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1.
J Chromatogr A ; 1524: 87-100, 2017 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-28989031

RESUMO

The purification of large viruses remains an important field of research and development. The development of efficient purification trains is restricted by limited analytical methods, as well as by the complexity of large viruses, as well as the high variability in starting material from cell culture. Vaccinia virus holds great potential as an oncolytic and immunotherapeutic vaccine against a broad spectrum of cancers. In this work, monolith-based capture and polishing chromatographic steps for vaccinia virus Lister strain has been developed. Virus produced in CV-1 cells was harvested and passed through a 0.8µm pre-filter before loading onto CIEX, AIEX and HIC CIM monoliths. Without the need for nuclease treatment, up to 99% of the total DNA loaded can be removed from the vaccinia feed stream by the CIM OH monolith, which also reduces the total protein concentration in the product pool to LLOQ levels, and achieves infectious virus recoveries of 90%. Binding capacities of greater than 1×109pfu of vaccinia per mL of matrix were obtained on both CIM SO3 and CIM OH monoliths. Multiple orthogonal analytical methods have been used to develop process knowledge and understanding.


Assuntos
Cromatografia , Vaccinia virus/isolamento & purificação , Virologia/métodos , DNA/isolamento & purificação , Virologia/instrumentação
2.
J Sep Sci ; 40(4): 979-990, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27928907

RESUMO

The downstream processing of enveloped virus-like particles is very challenging because of the biophysical and structural similarity between correctly assembled particles and contaminating vesicular particles present in the feedstock. We used hydroxyl-functionalized polymethacrylate monoliths, providing hydrophobic and electrostatic binding contributions, for the purification of HIV-1 gag virus-like particles. The clarified culture supernatant was conditioned with ammonium sulfate and after membrane filtration loaded onto a 1 mL monolith. The binding capacity was 2 × 1012 /mL monolith and was only limited by the pressure drop. By applying either a linear or a step gradient elution, to decrease the ammonium sulfate concentration, the majority of double-stranded DNA (88-90%) and host cell protein impurities (39-61%) could be removed while the particles could be separated into two fractions. Proteomic analysis and evaluation of the p24 concentration showed that one fraction contained majority of the HIV-1 gag and the other fraction was less contaminated with proteins originated from intracellular compartments. We were able to process up to 92 bed volumes of conditioned loading material within 3 h and eluted in average 7.3 × 1011 particles per particle fraction, which is equivalent to 730 vaccination doses of 1 × 109 particles.


Assuntos
Técnicas de Química Analítica/métodos , Produtos do Gene gag/isolamento & purificação , HIV-1/isolamento & purificação , Células Cultivadas , Produtos do Gene gag/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Radical Hidroxila/metabolismo , Proteômica , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação
3.
Virol J ; 9: 265, 2012 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-23140220

RESUMO

The NanoSight LM10 with Nanoparticle tracking analysis (NTA) software was evaluated for the quantification of latex particles, adenovirus 5, and influenza virus. The inter-day variability was determined by measuring the same sample over several consecutive days and the method's accuracy was demonstrated by using known concentrations of the subject particles. NTA analysis was also used to quantify chromatographic fractions of adenovirus and influenza virus after purification on a CIM monolithic column. NTA results were compared and evaluated against hemagglutination (HA) and end point dilution assay, determining total and infection virus particle number, respectively. The results demonstrated that nanoparticle tracking analysis is a method for fast estimation of virus concentration in different samples. In addition, it can provide a better insight into the sample status, regarding the level of virus aggregation.


Assuntos
Adenoviridae/isolamento & purificação , Nanopartículas , Orthomyxoviridae/isolamento & purificação , Carga Viral/métodos , Reprodutibilidade dos Testes , Software , Vírion/isolamento & purificação
4.
J Chromatogr A ; 1144(1): 143-9, 2007 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-17097098

RESUMO

Drawbacks of conventional virus purification methods have led to the development of new, mostly chromatography-based methods. Short monolithic columns are stationary phases intended for purification of large molecules. In this work efficient chromatographic purification of tomato mosaic virus (ToMV) from plant material is described. Based on short monolithic column, the purification process was shortened from 5 days to 2 hours. High viral purity was achieved and recovery of chromatographic step was up to 90%. In addition, these columns enabled preliminary quantification of the virus in just a few minutes, much faster than other quantification methods (e.g. enzyme-linked immunosorbent assay or real-time polymerase chain reaction) which take 1-2 days. These results demonstrate the potential of short monolith column technology for purification and analysis of different viruses.


Assuntos
Cromatografia por Troca Iônica/instrumentação , Cromatografia por Troca Iônica/métodos , Vírus do Mosaico do Tabaco/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Reprodutibilidade dos Testes , Fatores de Tempo
5.
J Virol Methods ; 120(1): 51-7, 2004 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-15234809

RESUMO

A new chromatographic medium, Convective Interaction Media(CIM) disk monolithic columns, was applied to plant virus concentration. The ability of the columns to concentrate highly diluted plant viruses was tested on a model plant virus, rod-shaped tomato mosaic virus (ToMV). Enzyme-linked immunosorbent assay (ELISA) was used for the quantitative analysis. The virus was concentrated using a strong anion exchanger, CIM quaternary amine (QA) disk monolithic column. A high salt concentration was used to elute the concentrated virus from the columns. It has been demonstrated that ToMV, which had been diluted considerably below the sensitivity of ELISA, was concentrated by several orders of magnitude in the one-step procedure. Concentrated virus preparations could be used directly for ELISA testing. In comparison with methods described for concentrating plant viruses from irrigation water, the above procedure may provide a much faster and more efficient way to concentrate highly diluted plant viruses. The procedure could be applied to the testing of other highly diluted plant viruses, and to concentrating viruses for antiserum production.


Assuntos
Cromatografia por Troca Iônica , Vírus do Mosaico/isolamento & purificação , Nicotiana/virologia , Vírus de Plantas/isolamento & purificação , Virologia/métodos , Resinas de Troca Aniônica , Ensaio de Imunoadsorção Enzimática , Vírus do Mosaico/imunologia , Vírus de Plantas/imunologia
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