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BACKGROUND: The development of safe, effective, and affordable vaccines has become a global effort due to its vast impact on overall world health conditions. A brief overview of vaccine characterization techniques, especially in the area of high-resolution mass spectrometry, is presented. It is highly conceivable that the proper use of advanced technologies such as high-resolution mass spectrometry, along with the appropriate chemical and physical property evaluations, will yield tremendous in-depth scientific understanding for the characterization of vaccines in various stages of vaccine development. This work presents the physicochemical and biological characterization of cancer vaccine Racotumomab/alumina, a murine anti-idiotypic antibody that mimics N-glycolyl-GM3 gangliosides. This antibody has been tested as an anti-idiotypic cancer vaccine, adjuvated in Al(OH)3, in several clinical trials for melanoma, breast, and lung cancer. METHODS: Racotumomab was obtained from ascites fluid, transferred to fermentation in stirred tank at 10 L and followed to a scale up to 41 L. The mass spectrometry was used for the determination of intact molecule, light and heavy chains masses; amino acids sequence analysis, N- and C-terminal, glycosylation and posttranslational modifications. Also we used the DLS for the size distribution and zeta potential analysis. The biological analyses were performed in mice and chickens. RESULTS: We observed differences in glycosylation pattern, charge heterogeneity and structural stability between in vivo-produced and bioreactor-obtained Racotumomab products. Interestingly, these modifications had no significant impact on the immune responses elicited in two different animal models. CONCLUSIONS: We are demonstrated that this approach could potentially be more efficient and effective for supporting vaccine research and development.
Assuntos
Anticorpos Anti-Idiotípicos/química , Anticorpos Monoclonais/química , Vacinas Anticâncer/química , Animais , Anticorpos Monoclonais Murinos , Reatores Biológicos , Galinhas , Cromatografia Líquida de Alta Pressão , Fermentação , Glicosilação , Espectrometria de Massas , Camundongos , Oxirredução , Tamanho da Partícula , Mapeamento de Peptídeos , Processamento de Proteína Pós-Traducional , Tecnologia Farmacêutica/métodos , Potência de VacinaAssuntos
Humanos , Masculino , Pessoa de Meia-Idade , Doenças da Medula Espinal/complicações , Doenças da Medula Espinal/diagnóstico , HIV/patogenicidade , Vírus Linfotrópico T Tipo 2 Humano/patogenicidade , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática , Ensaio de Imunoadsorção Enzimática/tendências , Citomegalovirus/patogenicidade , Herpesviridae/patogenicidade , Imageamento por Ressonância MagnéticaRESUMO
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Idoso , Humanos , Polimialgia Reumática/diagnóstico , Arterite de Células Gigantes/diagnóstico , Amaurose Fugaz/etiologiaRESUMO
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Masculino , Pessoa de Meia-Idade , Humanos , Osteocondromatose/fisiopatologia , Estenose Traqueal/fisiopatologia , Tomografia Computadorizada por Raios XRESUMO
The breakthrough performance of stacks of dye-cellulosic fabric in affinity chromatography of lysozyme was investigated in batch and flow experiments. Breakthrough curves were significantly affected by flow rate and were not dependent on the feed solution concentration. System dispersion curves could not explain the flow-rate dependence. Breakthrough curves were analyzed by coupling the kinetic model for pore mass transfer as the only controlling resistance and a system dispersion model. From the analysis, pore film mass transfer resistance was found to be the leading rate-limiting factor when the residence time in the column is greater than 5 min. The model was used to predict the operating and design parameters needed to obtain sharp breakthrough curves. Selectivity studies using lysozyme and bovine serum albumin mixtures showed a high system selectivity for lysozyme.
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The scaleup and optimization of large-scale affinity-chromatographic operations in the recovery, separation and purification of biochemical components is of major industrial importance. The development of mathematical models to describe affinity-chromatographic processes, and the use of these models in computer programs to predict column performance is an engineering approach that can help to attain these bioprocess engineering tasks successfully. Most affinity-chromatographic separations are operated in the frontal mode, using fixed-bed columns. Purely diffusive and perfusion particles and membrane-based affinity chromatography are among the main commercially available technologies for these separations. For a particular application, a basic understanding of the main similarities and differences between particle and membrane frontal affinity chromatography and how these characteristics are reflected in the transport models is of fundamental relevance. This review presents the basic theoretical considerations used in the development of particle and membrane affinity chromatography models that can be applied in the design and operation of large-scale affinity separations in fixed-bed columns. A transport model for column affinity chromatography that considers column dispersion, particle internal convection, external film resistance, finite kinetic rate, plus macropore and micropore resistances is analyzed as a framework for exploring further the mathematical analysis. Such models provide a general realistic description of almost all practical systems. Specific mathematical models that take into account geometric considerations and transport effects have been developed for both particle and membrane affinity chromatography systems. Some of the most common simplified models, based on linear driving-force (LDF) and equilibrium assumptions, are emphasized. Analytical solutions of the corresponding simplified dimensionless affinity models are presented. Particular methods for estimating the parameters that characterize the mass-transfer and adsorption mechanisms in affinity systems are described.
