Samah Alharthi

King Abdullah University of Science and Technology

 Revealing the dynamic interactome of human serum albumin by high-resolution NMR

 Among many other transporting proteins the human serum albumin (HSA) handles numerous exogenous and endogenous ligands as well as life-essential metal ions, like Zn(II), in the blood plasma. HSA displays multiple ligand binding sites with extraordinary binding capacity for a wide range of ions and molecules including fatty acids and HSA’s dynamic structures being modulated by them (Al-Harthi et al., 2019). Hence, HSA controls the availability and distribution of those molecules throughout the body. Based on the current knowledge of HSA’s atomic level 3D structures coming from single crystal X-ray diffraction techniques, there are only two main distinct conformations recognized so far, i.e. fatted and defatted HSA. To gain more atomic level insights into the interplay among short, long and medium carbon tail length fatty acids and Zn(II), thus their impact on solution HSA conformation, we followed the spectral changes of HAS’s methyl groups by a series of high resolution NMR SOFAST- methyl-TROSY experiments (Emwas et al., 2019). Besides, the still challenging nature for solution NMR techniques of HSA being 66.5 kDa, the obtained high-resolution and excellent quality spectra of methyl fingerprint region of natural abundance HSA demonstrated unequivocally the binding of ligands and fatty-acids. The analysis of changes of 1H/13C resonance positions of methyl groups points to the substantial conformational changes occurring within the HSA protein (Al-Harthi, in preparation). Subsequent studies aim to combine the state-of-the-art molecular docking and simulations guided by experimental NMR-based information to reveal the dynamic structures of HSA and the mechanism of the interplay between different fatty acids and zinc ions. These results might help in more rational search and design of drugs targeting HSA against numerous human disorders, like diabetes, where equilibrium in blood of fatty acids and metal ions is impaired.

 

Juan Araneda

Nanalysis

 Quantitative analysis of cannabinoids using Benchtop NMR instruments

 Liquid chromatographic methods are traditionally used in the cannabis industry to separate and quantify the multiple components present in cannabis samples. Even though these methods are the gold standard in the industry, they could be overkill for some applications because they are expensive, both in initial capital cost as well as in recurring operating expenditures, and take a relatively long time.

 Nuclear Magnetic Resonance (NMR) Spectroscopy has proven to be an attractive alternative, except for the costs associated with traditional super-conducting NMR spectrometers. The emergence of a new class of instrument: high-resolution benchtop NMR spectrometers offer a fast, easy-to-use low-maintenance alternative to quantify some key components of cannabis samples. Herein we will discuss our most recent results using 60 MHz NMR spectrometers to determine the ratio of tetrahydrocannabinol (THC) and cannabidiol (CBD) in cannabis products.

 

Victor Beaumont

Pfizer

 Using NMR Chemometrics to Detect and Quantitate Structural Differences in Monoclonal Antibodies Under Forced Degradation Conditions

 Therapeutic monoclonal antibodies (mAbs) are increasingly occupying a larger share of the pharmaceutical market because of their proven efficacy against diseases such as cancers, autoimmune diseases, cardiovascular disease, and inflammation. Heightened characterization of protein structure of the active mAb biologic is important during process and product development to ensure that quality, safety, and efficacy is maintained for clinical drug substance materials. Forced degradation studies using pH extremes, chemical oxidation, light, and elevated temperature are used to identify potential aggregation and fragmentation events, protein modifications such as deamidation, isomerization, oxidation, and changes in biological activity. For assessment of higher order structure (HOS), the common analytical techniques CD, IR, DSC, and fluorescence range in sensitivity for detection of potential structural changes following forced degradation. Nuclear magnetic resonance (NMR) spectroscopy provides high sensitivity and high resolution at the atomic level, and in recent years, high-field NMR has been used to characterize higher order structure differences for unlabeled therapeutic mAbs (1). More recent developments in pulse sequences (2, 3), software (4), and chemometric tools (5, 6) now permit NMR to be used more quantitatively for comparative HOS analysis, similar to CD, but with much higher sensitivity and resolution.

 In this study, mAb-1 and mAb-2 were buffer exchanged into three solutions with pH values of 4.5, 5.8 and 7.4, and then subjected to elevated temperatures (40 °C) for 4 weeks. Liquid chromatography-mass spectrometry (LC-MS) studies show that mAb-1 does not significantly degrade relative to the unstressed material, but mAb-2 shows significant deamidation and isomerization. For high-quality NMR spectra of an intact mAb (~150 kDa) in the presence of formulation excipients, replicates of the 1D 1H PGSTE and 1H-13C 2D ALSOFAST-HMQC experiments for each sample were collected and processed. The new software MBioHOS provided robust and easy chemometric and statistical analysis by implementing Protein Fingerprint by Lineshape Enhancement (PROFILE), Principal Component Analysis (PCA), and Easy Comparability of HOS (ECHOS).

