Method for preparing lyophilized hepatitis A attenuated live vaccine using cell factory

A technology of live attenuated vaccines and hepatitis A, which is applied to medical preparations containing active ingredients, antiviral agents, pharmaceutical formulas, etc. It can solve the problems of unsatisfactory effect and long proliferation cycle of hepatitis virus, and achieve easy scale-up Easy to apply, conducive to attachment and growth, and guaranteed sterility

Active Publication Date: 2011-08-31
长春生物制品研究所有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Reactor adherent culture usually adopts sheet carrier and basket stirring system, which can provide higher cell density, and is generally suitable for multiple harvests of culture products. Some domestic researchers have tried to use reactor culture cells to prepare hepatitis A vaccine. None ideal
The reason is that the hepatitis virus has a long cycle of proliferation in the cell and can only be harvested once

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The CELL FACTORIES 10 layers and 36 blocks produced by NUNC Company of Denmark were selected, and the preparation process of freeze-dried live attenuated hepatitis A vaccine was prepared by single-layer infection method.

[0036] 1.2BS cell preparation

[0037] Wash the surface of the selected cells with Hank's solution, then digest the cells with 0.25% trypsin, mix the digested cells with MEM culture medium containing 10% calf serum to make a cell suspension, and divide according to an appropriate ratio Seeds (1:5), aseptically added to the cell factory, 200ml / layer, placed at 37°C±0.5°C for 7 days.

[0038] 2. Single layer infection:

[0039] Carefully check the cells to confirm that the cells are monolayer and free of pollution, discard the old solution, wash the cell surface gently with Hank's solution, control it, add the virus seed dilution (0.03MOI), culture at 35°C±0.5°C for 3.5 hours, replenish Add maintenance solution (MEM culture solution containing 2% calf...

Embodiment 2

[0053] The Cell STACK, 10 layers, 36 pieces produced by the American CORNING company was selected, and the preparation process of the freeze-dried hepatitis A live attenuated vaccine was prepared by a single-layer infection method.

[0054] 1.2BS cell preparation

[0055] Wash the surface of the selected cells with Hank's solution, then digest the cells with 0.25% trypsin, mix the digested cells with MEM culture medium containing 10% calf serum to make a cell suspension, and divide according to an appropriate ratio Seeds (1:5), aseptically added to the cell factory, 200ml / layer, placed at 37°C±0.5°C for 7 days.

[0056] 2. Single layer infection:

[0057] Confirm that the cells are monolayer and free of pollution, discard the old solution, gently wash the cell surface with Hank's solution, control it, add virus seed dilution (0.04MOI), culture at 35°C±0.5°C for 3.5 hours, add maintenance solution ( MEM culture fluid containing 2% calf serum) to 200ml / layer, cultured at 35°C±...

Embodiment 3

[0071] Choose CELLFACTORIES produced by NUNC Company, 10 layers and 12 pieces, and Cell STACK produced by American CORNING Company, 40 layers and 6 pieces, and adopt the suspension infection method to prepare the preparation process of freeze-dried hepatitis A live attenuated vaccine.

[0072] 1. Suspension infection

[0073] Select the cell species, discard the culture medium in the cell factory, gently wash the cell surface with Hank's solution, control it, add digestive juice to digest, and when the cell surface is loose, add the mixed suspension of cells and virus species (0.05MOI) , sort according to the appropriate ratio (1:5), place at 35°C±0.5°C for 2.5 hours to absorb, add growth medium (MEM culture solution containing 15% calf serum) to 220ml / layer, place at 35°C±0.5°C for cultivation, The medium was changed after 7 days.

[0074] 2. Replace maintenance fluid

[0075] The liquid in the cell factory was discarded, and 220ml / layer of maintenance solution was added, a...

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PUM

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Abstract

The invention discloses a method for preparing a freeze-dried live attenuated hepatitis A vaccine by utilizing a cell factory. The method has good process controllability, is uneasy to pollute environment, is suitable for industrialized and mass production, improves the utilization rate of a culture space by more than 3 times, ensures that the growth density of cells reaches between 4.0x10<6> and 5.0x10<6> cells per ml, assures the multiplication space of viruses, and also effectively solves the problem of cell residue after the cell factory digests through methods such as sterile washing, centrifugal treatment and so on, so as to continuously use the cell factory for cell culture and improve the reuse rate of the cell factory.

Description

Technical field: [0001] The invention relates to a method for preparing a freeze-dried live attenuated hepatitis A vaccine, in particular discloses a method for preparing a freeze-dried live attenuated hepatitis A vaccine by using a cell factory, and belongs to the technical field of biopharmaceuticals. Background technique: [0002] The key to the preparation of traditional viral vaccines is to provide the virus with a suitable cell matrix for its proliferation, and the growth method of adherent culture is usually adopted. Adherent culture systems mainly include spinner bottles, hollow fibers, glass beads, and microcarrier systems. Roller bottle culture is generally used for the transition from small-scale culture to large-scale culture, or as a preparation method for inoculating cells in bioreactors. It is characterized by simple structure, low investment, mature technology, good repeatability, easy expansion, etc., but also has disadvantages such as low cell growth densi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/29A61P1/16A61P31/14
CPCY02A50/30
Inventor 刘景晔谢宝生周妍屈翠波占永生王云霞
Owner 长春生物制品研究所有限责任公司
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