RESUMO
CHARMM is an academic research program used widely for macromolecular mechanics and dynamics with versatile analysis and manipulation tools of atomic coordinates and dynamics trajectories. CHARMM-GUI, http://www.charmm-gui.org, has been developed to provide a web-based graphical user interface to generate various input files and molecular systems to facilitate and standardize the usage of common and advanced simulation techniques in CHARMM. The web environment provides an ideal platform to build and validate a molecular model system in an interactive fashion such that, if a problem is found through visual inspection, one can go back to the previous setup and regenerate the whole system again. In this article, we describe the currently available functional modules of CHARMM-GUI Input Generator that form a basis for the advanced simulation techniques. Future directions of the CHARMM-GUI development project are also discussed briefly together with other features in the CHARMM-GUI website, such as Archive and Movie Gallery.
Assuntos
Gráficos por Computador , Internet , Interface Usuário-Computador , Conformação Proteica , Proteínas/química , Solventes/química , Eletricidade EstáticaRESUMO
The effects of 2 squalene-based emulsion adjuvant systems (MedImmune emulsion 0 [ME.0] and Stable Emulsion [SE]) on the structure and stability of the recombinant protein antigen alpha-toxin (AT), a potential vaccine candidate for Staphylococcus aureus infection, were investigated using Fourier-transform infrared spectroscopy and both steady-state and time-resolved intrinsic fluorescence spectroscopy as well as differential scanning calorimetry (DSC). A component study, performed to identify the effects of the individual emulsion's components, showed negligible interactions between AT and ME.0. DSC analysis showed the ME.0 emulsion thermally destabilized AT, probably because of changes in the buffer composition of AT upon mixing. The SE emulsion caused increased alpha-helix and decreased beta-sheet content in AT, and a significant blue shift in the fluorescence spectra relative to that of AT in solution. DSC analysis showed SE exerted a dramatic thermal stabilization effect on AT, probably attributable to an interaction between AT and SE. Size exclusion chromatography showed a complete loss in the recovery of AT when mixed with SE, but not ME.0, indicating a high degree of interaction with SE. This work successfully characterized the biophysical properties of AT in the presence of 2 emulsion adjuvants including a component study to rationalize how emulsion components affect protein antigen stability.
Assuntos
Adjuvantes Imunológicos/química , Toxinas Bacterianas/química , Emulsões/química , Proteínas Hemolisinas/química , Temperatura Alta , Adjuvantes Imunológicos/análise , Adjuvantes Farmacêuticos/análise , Adjuvantes Farmacêuticos/química , Toxinas Bacterianas/análise , Emulsões/análise , Proteínas Hemolisinas/análise , Temperatura Alta/efeitos adversos , Estabilidade Proteica , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Staphylococcus aureusRESUMO
To generate potent vaccine responses, subunit protein antigens typically require coformulation with an adjuvant. Oil-in-water emulsions are among the most widely investigated adjuvants, based on their demonstrated ability to elicit robust antibody and cellular immune responses in the clinic. However, most emulsions cannot be readily frozen or lyophilized, on account of the risk of phase separation, and may have a deleterious effect on protein antigen stability when stored long term as a liquid coformulation. To circumvent this, current emulsion-formulated vaccines generally require a complex multivial presentation with obvious drawbacks, making a single-vial presentation for such products highly desirable. We describe the development of a stable, lyophilized squalene emulsion adjuvant through innovative formulation and process development approaches. On reconstitution, freeze-dried emulsion preparations were found to have a minimal increase in particle size of â¼20 nm and conferred immunogenicity in BALB/c mice similar in potency to freshly prepared emulsion coformulations in liquid form.
Assuntos
Adjuvantes Imunológicos/química , Emulsões/química , Liofilização/métodos , Esqualeno/química , Vacinas Virais/química , Adjuvantes Imunológicos/farmacologia , Animais , Linfócitos B/imunologia , Emulsões/farmacologia , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/prevenção & controle , Feminino , Herpesvirus Humano 4/imunologia , Imunidade Celular , Camundongos Endogâmicos C57BL , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vírus Sinciciais Respiratórios/imunologia , Esqualeno/farmacologia , Linfócitos T/imunologia , Vacinas Virais/imunologia , Vacinas Virais/farmacologiaRESUMO
For therapeutic protein analytical studies related to evaluating lot-to-lot variability, different processes and/or formulations, or biosimilars, there is growing interest in applying novel data visualization tools for fingerprint analysis to identify statistically significant differences between 2 samples. Comparative Signature Diagrams (CSDs) were previously developed to display such differences as colored contour plots using a variety of biophysical data sets. In this study, several improvements are proposed to enhance readability and quantitative determinations of CSDs using protein stability data from more commonly used analytical methods such as size exclusion chromatography and capillary isoelectric focusing. To demonstrate the effectiveness of improved CSDs for data visualization, an accelerated and real-time stability study was set up for an IgG1 mAb (mAb A) and its corresponding triple mutant (mAb E). The stability profiles of both mAbs were compared for differences in aggregation (size exclusion chromatography) and charge heterogeneity (capillary isoelectric focusing) profiles over time. Both traditional data analysis and the improved CSDs conclude that the triple mutant mAb E is more susceptible to physicochemical degradation than mAb A under accelerated conditions. The current abilities and limitations of CSDs to provide fingerprint analysis of protein stability profiles to facilitate the determination of similarity versus nonsimilarity between samples is discussed.