 Results from the PROFILE, ECHOS, and PCA of mAb-1 do not show significant differences between the thermally perturbed samples at different pH conditions and the unstressed starting material. However, mAb-2 shows significant differences between the perturbed samples and the unstressed starting material, especially in the 1D amide region. The overlay of the 2D spectra of mAb-2 indicate that the global 3D structure is consistent but chemical shift and peak intensity differences in only a few resonances are a result of local perturbations. These results are consistent with results from LC-MS and further validate NMR as a useful analytical tool in HOS comparability. In summary, these NMR experiments were conducted directly on unlabeled mAbs in formulation buffer. The high-quality spectra obtained demonstrates the strength of this method when particular pulse sequences, software, and chemometric tools are combined. The proposed high-field NMR method is sensitive enough to detect small local changes and the inherent atomic resolution is capable of pinpointing where these pH-induced modifications occur for proteins that have published/known NMR assignments or in the case of isotopically labeled mAbs.

 Robert G. Brinson, et al. mAbs 2019, 11 (1), 94-105 2. Phillip Zhe Sun, et al. JMR 2003, 161 (2), 168-173 3. Paul Schanda and Bernhard Brutscher JACS 2005, 127 (22), 8014-8015 4. MBioHOS, Santiago de Compostela, Spain, MestreLab Research 2021, www.mestrelab.com 5. Leszek Poppe, et al. Analytical Chemistry 2013, 85 (20), 9623-9629 6. Luke W. Arbogast, et al. Analytical Chemistry 2017, 89 (21), 11839-11845

Kaitlyn Doolittle

Genentech

 qNMR of Small Molecule Process Impurities in Biopharmaceuticals at Genentech

The Small Molecule Process Impurity NMR group in Genentech’s pharmaceutical technical development group faces unique challenges in the quantitative NMR field. Our lab supports pharmaceutical manufacturing and development through tracking low levels of small molecules present as impurities originating from raw materials or degradation, as well as those that are introduced into the product as extractables and leachables. These impurities are critical to track for both toxicity and stability purposes, in order to ensure patient safety and product effectiveness. The majority of pharmaceuticals at Genentech are large proteins, such as monoclonal antibodies, which pose significant challenges in terms of resolving and detecting the signals corresponding to low-level, small molecule impurities. Similarly, the aqueous nature of the drug products would result in limited detection of the impurities if the large water signal were not addressed. The methodology utilized in our group integrates water suppression and protein signal suppression techniques, along with internal and external standards to detect and quantify species that would otherwise be considered untenable given their low levels in these solutions. This poster provides an overview of the standard NMR techniques and general workflows in our lab, as well as some salient examples illustrating their implementation as a practical means by which relevant analytical challenges in biomanufacturing are addressed.

 

David Emmons

AstraZeneca

 Accelerated Make-to-Test process with liquid submission workflow

 Compound profiling requires pure compound of an accurately known amount. By submitting liquid samples post-synthesis, rather than solids, to compound management, manual weighing steps were made obsolete, saving time. The amount of compound is non-destructively measured, using qNMR, directly off the liquid submission sample, increasing the accuracy of the stock solution concentration. This quantitation step simultaneously provides the traditional NMR data for structural identification and relative purity of the compound, while verifying compound solubility in DMSO. The same analytical sample is diluted to 10 mM in compound management and fed forward for compound profiling assays, reusing the material, reducing sample waste, and avoiding false negatives due to undetected DMSO solubility issues.

 

Henry R. N. B. Enninful

Leipzig University

 Effect of Geometric Disorder on Phase Transitions in Disordered Mesoporous Solids:

 Majority of porous solids used in industrial processes such as energy storage, separations and catalysis possess structural disorder over varying length scales. These disorder effects strongly affect the properties of the confining fluids in the pores. Hence, detailed quantification of structural disorder with correlation to fluid phase behavior is a necessary step towards optimization for practical applications.

 Employing the serially connected pore model, (SCPM), we have determined the impact of a number of disorder-related parameters, including effect of pore chain length, “powder effect” and interconnectivity effect on phase transitions in disordered mesopore spaces. Additionally, we have showed experimental results from solid-liquid phase transitions obtained by NMR cryoporometry and gas-liquid transitions observed from nitrogen sorption experiments to corroborate the theoretical predictions from the SCPM.

 We conclude that, the SCPM has the potential of explaining many features of experimentally observed phase transitions in disordered mesoporous solids.

