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Process for producing yellow fever attenuated live vaccine by using SPF chicken embryo cells

A technology of attenuated live vaccines and chicken embryo cells, which is applied in the biological field and can solve problems such as generation limitations, inability to meet production needs, and limited sources

Inactive Publication Date: 2018-08-28
无锡鑫连鑫生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Diploid cells come from normal human tissues and have been proved to be the safest cells after strict testing. However, due to limited sources, they cannot meet the needs of mass production.
[0005] African green monkey kidney transformed cells are potentially tumorigenic. Although WHO allows them to be used in vaccine production, there are strict generation restrictions, and the country has also established strict standards for residual DNA control.

Method used

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  • Process for producing yellow fever attenuated live vaccine by using SPF chicken embryo cells
  • Process for producing yellow fever attenuated live vaccine by using SPF chicken embryo cells
  • Process for producing yellow fever attenuated live vaccine by using SPF chicken embryo cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, chicken embryo cell digestion

[0043] Chicken embryo cells come from domestic SPF chicken embryos. Take 9-10-day-old chicken embryos with normal development, take out the chicken embryos by aseptic operation, remove the head and viscera, wash them three times with PBS, and cut them with sterilized medical surgical scissors. Form 2-3mm tissue pieces, add 0.25% trypsin solution to digest for 20 minutes, and gently shake for 10 minutes in the middle to make trypsin fully contact with the tissue pieces.

[0044] When the digestion is terminated, discard the trypsin solution. Then use a blowing straw to blow and blow the tissue block 4 times at 20, 30, 50, and 40, and the strength gradually increases from low to high. After beating each time, 30ml of cell culture medium is added to each embryo to form a cell suspension.

[0045] Implementation example 2, chicken embryo cells are cultivated and inoculated with virus on the cell factory

[0046] 1) The virus ...

Embodiment 3

[0055] Embodiment 3, virus harvest

[0056] 72 to 96 hours after virus inoculation, when the lesion develops to +++~+++ + Viruses can be harvested.

[0057] Implementation Example 4: Preparation of Virus Seeds

[0058] Carry out virus continuous subculture with above method and prepare 17D yellow fever vaccine virus seed, when titer reaches 5.6lgPFU / ml can be considered to obtain the 17D virus strain that chicken embryo cell adapts, carry out each index examination qualified and be virus seed.

[0059] Verification of poisonous seeds in batches of seeds

[0060] Master seed lots shall undergo the following comprehensive tests:

[0061] 1) Identification test

[0062] The identification test was carried out by the plaque method. Dilute the virus to 50-100PFU / 0.4ml, mix it with yellow fever virus-specific immune serum and non-immune serum in equal amounts, neutralize it in a water bath at 37±1°C for 60 minutes, and culture it at 35±1°C for 6 days. The reduction rate of the...

Embodiment 5

[0085] Embodiment 5, virus is harvested

[0086] The harvested virus is filtered through a 1 μm or 0.6 μm filter membrane, sterilized with a 0.2 μm filter membrane to remove cell debris host proteins, and the stock solution is obtained by adding a lyoprotectant, which should be stored at -20°C. After the titer is qualified, freeze-dry in time and store at 2°C to 8°C.

[0087] With the preparation method of the present invention, the main quality index of the vaccine is:

[0088] The titer of toxic finished product is not lower than 4.5IU / ml / dose

[0089] Vaccine immunization dose: 0.5ml / person

[0090] Ovalbumin content: ≤100ng / dose.

[0091] Antibiotic residue: ≤50ng / dose

[0092] Bovine serum residue: ≤50ng / dose

[0093] Pyrogen: ≤5EU / dose

[0094] Vaccine pH: 7.2-8.0

[0095] Freeze-dried moisture: ≤3%

[0096] Thermal stability: After 2 weeks at 37°C, it should not be lower than 4.5LgPFU / ml / dose, and after 2 weeks at 37°C, the titer drop should not exceed 1.0LgPFU / ...

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Abstract

The invention belongs to the field of biological products, and particularly relates to a process for producing a yellow fever attenuated live vaccine by using SPF chicken embryo cells. According to the process, a 17D attenuated vaccine strain provided by WHO is used, and is subjected to passage adaptation by using chicken embryo cells to prepare virulent seeds, chicken embryo cells cultured by cell factory are inoculated as matrix cells for virus culture, culture is performed, the virus solution is collected, sterilization is performed, a gelatin-free protection agent is added, and freeze drying is performed to prepare the finished product vaccine. According to the present invention, the efficacy index of vaccine meets the WHO standard, other quality test standards meet the requirement ofthe Pharmacopoeia of the People's Republic of China Volume 3 (2015 edition), and the product is used for preventing yellow fever infection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a process for producing live attenuated yellow fever vaccine with SPF chicken embryo cells. Background technique: [0002] Yellow fever is a severe viscerophilic and neurotropic viral infectious disease transmitted by Aedes mosquitoes. It has a high fatality rate and is a public health issue of great concern in many countries in Africa and South America. In Africa, the disease poses a serious threat to 508 million people in 33 countries, mostly in impoverished sub-Saharan Africa. In endemic areas such as South America and Africa, at least more than 200,000 cases and more than 30,000 deaths are reported every year. Although our country is not in an epidemic area, with the opening up of our country's foreign economy, more and more people travel to and from tropical areas of Africa and South America every year (working out, official visits, and visiting relatives). The vect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12A61P31/14C12N7/00
Inventor 万里明鞠长军王建梅承静许定花吕品
Owner 无锡鑫连鑫生物医药科技有限公司
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