Ranking mAb-excipient interactions in biologics formulations by NMR spectroscopy and computational approaches.

Excipients are added to biopharmaceutical formulations to enhance protein stability and enable the development of robust formulations with acceptable physicochemical properties, but the mechanism by which they confer stability is not fully understood. Here, we aimed to elucidate the mechanism through direct experimental evidence of the binding affinity of an excipient to a monoclonal antibody (mAb), using saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopic method. We ranked a series of excipients with respect to their dissociation constant (KD) and nonspecific binding constants (Ns). In parallel, molecular dynamic and site identification by ligand competitive saturation (SILCS)-Monte Carlo simulations were done to rank the excipient proximity to the proteins, thereby corroborating the ranking by STD NMR. Finally, the excipient ranking by NMR was correlated with mAb conformational and colloidal stability. Our approach can aid excipient selection in biologic formulations by providing insights into mAb-excipient affinities before conventional and time-consuming excipient screening studies are conducted.

Differential Surface Adsorption Phenomena for Conventional and Novel Surfactants Correlates with Changes in Interfacial mAb Stabilization.

Protein adsorption on surfaces can result in loss of drug product stability and efficacy during the production, storage, and administration of protein-based therapeutics. Surface-active agents (excipients) are typically added in protein formulations to prevent undesired interactions of proteins on surfaces and protein particle formation/aggregation in solution. The objective of this work is to understand the molecular-level competitive adsorption mechanism between the monoclonal antibody (mAb) and a commercially used excipient, polysorbate 80 (PS80), and a novel excipient, N-myristoyl phenylalanine-N-polyetheramine diamide (FM1000). The relative rate of adsorption of PS80 and FM1000 was studied by pendant bubble tensiometry. We find that FM1000 saturates the interface faster than PS80. Additionally, the surface-adsorbed amounts from X-ray reflectivity (XRR) measurements show that FM1000 blocks a larger percentage of interfacial area than PS80, indicating that a lower bulk FM1000 surface concentration is sufficient to prevent protein adsorption onto the air/water interface. XRR models reveal that with an increase in mAb concentration (0.5-2.5 mg/mL: IV based formulations), an increased amount of PS80 concentration (below critical micelle concentration, CMC) is required, whereas a fixed value of FM1000 concentration (above its relatively lower CMC) is sufficient to inhibit mAb adsorption, preventing mAb from co-existing with surfactants on the surface layer. With this observation, we show that the CMC of the surfactant is not the critical factor to indicate its ability to inhibit protein adsorption, especially for chemically different surfactants, PS80 and FM1000. Additionally, interface-induced aggregation studies indicate that at minimum surfactant concentration levels in protein formulations, fewer protein particles form in the presence of FM1000. Our results provide a mechanistic link between the adsorption of mAbs at the air/water interface and the aggregation induced by agitation in the presence of surfactants.

No ordinary proteins: Adsorption and molecular orientation of monoclonal antibodies.

The interaction of monoclonal antibodies (mAbs) with air/water interfaces plays a crucial role in their overall stability in solution. We aim to understand this behavior using pendant bubble measurements to track the dynamic tension reduction and x-ray reflectivity to obtain the electron density profiles (EDPs) at the surface. Native immunoglobulin G mAb is a rigid molecule with a flat, "Y" shape, and simulated EDPs are obtained by rotating a homology construct at the surface. Comparing simulations with experimental EDPs, we obtain surface orientation probability maps showing mAbs transition from flat-on Y-shape configurations to side-on or end-on configurations with increasing concentration. The modeling also shows the presence of ß sheets at the surface. Overall, the experiments and the homology modeling elucidate the orientational phase space during different stages of adsorption of mAbs at the air/water interface. These finding will help define new strategies for the manufacture and storage of antibody-based therapeutics.

Investigation of anomalous charge variant profile reveals discrete pH-dependent conformations and conformation-dependent charge states within the CDR3 loop of a therapeutic mAb.

