Water Proton Transverse Relaxation Rate Research Articles

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.

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.

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.

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.

Linear dependence of the water proton transverse relaxation rate on the shear modulus of hydrogels.

It is found that hydrogelation of peptides enhances the transverse relaxation rate R2 of water protons but has no effect on the longitudinal relaxation rate R1 and the diffusion coefficient D. The magnitude of water proton R2 enhancement increases linearly with the shear modulus G of hydrogels.

Toward understanding transverse relaxation in human brain through its field dependence.

Apparent transverse-relaxation rate constants (R₂⁺ = 1/T₂⁺) were measured in various regions of the healthy human brain using a multiecho adiabatic spin-echo sequence at five different magnetic fields, 1.5, 1.9, 3, 4.7, and 7 T. The R₂⁺ values showed a clear dependence on magnetic field strength (B(0) ). The regional distribution of the R ₂⁺ was well explained by the sum of three components: (1) regional nonhemin iron concentration ([Fe]), (2) regional macromolecular mass fraction (f(M) ), and (3) a region-independent factor. Accordingly, R₂⁺ = α[Fe] + βf(M) + γ, where coefficients α, β, and γ were experimentally determined at each magnetic field by a least square fitting method using multiple regression analysis. Although the coefficient α linearly increased with B(0) , β showed a quadratic dependence on top of a field-independent component. The coefficient γ also increased slightly with B(0) on top of a field-independent component. The linear dependence of α on B(0) was consistent with that observed for the transverse-relaxation rate of water protons in ferritin solutions as found previously by others. The quadratic dependence of β on B(0) was accounted for by isochronous and anisochronous exchange mechanisms using intrinsic-relaxation parameters obtained from the literature.

On the strong field dependence and nonlinear response to gadolinium contrast agent of proton transverse relaxation rates in dairy cream

Dairy cream, as a suspension of lipid droplets in water, is a potentially useful magnetic resonance imaging (MRI) phantom material and an interesting material for studying fundamental relaxation mechanisms. Here we report a strong increase in the transverse relaxation rates with field strength for both the water and lipid protons in dairy cream. Also, studies at 4.7 T reveal a nonlinear response of transverse relaxation rates with increasing concentration of a common gadolinium (Gd)-based contrast agent, including an initial decrease of water relaxation rates as measured with Hahn spin echoes at the lower Gd concentrations. The results are treated within the framework of a model in which the magnetic susceptibility difference between the lipid droplets and the aqueous phase plays the prominent role for transverse relaxation. Second-order polynomial fits of the water proton transverse relaxation rate dependence on field strength and on Gd concentration at 4.7 T provided experimental parameters from which model parameters are extracted and compared with expectations available from the literature.

Gastric Response to Increased Meal Viscosity Assessed by Echo-Planar Magnetic Resonance Imaging in Humans

Normal meals are highly viscous, and viscosity is a key factor in influencing gastric emptying of food. However, the process of meal dilution and mixing is difficult to assess with the use of conventional methods. The aim of this study was to validate an in vivo, novel, noninvasive, echo-planar magnetic resonance imaging (EPI) technique, capable of monitoring the viscosity of a model meal, and to use this to investigate the effects of viscosity on gastric emptying, meal dilution and satiety. Healthy volunteers (n = 8) ingested 500 mL of locust bean gum (0.25, 0.5, 1.0 or 1.5 g/100 g), nonnutrient, liquid meals of varying viscosities, and labeled with a nonabsorbable marker, phenol red. Meal viscosity was calibrated against the water proton transverse relaxation rate (T(2)(-1)) in vitro before ingestion, thus viscosity was measured in vivo via EPI measurements of T(2)(-1). Viscosity and dilution were also measured directly using nasogastric aspirates. Gastric volumes as measured by EPI, fullness, appetite and hunger were also assessed serially. Before ingestion, the log of initial meal viscosity was linearly related to T(2)(-1) (n = 8, r(2) = 0.95). Similarly, T(2)(-1) measured in vivo was also linearly related to the viscosity of the aspirates (r(2) = 0.88). All meals underwent rapid dilution, leading to a reduction in viscosity, which was greatest for the most viscous meal (P < 0.01). Surprisingly, despite the fact that the initial meal viscosity varied 1000-fold, there was only a small delay in gastric emptying (P for trend < 0.05). The area under the curve for satiety increased with initial meal viscosity, whereas that for hunger decreased (P < 0.05). In conclusion, the viscosity of a meal in vivo can be measured noninvasively using EPI. The stomach responds to meal ingestion by rapid intragastric dilution, causing a reduction of meal viscosity, and gastric emptying is minimally delayed. However, increased viscosity is associated with more prolonged satiety.