increases in both soluble and insoluble aggregates, including subvisible particles. The simplicity of wNMR, its sensitivity, and possibility of noninvasive measurements are unique advantages that would permit its application for more efficient and higher throughput optimization of protein formulations.

Aggregation and gelation of oat β-glucan in aqueous solution probed by NMR relaxometry

Nuclear magnetic resonance was used to study water proton transverse relaxation in oat β-glucan aqueous solutions during storage. The molecular weights of the samples ranged from 112 to 494kDa. The polysaccharide structure was identified by IR, GC–MS, NMR spectroscopy, and high performance anion exchange chromatography. In 1% solutions, 494kDa β-glucan formed stable solution phase. Aggregates were generated in the solution of low molecular weight polysaccharide. The transverse relaxation time of water associated with the aggregate microphase was about 15ms. The majority of the solution was bulk water, with a relaxation time of 800–1600ms. The proton exchange rate was about 103s−1, calculated from two-site exchange model. The proportion of β-glucan in the aggregate microphase increased from 0 to over 80% of the total β-glucan during storage of 112kDa samples. In the 4% solutions, the evolution of water proton relaxation associated with the gel network can be explained by the fractal structure theory. The relaxation behavior suggested the growth and syneresis of the gel network during the gelation of the low molecular weight β-glucan solutions.

Dextran‐Coated Ultrasmall Gd2O3 Nanoparticles as Potential T1 MRI Contrast Agent

AbstractUltrasmall gadolinium oxide (Gd2O3) nanoparticles coated with dextran ( r=1500 amu) were prepared through one‐pot synthesis and their potential to act as a T1 MRI contrast agent was investigated. The dextran‐coated ultrasmall Gd2O3 nanoparticles were highly water‐dispersible, with an average particle diameter of 1.5 nm and an average hydrodynamic diameter of 12.4 nm, and non‐toxic, as determined in a cellular cytotoxicity test. The longitudinal (r1) and transverse (r2) water proton relaxivities were estimated to be 12.2 and 29.3 s−1mM−1 (r2/r1=2.4), respectively, larger than those of commercial Gd‐chelates. This r1 value is also greater than those for previously reported dextran‐coated large Gd2O3 nanoparticles with particle diameter bigger than 20 nm. Positive contrast magnetic resonance (MR) images were obtained after intravenous injection of a sample solution into a mouse tail, demonstrating the potential of dextran‐coated ultrasmall Gd2O3 nanoparticles as a T1 MRI contrast agent.

Ligand-size and ligand-chain hydrophilicity effects on the relaxometric properties of ultrasmall Gd2O3 nanoparticles

The relaxometric properties of ultrasmall Gd2O3 nanoparticles coated with various ligands were investigated. These ligands include small diacids with hydrophobic chains, namely, succinic acid (Mw = 118.09 amu), glutaric acid (Mw = 132.12 amu), and terephthalic acid (Mw = 166.13 amu), and large polyethylenimines (PEIs) with hydrophilic chains, namely, PEI-1300 (M¯n=1300) and PEI-10000 (M¯n=10000). Ligand-size and ligand-chain hydrophilicity effects were observed. The longitudinal (r1) and transverse (r2) water proton relaxivities generally decreased with increasing ligand-size (the ligand-size effect). The ligand-size effect was weaker for PEI because its hydrophilic chains allow water molecules to access the nanoparticle (the ligand-chain hydrophilicity effect). This result was explained on the basis of the magnetic dipole interaction between the dipoles of the nanoparticle and water proton. In addition, all samples were found to be non-toxic in cellular cytotoxicity tests.

Synthesis, Magnetic Properties, Map Images, and Water Proton Relaxivities of D-Glucuronic Acid Coated Ln2O3 Nanoparticles (Ln = Ho and Er).

T2 MRI contrast agents cannot be synthesized by using molecules but nanoparticles because appreciable magnetic moments at room temperature are needed. Recently, some of lanthanide (Ln) oxide nanoparticles have shown decent magnetic moments at room temperature and even at ultrasmall particle diameters. In this study, we explored D-glucuronic acid coated Ln2O3 nanoparticles (Ln = Ho and Er) with ultrasmall particle diameters. They showed decent magnetic moments at room temperature and as a result, appreciable transverse water proton relaxivities (r2s) at 1.5 tesla MR field. Clear dose-dependent contrast enhancements in R2 map images were observed in both samples. These results showed that D-glucuronic acid coated Ln2O3 nanoparticles (Ln = Ho and Er) would be potential T2 MRI contrast agents at high MR fields.