During the development of a therapeutic monoclonal antibody (mAb-1), the charge variant profile obtained by pH-gradient cation exchange chromatography (CEX) contained two main peaks, each of which exhibited a unique intrinsic fluorescence profile and demonstrated inter-convertibility upon reinjection of isolated peak fractions. Domain analysis of mAb-1 by CEX and liquid chromatography-mass spectrometry indicated that the antigen-binding fragment chromatographed as two separate peaks that had identical mass. Surface plasmon resonance binding analysis to antigen demonstrated comparable kinetics/affinity between these fractionated peaks and unfractionated starting material. Subsequent molecular modeling studies revealed that the relatively long and flexible complementarity-determining region 3 (CDR3) loop on the heavy chain could adopt two discrete pH-dependent conformations: an "open" conformation at neutral pH where the HC-CDR3 is largely solvent exposed, and a "closed" conformation at lower pH where the solvent exposure of a neighboring tryptophan in the light chain is reduced and two aspartic acid residues near the ends of the HC-CDR3 loop have atypical pKa values. The pH-dependent equilibrium between "open" and "closed" conformations of the HC-CDR3, and its proposed role in the anomalous charge variant profile of mAb-1, were supported by further CEX and hydrophobic interaction chromatography studies. This work is an example of how pH-dependent conformational changes and conformation-dependent changes to net charge can unexpectedly contribute to perceived instability and require thorough analytical, biophysical, and functional characterization during biopharmaceutical drug product development.

Armoring the Interface with Surfactants to Prevent the Adsorption of Monoclonal Antibodies.

The pharmaceutical industry uses surface-active agents (excipients) in protein drug formulations to prevent the aggregation, denaturation, and unwanted immunological response of therapeutic drugs in solution as well as at the air/water interface. However, the mechanism of adsorption, desorption, and aggregation of proteins at the interface in the presence of excipients remains poorly understood. The objective of this work is to explore the molecular-scale competitive adsorption process between surfactant-based excipients and two monoclonal antibody (mAb) proteins, mAb-1 and mAb-2. We use pendant bubble tensiometry to measure the ensemble average adsorption dynamics of mAbs with and without the excipient. The surface tension measurements allow us to quantify the rate at which the molecules "race" to the interface in single-component and mixed systems. These results define the phase space, where coadsorption of both mAbs and excipients occurs onto the air/water interface. In parallel, we use X-ray reflectivity (XR) measurements to understand the molecular-scale dynamics of competitive adsorption, revealing the surface-adsorbed amounts of the antibody and excipient. XR has revealed that at a sufficiently high surface concentration of the excipient, mAb adsorption to the surface and subsurface domains was inhibited. In addition, despite the fact that both mAbs adsorb via a similar mechanistic pathway and with similar dynamics, a key finding is that the competition for the interface directly correlates with the surface activity of the two mAbs, resulting in a fivefold difference in the concentration of the excipient needed to displace the antibody.

Diagnosing current distributions in batteries with magnetic resonance imaging.

Batteries and their defects are notoriously difficult to analyze non-destructively, and consequently, many defects and failures remain little noticed and characterized until they cause grave damage. The measurement of the current density distributions inside a battery could reveal information about deviations from ideal cell behavior, and could thus provide early signs of deterioration or failures. Here, we describe methodology for fast nondestructive assessment and visualization of the effects of current distributions inside Li-ion pouch cells. The technique, based on magnetic resonance imaging (MRI), allows measuring magnetic field maps during charging/discharging. Marked changes in the distributions are observed as a function of the state of charge, and also upon sustaining damage. In particular, it is shown that nonlinearities and asymmetries of current distributions could be mapped at different charge states. Furthermore, hotspots of current flow are also shown to correlate with hotspots in charge storage. This technique could potentially be of great utility in diagnosing the health of cells and their behavior under different charging or environmental conditions.

Stereospecific interactions between histidine and monoclonal antibodies.

Histidine is a frequently used buffer in the final formulation of many commercialized monoclonal antibodies (mAbs), with histidine helping to stabilize the antibody during storage in addition to its buffering function. The objective of this study was to examine the stereospecificity of any histidine-antibody interactions using a combination of experimental studies and molecular dynamics simulations. Isothermal titration calorimetry provided evidence of weak stereospecific interactions, with the antibody showing approximately two to four additional interaction sites for d- versus l-histidine. The greater interactions with d-histidine were confirmed by measurements of the net protein charge using electrophoretic light scattering. The reduction in the net negative charge of the antibody in d-histidine led to significantly different behavior during diafiltration due to Donnan exclusion effects. Molecular dynamics simulations corroborated the presence of additional d-histidine interaction sites. These results provide the first demonstration of weak stereospecific interactions between l- and d-histidine and a mAb and the implications of these interactions for antibody formulation.

