RESUMO
PURPOSE: This study is the first vaccine candidate in vitro investigation with a focus on finding a correlation between the spray characteristics and the delivery efficiency of the local deposition in the nasal airways of infants under 24 months using various intranasal devices. METHODS: In vitro tests were developed to measure the spray characteristics of four intranasal delivery devices and how they regionally deliver a candidate vaccine formulation matrix in five nasal airway replicas (3 to 24 months). The correlation between the spray performance, geometric parameters, and delivery efficiency were assessed. RESULTS: All four devices performed consistently in terms of spray characteristics and were capable of delivering a high percentage of the candidate vaccine to the target areas, with a minimum delivery efficiency of 80%. Moreover, the delivery efficiency was affected by either the spray droplet size distribution or the distance between the nozzle tip and the internal nasal valve. Correlations between the spray performance and the in vitro local dose deposition were established. CONCLUSION: The infant nasal model tests can be complementary to device spray performance evaluation. In the absence of in vivo correlations, they can also facilitate the process of new product development by estimating delivery a priori.
Assuntos
Sistemas de Liberação de Medicamentos , Nebulizadores e Vaporizadores , Humanos , Aerossóis , Administração Intranasal , Nariz , Sprays NasaisRESUMO
PURPOSE: Nasal delivery is a favorable route for vaccination against most respiratory infections, as antigen deposited in the nasal turbinate and Waldeyer's ring areas induce mucosal and systemic immune responses. However, little is known about the nasal distribution of the vaccines, specifically for infants. METHODS: Anatomical nasal replicas of five subjects, 3-24 months, were developed to assess local intranasal vaccine delivery using MAD Nasal™ device, and understand impact of breathing conditions and administration parameters. High performance liquid chromatography was used to quantify the deposition pattern and determine the delivery efficiency. RESULTS: The delivery efficiency on average for all models was found to be 86.57±14.23%. There were no significant differences in the total delivery efficiency between the models in all cases. However, the regional deposition pattern was altered based on the model and subsequent administration. Furthermore, removing the foam tip from the MAD Nasal™ device, to study the impact of insertion length, did not significantly increase the efficiency within the two models tested, 5- and 16-month. CONCLUSION: Incorporating nasal replicas in testing provided a benchmark to determine the efficiency of a common intranasal vaccine delivery combination product. This proposed platform would allow comparing other potential nasal vaccine delivery devices.
Assuntos
Modelos Anatômicos , Mucosa Nasal/metabolismo , Vacinação/métodos , Vacinas/farmacocinética , Administração Intranasal , Pré-Escolar , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Lactente , Nariz/anatomia & histologia , Nariz/diagnóstico por imagem , Impressão Tridimensional , Tomografia Computadorizada por Raios X , Vacinas/administração & dosagemRESUMO
We have investigated the effects of site specific "hinge" polyethylene glycol conjugation (PEGylation) on thermal, pH, and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab') using a variety of biophysical techniques. The results obtained by circular dichroism (CD), ultraviolet (UV) absorbance, and fluorescence spectroscopy suggested that the physical stability of the Fab' is maximized at pH 6-7 with no apparent differences due to PEGylation. Temperature-induced aggregation experiments revealed that PEGylation was able to increase the transition temperature, as well as prevent the formation of visible and subvisible aggregates. Statistical comparison of the three-index empirical phase diagram (EPD) revealed significant differences in thermal and pH stability signatures between Fab' and PEG-Fab'. Upon mechanical stress, micro-flow imaging (MFI) and measurement of the optical density at 360 nm showed that the PEG-Fab' had significantly higher resistance to surface-induced aggregation compared to the Fab'. Analysis of the interaction parameter, kD, indicated repulsive intermolecular forces for PEG-Fab' and attractive forces for Fab'. In conclusion, PEGylation appears to protect Fab' against thermal and mechanical stress-induced aggregation, likely due to a steric hindrance mechanism.
