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Method for removing host DNA in rabies vaccine

A rabies vaccine and host technology, applied in the field of vaccine purification, can solve the problems of unsatisfactory impurity removal effect, affecting vaccine yield, serious virus loss, etc., and achieve the effects of optimizing production and processing methods, reasonable process design, and improved quality

Pending Publication Date: 2022-01-04
江苏金迪克生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] A variety of methods for removing residual DNA from rabies vaccines have been developed in the prior art, such as adding protamine sulfate to the virus harvest solution to remove DNA. In this program, the virus loss is serious and the recovery rate is low; conventional gel filtration The actual impurity removal effect of chromatography cannot meet the requirements of the Chinese Pharmacopoeia, and it will affect the vaccine production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] A method for removing host DNA in rabies vaccine, comprising the steps of:

[0035] (1) Select Vero cells with clear cell borders and few senescent cells. After cell counting, subculture and adjust to the required cell density, the total number of cells is controlled to 3.1×10 8 , discard the cell growth medium, inoculate the virus seeds, supplement the maintenance medium, transfer to 34°C for culture, and harvest the virus liquid after 7 days of culture;

[0036] The maintenance solution is 199 culture solution containing 0.2% human serum albumin. The preparation method is: take 89mL water for injection and 10mL199 mother solution, stir and dissolve, add 1mL of 20% human serum albumin, and adjust the pH to 7.8, 0.2 with sodium bicarbonate. Sterile filtration with μm filter membrane. Store in the dark at 2-8°C.

[0037] Take the virus liquid and EDTA solution, mix them evenly, disperse them ultrasonically for 5 minutes, and then homogenize them under high pressure at ...

Embodiment 2

[0042] A method for removing host DNA in rabies vaccine, comprising the steps of:

[0043] (1) Select Vero cells with clear cell borders and few senescent cells. After cell counting, subculture and adjust to the required cell density, the total number of cells is controlled to 3.1×10 8 , discard the cell growth medium, inoculate the virus seeds, supplement the maintenance medium, transfer to culture at 33°C, and harvest the virus liquid after 6 days of culture;

[0044] The maintenance solution is 199 culture solution containing 0.2% human serum albumin. The preparation method is: take 89mL water for injection and 10mL199 mother solution, stir and dissolve, add 1mL of 20% human serum albumin, and adjust the pH to 7.8, 0.2 with sodium bicarbonate. Sterile filtration with μm filter membrane. Store in the dark at 2-8°C.

[0045] Take the virus liquid and EDTA solution, mix them evenly, disperse them ultrasonically for 8 minutes, and then homogenize them under high pressure at a...

Embodiment 3

[0050] A method for removing host DNA in rabies vaccine, comprising the steps of:

[0051] (1) Select Vero cells with clear cell borders and few senescent cells. After cell counting, subculture and adjust to the required cell density, the total number of cells is controlled to 3.1×10 8 , discard the cell growth medium, inoculate the virus seeds, supplement the maintenance medium, transfer to culture at 35°C, and harvest the virus fluid after 5 days of culture;

[0052] The maintenance solution is 199 culture solution containing 0.2% human serum albumin. The preparation method is: take 89mL water for injection and 10mL199 mother solution, stir and dissolve, add 1mL of 20% human serum albumin, and adjust the pH to 7.8, 0.2 with sodium bicarbonate. Sterile filtration with μm filter membrane. Store in the dark at 2-8°C.

[0053] Take the virus liquid and EDTA solution, mix them evenly, disperse them ultrasonically for 10 minutes, and then homogenize them under high pressure at a...

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Abstract

The invention discloses a method for removing host DNA in a rabies vaccine. The method comprises the following steps of: firstly selecting Vero cells with clear cell boundaries and few senile cells in a production process, carrying out subculture after cell counting, adjusting to the required cell density, then carrying out virus inoculation, and harvesting a virus solution about 6 days after virus inoculation; on the basis, uniformly mixing the virus solution with EDTA, then carrying out ultrasonic dispersion, wherein EDTA is used as a metal chelating agent and can chelate divalent cations to disperse DNA; then carrying out ultrasonic dispersion and high-pressure homogenization treatment on the basis of the step, wherein ultrasonic dispersion and high-pressure homogenization treatment are combined with each other to shear the host DNA, so that a host DNA chain is broken and broken, and the removal efficiency of the subsequent residual host DNA is improved. According to the scheme, magnetic silicon dioxide is introduced, separation of the magnetic silicon dioxide and the vaccine can be realized through an external magnetic field, the residual DNA is adsorbed in the process for purification, new impurities do not need to be introduced, the quality of the vaccine is greatly improved, and the practicability is higher.

Description

technical field [0001] The invention relates to the technical field of vaccine purification, in particular to a method for removing host DNA in rabies vaccines. Background technique [0002] As we all know, during the processing and production of rabies vaccine, the culture supernatant of virus-infected cells contains not only rabies virus, but also host cell fragments and host DNA. To ensure the safety of rabies vaccine, the Chinese Pharmacopoeia stipulates that freeze-dried human The residual amount of host DNA in rabies vaccine (Vero) cells shall not be higher than 3ng / dose. [0003] A variety of methods for removing residual DNA from rabies vaccines have been developed in the prior art, such as adding protamine sulfate to the virus harvest solution to remove DNA. In this program, the virus loss is serious and the recovery rate is low; conventional gel filtration The actual impurity removal effect of chromatography cannot meet the requirements of the Chinese Pharmacopoei...

Claims

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Application Information

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IPC IPC(8): C12N7/02
CPCC12N7/00C12N2760/20151
Inventor 杨骏宇唐阳余军李凡望朔朱实惠杨文彬
Owner 江苏金迪克生物技术股份有限公司
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