Concentration and purification of rubella virus using monolithic chromatographic support
Introduction
The production of viral vaccines requires viruses to be grown in living cells, harvested and then purified. In order to manufacture economically acceptable viral vaccine of high quality a development of a simple and efficient method for purification and concentration of viral particles is required. Conventional procedures for virus concentration and purification are based on either (a) cycles of differential centrifugation (ultracentrifugation, density gradient centrifugation), or (b) clarification with organic solvent and precipitation, or (c) ultrafiltration, or (d) combination of these techniques. Such methods are time consuming, they require expensive reagents and equipment and numerous problems are met when large volume samples need to be processed. Furthermore, many viruses are very sensitive to inactivation by physical and chemical agents.
In recent years chromatography has been intensively investigated as a method for efficient virus concentration and purification [1], [2], [3], [4], [5], [6], [7], [8]. Most of conventional, particle-based chromatographic columns are inappropriate for purification of large biomolecules such as virus particles, due to small pore sizes and high shearing forces that develop inside the columns. Monolithic chromatographic supports are characterized by highly interconnected network of large diameter channels that enable fast separations, laminar, nonturbulent flow of mobile phase and low pressure drops even at very high flow rates [9]. Based on these characteristics, monolithic supports were successfully used for concentration and purification of different types of viruses [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25].
Rubella virus (RV) belongs to the Togaviridae family and is the only member of the genus Rubivirus [16]. RV is an enveloped virus with a positive-polarity, single-stranded RNA genome. Its genome is approx. 10 000 nucleotides in length, contains an unusually high proportion of guanine (G) and cytidine (C) residues and it consists of two nonoverlapping open reading frames (ORFs) [17]. The 5′ proximal ORF encompasses two thirds of the genome and encodes non-structural proteins. The 3′ proximal ORF encompasses the remaining third of the genome and encodes three structural proteins: capsid protein (C) and two glycoproteins, E1 and E2. The glycoproteins are embedded in the lipid envelope and form a heterodimer in which E1 is more exposed and against which the majority of the host's humoral response is directed [16], [17].
The rubella virion is formed by budding from both intracellular membranes (primarily within the Golgi) and the plasma membrane [17], [18]. The virus particle has a diameter of about 60 nm [17], [19]. Humans are the only known host and infection caused by RV in adults and children is usually mild or asymptomatic. However, infection during pregnancy often leads to severe birth defects known as congenital rubella syndrome (CRS). In order to prevent CRS, live attenuated rubella vaccines were developed by serial passage of wild virus in cell culture [20]. The most commonly used vaccine strain is RA27/3 [21] developed in late 1960s.
By the end of 2006, vaccination with live RV was introduced in routine immunization programmes in 123 countries and great progress has been achieved toward elimination of rubella and congenital rubella syndrome. As large amounts of rubella vaccine doses are required worldwide, all methods enabling efficient RV purification and concentration could have great potential in vaccine production.
Most methods currently used for concentration and purification of RV from cell culture suspension involve different types of gradient ultracentrifugation and gel filtration [22], [23], [24]. All of them are time-consuming and often not very successful. The only written report on the purification of RV by the usage of anion exchange chromatography is the one of Magnuson et al. who used the DEAE Sephadex A25 support [25]. The 90% of the virus viability was kept when the elution was performed with linear gradient 0.3–0.5 M MgSO4. Although only 10 ml of viral suspension was applied, the chromatographic procedure lasted more than 10 h at 17 °C [25].
The aim of this study was to establish an efficient method based on usage of monolithic chromatographic support for concentration and purification of RV. Besides developing a fast and simple procedure, the key goal was to preserve virus infectivity during the process.
Section snippets
Cells and virus preparation
MRC-5 cells (human lung fibroblasts), obtained from the European Collection of Animal Cell Culture (ECACC, Salisbury, UK), were grown in Minimum Essential Medium with Hank's salts (MEM-H) (AppliChem, Darmstadt, Germany) supplemented with 10% fetal calf serum (FCS) (Moregate, Bulimba, Australia) and 50 μg/ml neomycin (Life Technologies Gibco-BRL, Carlsbad, CA, USA). RK13 cells (rabbit kidney cells) were also obtained from the ECACC and maintained in RPMI 1640 (Life Technologies Gibco-BRL,
Results and discussion
Commercial production of viral vaccines requires large quantities of virus as an antigen source. Vaccine virus production is achieved by replication of a seed virus in a cell culture system. Besides maximizing overall process productivity, the additional aim during the development of production process is to meet the product purity requirements. In many cases, including RV, in spite of optimization of conditions for virus replication the virus grows in low titers in acceptable cell systems (a
Conclusion
We presented highly reproducible and simple chromatographic method using QA monolithic column for efficient purification and concentration of RV from a complex biological suspension. Purified and concentrated viruses maintained their infectivity and high viral recovery was achieved. Furthermore, eluted fractions were depleted of proteins and residual host cell DNA. The results of this investigation represent a basis for future scale up studies. The work indicates the possibility of using
Acknowledgment
This work was supported by the Ministry of Science, Education and Sports of the Republic of Croatia, Grant no. 021-0212432-2033 (to B.H.).
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