Quantitative Comparison and Clustering of Circular Dichroism Spectra Using a Symmetrized Weighted Spectral Difference.

Spectroscopy (UV-visible, circular dichroism, infrared, Raman, fluorescence, etc.) is of fundamental importance to determine the structures of macromolecules and monitor their stability, especially for drug products, based on proteins or nucleic acids. In their 2014 article, Dinh et al.

Development of automated metabolite control using mid-infrared probe for bioprocesses and vaccine manufacturing.

Unlabelled: Automation of metabolite control in fermenters is fundamental to develop vaccine manufacturing processes more quickly and robustly. We created an end-to-end process analytical technology and quality by design-focused process by replacing manual control of metabolites during the development of fed-batch bioprocesses with a system that is highly adaptable and automation-enabled. Mid-infrared spectroscopy with an attenuated total reflectance probe in-line, and simple linear regression using the Beer-Lambert Law, were developed to quantitate key metabolites (glucose and glutamate) from spectral data that measured complex media during fermentation.

Assessment of Tunable Resistive Pulse Sensing (TRPS) Technology for Particle Size Distribution in Vaccine Formulations - A Comparative Study with Dynamic Light Scattering.

Purpose: A comparative assessment was performed to evaluate the potential of particle sizing by an ensemble based conventional dynamic light scattering (DLS) technique and an emerging technology based on tunable resistive pulse sensing (TRPS) using particle by particle approach by evaluating three different types of vaccine formulations representing three case studies and showing the limitation of each technique, instrument variability, sensitivity, and the resolution in mixed population.

Methods: Three types of in-house vaccine formulations- a protein antigen, an outer membrane vesicle and viral particles were simultaneously evaluated by TRPS based Exoid and two DLS instruments-Zetatrac and Zetasizer for particle size distribution, aggregates, and resolution of polydisperse species.

Results: The data from first case study show the risk of possible size overestimation and size averaging in polydisperse samples in DLS measurements which can be addressed by the TRPS analysis.

Application of PAT Probes in Aluminum Phosphate Adjuvant Manufacturing.

Purpose: This study is focused on monitoring process parameters and quality attributes of aluminum phosphate (AlPO) using multiple in-line probes incorporated into an industrial-scale adjuvant suspension manufacturing unit.

Methods: The manufacturing of aluminum adjuvant suspension was monitored at manufacturing scale using conductivity, turbidity, infrared, and particle sizing and count probes to follow the continuous evolution of particle formation and size distribution, and the reaction kinetics during the synthesis of AlPO.

Results: The data showed that AlPO forms large particles at the early stages of mixing, followed by a decrease in size and then stabilization towards the later stages of mixing and pH adjustment.

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 AlPO adjuvant microparticles, including the fully dispersed state and the sedimentation process.

Process Analytical Technologies - Advances in bioprocess integration and future perspectives.

Process Analytical Technology (PAT) instruments include analyzers capable of measuring physical and chemical process parameters and key attributes with the goal of optimizing process controls. PAT in the form of a probe or sensor is designed to integrate within the pharmaceutical manufacturing line and is coupled with computing equipment to perform chemometric modeling for result interpretation and multilayer statistical control of processes. PAT solutions are intended for understanding bioprocesses with a goal to control quality at all stages of product manufacturing and achieve quality by design (QbD).

Quantitative microcapillary electrophoresis immunoassay (mCE IA) for end-to-end analysis of pertactin within in-process samples and Quadracel® vaccine.

Protein concentration is an important attribute in the production of subunit or component-based vaccine antigens. Rigorous monitoring of protein concentration is required to identify potential areas for yield improvement. The current GMP method for quantitation is the plate-based ELISA which requires numerous hands-on steps and has low sensitivity in comparison to new microfluidic systems.

In-line monitoring of surfactant clearance in viral vaccine downstream processing.

Purpose: The goal of this study is to examine the suitability of in-line infrared measurements to monitor, in real-time, surfactant concentration in the viral vaccine drug substance during a 50KDa tangential flow filtration (TFF) process.

Methods: A ReactIR™ 702L instrument was used to gather spectra of process off-line samples and reference materials to assess the feasibility of monitoring surfactant concentration during a TFF process in real-time. Both univariate and multivariate models were used to evaluate the off-line sample data and were found to be in good agreement with surfactant concentration values obtained by HPLC.

PAT solutions to monitor adsorption of Tetanus Toxoid with aluminum adjuvants.

