CN1129451C

CN1129451C - Process for preparing injection liquid of brain protein hydrolyzate

Info

Publication number: CN1129451C
Application number: CN01130523A
Authority: CN
Inventors: 张素; 戴毅; 赵守田
Original assignee: SIDA PHARMACEUTICAL CO Ltd HAINAN
Current assignee: SIDA PHARMACEUTICAL CO Ltd HAINAN
Priority date: 2001-11-24
Filing date: 2001-11-24
Publication date: 2003-12-03
Anticipated expiration: 2021-11-24
Also published as: CN1356141A

Abstract

The present invention relates to a technology for producing a brain protein hydrolysate injection, which is characterized in that in the technology for producing brain protein hydrolysate injection, water with volume of 2 times of the volume of fresh pig brain is added to the fresh pig brain to be homogenized and filtered, and filtrate is hydrolyzed by pepsin and then hydrolyzed by using enzyme; a pH value is adjusted, and the hydrolyzed filtrate is processed through still standing, filtration at a room temperature, and water addition, and a pH value is adjusted to be neutral; the hydrolyzed filtrate is filtered and clarified by a microporous filter membrane, and ultrafiltered by using ultrafiltration membranes with different interception molecular weight to obtain concentrated solution; and the concentrated solution is heated, then cooled and ultrafiltered by an ultrafiltration membrane to a refined product of mixing polypeptide and amino acid solution of hydrolyzed brain protein.

Description

The brain protein hydrolysate injection production method

Technical field

The present invention is the production method that belongs to biochemical drug.

Background technology

Prior art: fresh Medulla sus domestica is removed the mucosa on surface and bloodstain etc., and homogenate adds the acetone precipitation of 5 times of volumes, and adding the washing of equivalent ether after the precipitate and separate again soaked 1 hour, refilters, vacuum drying is made the brain powder; The brain powder with water dissolution after, use pepsin, pancreatin double-enzyme hydrolysis successively, hydrolyzed solution centrifuging and taking supernatant, ebuillition of heated and rapidly cooling get hydrolyzed solution through plate-and-frame filtration, ultrafiltration and concentrating under reduced pressure then; The hydrolyzed solution branch is pulled on Sephadex G-25 gel chromatography column chromatography, liquid is collected at active peak made elaboration liquid through concentrating under reduced pressure, 0.45 microporous filter; According to required amount of liquid medicine, drug standard (numbering WS1-XG-25-2000) by the promulgation on the 28th of National Drug Administration's December in 2000 calculates the aminoacid consumption, add corresponding amino acid ligand and make concentrated wiring liquid, rare then equipped pin finished product medicinal liquid becomes sterile preparation through steam sterilization.

(1), uses a large amount of organic chemistry solvents

As everyone knows, the boiling point of acetone and ether is extremely low, inflammable and explosive, be deleterious organic chemicals, ether also is used as anesthetis, use a large amount of acetone and ether earlier the brain albumen precipitation to be come out to make the brain powder, this step is abnormally dangerous, produces troublesome poeration in must be between hoolivan, contaminated environment, the infringement health.

(2), equipment investment is big, production efficiency is low, the production cycle is long

With the chromatography method effective ingredient of purifying, each process need 30 hours, the production cycle is long.Use concentrating under reduced pressure and centrifugal step, production efficiency is low, is unfavorable for the control to the medicine quality.

Summary of the invention

The object of the invention provides a kind of organic chemistry solvent that do not use, and new method uses the physical method substituted chemistry reagent sedimentation method to extract the proteic brain protein hydrolysate injection production method of brain.

Brain protein hydrolysate injection production method of the present invention is: fresh Medulla sus domestica is removed surperficial mucosa and bloodstain etc., add the water homogenate of 2 times of volumes, filter with double gauze, filtrate is used earlier pepsin hydrolysis, transfer PH to 1.5-1.6 with hydrochloric acid, bath temperature 40--45 ℃, time is 6-8 hour, the reuse sodium hydroxide transfers PH to 7.6-7.7, add in the calcium chloride solution be dissolved in 0.02N in advance in 4 ℃ of pancreatin hydrolysis of 30 minutes of activation down, 40--45 ℃ of water-bath hydrolysis 4-6 hour obtains hydrolyzed solution, add hydrochloric acid and transfer pH value 2.5-3.0, left standstill 10-30 hour under-20～-10 ℃; At ambient temperature, filter with double gauze, the water that adds 1/2 volume again, hydro-oxidation sodium transfers pH value to neutral, with the aperture is the filtering with microporous membrane clarification of 0.5-1.0um, get clear liquor, be 70,000 and 10,000 ultrafilter membrane ultrafiltration with the molecular weight that dams successively, the ultrafiltrate reuse that the obtains molecular weight that dams is that 1000 ultrafilter membrane concentrates, the concentrated solution that obtains is heated to 100 ℃ of boilings 15 minutes, be cooled to below 25 ℃ then rapidly, be 70,000 and 10,000 ultrafilter membrane more successively through the microporous filter membrane in 0.5-1um aperture and the molecular weight that dams, the degerming solution that the ultrafiltrate that obtains obtained through 121 ℃ of steam sterilizations in 30 minutes again is called the proteoclastic mixed polypeptide of brain, the Freamine highly finished product; According to required amount of liquid medicine, drug standard (numbering WS1-XG-25-2000) by the promulgation on the 28th of National Drug Administration's December in 2000 calculates the aminoacid consumption that need add, be mixed with concentrated wiring liquid with above-mentioned solution, rare then equipped pin finished product medicinal liquid became sterile preparation in 30 minutes through 121 ℃ of steam sterilizations.