Assuntos
Anticorpos Monoclonais/química , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Imunoglobulina G/química , Estabilidade Proteica , Medicamentos Biossimilares/química , Química Farmacêutica/métodos , Cromatografia em Gel/métodosRESUMO
Humans and higher primates are unique in that they lack uricase, the enzyme capable of oxidizing uric acid. As a consequence of this enzyme deficiency, humans have high serum uric acid levels. In some people, uric acid levels rise above the solubility limit resulting in crystallization in joints, acute inflammation in response to those crystals causes severe pain; a condition known as gout. Treatment for severe gout includes injection of non-human uricase to reduce serum uric acid levels. Krystexxa® is a hyper-PEGylated pig-baboon chimeric uricase indicated for chronic refractory gout that induces an immunogenic response in 91% of treated patients, including infusion reactions (26%) and anaphylaxis (6.5%). These properties limit its use and effectiveness. An innovative approach has been used to develop a therapeutic uricase with improved properties such as: soluble expression, neutral pH solubility, high E. coli expression level, thermal stability, and excellent activity. More than 200 diverse uricase sequences were aligned to guide protein engineering and reduce putative sequence liabilities. A single uricase lead candidate was identified, which showed low potential for immunogenicity in >200 human donor samples selected to represent diverse HLA haplotypes. Cysteines were engineered into the lead sequence for site specific PEGylation and studies demonstrated >95% PEGylation efficiency. PEGylated uricase retains enzymatic activity in vitro at neutral pH, in human serum and in vivo (rats and canines) and has an extended half-life. In canines, an 85% reduction in serum uric acid levels was observed with a single subcutaneous injection. This PEGylated, non-immunogenic uricase has the potential to provide meaningful benefits to patients with gout.
Assuntos
Gota/tratamento farmacológico , Urato Oxidase/uso terapêutico , Animais , Varredura Diferencial de Calorimetria , Cães , Escherichia coli/metabolismo , Meia-Vida , Humanos , Concentração de Íons de Hidrogênio , Cinética , Papio , Polietilenoglicóis/química , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapêutico , Especificidade por Substrato , Suínos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Urato Oxidase/efeitos adversos , Urato Oxidase/imunologiaRESUMO
Oil-in-water emulsions have gained consideration as vaccine adjuvants in recent years due to their ability to elicit a differentiated immunogenic response compared to traditional aluminum salt adjuvants. Squalene, a cholesterol precursor, is a natural product with immunostimulatory properties, making it an ideal candidate for such oil-in-water emulsions. Particle size is a key parameter of these emulsions and its relationship to stability and adjuvanticity has not been extensively studied. This study evaluates the effect of particle size on the stability and immunogenicity of squalene emulsions. We investigated the effect of formulation parameters such as surfactant concentration on particle size, resulting in particles with average diameter of 80 nm, 100 nm, 150 nm, 200 nm, or 250 nm. Emulsions were exposed to shear and temperature stresses, and stability parameters such as pH, osmolarity, size, and in-depth visual appearance were monitored over time. In addition, adjuvanticity of different particle size was assessed in a mouse model using Respiratory Syncytial Virus Fusion protein (RSV-F) as a model antigen. Temperature dependent phase separation appeared to be the most common route of degradation occurring in the higher particle sizes emulsions. The emulsions below 150 nm size maintained stability at either 5 °C or 25 °C, and the 80 nm diameter ones showed no measurable changes in size even after one month at 40 °C. In vivo studies using the emulsions as an adjuvant with RSV F antigen revealed that superior immunogenicity could be achieved with the 80 nm particle size emulsion.
