Preventive Pharmacovigilance: timely and precise prevention of adverse events through person-level patient screening and dose-level product surveillance.

The goal of pharmacovigilance (PV) is to prevent adverse events (AEs) associated with drugs and vaccines. Current PV programs are of a reactive nature and rest entirely on data science, i.e., detecting and analyzing AE data from provider/patient reports, health records and even social media. The ensuing preventive actions are too late for people who have experienced AEs and often overly broad, as responses include entire product withdrawals, batch recalls, or contraindications of subpopulations. To prevent AEs in a timely and precise manner, it is necessary to go beyond data science and incorporate measurement science into PV efforts through person-level patient screening and dose-level product surveillance. Measurement-based PV may be called 'preventive pharmacovigilance', the goal of which is to identify susceptible individuals and defective doses to prevent AEs. A comprehensive PV program should contain both reactive and preventive components by integrating data science and measurement science.

Correlated analytical and functional evaluation of higher order structure perturbations from oxidation of NISTmAb.

The clinical efficacy and safety of protein-based drugs such as monoclonal antibodies (mAbs) rely on the integrity of the protein higher order structure (HOS) during product development, manufacturing, storage, and patient administration. As mAb-based drugs are becoming more prevalent in the treatment of many illnesses, the need to establish metrics for quality attributes of mAb therapeutics through high-resolution techniques is also becoming evident. To this end, here we used a forced degradation method, time-dependent oxidation by hydrogen peroxide, on the model biotherapeutic NISTmAb and evaluated the effects on HOS with orthogonal analytical methods and a functional assay. To monitor the oxidation process, the experimental workflow involved incubation of NISTmAb with hydrogen peroxide in a benchtop nuclear magnetic resonance spectrometer (NMR) that followed the reaction kinetics, in real-time through the water proton transverse relaxation rate R2(1H2O). Aliquots taken at defined time points were further analyzed by high-field 2D 1H-13C methyl correlation fingerprint spectra in parallel with other analytical techniques, including thermal unfolding, size-exclusion chromatography, and surface plasmon resonance, to assess changes in stability, heterogeneity, and binding affinities. The complementary measurement outputs from the different techniques demonstrate the utility of combining NMR with other analytical tools to monitor oxidation kinetics and extract the resulting structural changes in mAbs that are functionally relevant, allowing rigorous assessment of HOS attributes relevant to the efficacy and safety of mAb-based drug products.

Assessing Antigen-Adjuvant Complex Stability Against Physical Stresses By wNMR.

This study applies an emerging analytical technology, wNMR (water proton nuclear magnetic resonance), to assess the stability of aluminum adjuvants and antigen-adjuvant complexes against physical stresses, including gravitation, flow and freeze/thaw. Results from wNMR are verified by conventional analytical technologies, including static light scattering and microfluidic imaging. The results show that wNMR can quickly and noninvasively determine whether an aluminum adjuvant or antigen-adjuvant complex sample has been altered by physical stresses.

Inspecting Insulin Products Using Water Proton NMR. I. Noninvasive vs Invasive Inspection.

BACKGROUND: There is a clear need to transition from batch-level to vial/syringe/pen-level quality control of biologic drugs, such as insulin. This could be achieved only by noninvasive and quantitative inspection technologies that maintain the integrity of the drug product. METHODS: Four insulin products for patient self-injection presented as prefilled pens have been noninvasively and quantitatively inspected using the water proton NMR technology. The inspection output is the water proton relaxation rate R2(1H2O), a continuous numerical variable rather than binary pass/fail. RESULTS: Ten pens of each product were inspected. R2(1H2O) displays insignificant variation among the 10 pens of each product, suggesting good insulin content uniformity in the inspected pens. It is also shown that transferring the insulin solution out of and then back into the insulin pen caused significant change in R2(1H2O), presumably due to exposure to O2 in air. CONCLUSIONS: Water proton NMR can noninvasively and quantitatively inspect insulin pens. wNMR can confirm product content uniformity, but not absolute content. Its sensitivity to sample transferring provides a way to detect drug product tampering. This opens the possibility of inspecting every pen/vial/syringe by manufacturers and end-users.

Rapid and Noninvasive Quantification of Capsid Gene Filling Level Using Water Proton Nuclear Magnetic Resonance.

The present work reports an enabling novel technology for quantifying the gene content in adeno-associated viral capsids. The method is based on the water proton nuclear magnetic resonance (wNMR) technique. Instead of analyzing the capsid directly, it utilizes water molecules to distinguish empty and full capsids, as water interacts with them differently. The transverse relaxation rate of water protons, R2(1H2O), readily distinguishes empty and full capsids and is capable of quantifying the fraction of full capsids in a mixture of full and empty ones. It involves no sample preparation and no reagents. Measurement is rapid (data collection takes 1-2 min), noninvasive (the capsid sample can stay inside the original sealed and labeled container to be used in other studies or administered to a patient), and performed using a wide-bore benchtop NMR instrument. The method can be readily implemented at a production plant for product release as part of product quality control.