 References [1] Schneider D.; Kondrashova D.; Valiullin R., 2017, “Phase transitions in disordered mesoporous solids”, Scientific Reports, 7, 7216. [2] Schneider, D. and Valiullin R., 2019, “Capillary condensation and evaporation in irregular channels: Sorption isotherm for serially connected pore model”, Journal of Physical Chemistry C, 123, 16239 [3] Enninful H.R.N.B., Schneider D., Hoppe A., König S., Fröba M., Enke D. and Valiullin R., 2019, “Comparative gas sorption and cryoporometry study of mesoporous glass structure: Application of the serially connected pore model”, Frontiers in Chemistry, doi: 10.3389/fchem.2019.00230. [4] Enninful H.R.N.B., Schneider D., Kohns R., Enke D. and Valiullin R., 2020, “A novel approach for advanced thermoporometry characterization of mesoporous solids: Transition kernels and the serially connected pore model”, Microporous and Mesoporous Materials 309, 110534. [5] Enninful H.R.N.B., Schneider D., Enke D. and Valiullin R., 2021, “Impact of Geometrical Disorder on Phase Equilibria of Fluids and Solids Confined in Mesoporous Materials”, Langmuir, Under Revision.

 

 

Piotr Garbacz

 University of Warsaw

 Direct discrimination between chiral molecules by antisymmetric spin-spin interactions

 Standard NMR spectra cannot provide directly information about molecular chirality – indirect methods are used instead: chiral solvents, chiral derivatizing agents and chiral lanthanide shift reagents. The disadvantages of these indirect NMR methods, which hinder their industrial application, are that the sample modification is required, and more importantly, their limited applicability as these methods are specific to a given class of compounds. This limitation may be lifted if an external electric field is applied. It is predicted that the electric oscillating at the frequency equal the difference between the spin precession frequencies of two nuclear spins induces chirality-sensitive zero-quantum coherences. It is anticipated that for one enantiomer an NMR line will be positive, while for the other enantiomer negative, i.e., shifted in the phase by the 180 degrees in respect of the first enantiomer, and there is no line expected for an achiral molecule. The magnitude of the effect is determined by antisymmetric parts of the tensors describing the spin-spin interactions. Moreover, the proposed effect can be used for determination of the three-dimensional structure of a chiral molecule because their magnitudes depend on local nuclear properties and the direction of the permeant electric dipole moment of the molecule. Therefore, it provides an alternative method of structure determination to those based on the nuclear Overhauser effect and the Karplus relation.

Derrick Green

Green Imaging Technologies

Measuring Relative Permeability With NMR

Relative permeability is one of the most important petrophysical parameters assessed in core analysis experiments. In the unsteady state relative permeability measurement, the core plug is saturated with one immiscible fluid while another fluid is then injected into the core and displaces the first fluid. The pressure across the core and the average saturation in the core are monitored as a function of time. The relative permeability is calculated from this pressure and saturation data following breakthrough of the injected fluid from the core. Breakthrough is marked by a sharp decrease in pressure across the core and it is crucial that this decrease in pressure be well corelated with the satuation data.

 In a recent paper, we presented an unsteady state relative permeability measurement where NMR data was employed to determine saturation. Breakthrough was precisely determined by monitoring the saturation in the core using one dimensional saturation profile measurements. Following breakthrough, the saturation of the core was tracted as a function of time using the T2 distribution. The T2 distribution is derived from the Carr-Purcell-Meiboom-Gill (CPMG) NMR pulse sequence. We believe this measurement is more accurate than the conventional method because it directly measures the in-situ saturation profiles in the core rather than relying on the material balance method, where factors such as dead volumes, grain loss and instrument uncertainties can lead to errors in estimating core saturations. While the accuracy of the NMR relative permeability measurement is better than traditional method, it still hinges on determining both the pressure and NMR core saturation simultaneously. In our previous paper, the pressure and core saturation were measured separately. These measurements were then corelated in post-experiment analysis using their independent time stamps. This method is open to error as the clock employed for the pressure and NMR measurements could be out of sync. A more accurate method would be to have the pressure measurement integrated into the NMR pulse sequence so each T2 distribution would have a pressure measurement associated with it. We set out to add this functionality to our pulse sequence this year and increase the accuracy of our relative permeability measurement.

 A development tool known as the Application Builder was employed to integrate accurately the NMR measurements with external measurements such as pressure. We demonstate in this paper a fully working integrated NMR/pressure system. In addition, we investigate the inaccuracies introduced if the breakthough point is incorrectly mapped and miss aligned wit the saturation data. The functionality we have built into our pulse sequence is only the beginning of what we can achieve using integrated measurements. We can measure other parameters like temperature, volume etc. In addition, we can also operate other equipment such as pressure pumps or heating apparatus.