Assuntos
Adjuvantes Imunológicos/química , Animais , Química Farmacêutica , Estabilidade de Medicamentos , Emulsões , Concentração de Íons de Hidrogênio , Imunidade Celular/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Óleos , Concentração Osmolar , Tamanho da Partícula , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/virologia , Vacinas contra Vírus Sincicial Respiratório/química , Vacinas contra Vírus Sincicial Respiratório/imunologia , Esqualeno/química , Temperatura , ÁguaRESUMO
Changes in the measurements of a macromolecular biopharmaceutical's physical form are often used to predict changes in the drug's long-term stability. These can in turn be used as important markers of changes to a drug's efficacy and safety. Such stability estimates traditionally require human judgment and are frequently tentative. We introduce methods for developing mathematical models that predict a drug's long-term storage stability profile from measurements of short-term physical form and behavior. We measured the long-term (2 year) chemical and colloidal stability of Granulocyte Colony Stimulating Factor (GCSF) in 16 different liquid formulations. Shortly after formulations were placed on stability, we also employed various spectroscopic techniques to characterize the short-term thermal unfolding response of GCSF in the 16 formulations. The short-term data were processed using several data reduction methods, including reduction to spectra at low temperature, to melt curves, and to transition temperatures. Least squares fitting was used to predict the long-term stability measurements from the reduced short-term spectroscopic measurements. On the basis of the cross-validation and a permutation test, many of the long-term stability predictions have less than 1% probability of occurring by chance.
Assuntos
Química Farmacêutica/métodos , Fator Estimulador de Colônias de Granulócitos/química , Fatores Imunológicos/química , Modelos Moleculares , Fenômenos Químicos , Dicroísmo Circular , Coloides , Estabilidade de Medicamentos , Excipientes/química , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Fatores Imunológicos/genética , Fatores Imunológicos/metabolismo , Análise dos Mínimos Quadrados , Nefelometria e Turbidimetria , Estabilidade Proteica , Estrutura Secundária de Proteína , Desdobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Espectrometria de Fluorescência , Temperatura de TransiçãoRESUMO
A solution to the problem of being able to show statistically significant differences in the measurements of various levels of higher-order protein structure has been an elusive one. We propose the use of comparative signature diagrams (CSDs) to this end. CSDs compare datasets from different biophysical techniques that fingerprint the secondary, tertiary, and quaternary structures of a protein molecule and display statistically significant differences in these datasets. In this paper, we explore the differences in the structures of two proteins (Granulocyte Colony Stimulating Factor [GCSF] and a monoclonal antibody [mAb]) in various formulations. These proteins were chosen based on the extent of differences in structure observed in the formulations. As an initial test, we utilize data from circular dichroism, 8-anilino-1-naphthalene-sulfonate and intrinsic fluorescence spectroscopy, and static light scattering measurements to fingerprint protein structure in the different formulations. Several layers of statistics were explored to visualize the regions of significant differences in the protein spectra. This approach provides a rapid, high-resolution methodology to compare various structural levels of proteins using standard biophysical instrumentation.
Assuntos
Anticorpos Monoclonais/química , Medicamentos Biossimilares/química , Fator Estimulador de Colônias de Granulócitos/química , Química Farmacêutica , Dicroísmo Circular , Bases de Dados de Proteínas , Luz , Modelos Estatísticos , Desnaturação Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espalhamento de Radiação , Espectrometria de Fluorescência , TemperaturaRESUMO
Dominant Negative Inhibitor (DNI) is a translocation-deficient homolog of recombinant protective antigen of Bacillus anthracis that is a candidate for a next generation anthrax vaccine. This study demonstrates that the biophysical characteristics of the DNI protein stored in lyophilized form at 4°C for 8 y were similar to recombinant Protective Antigen (rPA). To provide information on the accelerated stability of DNI, samples in the lyophilized form were subjected to thermal stress (40°C and 70°C for up to 4 weeks) and thoroughly evaluated using various biophysical and chemical characterization techniques. Results demonstrate preserved structural stability of the DNI protein under extreme conditions, suggesting long-term stability can be achieved for a vaccine that employs DNI, as desired for a biodefense countermeasure. Furthermore, the biological activity of the stressed DNI bound to the adjuvant Alhydrogel (®) was evaluated in mice and it was found that the immunogenicity DNI was not affected by thermal stress.
Assuntos
Vacinas contra Antraz/imunologia , Antraz/prevenção & controle , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Toxinas Bacterianas/química , Toxinas Bacterianas/imunologia , Imunização/métodos , Adjuvantes Imunológicos/administração & dosagem , Hidróxido de Alumínio/administração & dosagem , Animais , Vacinas contra Antraz/administração & dosagem , Fenômenos Biofísicos , Estabilidade de Medicamentos , Liofilização , Camundongos Endogâmicos BALB C , Conformação Proteica , Estabilidade Proteica/efeitos da radiação , Subunidades Proteicas/química , Subunidades Proteicas/imunologia , Temperatura , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
The empirical phase diagram (EPD) is a colored representation of overall structural integrity and conformational stability of macromolecules in response to various environmental perturbations. Numerous proteins and macromolecular complexes have been analyzed by EPDs to summarize results from large data sets from multiple biophysical techniques. The current EPD method suffers from a number of deficiencies including lack of a meaningful relationship between color and actual molecular features, difficulties in identifying contributions from individual techniques, and a limited ability to be interpreted by color-blind individuals. In this work, three improved data visualization approaches are proposed as techniques complementary to the EPD. The secondary, tertiary, and quaternary structural changes of multiple proteins as a function of environmental stress were first measured using circular dichroism, intrinsic fluorescence spectroscopy, and static light scattering, respectively. Data sets were then visualized as (1) RGB colors using three-index EPDs, (2) equiangular polygons using radar charts, and (3) human facial features using Chernoff face diagrams. Data as a function of temperature and pH for bovine serum albumin, aldolase, and chymotrypsin as well as candidate protein vaccine antigens including a serine threonine kinase protein (SP1732) and surface antigen A (SP1650) from S. pneumoniae and hemagglutinin from an H1N1 influenza virus are used to illustrate the advantages and disadvantages of each type of data visualization technique.
