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Method for preparing mouse nerve growth factor and method for preparing mouse nerve growth factor for injection

A technology for nerve growth factor and injection, which is applied in the field of preparation of mouse nerve growth factor, can solve the problems of large nerve growth factor protein activity damage, unsuitable virus removal, and increased risk of product contamination, achieving high safety and high ratio live effect

Active Publication Date: 2009-12-30
SINOBIOWAY BIOMEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has no removal effect on non-lipid enveloped viruses, and artificially introduces organic solvents and detergents, which are very difficult to completely remove organic solvents and detergents, thus increasing the risk of product contamination; nano-membrane filtration The method mainly uses the size difference between virus particles and protein molecules to remove viruses through nano-scale filter membranes, which is suitable for proteins with smaller molecular weight (smaller diameter), but not suitable for virus removal of protein products with larger diameters, and nano-membranes are expensive The photochemical method is to use some photosensitizers to have a strong affinity for the surface of the virus and the structure of the viral nucleic acid, which is easily activated under the light of an appropriate wavelength, thereby destroying the virus structure in contact with it through photochemical action, but the photochemical method has a strong affinity for the nerve growth factor. The activity of the protein is greatly damaged; pasteurization is a heat treatment inactivation virus method, that is, the virus protein is denatured by heat, the virus structure is destroyed and the virus is inactivated, and both lipid membrane viruses and non-lipid membrane viruses can be effectively removed , clinical records show that pasteurization is the most reliable method of virus inactivation at present
However, heat often denatures protein products or reduces biological activity.

Method used

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  • Method for preparing mouse nerve growth factor and method for preparing mouse nerve growth factor for injection
  • Method for preparing mouse nerve growth factor and method for preparing mouse nerve growth factor for injection
  • Method for preparing mouse nerve growth factor and method for preparing mouse nerve growth factor for injection

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Homogenate mouse submandibular gland tissue, freeze and thaw repeatedly to break up cells, centrifuge to remove sediment, and obtain homogenate supernatant; remove cell debris from homogenate supernatant, apply to CM-Cellulose 52 column, collect flow-through, and carry out Dialysis, acidification and dissociation with pH 4.0 acetate buffer for 10 minutes, centrifugation to obtain the acid hydrolysis supernatant; put the acid hydrolysis supernatant on a CM-Cellulose52 column, wash with Tris-HCl and then elute with Tris-HCl-NaCl solution Collect target protein peaks to obtain protein collection solution.

[0023] Use a 100KD ultrafiltration membrane to perform ultrafiltration on the protein collection solution to remove macromolecular viruses; the 100KD ultrafiltration membrane filtrate is diluted to 5 μg / ml with water for injection, kept at a constant temperature in a 60°C water bath for 10 hours, and then passed through a 5KD ultrafiltration membrane The stock solution ...

Embodiment 2

[0025] The difference from Example 1 is that the 100KD ultrafiltration membrane filtrate is diluted with water for injection to 10 μg / ml, kept at a constant temperature in a water bath at 55°C for 12 hours, and then subjected to ultrafiltration and filtration sterilization through a 5KD ultrafiltration membrane, that is Rat nerve growth factor stock solution was obtained. The detection data of mouse nerve growth factor are shown in Table 1.

Embodiment 3

[0027] The difference from Example 1 is that the 100KD ultrafiltration membrane filtrate is diluted to 20 μg / ml with physiological saline for injection, kept at a constant temperature in a water bath at 60°C for 10 hours, and then ultrafiltered and filtered through a 5KD ultrafiltration membrane. , to obtain the stock solution of mouse nerve growth factor. The detection data of mouse nerve growth factor are shown in Table 1.

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Abstract

The invention discloses a method for preparing a mouse nerve growth factor (NGF) and a method for preparing a mouse nerve growth factor for injection. The method for preparing the mouse nerve growth factor adopts 100KD and 5KD of ultrafiltration membrane filtration and pasteurization processes on the basis of the conventional process, can effectively filter a macromolecular virus, can further inactivate potential viruses, such as pseudo rabies viruses (PRV), Sindbis viruses, encephalomyo-carditis (EMC) viruses and the like by pasteurization at the same time, does not introduce any pollutant, such as a foreign organic solvent, a detergent and the like, and can keep high purity, high specific activity and high safety of the mouse nerve growth factor.

Description

technical field [0001] The invention relates to a preparation method of mouse nerve growth factor and a preparation method of mouse nerve growth factor for injection, in particular to a method for preparing mouse nerve growth factor by using a novel virus inactivation method. Background technique [0002] Mouse nerve growth factor (NGF) is a freeze-dried product of nerve growth factor isolated and purified from the mouse submandibular gland. Its traditional preparation process is: homogenate the mouse submandibular gland tissue to obtain the homogenate supernatant; After centrifugation and removal of precipitation, ion-exchange chromatography I was used to collect the flow-through; the flow-through was subjected to acidification, dissociation, and centrifugation to obtain the acid hydrolysis supernatant; the acid hydrolysis supernatant was subjected to ion-exchange chromatography to collect the peak of the target protein to obtain Nerve growth factor stock solution; add exci...

Claims

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Application Information

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IPC IPC(8): C07K1/34C07K1/18C07K14/48A61K9/19A61K38/18
Inventor 熊玲媛任宏伟孙朗陈远志马凌燕杨佑瑶
Owner SINOBIOWAY BIOMEDICINE
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