Assuntos
Anticorpos Monoclonais/química , Fragmentos Fab das Imunoglobulinas/química , Polietilenoglicóis/química , Dicroísmo Circular , Difusão Dinâmica da Luz , Concentração de Íons de Hidrogênio , Conformação Proteica , Estabilidade Proteica , Espectrometria de FluorescênciaRESUMO
Deamidation is a post-translational chemical modification that occurs within proteins and can be influenced by many factors, including temperature and pH. In vaccines, deamidation is considered undesirable as it may lead to changes in structure, function, stability, and immunogenicity. Detecting deamidation in vaccines, especially adjuvanted vaccines, can be challenging due to the lack of simple quantitative techniques. In this study, the quantification of isoaspartic acid (isoAsp) was used to assess deamidation in model antigens in the presence and absence of common vaccine adjuvants. This study shows that the detection of isoAsp was possible in the presence of various types of adjuvants with little to no interference. High levels of isoAsp were detected in thermally and pH-stressed adjuvanted vaccines, suggesting significant deamidation and highlighting the stability-indicating capabilities of the assay. The quantification of isoAsp in stability programs of a vaccine drug product could possibly find applications in product shelf-life determination, using thermal kinetic modeling to predict deamidation over time. The ability to detect deamidation early in vaccine development enhances process improvements and ultimately improves the vaccine's stability. To summarize, this paper describes a rapid and simple method to determine deamidation in adjuvanted vaccines. This method could be applicable to formulation development, stability assessment, or shelf-life determination.
RESUMO
Current influenza vaccines could be augmented by including recombinant neuraminidase (rNA) protein antigen to broaden protective immunity and improve efficacy. Toward this goal, we investigated formulation conditions to optimize rNA physicochemical stability. When rNA in sodium phosphate saline buffer (NaPBS) was frozen and thawed (F/T), the tetrameric structure transitioned from a "closed" to an "open" conformation, negatively impacting functional activity. Hydrogen deuterium exchange experiments identified differences in anchorage binding sites at the base of the open tetramer, offering a structural mechanistic explanation for the change in conformation and decreased functional activity. Change to the open configuration was triggered by the combined stresses of acidic pH and F/T. The desired closed conformation was preserved in a potassium phosphate buffer (KP), minimizing pH drop upon freezing and including 10% sucrose to control F/T stress. Stability was further evaluated in thermal stress studies where changes in conformation were readily detected by ELISA and size exclusion chromatography (SEC). Both tests were suitable indicators of stability and antigenicity and considered potential critical quality attributes (pCQAs). To understand longer-term stability, the pCQA profiles from thermally stressed rNA at 6 months were modeled to predict stability of at least 24-months at 5°C storage. In summary, a desired rNA closed tetramer was maintained by formulation selection and monitoring of pCQAs to produce a stable rNA vaccine candidate. The study highlights the importance of understanding and controlling vaccine protein structural and functional integrity.
Assuntos
Vacinas contra Influenza , Influenza Humana , Humanos , Influenza Humana/prevenção & controle , Neuraminidase/genética , Vacinas Sintéticas/genética , RNARESUMO
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 (AlPO4) 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 AlPO4 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. ParticleTrack distribution results and kinetic measurements were also supported by observations made using EasyViewer. In addition to EasyMax, BioBLU reactor was also used for the adsorption experiments. ReactIR with ATR-Fiber probe was effectively able to monitor adsorption progress of TT to AlOOH and to AlPO4. ReactIR, EasyViewer, and ParticleTrack provided detailed mechanistic and kinetic information for reaction of TT with AlPO4 and AlOOH. These in-situ measurements revealed a possible multi-step process for TT to AlPO4 which may be an indication of antigen adsorption.
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Adjuvantes Imunológicos , Alumínio , Adsorção , Tamanho da Partícula , Toxoide TetânicoRESUMO
This work demonstrates that an HSV-2 candidate vaccine can be thermostabilized by spray drying to reduce cold chain demands. This work is also to optimize the process responses by varying spray dry parameters for pre-screened suitable excipients; and to determine the validity of current prescreening techniques. Vaccine activity losses were measured by in vitro plaque forming assay with Vero cell line. An accelerated storage condition of 45⯰C for 10â¯days was used to determine spray dried sample stability. Prescreening studies demonstrated that trehalose and sucrose were superior to other tested excipients spray dry thermal stabilization of HSV-2. Subsequent optimization by design of experiments (DOE) of activity responses to spray dry parameter changes demonstrated significant differences between trehalose and sucrose for stability of the viral vaccine. Model parameters included the drying conditions inlet temperature, spray gas flow rate, and solids concentration for the model responses of vaccine stabilization. Trehalose was an effective and robust stabilizing excipient for spray drying HSV-2 vaccine. In contrast, stabilization by sucrose was greatly dependent on the spray dry process parameters. These DOE differences indicated inadequate excipient selection by prescreening methods and the variability demonstrated current prescreening techniques may not be adequate for determining optimal excipients.
