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Formulation of Sugar Solutions for Continuous Ultracentrifugation for Virus Purification

a technology of sugar solution and purification virus, which is applied in the field of virus purification, can solve the problems of loss of viral antigen, inhibit viral inactivation steps, and inhibit the yield of chromatographic processes

Inactive Publication Date: 2010-09-23
BAXTER INT INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for purifying viruses or virus antigens by using a sugar gradient established through centrifugation. The method involves using a first buffered sugar solution and a second buffered sugar solution with different concentrations of sugar. A virus preparation is then added to the sugar gradient, and the mixture is centrifuged to create a peak pool. The peak pool is then extracted to obtain the virus or virus antigen. The technical effect of this method is to provide a reliable and efficient way to purify viruses or virus antigens.

Problems solved by technology

Such methods also suffer from antigen aggregation, which can lead to a loss of viral antigen, or inhibit viral inactivation steps.
Viral aggregation can also inhibit the yield of chromatographic processes, which are time and cost intensive and difficult to adapt to a large scale production.

Method used

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  • Formulation of Sugar Solutions for Continuous Ultracentrifugation for Virus Purification
  • Formulation of Sugar Solutions for Continuous Ultracentrifugation for Virus Purification

Examples

Experimental program
Comparison scheme
Effect test

example 1

Materials and Methods

[0033]A continuous flow ultracentrifuge CC40S with a C40CTS rotor (rotor volume 1,600 mL) or an Alfa Wassermann ultracentrifuge RK6 with an equivalent rotor was used. Sucrose gradient solutions were loaded to the rotor and then accelerated to the rotational speed of 20,000 to 35,000 rpm. After continuous loading with the inactivated harvest, the rotor was flushed with buffer to remove residual protein, which had not entered the gradient. After flushing, the rotor was decelerated and the ultracentrifugation was stopped, allowing the gradient to shift from the radial to axial direction of the rotor. Elution and fractionation was carried according to sucrose concentrations. A Coriolis type density detection unit and a UV 254 nm detection unit were used to monitor the sucrose and protein concentration.

[0034]Samples of the purified inactivated harvests were measured for total protein concentration, HA-SRD and Vero Antigen by ELISA according to standardized procedures...

example 2

Initial Experiments with a TBS-Sucrose Gradient

[0035]A number of small scale purification runs with A / Panama / 2007 / 99 monovalent virus harvests indicated that the use of a sucrose gradient produced from a mixture of 50% w / w sucrose with 50% w / w Tris buffered saline (20 mmol / kg TRIS, 8 g / kg NaCl) (final concentration 10 mmol / kg TRIS, 4 g / kg NaCl) had several advantages over the standard sucrose / water system. A laboratory ultracentrifuge model RK-6 was used to purify 25 liter and 50 liter MVH aliquots under different conditions. An overview of the parameter setup and the results for purification runs with the sucrose / water and sucrose / TBS system is given in Table 1.

TABLE 1Comparison of Influenza A / Panama / 2007 / 99 antigen yield and PMVH appearance. Sucrosegradient purified virus from ultracentrifugation experiments with preclarifier at 30,000 g.HA-PeakMVHSRD / proteinPoolAntigen yieldPurificationConditions / LoadratioVolume(mg antigen / PMVHRunSetup(liter)(mg / mg)(mL)liter harvest)(appearance)1...

example 3

Development of a Two-Step Sucrose / TBS Gradient

[0038]This example shows an exemplary embodiment in which a sucrose gradient was modified to increase the volume of the peak pool fraction without changing the limits for fractionation. A sucrose gradient with a reduced concentration of 42% (w / w %) was used, which resulted in a less steep sucrose gradient eluted from the ultracentrifuge. In order to ensure a sufficiently high sucrose concentration in the gradient, a more concentrated sucrose / TBS solution of 50% (w / w %) was loaded subsequent to the 42% (w / w %) solution, which, as a higher concentration solution, formed a “cushion” to prevent from sedimentation of antigen on the wall of the rotor. Table 2 shows purification runs using such a two-step gradient with 900 mL of a less concentrated sucrose / TBS solution (42% w / w sucrose, 11.7 mmol / kg TRIS, 4.7 g / kg NaCl) and 100 mL of a more concentrated sucrose / TBS solution (50% w / w, 10 mmol / kg TRIS, 4 g / kg NaCl), compared to a purification run...

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Abstract

The present invention provides a method for purification of a virus or virus antigen comprising providing a virus preparation and centrifugation of said virus preparation in a gradient of a sugar established by the addition of two or more buffered sugar layers of different concentration. The method leads to higher yields and reduces unwanted aggregation of the virus or virus antigen by increasing the volume of the peak pool.

Description

[0001]This application claims the benefit of priority to U.S. Provisional Application No. 60 / 927,692, filed May 4, 2007, the contents of which are incorporated by reference in their entirety.FIELD OF THE INVENTION[0002]The present invention relates to the field of virus purification.BACKGROUND OF THE INVENTION[0003]Viruses, either those occurring in nature, or recombinant versions thereof, are used for vaccination and in the field of gene therapy. It is possible for many viruses or virus-like particles to safely and efficiently propagate in host cells. Several publications describe the purification of viruses from host cells, mostly concentrating on the use of specific chromatographic matrices for purification of the virus from a host cell lysate (see, e.g. U.S. Pat. No. 6,008,036). Other methods as described, for example, in U.S. Pat. No. 6,048,537 employ continuous sucrose gradient centrifugation, which delivers products with less antigen purity and requires further purification s...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/145C12N7/02A61K39/12A61P37/04A61P31/16A61P31/14
CPCA61K39/145C12N7/00C12N2760/16134A61K39/00C12N2760/16234C12N2760/16251C12N2760/16151A61K39/12A61P31/12A61P31/14A61P31/16A61P37/04C12N2750/14351C12N2760/16152C12N2760/16252
Inventor REITER, MANFREDGRILLBERGER, LEOPOLDMUNDT, WOLFGANGMITTERER, ARTURSCHAFHAUSER, HORST
Owner BAXTER INT INC
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