Mark Irving

 Rigel Pharmaceuticals

 Dual binding modes of R406 in kinases active site are due to molecular conformation state change

 Establishing structural relationships between biologically active molecules and protein binding sites is a driving force behind drug discovery process. Understanding three-dimensional molecular structure plays an important and necessary role in the early drug development phase. We demonstrate here how biological assay readouts, density function theory (DFT), protein structure and nuclear magnetic resonance (NMR) theory can map small molecule drug-protein interactions. More specifically, we examined an active pharmaceutical ingredient (R940406) using the above techniques and propose the biological activity measured in both spleen tyrosine kinase (SYK) and vascular endothelial growth factor receptor 2 (VEGFR-2) come from two distinct conformational states of the molecule. The probability (energy) of each state appears loosely correlated to cellular biological activity.

Jessica Kelz

University of California, Irvine

 Accessible Method to Achieve Optimized and Intricate Wire Transceiver Coil Designs

 3D printing offers a faster, less expensive, and more accessible approach to hardware conventionally manufactured by hand. It has been used to generate many components of probes with a promising future in enabling modular approaches to instrumentation which will readily expand the capabilities available to experimentalists posed with ever changing challenges.

 1 The central component of an NMR probe is the transceiver coil, and one component of its effectiveness at interfacing with a sample is the radiofrequency (rf) homogeneity of the B1 field it produces. Traditionally, wire transceiver coils have been made by hand, generally requiring many repetitions to yield an adequate result and limiting achievable designs either due to lack of experience or inability to accurately produce them by eye. While solenoids are relatively easy to make and widely used in NMR because of their large filling factor and high sensitivity, the axial field profile drops off sharply toward the coil ends. The axial length of the homogeneous region of the coil can be extended if the current density is increased by decreasing the spacing between turns at the ends, otherwise known as variable pitch. In order to fabricate transceiver coils that accurately reflect design specifications that are difficult or impossible to achieve by hand, we developed a method to make 3D-printed polymer forms referred to as dissolvable inserts for achieving performance enhanced resonators (DIAPERs). These forms, guide the wire as it is wrapped and can be removed by dissolving in an appropriate solvent, allowing recovery of the coil with minimal risk of deformation. The utility of DIAPERs to enable accurate manufacture of several different variable-pitch solenoids, has been demonstrated by comparing experimental results to theory-driven modeling predictions.

 2 This work was then expanded upon through a collaborative effort with the Department of Electrical Engineering and Computer Sciences to design and fabricate coils with optimized magnetic field profiles. Experimental considerations specific to coil design such as material, wire gauge, minimum allowable spacing between turns, maximum axial length, and inner/outer diameters were defined as constraints in an effort to control for overall performance such as the Q factor of the coil. The parameterization was composed in a MATLAB script that enables the creation of a parameter space that encompasses all possible combinations that yield viable results based on user-defined constraints specific to their system. From here, a rough Biot-Savart calculation using the thin-wire approximation can be completed in order to quickly identify areas of the parameter space with desired field profiles, whether this be maximum B1, maximum length of axial homogeneity or uniformity of the field profile. After choosing a design, the profile is verified by running a more rigorous simulation in Computer Simulations Technology MicroWave Studio Suite at a representative tuned frequency and then exported for use in Autodesk Inventor Computer Aided Design (CAD) software. The surface of the wire is cut from a template geometry such as a cylinder and saved as a .STL file which is compatible with 3D-printing slicer software. This was performed using a modification to a previously published optimized design3 for a larger diameter and shorter axial length coil than those for the MAS probe that the coil will be implemented into for experimental validation. This validated that the developed method could at least meet or improve upon a previously published “optimized” design 3 for axial homogeneity. Further analysis determined that this could be further simplified from a three variable exponential parameterization of the pitch in the original work3 to a two-variable gaussian parameterization. We will present the characterization of an optimized variable-pitch solenoid for a magic-angle spinning (MAS) probe made using this method using both benchtop and experimental analyses. Progress toward expanding this method for use in switched-angle spinning (SAS) and solution-state probes through templates for transverse resonators such as saddle-coils or tilted solenoids will also be presented. This work is focused on providing a fast, customizable, and readily implemented approach based on experimentally driven objectives for transceiver performance. Spectroscopists will be empowered with a reproducible, scalable and generalized method for design and fabrication which has the potential to contribute broadly to the field of magnetic resonance both in improving efficiency of large-scale production and further enabling development of coil forms for novel applications.

