A method for the inactivation and removal of dengue virus from biological samples

A dengue virus, virus inactivation technology

Inactive Publication Date: 2007-08-29
ADVANTEK SERUM LAB LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

None of the above-mentioned methods have been verified for the removal of dengue virus, so they have never been applied in the actual operation of dengue virus removal

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Dengue virus inactivation and removal by immunoglobulin process

[0027] Step 1: Virus Removal Filtration

[0028] The preliminarily purified immunoglobulin solution was passed through a 0.22 μm or 0.1 μm filter (Millipore Steritop TM ) pre-filtered to remove viral aggregates. The immunoglobulin solution was kept at room temperature and under a constant pressure of 80kPa, and then a 35nm filter (Asahi KaseiPlanova  ) for virus removal filtration.

[0029] Step 2: Treatment with Organic Solvents and Detergents

[0030] The virus-removed and filtered immunoglobulin solution was heated to 28°C, and then octylphenol polyoxyethylene ether (Triton X-100  ) and tri-n-butyl phosphate to final concentrations of 1% and 0.3%. Viral inactivation of the immunoglobulin solution was maintained for sixteen hours at 28°C-30°C.

[0031] Step 3: Ion Exchange Chromatography

[0032] The cross-linked agarose resin with carboxymethyl groups (Pharmacia CM Sepharose) was re...

Embodiment 2

[0061] Example 2: Preliminary purification of immunoglobulins

[0062] Plasma fractions II+III were obtained from frozen human plasma by frozen ethanol precipitation (Cohn et al., J. Am. Chem. Soc. 1946; 68: 459-475). Human plasma was first subjected to 8.0% ethanol precipitation at pH 7.1, the resulting supernatant was subjected to 19.0% ethanol precipitation at pH 5.85, and fractions II+III were obtained by centrifugation at 2,300 xg. Add 0.8M sodium acetate / 4.0M acetate buffer (pH 3.9) to the dissolved fraction II+III to adjust to pH 5.1, then add 95% ethanol to a final concentration of 15.0%, and at -5°C to -5.5°C Gentle agitation in the range for 1.5 hours. The pellet was removed by centrifugation at 2,300 x g to obtain a supernatant containing immunoglobulins.

Embodiment 3

[0063] Example 3: Purification of human plasma albumin free from infectious dengue virus

[0064] Step 1: Precipitation of Component IV

[0065] As described in Example 2, supernatants II+III were obtained by two consecutive steps of ethanol precipitation (8.0% and 19.0%). 95% ethanol was then added to the supernatant to a final concentration of 40.0% and gentle stirring was performed in the range of -5°C to -5.5°C for 1 hour. Fraction IV was removed by centrifugation at 2,300 xg to obtain supernatant IV containing albumin.

[0066] Step 2: Pasteurization

[0067] The purified albumin solution was diafiltered with 8 volumes of purified water using Millipore's 30kD ultrafiltration membrane package, and then concentrated to a final albumin concentration of 22%. Pharmaceutical grade sodium caprylate was added to the concentrated albumin to a final concentration of 0.032M, then adjusted to pH 6.8 and final albumin concentration of 20%. Next, the 20% albumin solution and the st...

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PUM

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Abstract

A method for inactivating and removing dengue virus from a biological sample comprising the dengue virus and at least one biomolecule of interest is characterized by: inactivating dengue virus by using an organic solvent lonely or combined with a nonionic detergent, and separating the dengue virus from the biomolecule of interest actually by using cation exchange chromatography. In another aspect of the present invention, a method is provided for producing human plasma albumin that is substantially free from infective dengue virus. According to the invention, dengue virus can be removed effectively to avoid danger of viral infection or immunological response caused by exogenetic virus or the genetic material thereof transmitted into human body. The invented method is provided with simplicity, economy and high efficiency.

Description

technical field [0001] The present invention relates to a virus inactivation and removal method, especially a method for inactivating and removing dengue virus from biological samples. Background technique [0002] Dengue virus (Dengue virus) belongs to the Flaviviridae family. Its morphological structure is similar to that of Japanese encephalitis virus, but its size is small, about 40-50 nm. Different strains also have antigenic differences. Among them, type 2 is the most widely spread, and the antigenicity of each type of virus is crossed, and some antigens are the same as Japanese encephalitis virus and West Nile virus. The virus can proliferate in mosquitoes and in Aedes albopictus passage cells (C6 / 36 cells), monkey kidney, hamster kidney primary and passage cells, and produce obvious cytopathic effects. [0003] Dengue virus infection causes dengue fever. The disease is prevalent in tropical and subtropical regions, especially Southeast Asia, the Western Pacific, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04C07K14/765C07K1/14
CPCC12N2770/24163A61L2/0011A61L2/0088C12N7/00C07K16/065
Inventor 黄炳镠谢亦武
Owner ADVANTEK SERUM LAB LTD
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