Assuntos
Biologia Computacional/métodos , Gráficos por Computador , Proteínas/química , Processamento de Sinais Assistido por Computador , Animais , Bovinos , Dicroísmo Circular , Análise por Conglomerados , Face/anatomia & histologia , Humanos , Concentração de Íons de Hidrogênio , Modelos Biológicos , Modelos Moleculares , TemperaturaRESUMO
Influenza is a prevalent, highly contagious and sometimes fatal respiratory disease. Vaccination provides an effective approach to control the disease, but because of frequent changes in the structure of the major surface proteins, there is great need for a technology that permits rapid preparation of new forms of the vaccine each year in sufficient quantities. Recently, using a safe, simple, time- and cost-effective plant viral vector-based transient expression system, the hemagglutinin antigen of H1N1 influenza A strain (HAC1), an H1N1 influenza vaccine candidate, has been produced in Nicotiana benthamiana plants. As a step toward the generation of a commercially viable subunit influenza vaccine, we developed HAC1 formulations in the presence and absence of an aluminum salt adjuvant (Alhydrogel(®)), analyzed their properties, and assessed immunogenicity in an animal model. Biophysical properties of HAC1 were evaluated using several spectroscopic and light scattering techniques as a function of pH and temperature combined with data analysis using an empirical phase diagram approach. Excipients that were potent stabilizers of the recombinant protein were identified using intrinsic fluorescence spectroscopy. The adsorptive capacity and thermal stability of the protein on the surface of Alhydrogel(®) were then examined in the presence and absence of selected stabilizers using UV absorbance after centrifugation and intrinsic fluorescence spectroscopy, respectively. Immunogenicity studies conducted in mice demonstrated that the highest level of serum immune responses (hemagglutination-inhibiting antibody titers), with a 100% seropositive rates, were induced by HAC1 in the presence of Alhydrogel(®), and this response was elicited regardless of the solution conditions of the formulation.
Assuntos
Excipientes/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Proteínas Recombinantes/imunologia , Adjuvantes Imunológicos/administração & dosagem , Hidróxido de Alumínio/administração & dosagem , Animais , Anticorpos Antivirais/sangue , Química Farmacêutica , Vetores Genéticos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Concentração de Íons de Hidrogênio , Vírus da Influenza A Subtipo H1N1/genética , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/química , Vacinas contra Influenza/genética , Camundongos , Vírus de Plantas/genética , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Análise Espectral , Temperatura , Nicotiana/genética , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/química , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/química , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
The preformulation of a trivalent recombinant protein-based vaccine candidate for protection against Streptococcus pneumoniae is described both in the presence and in the absence of aluminum salt adjuvants. The biophysical properties of the three protein-based antigens, fragments of pneumococcal surface adhesion A (PsaA), serine-threonine protein kinase (StkP), and protein required for cell wall separation of group B streptococcus (PcsB), were studied using several spectroscopic and light scattering techniques. An empirical phase diagram was constructed to assess the overall conformational stability of the three antigens as a function of pH and temperatures. A variety of excipients were screened on the basis of their ability to stabilize each antigen using intrinsic fluorescence spectroscopy and circular dichroism spectroscopy. Sorbitol, sucrose, and trehalose stabilized the three proteins in solution. The addition of manganese also showed a drastic increase in the thermal stability of SP1650 in solution. The adsorption and desorption processes of each of the antigens to aluminum salt adjuvants were evaluated, and the stability of the adsorbed proteins was then assessed using intrinsic fluorescence spectroscopy and Fourier transform infrared spectroscopy. All the three proteins showed good adsorption to Alhydrogel. PsaA was destabilized when adsorbed onto Alhydrogel® and adding sodium phosphate showed a stabilizing effect. PcsB was found to be stabilized when adsorbed to Alhydrogel®, and no destabilizing or stabilizing effects were seen in the case of StkP.