 References: (1) Kelz, J. I.; Uribe, J. L.; Martin, R. W. Reimagining Magnetic Resonance Instrumentation Using Open Maker Tools and Hardware as Protocol. J. Magn. Reson. Open 2021, 6-7, 100011. https://doi.org/10.1016/j.jmro.2021.100011. (2) Kelz, J. I.; Kelly, J. E.; Martin, R. W. 3D-Printed Dissolvable Inserts for Efficient and Customizable Fabrication of NMR Transceiver Coils. J. Magn. Reson. 2019, 305, 89-92. https://doi.org/10.1016/j.jmr.2019.06.008. (3) Idziak, S.; Haeberlen, U. Design and Construction of a High Homogeneity Rf Coil for Solid-State Multiple-Pulse NMR. J. Magn. Reson. 1969 1982, 50 (2), 281-288.

 

Maxwell Marple

Lawrence Livermore National Laboratory

 Fluoropolymer Crystalline Domain Size Determination by 19F Spin Diffusion and Relaxometry

Fluoropolymers are an important group of polymers to many technological sectors due to their excellent physical properties and stability against chemical and thermal degradation. Many fluoropolymers are semi-crystalline, and their resulting mechanical properties are largely dictated by the degree of crystallinity and the morphology of the crystalline domains. Detailed characterization of the crystalline content and crystalline domains is then required for developing models relating the microscopic structure to macroscopic mechanical properties especially in the cases of polymer aging and degradation. Proton driven spin diffusion NMR has been the preeminent technique for characterizing polymer domain sizes. However, in the case of perfluorinated polymers and co-polymer blends, the absence of proton spins prevents traditional proton spin diffusion from being used. Alternatively, 19F is a 100% natural abundance, high γ nuclei, with relatively high receptivity compared to 1H, and is therefore amenable to spin diffusion studies in fluoropolymers. Fluorine driven spin diffusion in polymers has not been as widely explored and may have key differences in the spin diffusion mechanism as fluorine tends to have much larger chemical shift anisotropy and weaker dipolar interactions relative to the almost entirely dipolar driven proton spin diffusion process. This work explores how fluorine driven spin diffusion can be used for the determination of domain sizes in perfluorinated polymers. Additionally, results from 19F relaxometry will be presented to show how NMR relaxation parameters can relatively easily provide information on the polymer crystalline content.

 

David McGarvey

U.S. Army Chemical Biological Center

 Solid and Liquid NMR Analysis of Chemical Agent Reaction Masses

 The Army requires the capability to destroy relatively small amounts of nerve agents in the field. These may take the form of munitions, or drums of these supertoxic materials. This research explores the analytical challenges presented by characterizing the reaction masses formed when nerve agents (VX, Sarin, Soman, and their precursors) react with lithium nitride and water to form heterogeneous mixtures of agent breakdown products. Some cases result in the complete solidification of the materials with minimal amounts of trapped liquid in the solid matrix, while other remain in a slurry form. Using both solid and liquid methods of NMR analysis, this research presents methods for the complete characterization of the complex mixtures.

 

Klas Meyer

Bundesanstalt für Materialforschung und -prüfung (BAM)

 Compact NMR Spectroscopy in the field: A Versatile Tool for Automated Continuous-Flow Production

Chemical companies must find new paths to successfully survive in a changing environment. The potential of digital technologies belongs to these. Flexible and modular chemical plants can produce various high-quality products using multi-purpose equipment with short downtimes between campaigns and reduce time to market for new products. Intensified continuous production plants allow for difficult to produce compounds. Therefore, fully automated “chemical” process control along with real-time quality control are prerequisites to such concepts and thus should be based on “chemical” information. A commercially available benchtop NMR spectrometer was integrated to the full requirements of an automated chemical production environment such as, e.g., explosion safety, field communication, and robust evaluation of sensor data. It was thereof used for direct loop advanced process control and real-time optimization of the process. Field studies in modular and conventional production plant setups show promising results gaining process knowledge for further optimization. NMR appeared as preeminent online analytical method and allow using a modular data analysis tool, which even served as reliable reference method for further PAT applications (e.g. NIR spectroscopy). In the future, such fully integrated and intelligently interconnecting “smart” systems and processes can speed up the high-quality production of specialty chemicals and pharmaceuticals.

 

Samantha Miller

Colorado State University

 Nanoconfinement Raises the Barrier to Hydrogen Atom Exchange between Water and Glucose

 In bulk aqueous environments, the exchange of protons between labile hydroxyl groups typically occurs easily and quickly. In a bulk mixture containing glucose and water, the timescale of exchange occurs so quickly that exchange broadening prevents the observation of the glucose hydroxyl peaks [1]. However, nanoconfinement can dramatically change this normally facile process [2]. Reverse micelles make a particularly ideal way to achieve a nanoscale environment because of extensive previous characterization and their ease of preparation [3,4].

