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.

Analysis of the Adsorbed Vaccine Formulations Using Water Proton Nuclear Magnetic Resonance-Comparison with Optical Analytics.

PURPOSE: To evaluate wNMR, an emerging noninvasive analytical technology, for characterizing aluminum-adjuvanted vaccine formulations. METHODS: wNMR stands for water proton nuclear magnetic resonance. In this work, wNMR and optical techniques (laser diffraction and laser scattering) were used to characterize vaccine formulations containing different antigen loads adsorbed onto AlPO4 adjuvant microparticles, including the fully dispersed state and the sedimentation process. All wNMR measurements were done noninvasively on sealed vials containing the adsorbed vaccine suspensions, while the optical techniques require transferring the adsorbed vaccine suspensions out of the original vial into specialized cuvette/tube for analysis. For analyzing fully dispersed suspensions, optical techniques also require sample dilution. RESULTS: wNMR outperformed laser diffraction in differentiating high- and low-dose formulations of the same vaccine, while wNMR and laser scattering achieved comparable results on vaccine sedimentation kinetics and the compactness of fully settled vaccines. CONCLUSION: wNMR could be used to analyze aluminum-adjuvanted formulations and to differentiate between formulations containing different antigen loads adsorbed onto aluminum adjuvant microparticles. The results demonstrate the capability of wNMR to characterize antigen-adjuvant complexes and to noninvasively inspect finished vaccine 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.

Supramolecular Protein-Polyelectrolyte Assembly at Near Physiological Conditions-Water Proton NMR, ITC, and DLS Study.

The in vivo potency of polyphosphazene immunoadjuvants is inherently linked to the ability of these ionic macromolecules to assemble with antigenic proteins in aqueous solutions and form physiologically stable supramolecular complexes. Therefore, in-depth knowledge of interactions in this biologically relevant system is a prerequisite for a better understanding of mechanism of immunoadjuvant activity. Present study explores a self-assembly of polyphosphazene immunoadjuvant-PCPP and a model antigen-lysozyme in a physiologically relevant environment-saline solution and neutral pH. Three analytical techniques were employed to characterize reaction thermodynamics, water-solute structural organization, and supramolecular dimensions: isothermal titration calorimetry (ITC), water proton nuclear magnetic resonance (wNMR), and dynamic light scattering (DLS). The formation of lysozyme-PCPP complexes at near physiological conditions was detected by all methods and the avidity was modulated by a physical state and dimensions of the assemblies. Thermodynamic analysis revealed the dissociation constant in micromolar range and the dominance of enthalpy factor in interactions, which is in line with previously suggested model of protein charge anisotropy and small persistence length of the polymer favoring the formation of high affinity complexes. The paper reports advantageous use of wNMR method for studying protein-polymer interactions, especially for low protein-load complexes.

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.

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.

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.

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.

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

Typesetting error occurred and author corrections to the equations and text edits at the proofing stage were not incorporated in the published article. The original article has been corrected.

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.

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.

An interplay between electrostatic and polar interactions in peptide hydrogels.

Inherent chemical programmability available in peptide-based hydrogels has allowed diversity in the development of these materials for use in biomedical applications. Within the 20 natural amino acids, a range of chemical moieties are present. Here we used a mixing-induced self-assembly of two oppositely charged peptide modules to form a peptide-based hydrogel. To investigate electrostatic and polar interactions in the hydrogel, we replace amino acids from the negatively charged acidic glutamic acid (E) to the uncharged polar glutamine (Q) on a negatively charged peptide module, while leaving the positively charged module unchanged. Using dynamic rheology, the mechanical properties of each hydrogel were investigated. It was found that the number, but not the location, of electrostatic interactions (E residues) dictate the elastic modulus (G') of the hydrogel, compared to polar interactions (Q residues). Increased electrostatic interactions also promote faster peptide assembly into the hydrogel matrix, and result in the decrease of T2 relaxation times of H2 O and trifluoroacetic acid. Small-angle X-ray scattering (SAXS) showed that changing from electrostatic to polar interactions affects the ability to form fibrous networks: from the formation of elongated fibers to no fiber assembly. This study reveals the systematic effects that the incorporation of electrostatic and polar interactions have when programmed into peptide-based hydrogel systems. These effects could be used to design peptide-based biomaterials with predetermined properties.

Split of chiral degeneracy in mechanical and structural properties of oligopeptide-polysaccharide biomaterials.

Enantiomeric biomaterials which are mirror images of each other are characterized by chiral degeneracy--identical structural characteristics and bulk material properties. The addition of another chiral component, D-polysaccharide, has been shown to split such degeneracy and result in two distinct biomaterials. Dynamic oscillatory rheometry and small-angle X-ray scattering demonstrate that the natural biochirality combination of L-peptides and D-polysaccharides assembles faster, has higher elastic moduli (G'), and is structurally more beneficial as opposed to the alternative D-peptide and D-polysaccharide combination. Chemical modifications of the OH-groups in α-D-glucose units in D-polysaccharides weaken such splitting of chiral degeneracy. These findings form a basis to design novel biomaterials and provide additional insight on why proteins and polysaccharides have oppoiste chirality in the biological world.

Using Small-Angle Scattering Techniques to Understand Mechanical Properties of Biopolymer-Based Biomaterials.

The design and engineering of innovative biopolymer-based biomaterials for a variety of biomedical applications should be based on the understanding of the relationship between their nanoscale structure and mechanical properties. Down the road, such understanding could be fundamental to tune the properties of engineered tissues, extracellular matrices for cell delivery and proliferation/differentiation, etc. In this tutorial review, we attempt to show in what way biomaterial structural data can help to understand the bulk material properties. We begin with some background on common types of biopolymers used in biomaterials research, discuss some typical mechanical testing techniques and then review how others in the field of biomaterials have utilized small-angle scattering for material characterization. Detailed examples are then used to show the full range of possible characterization techniques available for biopolymer-based biomaterials. Future developments in the area of material characterization by small-angle scattering will undoubtedly facilitate the use of structural data to control the kinetics of assembly and final properties of prospective biomaterials.