Evaluation of the Physicochemical Properties of the Iron Nanoparticle Drug Products: Brand and Generic Sodium Ferric Gluconate.

Complex iron nanoparticle-based drugs are one of the oldest and most frequently administered classes of nanomedicines. In the US, there are seven FDA-approved iron nanoparticle reference drug products, of which one also has an approved generic drug product (i.e., sodium ferric gluconate (SFG)). These products are indicated for the treatment of iron deficiency anemia and are administered intravenously. On the molecular level, iron nanomedicines are colloids composed of an iron oxide core with a carbohydrate coating. This formulation makes nanomedicines more complex than conventional small molecule drugs. As such, these products are often referred to as nonbiological complex drugs (e.g., by the nonbiological complex drugs (NBCD) working group) or complex drug products (e.g., by the FDA). Herein, we report a comprehensive study of the physiochemical properties of the iron nanoparticle product SFG. SFG is the single drug for which both an innovator (Ferrlecit) and generic product are available in the US, allowing for comparative studies to be performed. Measurements focused on the iron core of SFG included optical spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS), X-ray powder diffraction (XRPD), 57Fe Mössbauer spectroscopy, and X-ray absorbance spectroscopy (XAS). The analysis revealed similar ferric-iron-oxide structures. Measurements focused on the carbohydrate shell comprised of the gluconate ligands included forced acid degradation, dynamic light scattering (DLS), analytical ultracentrifugation (AUC), and gel permeation chromatography (GPC). Such analysis revealed differences in composition for the innovator versus the generic SFG. These studies have the potential to contribute to future quality assessment of iron complex products and will inform on a pharmacokinetic study of two therapeutically equivalent iron gluconate products.

Grand Challenges in Pharmaceutical Research Series: Ridding the Cold Chain for Biologics.

Biologics are complex pharmaceuticals that include formulated proteins, plasma products, vaccines, cell and gene therapy products, and biological tissues. These products are fragile and typically require cold chain for their delivery and storage. Delivering biologics, while maintaining the cold chain, whether standard (2°C to 8°C) or deepfreeze (as cold as -70°C), requires extensive infrastructure that is expensive to build and maintain. This poses a huge challenge to equitable healthcare delivery, especially during a global pandemic. Even when the infrastructure is in place, breaches of the cold chain are common. Such breaches may damage the product, making therapeutics and vaccines ineffective or even harmful. Rather than strengthening the cold chain through building more infrastructure and imposing more stringent guidelines, we suggest that money and effort are best spent on making the cold chain unnecessary for biologics delivery and storage. To meet this grand challenge in pharmaceutical research, we highlight areas where innovations are needed in the design, formulation and biomanufacturing of biologics, including point-of-care manufacturing and inspection. These technological innovations would rely on fundamental advances in our understanding of biomolecules and cells.

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.

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.

Monitoring dendrimer conformational transition using 19 F and 1 H2 O NMR.

The conformational transition of a fluorinated amphiphilic dendrimer is monitored by the 1 H signal from water, alongside the 19 F signal from the dendrimer. High-field NMR data (chemical shift δ, self-diffusion coefficient D, longitudinal relaxation rate R1 , and transverse relaxation rate R2 ) for both dendrimer (19 F) and water (1 H) match each other in detecting the conformational transition. Among all parameters for both nuclei, the water proton transverse-relaxation rate R2 (1 H2 O) displays the highest relative scale of change upon conformational transition of the dendrimer. Hydrogen/deuterium-exchange mass spectrometry reveals that the compact form of the dendrimer has slower proton exchange with water than the extended form. This result suggests that the sensitivity of R2 (1 H2 O) toward dendrimer conformation originates, at least partially, from the difference in proton exchange efficiency between different dendrimer conformations. Finally, we also demonstrated that this conformational transition could be conveniently monitored using a low-field benchtop NMR spectrometer via R2 (1 H2 O). The 1 H2 O signal thus offers a simple way to monitor structural changes of macromolecules using benchtop time-domain NMR.

Conformational transition of a non-associative fluorinated amphiphile in aqueous solution. II. Conformational transition vs. supramolecular assembly.

Unlike many known amphiphiles, the fluorinated amphiphilic dendrimer studied in this work demonstrated a concentration-dependent conformational transition rather than micellization or assembly. Hydrophobic and hydrophilic interactions with water were suggested as the most probable driving force of this transition. This assumption was consistent with the observed 19F chemical shift changes of the dendrimer compared to a known micelle-forming fluorinated amphiphile. Since water is an important factor in the process, trends of the concentration-dependent changes in water proton transverse relaxation rate served as an indicator of structural changes and/or supramolecular assembly. The conformational transition process was also confirmed by ion-mobility mass-spectrometry. We suggested that structural features, namely, steric hindrances, prevented the micellization/assembly of the dendrimer of this study. This conclusion might inform the approach to develop novel unconventional amphiphiles.