 Our experiments utilize exchange spectroscopy (EXSY) NMR in order to quantify the kinetics of hydrogen exchange. We observe that the nanoconfinement of glucose and water within our AOT (sodium bis(2-ethylhexyl) sulfosuccinate) reverse micelles raises the energy barrier to labile hydrogen exchange which suggests a disruption of the hydrogen bond network. Near room temperature, the barrier is high enough to slow the process by as much as two orders of magnitude. Although exchange rates slow with decreasing temperatures, the barrier we measure below ~285 K is three to five times lower than the barrier measured at room temperature, indicating a change in mechanism for the process. These findings suggest the possibility of hydrogen tunneling at a surprisingly high temperature threshold. Literature precedent for this behavior exists, but at temperatures far lower than we have shown here [5]. Furthermore, our experiment investigated all hydroxyl groups on the glucose pyranose ring, allowing us to hypothesize that nanoconfinement favors a specific orientation of the glucose molecule at the reverse micelle interface.

 Wiebenga-Sanford, B. et al., J. Phys. Chem. Lett., 7(22), 4597-4601, 2016 2. Wiebenga-Sanford, B. et al., J. Phys. Chem. B, 122(41), 9555-9566, 2018 3. Suzuki, A. et al., Langmuir, 30, 7274-7282, 2014 4. Eskisi, G. et al. J. Phys. Chem. B, 120(44), 11337-1347, 2016 5. Meng, X. et al., Nat. Phys., 113(1), 235-239, 2015.

 

Cameron Robertson / Tomris Coban

Kingston University

Complex mixture NMR analysis; Characterization and Quantitation of compound interaction in consumer healthcare

The accurate characterization and quantitation of biologically active compounds and their interactions in formulations is of considerable importance when evaluating consumer health products for efficacy and activity. We share some of our group’s methodologies used in projects in collaboration with industry on this topic, ranging from analysis of biofuels all the way to vitamin supplementation. Using qNMR techniques augmented with DOSY and STD acquisitions we can reliably measure the abundances of excipients in various complex mixtures and how they interplay. NMR is of particular value to biological, consumer mixtures as it is non-destructive, uses a primary ratio method for quantification, and tolerates a wide variety of hydrophilic and hydrophobic components within a given matrix. In our investigations we have observed trueness levels <10% RSD, repeatability values of <1% RSD and brought the limit of quantitation down to 100nM (?limit of baseline range for several formulation targets and excipients) for our mainstream NMR instrument. Our studies show the potential of novel NMR techniques in complex mixture analysis from an industry point of view, with formulation excipient interactions explored, how these interactions may affect delivery of actives to consumers and how this delivery subsequently affects biomarkers of health in the consumer. Successful project fulfilment with industry partners has further validated this approach through increased interest and quantifiable impact being undertaken evidenced by industrial partners to highlight the significance of NMR for innovation.

 

Alexander Rueck

Sigma-Aldrich Production GmbH

 Certification of gravimetrically prepared organic solutions by quantitative NMR

 Quantitative NMR (qNMR) spectroscopy has become an important tool for the content determination of organic substances and the quantitative evaluation of impurities. Since the signal intensity is directly proportional to the number of protons contributing to the resonance, qNMR is considered as a relative primary method [1-3]. We are using the method under ISO/IEC 17025 and ISO 17034 [4,5] for the development of certified reference material (CRM). Metrological traceability to the SI is achieved using primary Reference Materials from the National Institute of Standards and Technology (NIST) [6] and the National Metrology Institute of Japan (NMIJ). Neat material CRM for qNMR is already well established for different nuclei (1H, 31P, 19F) [7]. Here we show the application of qNMR for the development of various CRM in deuterated solvents and a comparison of the results of gravimetrically prepared organic solutions with the corresponding qNMR measurements.

 [1] Malz F, Jancke H, Journal of Pharmaceutical and Biomedical Analysis, 38(5), 813-823, 2005 [2] Saito T, Ihara T, Koike M, Kinugasa S, Fujimine Y, Nose K, Hirai T, Accreditation and Quality Assurance, 14(2), 79-86, 2009 [3] De Bievre P, Dybkaer R, Fajgelj A, Hibbert BD, Pure and Applied Chemistry, 83(10), 1873-1935, 2011 [4] ISO/IEC 17025:2017, “General requirements for the competence of testing and calibration laboratories” [5] ISO 17034:2016, “General requirements for the competence of reference material producers” [6] M. A. Nelson, J. F. Waters, B. Toman, B. E. Lang, A. Rück, K. Breitruck, M. Obkircher, A. Windust, and K. A. Lippa, Analytical Chemistry 2018 90 (17), 10510-10517 [7] Rigger R, Hellriegel C, Rueck A, Sauermoser R, Morf F, Breitruck K, Obkircher M, Journal of AOAC International, 100(5), 1365-1375, 2017