Small-Scale Tools to Assess the Impact of Interfacial and Shear Stress on Biologic Drug Products.

Proper risk analysis needs to be in place to understand the susceptibility of protein to unfold and aggregate in the presence of interfacial and/or shear stress. Certain techniques, such as agitation/shaking studies, have been traditionally used to understand the impact of these stresses on the protein physical stability. However, the stresses applied in these systems are convoluted, making it difficult to define the control strategy (i.e., adjustment in process parameters to reduce foaming/bubble formation, change pump type). We have developed two small-scale tools that allow for the isolation of interfacial and shear stress, respectively. These systems, in combination with computational fluid dynamics and numerical approximations, help simulate the normal operating ranges as well as the proven acceptable ranges for different unit operations such as tangential flow filtration (TFF), mixing, and filling.

Rechargeable lithium-ion cell state of charge and defect detection by in-situ inside-out magnetic resonance imaging.

When and why does a rechargeable battery lose capacity or go bad? This is a question that is surprisingly difficult to answer; yet, it lies at the heart of progress in the fields of consumer electronics, electric vehicles, and electrical storage. The difficulty is related to the limited amount of information one can obtain from a cell without taking it apart and analyzing it destructively. Here, we demonstrate that the measurement of tiny induced magnetic field changes within a cell can be used to assess the level of lithium incorporation into the electrode materials, and diagnose certain cell flaws that could arise from assembly. The measurements are fast, can be performed on finished and unfinished cells, and most importantly, can be done nondestructively with cells that are compatible with commercial design requirements with conductive enclosures.

Super-resolution Surface Microscopy of Conductors using Magnetic Resonance.

The spatial resolution of traditional Magnetic Resonance Imaging (MRI) techniques is typically dictated by the strength of the applied magnetic field gradients, resulting in hard resolution limits of the order of 20-50 µm in favorable circumstances. We demonstrate here a technique which is suitable for the interrogation of regions at specified distances below the surface of conducting objects with a resolution well below these limiting values. This approach does not rely on magnetic field gradients, but rather on the spatial variation of the radiofrequency field within a conductor. Samples of aluminium and lithium metal with different sizes and morphologies are examined with this technique using 27Al and 7Li NMR. In this implementation, the slice selectivity depends on the conductivity of the material, as well as on the frequency of operation, although in the most general case, the technique could also be used to provide spatial selectivity with arbitrary B 1 field distributions in non-conductors.

1 H NMR study and multivariate data analysis of reindeer skin tanning methods.

Reindeer skin clothing has been an essential component in the lives of indigenous people of the arctic and sub-arctic regions, keeping them warm during harsh winters. However, the skin processing technology, which often conveys the history and tradition of the indigenous group, has not been well documented. In this study, NMR spectra and relaxation behaviors of reindeer skin samples treated with a variety of vegetable tannin extracts, oils and fatty substances are studied and compared. With the assistance of principal component analysis (PCA), one can recognize patterns and identify groupings of differently treated samples. These methods could be important aids in efforts to conserve museum leather artifacts with unknown treatment methods and in the analysis of reindeer skin tanning processes. Copyright © 2016 John Wiley & Sons, Ltd.

Low-power suppression of fast-motion spin 3/2 signals.

Triple Quantum Filters (TQFs) are frequently used for the selection of bi-exponentially relaxing spin 3/2 nuclei (in particular 23Na) in ordered environments, such as biological tissues. These methods provide an excellent selection of slow-motion spins, but their sensitivity is generally low, and coherence selection requirements may lead to long experiments when applied in vivo. Alternative methods, such as 2P DIM, have demonstrated that the sensitivities of the signals from bi-exponentially relaxing sodium can be significantly increased using strategies other than TQFs. A shortcoming of the 2P DIM method is its strong dependence on B0 inhomogeneities. We describe here a method, which is sensitive to the slow-motion regime, while the signal from spins in the fast-motion regime is suppressed. This method is shown to be more effective than TQFs, requires minimal phase cycling for the suppression of the influence of rf inhomogeneity, and has less dependence on resonance offsets and B0-inhomogeneity than 2P DIM.

Real-time 3D imaging of microstructure growth in battery cells using indirect MRI.