Improving Biopharmaceutical Safety through Verification-Based Quality Control.

Biopharmaceuticals and small-molecule drugs have different approval pathways but the same quality control (QC) paradigm, where the quality of released but untested units is inferred from that of tested but destroyed units. This inference-based QC will likely miss rare prerelease defects, and defects emerging after product release. The likelihood for such defects is heightened for biopharmaceuticals due to their complexity, which makes manufacturing errors more likely, and fragility, which makes postrelease damage more likely. To improve biopharmaceutical safety, we suggest transitioning their QC from inference- to verification-based practice by developing inspection technologies that can nondestructively verify the quality of every vial from the point of release to the point of care. One candidate, water proton NMR (wNMR), is briefly discussed.

Water Proton NMR for In Situ Detection of Insulin Aggregates.

The need for quality control during the manufacturing and distribution of biopharmaceuticals is becoming increasingly necessary. At present, detecting drug degradation through the monitoring of active factor aggregation is accomplished through "invasive" techniques, such as size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), and so on. Unfortunately, these analytical methods require sampling the drug by opening the drug container that renders the remaining drug unusable regardless of the outcome of the test. Visual inspection, the current non-invasive quality control method is qualitative and can only detect visible particulates. Thus, it will miss sub-visible protein aggregates. In this paper, human insulin preparations were used to demonstrate that the transverse relaxation rate of water protons R2 ((1) H2 O) can serve as a sensitive and reliable indicator to detect and quantify both visible and sub-visible protein aggregates. R2 ((1) H2 O) is measured using a wide-bore low-field bench-top NMR instrument with permanent magnets. Such analysis could be carried out without opening the drug container, thus saving a drug for further use. The results suggest a novel, economical, non-destructive in situ analytical technique that allows for on-the-site quantification of protein aggregation in biopharmaceutical products.

Water proton NMR-a sensitive probe for solute association.

It is found that the transverse relaxation rate R2 of the water protons can be used to quantify protein aggregation and surfactant micellization in water. The simplicity and high intensity of the water proton signal enables non-invasive chemical analysis not readily achievable through solute proton signals, such as inspecting finished biologic products.

Optimize the Separation of Fluorinated Amphiles Using High-Performance Liquid Chromatography.

Using the set of fluorinated amphiles that contain the same fluorocarbon moiety but differ in their fluorine content percentage F% (25-45%), the optimal condition for a F%-based separation of these analytes using reverse-phase chromatography was explored. It is found that optimal separation can be achieved by pairing a regular reverse-phase column (such as C8) with a fluorinated eluent (such as trifluoroethanol). Separation is further improved at higher chromatographic temperature with baseline separation achieved at 45°C. This result indicates that the separation of fluorocarbon-tagged molecules can be based on the fluorine content percentage rather than the number of fluorine atoms.

Fluorinated dendrimers as imaging agents for ¹9F MRI.

¹9F is the second most sensitive stable nucleus for magnetic resonance imaging (MRI). Because there is no endogenous ¹9F signal, ¹9F MRI is much more suited for quantification and tracking than ¹H MRI. However, ¹9F MRI is not in clinical use because in spite of more than three decades of research, there are no approved ¹9F imaging agents. New approaches and new methodologies are needed to move the field forward. Water-soluble fluorinated dendrimers present a promising alternative to conventional perfluorocarbon emulsions. This article outlines recent development of fluorinated dendrimers as ¹9F imaging agents. This is not meant to be a comprehensive review of ¹9F imaging agents, for which there is an excellent recent review by Knight et al. Rather, the article aims to give an insider's account on research efforts in this exciting and challenging field.

Avoiding steric congestion in dendrimer growth through proportionate branching: a twist on da Vinci's rule of tree branching.

Making defect-free macromolecules is a challenging issue in chemical synthesis. This challenge is especially pronounced in dendrimer synthesis where exponential growth quickly leads to steric congestion. To overcome this difficulty, proportionate branching in dendrimer growth is proposed. In proportionate branching, both the number and the length of branches increase exponentially but in opposite directions to mimic tree growth. The effectiveness of this strategy is demonstrated through the synthesis of a fluorocarbon dendron containing 243 chemically identical fluorine atoms with a MW of 9082 Da. Monodispersity is confirmed by nuclear magnetic resonance spectroscopy, mass spectrometry, and small-angle X-ray scattering. Growing different parts proportionately, as nature does, could be a general strategy to achieve defect-free synthesis of macromolecules.