 

James Sagar

Oxford Instruments

 Broadband Benchtop NMR for Rapid Development and Quality Control of Battery Electrolytes

Currently, all commercial batteries and those technologies expected to come into production in the near future are based on electrolytes containing solvated ions. Due to the wide variety of salts, organic solvents and additives, electrolytes may have significant differences in thermal resistance, chemical stability, ionic conductivity, and electrode compatibility. These variations can greatly affect battery performance, lifetime, safety, and application scope. Therefore, accurately and comprehensively characterizing the electrolyte as well as understanding and controlling its method of action are indispensable in the development and quality control of battery technology.

 While wet chemistry and electrochemical techniques are commonly used for electrolyte analysis, new benchtop NMR instruments have the potential to provide faster, qualitative, and quantitative results with a high degree of accuracy and chemical specificity, allowing monitoring of a wide range of electrolyte properties on a single instrument. Specific examples of great importance in battery electrolyte design and QA/QC, include monitoring electrolyte degradation via standard one-dimensional NMR, identification, and quantification of electrolyte components via multinuclear one-dimensional, 1H, 19F, 7Li, 31P and 11B NMR, and the use of pulsed field gradient NMR to measure properties such as ionic conductivity and the transference numbers will be examined.

 

Thilini Oshadhi Senarath Ukwaththage

Louisiana State University

 Investigations of the structure and protein-protein interactions of chlamydia trachomatis scc4

 Being the most common sexually transmitted bacterial disease, Chlamydia trachomatis (CT) infects 1.7 million people in the United States and 90 million infections globally in 2017. Specific chlamydia chaperone 4 (Scc4) is a bi-functional, unique protein in the CT developmental cycle. As a type three secretion system (T3SS) chaperone, it interacts with the specific chlamydia chaperone 1 (Scc1) and chlamydial outer protein N (CopN) which is an essential effector in bacterial virulence. This Scc1:Scc4 complex is important to regulate and release virulence factor CopN through the T3SS needle. Also, Scc4 directly alters the σ66 dependent transcription in CT by interacting with RNA polymerase holoenzyme (RNAP). Due to these essential and multiple roles of Scc4 in CT propagation and pathogenesis, Scc4 is a significant virulence target for therapeutic approaches for treating Chlamydial infections. In this study, the 3D structure of Scc4 was investigated using solution NMR spectroscopy. The poor-quality NMR spectra produced by Scc4 with the 6X-histidine affinity purification tag led us to develop a novel strategy to purify milligram quantities of tag-free Scc4. Using triple resonance (1H, 15N, 13C) NMR experiments, resonances from 89% of the amino acids in Scc4 were assigned in the protein backbone and side chains. From these assignments and nuclear Overhauser effect spectra, distance and angle restraint data were calculated and analyzed to complete the 3D structure of Scc4. With the Scc4 structure, the identification of small molecular inhibitors by in silico molecular docking with fragment libraries will be investigated in the future. Also, the Scc4 interactions with its binding partner of Scc1 were investigated. These interactions showed that the Scc4 total conformational rearrangement was necessary to function as a T3SS chaperone protein with Scc1.

 

Tsega Solomon

 National Institutes of Standards and Technology

 Integration of 2D NMR Fingerprinting with other Analytical and Functional Methods: A case study of Oxidation of the NISTmAb

 The clinical efficacy and safety of protein-based drugs such as monoclonal antibodies rely on the integrity of the protein structure from development and manufacturing processes to storage and patient administration. Since misfolding, aggregation, or chemical modifications of mAbs affect the biological activity and safety of the drugs, the quality attributes of therapeutic mAbs are rigorously evaluated under pharmaceutically relevant conditions including forced degradation studies. While low to moderate resolution analytical techniques are commonly used in the pharmaceutical industry to assess the activity, quality, and integrity of mAbs, integration of high-resolution NMR with these methods provides atomic level structural information that enables reliable and precise characterization of mAb drugs. The present study demonstrates how high resolution NMR fingerprinting and a number of analytical techniques including low-field benchtop NMR, Tycho, SPR and SEC can be implemented in parallel to evaluate a model mAb therapeutic, NISTmAb, subjected to an oxidative stress condition. This case study involves monitoring the time dependent oxidation of methionine residues of NISTmAb by hydrogen peroxide in a benchtop NMR instrument and analyzing time course aliquots through 2D 1H-13C HSQC Methyl fingerprint spectra in addition to the different analytical methods. The measurement outputs from the different techniques show higher order structural changes of oxidized NISTmAb that are complementary to stability and functional changes and correlate with oxidation time course. The result indicate that the combined approach of analytical tools can be used to extract structural changes of therapeutic mAbs that are functionally relevant and reliably decide the efficacy and safety of mAb drugs.

