Method to increase salt ion concentration of buffer to purify virus

A technology of salt ions and buffer solution, which is applied in the field of biopharmaceutical technology, can solve the problems of high cost and unsuitability for industrial production, and achieve the effects of reducing load, increasing load capacity, and good application prospects

Inactive Publication Date: 2018-01-19
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The technical problem to be solved by the present invention is: the existing method for purifying virus is high in cost and unsuitable for industrialized production

Method used

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  • Method to increase salt ion concentration of buffer to purify virus
  • Method to increase salt ion concentration of buffer to purify virus
  • Method to increase salt ion concentration of buffer to purify virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Amplification of Ad-HBx-hIL12 recombinant adenovirus

[0056] a) Expansion of cell seeds: take a cell from the HEK293 cell working bank stored in liquid nitrogen, and resuscitate to T175cm 2 square bottle, at 37°C, 5% CO 2 Cultivate in an incubator, and pass to the cell factory step by step after the cells are full.

[0057] b) Expansion of cells in bioreactor: First, add cell culture medium into the bioreactor, when the operating conditions are stable at 37°C, pH 7.0, DO 50%, 50rpm, digest and collect HEK293 cells amplified by the cell factory, and inoculate in In the bioreactor, the inoculated cell density was 1.5×10 5 Cells / ml, add cell culture medium to 5L, the conditions of reactor cell culture are temperature 37℃, rotating speed 45rpm, pH 7.15-7.25, DO 30-50%, samples are taken every day to detect glucose concentration, cell density and micro Morphology of the cells on the vector.

[0058] c) Virus inoculation and virus amplification: when the cell d...

Embodiment 2

[0061] Example 2, Ultrafiltration Concentration and Filter Washing of Ad-HBx-hIL12 Recombinant Adenovirus

[0062] Take the virus supernatant collected in Example 1 at 6000rpm, centrifuge at a high speed at 4°C, discard the precipitate, keep the supernatant, and filter and clarify the centrifuged virus supernatant through a Burst filter; filter the clarified virus liquid with 300KD The membrane bag was concentrated, and the virus concentrate and filtrate were collected separately. Divide the concentrated sample into two parts, carry out 10 times volume filtration with low-salt filter buffer (100mM) and high-salt buffer (300mM) respectively, collect filter-wash samples and filter-wash samples respectively; Collect clarified Take 2ml of sample, concentrated sample, filtered sample, low-salt filtered sample, low-salt filtered sample, high-salt filtered sample, and high-salt filtered sample, add it to a glass tube, and observe the sample under natural light The change of phenol r...

Embodiment 3

[0064] Example 3 Optimum Filtration and Washing Volume of Recombinant Adenovirus Ad-HBx-hIL12

[0065]The steps of ultrafiltration concentration are the same as in Example 2, the difference being: the concentrated sample is subjected to 5 times, 10 times, 15 times, and 20 times conditional washing with high-salt filter washing buffer, and 5 times, 10 times, and 20 times are collected respectively. 15-fold and 20-fold filtered samples, 50mM Tris-HCl, 2mM MgCl 2 After dialysis with pH 8.0 buffer solution, 12.5% ​​SDS-PAGE gel electrophoresis and Source15Q chromatographic analysis were used to detect the removal effect of impurity proteins; antibody staining and TCID50 method were used to detect the influence of filtration volume on virus activity,

[0066] Experimental results such as Figure 4 : With the increase of the volume of filtration and washing, the removal of impurity protein increases, and the activity of the virus will not change with the increase of the volume of f...

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Abstract

The present invention pertains to the field of biopharmaceutical processes, more specifically relates to a method to increase salt ion concentration of a buffer to purify a virus, and aims at the problems that virus purifying methods in the prior art are high in cost and not suitable for industrialized production, and the method comprises the following steps: A, inoculation and culture of host cells; B, amplification and lysis of the virus-infected host cells to obtain virus-containing lysate; C, concentration of the lysate to obtain concentrated lysate; D, use of a high salt ion buffer to filter the concentrated lysate, and use of ion exchange chromatography for purifying to obtain the purified virus. The method can obviously remove a large number of impure proteins such as serum and hostcell proteins, increases the sample loading size of the chromatography, and increases the carrying capacity of packing. The method is simple, economical and efficient, is suitable for industrialization and has a good application prospect.

Description

technical field [0001] The invention belongs to the field of biopharmaceutical technology, and in particular relates to a method for purifying viruses by increasing the salt ion concentration of a buffer solution. Background technique [0002] In recent years, with the rapid development of molecular biology, immunology, and cell biology, biological therapy represented by immunotherapy and gene therapy is expected to become a new means of treating diseases. Gene therapy refers to a new biomedical technology that introduces human normal genes or genes with therapeutic effects into human target cells in a certain way to correct gene defects or exert therapeutic effects, so as to achieve the purpose of treating diseases. The most common gene therapy delivery systems include non-viral vectors and viral vectors. [0003] The non-viral vector delivery system uses the physical and chemical properties of non-viral carrier materials to mediate gene transfer, mainly including liposome...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/02
Inventor 程平魏于全
Owner SICHUAN UNIV
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