The focus of this study was to examine the small-scale adsorption process of Tetanus Toxoid (TT) as a model protein antigen to aluminum phosphate (AlPO) and aluminum oxyhydroxide (AlOOH) adjuvants with real-time monitoring by in-line ReactIR™, ParticleTrack™ based on Focused Beam Reflectance Measurement (FBRM) and EasyViewer™ probes. The adsorption process of AlPO and AlOOH with TT using was monitored in the small-scale reactors. Conformational changes in TT were monitored using in-line infrared probe ReactIR, whereas particle formation associated with protein adsorption were measured by particle size, count, and imaging tools, such as ParticleTrack with FBRM and EasyViewer probes.

Structure and compositional analysis of aluminum oxyhydroxide adsorbed pertussis vaccine.

Purpose: The goal of this study was to characterize an acellular pertussis vaccine (Tdap) containing genetically modified pertussis toxin (gdPT) and TLR agonist adsorbed to AlOOH adjuvant.

Methods: Several analytical tools including nanoDSF, FTIR, and LD were used to examine the conformation of novel gdPT and the composition of AlOOH adjuvant formulations adsorbed to pertussis vaccine.

Results: DLS particle size results were 9.

Aluminum Phosphate Vaccine Adjuvant: Analysis of Composition and Size Using Off-Line and In-Line Tools.

Purpose: Aluminum-based adjuvants including aluminum phosphate (AlPO) are commonly used in many human vaccines to enhance immune response. The interaction between the antigen and adjuvant, including the physical adsorption of antigen, may play a role in vaccine immunogenicity and is a useful marker of vaccine product quality and consistency. Thus, it is important to study the physicochemical properties of AlPO, such as particle size and chemical composition.

Identity, Structure and Compositional Analysis of Aluminum Phosphate Adsorbed Pediatric Quadrivalent and Pentavalent Vaccines.

Purpose: The goal of this study is to set an empirical baseline to map the structure-function relation of the antigens from the commercialized vaccine products.

Methods: To study the structural changes of protein antigens after adsorption several analytical tools including DLS, FTIR, Fluorescence, LD, and SEM have been used.

Results: All antigens have shown wide range of hydrodynamic diameter from 7 nm to 182 nm.

Al and P NMR spectroscopy method development to quantify aluminum phosphate in adjuvanted vaccine formulations.

A novel qNMR method is described for the quantitative determination of total aluminum and phosphate in aluminum phosphate (AlPO) adjuvanted vaccine samples using solution Al and P nuclear magnetic resonance (NMR) spectroscopy. External standard calibrations of AlPO solutions established excellent linearity in the range of 15-40 × 10 M and additional studies determined the level of detection for both nuclei. A commercialized combination vaccine product (Quadracel®), along with several individual adsorbed antigen components used in the vaccine were employed as model systems for method development.

Tuberculosis vaccine candidate: Characterization of H4-IC31 formulation and H4 antigen conformation.

Tuberculosis (TB) is one of the leading causes of death worldwide, making the development of effective TB vaccines a global priority. A TB vaccine consisting of a recombinant fusion protein, H4, combined with a novel synthetic cationic adjuvant, IC31, is currently being developed. The H4 fusion protein consists of two immunogenic mycobacterial antigens, Ag85 B and TB10.

Characterization of Inherent Particles and Mechanism of Thermal Stress Induced Particle Formation in HSV-2 Viral Vaccine Candidate.

Background: Vaccine formulations may contain visible and/or subvisible particles, which can vary in both size and morphology. Extrinsic particles, which are particles not part of the product such as foreign contaminants, are generally considered undesirable and should be eliminated or controlled in injectable products. However, biological products, in particular vaccines, may also contain particles that are inherent to the product.

Nuclear Magnetic Resonance (NMR) Study for the Detection and Quantitation of Cholesterol in HSV529 Therapeutic Vaccine Candidate.

This study describes the NMR-based method to determine the limit of quantitation (LOQ) and limit of detection (LOD) of cholesterol, a process-related impurity in the replication-deficient Herpes Simplex Virus (HSV) type 2 candidate vaccine HSV529. Three signature peaks from the 1D H NMR of a cholesterol reference spectrum were selected for the identification of cholesterol. The LOQ for a cholesterol working standard was found to be 1 μg/mL, and the LOD was found to be 0.

Biophysical Characterization and Thermal Stability of Pneumococcal Histidine Triad Protein D in the Presence of Zinc and Manganese.

