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

PURPOSE: Aluminum-based adjuvants including aluminum phosphate (AlPO4) 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 AlPO4, such as particle size and chemical composition. Control of the vaccine adjuvant throughout the manufacturing process, including raw materials and the intermediate and final product stages, can be effectively achieved through monitoring of such key product attributes to help ensure product quality. METHODS: This study focuses on the compositional analysis of AlPO4 adjuvant at the intermediate and final manufacturing stages using the off-line methods Fourier-Transform Infrared (FTIR) and Raman spectroscopy, X-ray Photoelectron Spectroscopy (XPS), and the in-line method Attenuated Total Reflectance (ATR). Particle size distribution of AlPO4 was measured off-line using Laser diffraction (LD) and in-line using Focused Beam Reflectance Measurement (FBRM®). RESULTS: There was no observable difference in size distribution between the intermediate and final stage AlPO4 by off-line and in-line analysis, in both small- or large-scale production samples. Consistent peak shifts were observed in off-line and in-line infrared (IR) spectroscopy as well as off-line XPS for both small- and large-scale AlPO4 manufacturing runs. Additionally, IR spectroscopy and FBRM® for size distribution were used as in-line process analytical technology (PAT) to monitor reaction progress in real-time during small-scale AlPO4 manufacturing from raw materials. The small-scale adsorption process of a model protein antigen (Tetanus toxoid) to AlPO4 adjuvant was also monitored by in-line ReactIR probe. CONCLUSION: This study demonstrated that in-line PAT can be used to monitor particle size and chemical composition for the various stages of adjuvant manufacturing from raw materials through intermediate to final adjuvant product stage. Similar approaches can be utilized to help assess lot-to-lot consistency during adjuvant manufacturing and vaccine product development. Moreover, the use of in-line PAT is highly conductive to advanced manufacturing strategies such as real-time product release testing and automated processes of the future.

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. Upon adjuvantation, the size distribution has become narrow, ranging from 10 to 12 µm, and has been driven by the derived diameter of aluminum phosphate (AlPO4) adjuvant. Further to examine size and morphology of adsorbed antigens, SEM has been used. The SEM results have demonstrated that the AlPO4 adjuvant suspension and adsorbed proteins consist of submicron particles that form a continuous porous surface. Diphtheria Toxoid (DT), Tetanus Toxoid (TT), and chemically-modified Filamentous Haemagglutinin (FHA) have shown surface adsorption to AlPO4. Secondary structure alpha-helix and beta-sheet content of DT and TT has increased after adsorption to AlPO4 adjuvant as shown by FTIR, whereas no significant changes were noted for other protein antigens. The results from Intrinsic Fluorescence have shown a structural rearrangement in DT and TT, consistent with the FTIR results. Multivalent vaccine product identity has been determined by FTIR as unique fingerprint spectrum. CONCLUSION: The globular proteins such as DT and TT have shown changes in secondary structure upon adsorption to AlPO4, whereas fibrillar protein FHA has not been affected by adsorption. FTIR can be used as a lean technique to confirm product identity at different manufacturing sites.

Functionalized porous silicon surfaces as DESI-MS substrates for small molecules analysis.

In desorption electrospray ionization mass spectrometry (DESI-MS), the type of surface in addition to low gas and solvent flow rates help to avoid the "splashing of solvent" or "washing effect", by which samples are promptly removed from the surface by the spray. These effects operate on smooth surfaces and generally result in unstable signals as the spray moves over the spot. The aim of this work is to compare the performance of functionalized porous silicon surfaces (pSi) for small molecules analysis with regard to the stability of the signal and the limits of detection (LODs) observed in DESI-MS. The results showed that functional groups, like 1-decene and heptadecafluoro-1,1,2,2-tetrahydrodecyl trimethoxysilane, on pSi surface provides a good alternative for dried spot analysis by DESI-MS, improving stability of the signal and the LODs. This improvement is possible because the dual process containing the weak sample-surface interactions of the hydrophobic characteristic of the functional groups and increasing the surface area of interaction between the sample and the thin solvent film created by the DESI spray, resulting in more effective dissolution of the analyte in the spray solvent without fast removal of the sample.

Global organization of a positive-strand RNA virus genome.

The genomes of plus-strand RNA viruses contain many regulatory sequences and structures that direct different viral processes. The traditional view of these RNA elements are as local structures present in non-coding regions. However, this view is changing due to the discovery of regulatory elements in coding regions and functional long-range intra-genomic base pairing interactions. The ∼4.8 kb long RNA genome of the tombusvirus tomato bushy stunt virus (TBSV) contains these types of structural features, including six different functional long-distance interactions. We hypothesized that to achieve these multiple interactions this viral genome must utilize a large-scale organizational strategy and, accordingly, we sought to assess the global conformation of the entire TBSV genome. Atomic force micrographs of the genome indicated a mostly condensed structure composed of interconnected protrusions extending from a central hub. This configuration was consistent with the genomic secondary structure model generated using high-throughput selective 2'-hydroxyl acylation analysed by primer extension (i.e. SHAPE), which predicted different sized RNA domains originating from a central region. Known RNA elements were identified in both domain and inter-domain regions, and novel structural features were predicted and functionally confirmed. Interestingly, only two of the six long-range interactions known to form were present in the structural model. However, for those interactions that did not form, complementary partner sequences were positioned relatively close to each other in the structure, suggesting that the secondary structure level of viral genome structure could provide a basic scaffold for the formation of different long-range interactions. The higher-order structural model for the TBSV RNA genome provides a snapshot of the complex framework that allows multiple functional components to operate in concert within a confined context.

Specific disruption of transthyretin(105-115) fibrilization using "stabilizing" inhibitors of transthyretin amyloidogenesis.

Transthyretin (TTR) is a cerebrospinal fluid and serum protein that undergoes ordered aggregation (amyloidogenesis) in familial amyloidotic polyneuropathy (FAP) and senile systemic amyloidosis (SSA). It is now widely accepted that dissociation of the native TTR tetramer is a precondition for amyloidogenesis; thus, molecules that stabilize the tetramer have received much attention as potential TTR amyloidosis inhibitors. Many of these inhibitors bind to the thyroxine (T(4)) binding pocket and interact specifically with a section of the TTR sequence, corresponding to residues 105-115, that is implicated in amyloidogenic propensity. In this work, we study the effects of "stabilizing" inhibitors on ordered aggregation of TTR(105-115) peptide. We show that molecules known to bind full-length TTR at the T(4) site are potent, specific inhibitors of ordered aggregation, while molecules that do not interact with TTR exhibit milder, nonspecific disruption through a "hyperbundling" effect. Our results suggest that, in addition to annealing the native tetramer, "stabilizing" inhibitors may also directly disrupt amyloidogenic aggregation of TTR monomers through specific interactions with the exposed TTR(105-115) sequence.