Virus in deactivated bioproduct and method of removing toxin in bacteria

A technology of bacterial endotoxin and biological products, applied in the direction of inactivation/attenuation, etc., can solve the problems of inability to use, large influence of cytokine biological activity, expensive equipment, etc., to maintain biological activity, good inactivation effect, and ensure safety effect

Inactive Publication Date: 2006-06-14
李法卿
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 1. Pasteurization method (pasteurization method) and dry heat method (heating at 80°C for 72 hours), the inactivation effect is relatively certain, but sometimes it cannot be used due to the greater impact on the biological activity of some cytokines
[0005] 2. The organic solvent / detergent (S / D) treatment method has strict requirements for the whole process of inactivating viruses, and is ineffective for non-enveloped viruses
[0006] 3. The nano-membrane filtration method can only be used if the membrane pore size is smaller than the effective diameter of the virus, which has great limitations. The effect of inactivating the virus is easily affected by many factors and must be used in combination with other methods
[0007] 4. Low pH incubation method, this method can inactivate several lipid-enveloped viruses, but the inactivation effect is difficult to control
[0008] None of the above virus inactivation methods can be applied to both lipid-enveloped viruses and non-lipid-enveloped viruses, and the effect is not complete. Therefore, it is difficult to inactivate all viruses at once. Generally, several methods need to be used in combination
The implementation effect of the above method is affected by many factors, the stability is poor, and the pyrogen cannot be removed at the same time, and the removal of endotoxin cannot be taken into account.
Bacterial endotoxins need to be removed by ultrafiltration or chromatography. The former is mainly suitable for small molecular weight infusion drugs, such as water for injection, etc.; the latter has a wide range of applications, but the equipment is expensive and costly. limited quantity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Inactivation test of embodiment 1 human nerve growth factor injection intermediate product pseudorabies virus

[0019] Indicating virus: pseudorabies virus, adding titer is 8.00LgTCID 50 / ml

[0020] Cells for culture: PK-15 cells.

[0021] 1. Virus inactivation according to the steps:

[0022] Take 45ml of each of the three batches of samples, add 5ml of pseudorabies virus respectively, and add endotoxin at the same time, the concentration is 4EU / ml, mix well, adjust the pH value to 12±0.1 with 4mol / L NaOH, and equally distribute to 5 centrifuge tubes , 8ml / tube, sealed, incubated in an incubator at 25°C±1°C, sampled at 15, 30, 60, 90, and 120 minutes, and immediately added 3mol / L HCl to adjust the pH to neutral.

[0023] The above treated samples were centrifuged at 4°C (4000rpm) for 30 minutes, and the supernatant was taken and filtered into small centrifuge tubes, 1.2ml / tube, 4 tubes / sampling point, and frozen at -70°C for detection.

[0024] 2. Neutral control ...

Embodiment 2

[0035] Carrying out the inactivation test of Sindbis virus, an intermediate product of 2 human nerve growth factor injections

[0036] Indicator virus: Sindbis, titer is 7.34LgTCID 50 / ml.

[0037] Cells used for culture: BHK-21, titration method: 6-well plate plaque method.

[0038] Follow the steps for virus inactivation:

[0039] Take 20ml of each of the three batches of samples and adjust to neutral with 3mol / L HCl.

[0040] Take 45ml of each of the three batches of samples, add 5ml of Sindbis virus, adjust the pH value to 12±0.1 with 4mol / L NaOH, divide each batch into 5 centrifuge tubes with 8ml / tube, incubate at 25°C±1°C, and incubate at 15 , 30, 60, 90, and 120 minutes later, samples were taken, and 80 μl of 3mol / L HCl was added immediately to adjust the pH value to neutral.

[0041] The above inactivated samples were centrifuged (4000rpm) for 30 minutes, filtered with a 0.22 μm membrane, and the supernatant was divided into small centrifuge tubes, 4 tubes per batc...

Embodiment 3

[0046] The inactivation test of embodiment 3 human nerve growth factor injection intermediate product EMCV virus

[0047] Indicator virus: EMCV, titer: 7.50LgTCID 50 / 0.1ml.

[0048] Cells used for culture: Vero, titration method: 96-well micro-lesion method.

[0049] Virus inactivation: with embodiment 2 (except that the added virus Sindbis is changed into EMCV, all the other are identical with embodiment 2).

[0050] Virus detection method: Vero cell 96-well micro-lesion method was used.

[0051] The results of virus titration showed that three batches of human nerve growth factor were added to the indicator virus EMCV, and the virus detection results after being inactivated by the method of the present invention are shown in Table 3.

[0052] Incubation time

(minute)

20030126 batches

20030214 batches

20030222 batches

residual titer

drop titer

residual titer

drop titer

residual titer

drop titer

15

30

60

...

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PUM

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Abstract

The invention relates to an inactivation biological product virus and removing bacterial endotoxin method. The biological product or middle product is adjusted its pH value to 12.0+/-0.2 by NaOH solution, hatched for at least one hour at 25+/-1 centigrade degree, then adjusted its pH value to need by HCl solution. The method has good inactivation effect for all fat enveloped virus or non. It has done a lot of test for many viruses. And all of them have good inactivation effect. It is qualified tested by TAL and rabbit detection pyrogen.

Description

technical field [0001] The invention relates to a method for inactivating viruses in biological products and simultaneously removing bacterial endotoxins. It belongs to the field of virus inactivation of biological products. Background technique [0002] Products such as medicines, food, and health care products made from extracts of living organisms, biological organs, and blood are called biological products. Organisms, biological organs, especially blood contain various types of infectious viruses and bacterial endotoxins. Viruses mainly include DNA and RNA. From the perspective of virus structure, there are lipid-enveloped viruses and non-lipid-enveloped viruses. Specific viruses include: HBV, HCV, HIV-1, HIV-2, HTLV, and parvovirus B19, etc., are numerous. In order to ensure the safety of the use of biological products, it is necessary to remove / inactivate viruses and remove bacterial endotoxins on intermediate or finished products during the production process of bio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/06
Inventor 李法卿李素芹李越希
Owner 李法卿
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