The pneumococcal histidine triad protein D (PhtD) is believed to play a central role in pneumococcal metal ion homeostasis and has been proposed as a promising vaccine candidate against pneumococcal disease. To investigate for potential stabilizers, a panel of physiologically relevant metals was screened using the thermal shift assay and it was found that only Zn and Mn were able to increase PhtD melting temperature. Differential scanning calorimetry analysis revealed a sequential unfolding of PhtD and the presence of at least 3 independent folding domains that can be stabilized by Zn and Mn.

Differential Scanning Calorimetry - A Method for Assessing the Thermal Stability and Conformation of Protein Antigen.

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the case of protein samples, DSC profiles provide information about thermal stability, and to some extent serves as a structural "fingerprint" that can be used to assess structural conformation. It is performed using a differential scanning calorimeter that measures the thermal transition temperature (melting temperature; Tm) and the energy required to disrupt the interactions stabilizing the tertiary structure (enthalpy; ∆H) of proteins.

Visible and subvisible particles in the BCG immunotherapeutic product Immucyst®.

Bacille Calmette-Guerin, BCG, is a live attenuated bovine tubercle bacillus used for the treatment of non-muscle invasive bladder cancer. In this study, an Electrical Sensing Zone (ESZ) method was developed to measure the particle count and the size of BCG immunotherapeutic (BCG IT), or ImmuCyst® product using a Coulter Counter Multisizer 4® instrument. The focus of this study was to establish a baseline for reconstituted lyophilized BCG IT product using visible and sub-visible particle concentration and size distribution as reportable values.

Molecular mechanisms initiating amyloid beta-fibril formation in Alzheimer's disease.

The deposition of aggregated amyloid beta-protein (Abeta) in the human brain is a major lesion in Alzheimer' disease (AD). The process of Abeta fibril formation is associated with a cascade of neuropathogenic events that induces brain neurodegeneration leading to the cognitive and behavioral decline characteristic of AD. Although a detailed knowledge of Abeta assembly is crucial for the development of new therapeutic approaches, our understanding of the molecular mechanisms underlying the initiation of Abeta fibril formation remains very incomplete.

A direct interaction between transforming growth factor (TGF)-betas and amyloid-beta protein affects fibrillogenesis in a TGF-beta receptor-independent manner.

Transforming growth factor-beta (TGF-beta) receptor-mediated signaling has been proposed to mediate both the beneficial and deleterious roles for this cytokine in amyloid-beta protein (Abeta) function. In order to assess receptor dependence of these events, we used PC12 cell cultures, which are devoid of TGF-beta receptors. Surprisingly, TGF-beta potentiated the neurotoxic effects of the 40-residue Abeta peptide, Abeta-(1-40), in this model suggesting that there may be a direct, receptor-independent interaction between TGF-beta and Abeta-(1-40).

Paradigm shifts in Alzheimer's disease and other neurodegenerative disorders: the emerging role of oligomeric assemblies.

Alzheimer's disease (AD) is a progressive, neurodegenerative disorder characterized by amyloid deposition in the cerebral neuropil and vasculature. These amyloid deposits comprise predominantly fragments and full-length (40 or 42 residue) forms of the amyloid beta-protein (Abeta) organized into fibrillar assemblies. Compelling evidence indicates that factors that increase overall Abeta production or the ratio of longer to shorter forms, or which facilitate deposition or inhibit elimination of amyloid deposits, cause AD or are risk factors for the disease.

Structure determination of micelle-like intermediates in amyloid beta -protein fibril assembly by using small angle neutron scattering.

Increasing evidence supports the hypothesis that amyloid beta-protein (Abeta) assembly is a key pathogenic feature of Alzheimer's disease. Thus, understanding the assembly process offers opportunities for the development of strategies for treating this devastating disease. In prior studies, Abeta was found to form micelle-like aggregates under acidic conditions.

Identification and characterization of key kinetic intermediates in amyloid beta-protein fibrillogenesis.

Amyloid beta-protein (Abeta) assembly into toxic oligomeric and fibrillar structures is a seminal event in Alzheimer's disease, therefore blocking this process could have significant therapeutic benefit. A rigorous mechanistic understanding of Abeta assembly would facilitate the targeting and design of fibrillogenesis inhibitors. Prior studies have shown that Abeta fibrillogenesis involves conformational changes leading to the formation of extended beta-sheets and that an alpha-helix-containing intermediate may be involved.