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Herpesvirus Humano 2/imunologia , Vacinas Virais/administração & dosagem , Animais , Chlorocebus aethiops , Dessecação , Composição de Medicamentos/métodos , Estabilidade de Medicamentos , Excipientes/administração & dosagem , Sacarose/administração & dosagem , Trealose/administração & dosagem , Células VeroRESUMO
Determination of protein concentration in vaccines containing aluminum salt adjuvant typically necessitates desorption of the protein prior to analysis. Here we describe a method based on the intrinsic fluorescence of tyrosine and tryptophan that requires no desorption of proteins. Adjuvanted formulations of three model Bordetella pertussis antigens were excited at 280â¯nm and their emission spectra collected from 290 to 400â¯nm. Emission spectra of protein antigens in the presence of aluminum salt adjuvants were able to be detected, the effects of adjuvants on the spectra were analyzed, and linear regressions were calculated. The fluorescence method proved to be very sensitive with a limit of quantification between 0.4 and 4.4⯵g/mL and limit of linearity between 100 and 200⯵g/mL, across the formulations tested. The fluorescence method was found to be influenced by adjuvant presence, type of adjuvant, adjuvant concentration, buffer and pH conditions. The method also demonstrated ability to monitor the percent adsorption of antigens to the adjuvants. Furthermore, intrinsic fluorescence showed good correlation with micro-Kjeldahl elemental assay in quantifying protein concentration. Being a non-invasive, quick and sensitive method, intrinsic fluorescence has the potential to be utilized as a high throughput tool for vaccine development and conceivably implemented in-line, using in-line fluorimeters, to monitor antigen concentration during formulation processing.
Assuntos
Adjuvantes Imunológicos/química , Hidróxido de Alumínio/química , Antígenos de Bactérias/análise , Proteínas de Bactérias/análise , Bordetella pertussis/química , Medições Luminescentes/métodos , Adesinas Bacterianas/análise , Adesinas Bacterianas/química , Proteínas da Membrana Bacteriana Externa/análise , Proteínas da Membrana Bacteriana Externa/química , Fímbrias Bacterianas/química , Fluorescência , Humanos , Triptofano/química , Tirosina/química , Vacinas/imunologia , Fatores de Virulência de Bordetella/análise , Fatores de Virulência de Bordetella/químicaRESUMO
Stressed stability testing is crucial to the understanding of mechanisms of degradation and the effects of external stress factors on adjuvant stability. These studies vastly help the development of stability indicating tests and the selection of stabilizing conditions for long term storage. In this chapter, we provide detailed protocols for the execution of forced degradation experiments that evaluate the robustness of adjuvant formulations against thermal, mechanical, freeze-thawing, and photo stresses.
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Adjuvantes Imunológicos/química , Composição de Medicamentos/métodos , Estabilidade de MedicamentosRESUMO
Monitoring the immunological functionality of vaccine formulations is critical for vaccine development. While the traditional approach using established animal models has been relatively effective, the use of animals is costly and cumbersome, and animal models are not always reflective of a human response. The development of a human-based approach would be a major step forward in understanding how vaccine formulations might behave in humans. Here, we describe a platform methodology using fresh human whole blood (hWB) to monitor adjuvant-modulated, antigen-specific responses to vaccine formulations, which is amenable to analysis by standard immunoassays as well as a variety of other analytical techniques.
Assuntos
Sangue , Composição de Medicamentos , Vacinas , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Vacinas/química , Vacinas/farmacologiaRESUMO
The restimulation of an immune memory response by in vitro culture of blood cells with a specific antigen has been used as a way to gauge immunity to vaccines for decades. In this commentary we discuss a less appreciated application to support vaccine process development. We report that human whole blood from pre-primed subjects can generate a profound adjuvant-modulated, antigen-specific response to several different vaccine formulations. The response is able to differentiate subtle changes in the quality of an immune memory response to vaccine formulations and can be used to select optimal conditions relating to a particular manufacture process step. While questions relating to closeness to in vivo vaccination remain, the approach is another big step nearer to the more relevant human response. It has special importance for new adjuvant development, complementing other preclinical in vivo and in vitro approaches to considerably de-risk progression of novel vaccines before and throughout early clinical development. Broader implications of the approach are discussed.