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Cromatografia de Afinidade/métodos , Membranas/ultraestrutura , Modelos Biológicos , Modelos Teóricos , Software , Processos EstocásticosRESUMO
The aspartic proteinase gene of Mucor pusillus rennin expressed in Pichia pastoris was characterized in terms of structural and conformational stability induced by temperature. This enzyme is 12% glycosylated, with a similar specific activity to the native fungal enzyme. The secondary structure determined by CD is mainly due to beta-sheet structures with an important contribution of aromatic components. The calorimetric studies were carried out in the temperature range in which the enzyme is most stable. The enzyme undergoes an irreversible, highly scan-rate-dependent thermal denaturation under all the experimental conditions investigated. Between pH 3.0 and 7.0, only one endotherm characterized the thermal denaturation of enzyme. At pH 5.0, the most stable condition found, the denaturation can be fitted to the two-state irreversible model. Thus the kinetic constant and activation parameters of the denaturation process could be obtained. Upon reaching pH 7.5, the denaturation is characterized by two endotherms. This evidence indicates the complex tridimensional structure of this enzyme. Finally, taking into account the conservative tertiary structure of the aspartic proteinase family we comment on our results with reference to the crystallographic structure of M. pusillus pepsin [Newman, Watson, Roychowdhury, Badasso, Cleasby, Wood, Tickle and Blundell (1993) J. Mol. Biol. 221, 1295-1309].
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Quimosina/química , Mucor/enzimologia , Biotecnologia , Varredura Diferencial de Calorimetria , Quimosina/genética , Quimosina/metabolismo , Dicroísmo Circular , Estabilidade Enzimática , Expressão Gênica , Genes Fúngicos , Glicosilação , Concentração de Íons de Hidrogênio , Peso Molecular , Mucor/genética , Pichia/genética , Conformação Proteica , Desnaturação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , TemperaturaRESUMO
The effect of in-place regeneration on equilibrium and kinetic characteristics of the adsorption of bovine serum albumin to a DEAE-cellulose anion exchanger has been determined. Regeneration with sodium hydroxide and time of exposure showed no effect on equilibrium behavior. Breakthrough curves were measured for protein adsorption on fixed-bed columns and analyzed by a simple model to determine the relevant rate constants for the adsorption process. It was found that forward adsorption rate constant decreased exponentially with the chemical treatment exposure time. The implications of the results on the design and optimization of ion-exchange chromatographic processes are discussed.
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Cromatografia DEAE-Celulose/métodos , Albumina Sérica/química , Hidróxido de Sódio/química , Adsorção , Animais , Bovinos , Cromatografia DEAE-Celulose/instrumentação , Ácido Clorídrico/química , Concentração de Íons de Hidrogênio , Modelos Químicos , Concentração Osmolar , Ligação Proteica , Albumina Sérica/metabolismoRESUMO
A DNA fragment containing a transcription regulating region of the alcohol oxidase (AOX1) gene from the methylotrophic yeast Pichia pastoris was used in the construction of a vector for the expression of heterologous proteins in the methylotrophic yeast Hansenula polymorpha. We used this vector to clone the SUC2 gene from Saccharomyces cerevisiae into H. polymorpha yeast. The culture conditions for invertase production using a fed-batch culture were studied. More than 1.5 x 10(3) U/ml of biologically active invertase (1 g/l) were secreted to the cellular periplasmic space. The fermentative process was scaled up to 50 l. Invertase produced from H. polymorpha was glycosylated, but it contained significantly less carbohydrate than protein produced by S. cerevisiae. Using the Western-blot technique, it was observed that invertase secreted from H. polymorpha and invertase secreted from S. cerevisiae showed common antigenic determinants.
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Glicosídeo Hidrolases/metabolismo , Pichia/metabolismo , Oxirredutases do Álcool/genética , Compartimento Celular , Meios de Cultura , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Vetores Genéticos , Glicerol/farmacologia , Glicosídeo Hidrolases/genética , Metanol/metabolismo , Pichia/efeitos dos fármacos , Pichia/genética , Pichia/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , beta-FrutofuranosidaseRESUMO
Recently, a gene coding for the Bm86 tick gut glycoprotein was cloned, expressed in Escherichia coli and shown to induce an immunological response in cattle to damage ticks engorging on these animals (Rand et al., 1989). We report here the increased expression of the Bm86 antigen from the cattle tick Boophilus microplus in the methylotrophic yeast Pichia pastoris. The recombinant protein was obtained with a purity higher than 95% by a procedure with a high yield. The conducted biochemical studies demonstrated the antigen to be glycosylated and found to form particles of around 17 to 45 nm in diameter with enhanced immunogenic properties. Ticks engorging on vaccinated cattle were significantly damaged as a result of the immune response against the recombinant antigen. This system permits the obtainment in a high yield of the tick Bm86 antigen, in a glycosylated and particulated form.