 

 Marc Taraban

University of Maryland

 Inspecting Insulin Products Using Water Proton NMR

The current practice of quality control inspection of finished drug products involves statistical sampling of a small fraction from a batch that is subjected to invasive analysis. However, statistical sampling could easily miss the defective drug product. In the well-known case of Novomix® 30 insulin pen recall, such statistical sampling failed to detect a fill-finish error resulting in millions of insulin pens recalled and a documented serious adverse event.

 Both the pharmaceutical industry and patient community would benefit from the quality control of every drug container. Yet, such inspection is possible only noninvasively—maintaining the integrity of a drug product to still be used by a patient. We have demonstrated the potential of water proton NMR (wNMR) to detect incorrect insulin dose level in prefilled insulin pens noninvasively in the intact pen. Four insulin products in prefilled pens—Lantus®, Basaglar®, Humalog® and Admelog®—have been noninvasively and quantitatively analyzed at 4°C by wNMR using a variable temperature low-field time-domain benchtop NMR instrument. Importantly, Lantus®/ Basaglar®, and Humalog®/Admelog® are the respective pairs of the innovator and follow-on products.

 Within a carton of each insulin product, insignificant variation of water proton transverse relaxation rate R2(1H2O) were observed which points to insulin content uniformity. On the other hand, differences between R2(1H2O) for innovator drug and corresponding follow-on product were detected. We also show that exposing the insulin solution to air results in significant R2(1H2O) changes.

 Our observations show that wNMR can noninvasively and quantitatively inspect insulin pens which could make it possible for the pharmaceutical industry to inspect every drug unit. wNMR is capable to distinguish between innovator drug and its follow-on product which might benefit the drug characterization for regulatory purposes. We also demonstrated that exposure to air, which is unavoidable during invasive analyses, could affect the results and outcomes of such analyses.

 

Jose Uribe

University of California, Irvine

 Method Automation to Achieve Reproducible Mapping of Transceiver Coils

 Achieving homogenous radio-frequency (rf) magnetic fields in solid-state NMR transceiver coils is crucial for maximizing sensitivity during experimentation. Methods to measure coil homogeneity successfully and accurately has been a time-consuming and error-prone task. Conventionally this has been performed using a manual ball-shift method to map the magnetic field of a resonant cavity, rf coil, by measuring perturbations in the tuning frequency during sequential advancements of a small conductor. However, with this manual setup it is difficult to achieve adequate precision to ensure uniform steps, as this is done by hand and turns are estimated by eyeballing. This human error causes large errors in data collection due to the high sensitivity of perturbations to small movements. Furthermore, assessing accurate results from this setup is difficult, thus, reproducibility is hard to attain. This work has developed an automated method which uses inexpensive and open-source equipment to create a modular, yet specialized tool. The manual method uses 4-40 thread, meaning 40 turns per inch, pushing the small conductor 0.025″ each turn. The benefit of micro-stepping using a stepper motor capable of 200 steps per revolution, using the same 4-40 thread, will be capable of 0.005″ increments, increasing resolution in field mapping. The stepper motor is fully controlled by an Arduino DUE with 12-bit analogue-to-digital converter (ADC) capabilities. The difference is not only smaller increments, but accurate results about rf coil inhomogeneity as well. Moreover, this automated method will serve as quality control for our theory-driven parameterized and optimized coil designs, which have similar appearance but very distinct field profiles. Once modeled, our coils are made using 3D-printed forms. A high-resolution benchtop method is required to test small changes in these coils. This automated method is fully customizable to each lab’s needs for probe testing. Changing the input-output devices controlled with an Arduino micro-controller allows for a wide range of modularity. Automating methods like this work can make testing of large-scale production or new coil designs a time-efficient task. Automating data collection is an important step in completing this testing package and will remove any human interaction in the test process. A mini-Vector Network Analyzer (VNA) is lower-cost and portable VNA which can potentially serve this use. We will present data using this device, comparing our optimized variable-pitch coil designs to theoretical modeling results. In addition, in-house manufacturing of solid-state NMR spinning assemblies is not commonly practiced due to expensive machine-methods or rate of production. Using automation methods and 3D-printing capabilities, we present achievable methods of modulating spinning assemblies.