Assuntos
Adjuvantes Imunológicos , Sangue/imunologia , Memória Imunológica , Vacinas/imunologia , Imunidade Adaptativa , Ensaios de Triagem em Larga Escala , Humanos , Interferon gama/biossíntese , Interferon gama/imunologia , Linfócitos T/imunologia , VacinaçãoRESUMO
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 Zn2+ and Mn2+ 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 Zn2+ and Mn2+. UV spectroscopy and fluorescence quenching studies showed significant Zn2+-induced tertiary structure changes in PhtD characterized by decreased accessibility of inner tryptophan residues to the aqueous solvent. Isothermal titration calorimetry data show no apparent binding to Mn2+ but revealed a Zn2+:PhtD exothermic interaction stoichiometry of 3:1 with strong enthalpic contribution, suggesting that 3 of the 5 histidine triads are accessible binding sites for Zn2+. Only Zn+2, but not Mn+2, was able to increase the thermal stability of PhtD in the presence of aluminum hydroxide adjuvant, making it a promising stabilizer excipient candidate in vaccine products containing PhtD.
Assuntos
Proteínas de Bactérias/química , Hidrolases/química , Manganês/química , Streptococcus pneumoniae/metabolismo , Zinco/química , Adjuvantes Imunológicos/química , Hidróxido de Alumínio/química , Anticorpos Antibacterianos/química , Proteínas de Transporte/química , Histidina/química , Vacinas Pneumocócicas/química , Triptofano/químicaRESUMO
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. Here we focus on the characterization of visible and subvisible particles in a live, replication-deficient viral vaccine candidate against HSV genital herpes in an early developmental stage. METHOD: HSV-2 viral vaccine was characterized using a panel of analytical methods, including Fourier transform infrared spectroscopy (FTIR), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, liquid chromatography-mass spectrometry (LC-MS), light microscopy, transmission electron microscopy (TEM), micro-flow imaging (MFI), dynamic light scattering (DLS), right angle light scattering (RALS), and intrinsic fluorescence. RESULTS: Particles in HSV-2 vaccine typically ranged from hundreds of nanometers to hundreds of micrometers in size and were determined to be inherent to the product. The infectious titer did not correlate with any trend in subvisible particle concentration and size distribution as shown by DLS, MFI, and TEM under stressed conditions. This suggested that particle changes in the submicron range were related to HSV-2 virion structure and had direct impact on biological activity. It was also observed that subvisible and visible particles could induce aggregation in the viral product. The temperature induced aggregation was observed by RALS, intrinsic fluorescence, and DLS. The increase of subvisible particle size with temperature could be fitted to a two-step thermokinetic model. CONCLUSION: Visible and subvisible particles were found to be inherent to the HSV-2 viral vaccine product. The mechanism of protein aggregation was discussed and a two-step thermokinetic aggregation profile was proposed. The approaches reported in this study may be applied to a variety of vaccines and other biological products, as a way to assess the consistency of the manufacturing process and identify key product quality attributes.
Assuntos
Composição de Medicamentos/métodos , Herpesvirus Humano 2/imunologia , Vacinas Virais/análise , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Eletroforese em Gel de Poliacrilamida , Congelamento , Herpesvirus Humano 2/química , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Agregados Proteicos , Estabilidade Proteica , Espectroscopia de Infravermelho com Transformada de Fourier , Vacinas Virais/normas , Vírion/ultraestruturaRESUMO
During the early stages of vaccine development, forced degradation studies are conducted to provide information about the degradation properties of vaccine formulations. In addition to supporting the development of analytical methods for the detection of degradation products, these stress studies are used to identify optimal long-term storage conditions and are part of the regulatory requirements for the submission of stability data. In this chapter, we provide detailed methods for forced degradation analysis under thermal, light, and mechanical stress conditions.
