Concentration and purification of rubella virus using monolithic chromatographic support

L-Glutamine (L-Gln) instability in liquid media is a well-known fact. Also, negative effect of ammonia, one of the L-Gln degradation products, on viability of many cell cultures and on replication of different viruses has been described. However, negative effects of ammonia have been reported in doses excessively exceeding those that could be generated in regularly used liquid culture media due to spontaneous L-Gln breakdown (below 2 mM). Traditional virus vaccine production processes have been established and registered involving L-Gln containing media use. Eventual culture media replacement in the regular production process belongs to the major regulative changes that require substantial financial expenses. The aim of this study was to evaluate the effect of storage of Minimum Essential Media with Hanks salts on their relevant biological functions during virus vaccine production process in relation to L-Gln decrease. Our results show a cell type dependent effect of spontaneous L-Gln degradation during medium storage. They also suggest that for cell cultures used in measles, mumps, and rubella virus production the media retain their functionality in respect to cell viability or virus growth over a certain time window despite L-Gln degradation.

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“…Rubella virus (RV) was titrated in microplates on RK 13 cells as described (Forcic et al 2011;Kutle et al 2010). …”

Section: Virus Titrationsmentioning

confidence: 99%

Stability of Minimum Essential Medium functionality despite l-glutamine decomposition

et al. 2015

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“…Highly porous material with interconnected flowthrough channels (average diameter of 1.5, 2 or even 6 m) enables convective mass transport of the molecules (in contrast to the diffusive transport of bead based supports), leading to flow independent dynamic binding capacity and separation [18,19]. Anion exchange monoliths (QA and DEAE) have proven to be very effective for fast separation or concentration of different viruses such as tomato mosaic virus [20,21], potato virus Y [22], orthoreovirus [23], rotaviruses [24,25], hepatitis A virus and caliciviruses [26], rubella virus [27], influenza virus [28], adenovirus [29], adeno-associated virus [30], lenti virus [31] bacteriophages [32][33][34] and virus-like particles [35,36]. They have also been successfully applied to the concentration of waterborne viruses [25,26] where fast flow rates and large binding capacity are essential.…”

Section: Introductionmentioning

confidence: 99%

Methacrylate monolith chromatography as a tool for waterborne virus removal

Rački

Kramberger

Steyer

et al. 2015

Journal of Chromatography A

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Enteric viruses are commonly present in environmental waters and represent the major cause of waterborne infections and outbreaks. Since traditional wastewater treatments fail to remove enteric viruses in the water purification process, they are released daily into environmental waters. Monolithic supports have enabled chromatography to enter the field of virology. They have been successfully used in virus purification and concentration. In this work quaternary amine (QA) methacrylate monoliths were exploited to remove enteric viruses from wastewater treatment plant effluent. Expectedly, chromatographic processing of such a complex medium was troublesome, even for monoliths, characterized by extremely large pore dimensions. This problem was solved by introducing a pre-step chromatography using hydroxyl (OH) methacrylate monoliths. This way, molecules, that would hinder virus binding to the anion-exchanger monolith, were removed. As a result, the OH pre-column reduced backpressure increase on the subsequent anion-exchanger column, and increased both QA column binding capacity and life time. Wastewater effluent samples were successfully purified from five waterborne enteric viruses (rotavirus, norovirus genogroup I and II, astrovirus, sapovirus), below the detection limit of RT-qPCR. The breakthrough of the rotavirus binding capacity was not reached for concentrations that significantly exceeded those expected in effluent waters. The obtained results confirm that methacrylate monoliths can be a valuable tool for simultaneous removal of different waterborne viruses from contaminated water sources.

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“…Os valores de F p foram relativamente baixos, quando comparados com outros métodos de purificação (FORCIC et al, 2011). Porém, há poucos estudos sobre extrações de Adenovirus, quando comparamos com biopartículas como anticorpos.…”

Section: Análise Estatística -Planejamentounclassified

Extração de adenovírus em sistemas micelares de duas fases aquosas

Molino¹

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Palavras-chave:Extração líquido-líquido. Adenovirus. Sistema micelar de duas fases aquosas. Biopartículas. ABSTRACTBiotechnological processes depend significantly on separation and purification techniques used to maintain cost-effective industrial-scale production of biotechnological products for commercial, industrial and therapeutic uses. The application of the aqueous two-phase micelar system (ATPMS) is proposed as an alternative for purification, since it allows the separation and analysis of biomolecules /bioparticles, often without loses of activity or their properties. This allows to perform a selective partition that enables high yields. This work aims to study the use of this methodology in the extraction and purification of adenovirus in micelle aqueous twophase formed by TritonX-114/Viral suspension. All assays were performed in 5 g systems following a full factorial design (2 3 ) with four central points. The studied factors were extraction temperature, pH of the viral suspension and concentration of the surfactant. Systems containing masses of 3g, 10g and 40g were evaluated.Extraction procedure effects over integrity and infectivity of adenovirus were also evaluated. Some of the parameters evaluated in the viral recovery process were viral potency (R Pv ) and recovery of viral specific potency (R Pvφ ). These two parameters measure the inactivation of Adenovirus by the extraction process and both showed improvement when compared with the viral suspension for some of the systems studied (i.e RPv: 341% and RPvφ 1466%). These results show that ATPMS selectively partition the infectious viral particles. According to the results of the experimental design is possible to increase, even further, these results controlling the surfactant concentration, viral suspension pH and temperature of extraction.

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Concentration and purification of rubella virus using monolithic chromatographic support

Cited by 34 publications

References 28 publications

Stability of Minimum Essential Medium functionality despite l-glutamine decomposition

Stability of Minimum Essential Medium functionality despite l-glutamine decomposition

Methacrylate monolith chromatography as a tool for waterborne virus removal

Extração de adenovírus em sistemas micelares de duas fases aquosas

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