Lithium metal is a promising anode material for Li-ion batteries due to its high theoretical specific capacity and low potential. The growth of dendrites is a major barrier to the development of high capacity, rechargeable Li batteries with lithium metal anodes, and hence, significant efforts have been undertaken to develop new electrolytes and separator materials that can prevent this process or promote smooth deposits at the anode. Central to these goals, and to the task of understanding the conditions that initiate and propagate dendrite growth, is the development of analytical and nondestructive techniques that can be applied in situ to functioning batteries. MRI has recently been demonstrated to provide noninvasive imaging methodology that can detect and localize microstructure buildup. However, until now, monitoring dendrite growth by MRI has been limited to observing the relatively insensitive metal nucleus directly, thus restricting the temporal and spatial resolution and requiring special hardware and acquisition modes. Here, we present an alternative approach to detect a broad class of metallic dendrite growth via the dendrites' indirect effects on the surrounding electrolyte, allowing for the application of fast 3D (1)H MRI experiments with high resolution. We use these experiments to reconstruct 3D images of growing Li dendrites from MRI, revealing details about the growth rate and fractal behavior. Radiofrequency and static magnetic field calculations are used alongside the images to quantify the amount of the growing structures.

Low-power slice selective imaging of broad signals.

One of the major challenges in using magnetic resonance imaging (MRI) to study immobile samples, such as solid materials or rigid tissues like bone or ligaments, is that the images appear dark due to these samples' short-lived signals. Although it is well known that narrowband signals can be excited in inhomogeneously-broadened lines, it is less well known that similar effects can be observed in dipolar-broadened systems. These long-lived signals have not been used much, mainly because their description frequently does not match intuition. While 3D imaging with these signals has previously been reported, here we focus on the demonstration of faster, 2D slice-selective imaging. The faster imaging provides more flexibility for visualizing these rigid objects. We also focus on the frequently-encountered regime wherein the maximum power achievable for rf pulses is significantly weaker than the linewidth. This regime is typically encountered in clinical MRI scans or large volume setups. When compared to UTE and conventional slice-selective spin echo methods, this technique provides better representations of the sample considered here (an eraser sample), and higher signal-to-noise ratios than spin-echo techniques in both the high and low power regimes.

Correlation of Phosphorus Cross-Linking to Hydration Rates in Sodium Starch Glycolate Tablet Disintegrants Using MRI.

Understanding the behavior of tablet disintegrants is valuable in the development of pharmaceutical solid dosage formulations. In this study, high-resolution magnetic resonance imaging has been used to understand the hydration behavior of a series of commercial sodium starch glycolate (SSG) samples, providing robust estimates of tablet disintegration rate that could be correlated with physicochemical properties of the SSGs, such as the extent of phosphorus (P) cross-linking as obtained from infra-red spectroscopy. Furthermore, elemental analysis together with powder X-ray diffraction has been used to quantify the presence of carboxymethyl groups and salt impurities, which also contribute to the disintegration behavior. The utility of Fast Low Angle SHot magnetic resonance imaging has been demonstrated as an approach to rapidly acquire approximations of the volume of a disintegrating tablet and, together with a robust voxel analysis routine, extract tablet disintegration rates. In this manner, a complete characterization of a series of SSG grades from different sources has been performed, showing the variability in their physicochemical properties and demonstrating a correlation between their disintegration rates and intrinsic characteristics. The insights obtained will be a valuable aid in the choice of disintegrant source as well as in managing SSG variability to ensure robustness of drug products containing SSG.

Correlating Microstructural Lithium Metal Growth with Electrolyte Salt Depletion in Lithium Batteries Using 7Li MRI.

Lithium dendrite growth in lithium ion and lithium rechargeable batteries is associated with severe safety concerns. To overcome these problems, a fundamental understanding of the growth mechanism of dendrites under working conditions is needed. In this work, in situ (7)Li magnetic resonance (MRI) is performed on both the electrolyte and lithium metal electrodes in symmetric lithium cells, allowing the behavior of the electrolyte concentration gradient to be studied and correlated with the type and rate of microstructure growth on the Li metal electrode. For this purpose, chemical shift (CS) imaging of the metal electrodes is a particularly sensitive diagnostic method, enabling a clear distinction to be made between different types of microstructural growth occurring at the electrode surface and the eventual dendrite growth between the electrodes. The CS imaging shows that mossy types of microstructure grow close to the surface of the anode from the beginning of charge in every cell studied, while dendritic growth is triggered much later. Simple metrics have been developed to interpret the MRI data sets and to compare results from a series of cells charged at different current densities. The results show that at high charge rates, there is a strong correlation between the onset time of dendrite growth and the local depletion of the electrolyte at the surface of the electrode observed both experimentally and predicted theoretical (via the Sand's time model). A separate mechanism of dendrite growth is observed at low currents, which is not governed by salt depletion in the bulk liquid electrolyte. The MRI approach presented here allows the rate and nature of a process that occurs in the solid electrode to be correlated with the concentrations of components in the electrolyte.