Assuntos
Vacinas/química , Química Farmacêutica , Estabilidade de Medicamentos , Fotólise , Proteínas/química , Estresse Mecânico , TemperaturaRESUMO
A tuberculosis (TB) vaccine consisting of a recombinant fusion protein (H4) and a novel TLR9 adjuvant (IC31) is in clinical development. To better understand the H4-IC31 ratio, we measured the binding capacity of IC31 for H4 protein and immunized mice with formulations that contained limiting to excess ratios of IC31 to H4. An immunomodulated H4-specific IFNγ response was only observed when IC31 was present in excess of H4. Since TLR expression is species-specific and the vaccine is intended to boost BCG-primed immunity, we questioned whether data in mice would translate to humans. To address this question, we used the fresh human Whole Blood (hWB) recovered from BCG-vaccinated subjects to screen H4-IC31 formulations. We found IC31 modulation in hWB to be quite distinct from the TLR4-Adjuvant. Unlike TLR4-Adjuvant, IC31 formulations did not induce the pro-inflammatory cytokine TNFα, but modulated a robust H4-specific IFNγ response after 12 d of culture. We then re-stimulated the fresh hWB of 5 BCG-primed subjects with formulations that had excess or limiting IC31 binding for H4 protein and again found that an immunomodulated H4-specific IFNγ response needed an excess of IC31. Finally, we monitored the zeta (ζ) potential of H4-IC31 formulations and found that the overall charge of H4-IC31 particles changes from negative to positive once IC31 is in greater than 9-fold excess. Using two diverse yet mutually supportive approaches, we confirm the need for an excess of IC31 adjuvant in H4 TB vaccine formulations and suggest surface potential may be an important factor.
Assuntos
Antígenos de Bactérias/imunologia , Imunomodulação , Oligodesoxirribonucleotídeos/farmacologia , Oligopeptídeos/farmacologia , Vacinas contra a Tuberculose/imunologia , Animais , Células Cultivadas , Química Farmacêutica , Combinação de Medicamentos , Humanos , Interferon gama/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Camundongos , Vacinação/métodosRESUMO
We have used a protein-based vaccine, a live virus vaccine, and an experimental adjuvant to evaluate the utility of an advanced kinetic modeling approach for stability prediction. The modeling approach uses a systematic and simple procedure for the selection of the most appropriate kinetic equation to describe the degradation rate of compounds subjected to accelerated conditions. One-step and two-step reactions with unlimited combinations of kinetic models were screened for the three products under evaluation. The most appropriate mathematical model for a given product was chosen based on the values of residual sum of squares and the weight parameter w. A relatively simple n-th order kinetic model best fitted the degradation of an adjuvanted protein vaccine with a prediction error lower than 10%. A more complex two-step model was required to describe inactivation of a live virus vaccine under normal and elevated storage temperatures. Finally, an autocatalytic-type kinetic model best fitted the degradation of an oil-in-water adjuvant formulation. The modeling approach described here could be used for vaccine stability prediction, expiry date estimation, and formulation selection. To the best of our knowledge, this is the first report describing a global kinetic analysis of degradation of vaccine components with high prediction accuracy.
Assuntos
Química Farmacêutica , Vacinas Virais , CinéticaRESUMO
Understanding the relevant biological activity of any pharmaceutical formulation destined for human use is crucial. For vaccine-based formulations, activity must reflect the expected immune response, while for non-vaccine therapeutic agents, such as monoclonal antibodies, a lack of immune response to the formulation is desired. During early formulation development, various biochemical and biophysical characteristics can be monitored in a high-throughput screening (HTS) format. However, it remains impractical and arguably unethical to screen samples in this way for immunological functionality in animal models. Furthermore, data for immunological functionality lag formulation design by months, making it cumbersome to relate back to formulations in real-time. It is also likely that animal testing may not accurately reflect the response in humans. For a more effective formulation screen, a human whole blood (hWB) approach can be used to assess immunological functionality. The functional activity relates directly to the human immune response to a complete formulation (adjuvant/antigen) and includes adjuvant response, antigen response, adjuvant-modulated antigen response, stability, and potentially safety. The following commentary discusses the hWB approach as a valuable new tool to de-risk manufacture, formulation design, and clinical progression.
Assuntos
Sangue/imunologia , Química Farmacêutica/métodos , Vacinas/imunologia , HumanosRESUMO
A systematic investigation of protein encapsulation in polylactic-co-glycolic-acid (PLGA) was carried out using the formation of a w/o/o emulsion followed by solvent removal. Various factors were studied, including composition of the suspension medium and the relative amounts of aqueous phase containing protein to polymer solution. High yields of microsphere fabrication were achieved by using silicon oil containing methylene chloride as a suspension medium instead of pure silicon oil, with minimal loss of polymer and protein drug (<2%). The amount of aqueous phase influenced the process and successful encapsulation was obtained if the volume ratios of aqueous phase to polymer solution were less than 5% (v/v) at a wide range of polymer concentration (2-15% g ml-1). Protein encapsulation by this w/o/o emulsion and solvent removal method has a high yield of microsphere fabrication and protein encapsulation (98%). In addition, it provides an easy way to control the release rate of protein encapsulated in microspheres by modulating their porosity in fabrication process.