2H and 27Al Solid-State NMR Study of the Local Environments in Al-Doped 2-Line Ferrihydrite, Goethite, and Lepidocrocite.

Although substitution of aluminum into iron oxides and oxyhydroxides has been extensively studied, it is difficult to obtain accurate incorporation levels. Assessing the distribution of dopants within these materials has proven especially challenging because bulk analytical techniques cannot typically determine whether dopants are substituted directly into the bulk iron oxide or oxyhydroxide phase or if they form separate, minor phase impurities. These differences have important implications for the chemistry of these iron-containing materials, which are ubiquitous in the environment. In this work, 27Al and 2H NMR experiments are performed on series of Al-substituted goethite, lepidocrocite, and 2-line ferrihydrite in order to develop an NMR method to track Al substitution. The extent of Al substitution into the structural frameworks of each compound is quantified by comparing quantitative 27Al MAS NMR results with those from elemental analysis. Magnetic measurements are performed for the goethite series to compare with NMR measurements. Static 27Al spin-echo mapping experiments are used to probe the local environments around the Al substituents, providing clear evidence that they are incorporated into the bulk iron phases. Predictions of the 2H and 27Al NMR hyperfine contact shifts in Al-doped goethite and lepidocrocite, obtained from a combined first-principles and empirical magnetic scaling approach, give further insight into the distribution of the dopants within these phases.

MRI and unilateral NMR study of reindeer skin tanning processes.

The study of arctic or subarctic indigenous skin clothing material, known for its design and ability to keep the body warm, provides information about the tanning materials and techniques. The study also provides clues about the culture that created it, since tanning processes are often specific to certain indigenous groups. Untreated skin samples and samples treated with willow (Salix sp) bark extract and cod liver oil are compared in this study using both MRI and unilateral NMR techniques. The two types of samples show different proton spatial distributions and different relaxation times, which may also provide information about the tanning technique and aging behavior.

Multinuclear in situ magnetic resonance imaging of electrochemical double-layer capacitors.

The last decade has seen an intensified interest in the development and use of electrochemical double-layer capacitors, fuelled by the availability of new electrode materials. The use of nanoporous carbons, in particular, with extremely high surface areas for ion adsorption has enabled the development of working devices with significantly increased capacitances that have become viable alternatives to lithium-ion batteries in certain applications. An understanding of the charge storage mechanism and the ion dynamics inside the nanopores is only just emerging, with the most compelling evidence coming from simulation. Here we present the first in situ magnetic resonance imaging experiments of electrochemical double-layer capacitors. These experiments overcome the limitations of other techniques and give spatially resolved chemical information about the electrolyte ions in real time for a working capacitor of standard geometry. The results provide insight into the predominant capacitive processes occurring at different states of charge and discharge.

Visualizing skin effects in conductors with MRI: (7)Li MRI experiments and calculations.

While experiments on metals have been performed since the early days of NMR (and DNP), the use of bulk metal is normally avoided. Instead, often powders have been used in combination with low fields, so that skin depth effects could be neglected. Another complicating factor of acquiring NMR spectra or MRI images of bulk metal is the strong signal dependence on the orientation between the sample and the radio frequency (rf) coil, leading to non-intuitive image distortions and inaccurate quantification. Such factors are particularly important for NMR and MRI of batteries and other electrochemical devices. Here, we show results from a systematic study combining rf field calculations with experimental MRI of (7)Li metal to visualize skin depth effects directly and to analyze the rf field orientation effect on MRI of bulk metal. It is shown that a certain degree of selectivity can be achieved for particular faces of the metal, simply based on the orientation of the sample. By combining rf field calculations with bulk magnetic susceptibility calculations accurate NMR spectra can be obtained from first principles. Such analyses will become valuable in many applications involving battery systems, but also metals, in general.