The technology of the present invention advantage: existing method comes out to make the brain powder with the brain albumen precipitation earlier with the chemical reagent sedimentation method; acetone and ether that chemical precipitation method is used are abnormally dangerous, produce troublesome poeration in must be between hoolivan; contaminated environment, the safeguard protection to the operator simultaneously requires very high.This method has been given up the chemical precipitation step, and direct hydrolysis reuse clarification mode is removed water-insoluble, can reach the separating effect of existing method.That this method has is safe, efficient, pollution-free, the characteristics of handled easily.

Existing method is with the chromatography method active ingredient of purifying, and the production cycle is long, and improve output will over-bought equipment, and equipment investment is big, and production efficiency is low.This method is with acid hydrolyzation, microfiltration and ultrafiltration, pyromorphism purification medicinal liquid, advantage be shorten greatly the production time, output improves, production environment requires to reduce, easy and simple to handle.Simultaneously, reduce production link just mean reduced medicine may contaminated chance, shortening just meaning of production time has lowered the rotten aborning probability of medicine.

Except that having above advantage, this method has reduced production cost, and the mode of production is sealed production substantially, adopts physical method, does not have leakage, pollution-free, GMP compatible.

The specific embodiment

1, fresh Medulla sus domestica is 100 kilograms, removes surperficial mucosa and bloodstain etc., and the distilled water (hereinafter to be referred as water) that homogenate limit, limit adds 200L filters homogenate with double gauze; Get and add in the filtrate after 2.16 kilograms of pepsin add aqueous solution, hydrochloric acid with 0.5N is transferred PH to 1.5,44 ℃ of water-bath hydrolysis 8 hours then, are transferred PH to 7.2 with the sodium hydroxide of 2N, getting 1.25 kilograms of pancreatin uses the calcium chloride of 0.02N to activate 30 minutes down in 4 ℃, add the back and continue to transfer PH to 7.6 with the sodium hydroxide of 2N, 44 ℃ of water-bath hydrolysis 6 hours obtain hydrolyzed solution, hydrochloric acid with 0.5 is transferred PH to 3.0 ,-10 ℃ of following standing over night; Filter once with double gauze, it is about 450L that the water that adds 150L again makes cumulative volume, transfer pH value to neutral with the sodium hydroxide of 2N, with the aperture is the filtering with microporous membrane of 0.6um, the clear liquor that obtains passes through the ultrafilter membrane ultrafiltration of dam molecular weight 70,000 and 10,000 more successively, the ultrafiltrate that obtains (volume is about 450L) is 1000 ultrafilter membrane ultrafiltration elimination ultrafiltrate with the molecular weight that dams, keep concentrated solution, to the remaining about 100L of concentrated solution stop, with concentrated solution be heated to 100 ℃ the boiling 15 minutes, be cooled to 25 ℃ with psychrolusia then, the reuse aperture is the 0.6um filtering with microporous membrane, the clear liquor that obtains is 70,000 and 10,000 ultrafilter membrane ultrafiltration more successively through the molecular weight that dams, obtain about 100L ultrafiltrate, in the glass container of packing into, sterilized 30 minutes down for 121 ℃, obtain the proteoclastic mixed polypeptide of brain of degerming, preserve under the Freamine highly finished product, room temperature; Before the preparation finished product dress pin, according to required amount of liquid medicine, drug standard (numbering WS1-XG-25-2000) interpolation amino acid ligand by the promulgation on the 28th of National Drug Administration's December in 2000 is made concentrated wiring liquid, and rare then equipped pin finished product became sterile preparation in 30 minutes through 121 ℃ of steam sterilizations.

Claims

1, the brain protein hydrolysate injection production method, it is characterized in that: the brain protein hydrolysate injection production method is: fresh Medulla sus domestica is removed surperficial mucosa and bloodstain etc., add the water homogenate of 2 times of volumes, filter with double gauze, filtrate is used earlier pepsin hydrolysis, transfer PH to 1.5-1.6 with hydrochloric acid, bath temperature 40-45 ℃, time is 6-8 hour, and the reuse sodium hydroxide transfers PH to 7.6-7.7, adds in the calcium chloride solution that is dissolved in 0.02N in advance in 4 ℃ of pancreatin hydrolysis that activate 30 minutes down, 40-45 ℃ of water-bath hydrolysis 4-6 hour, obtain hydrolyzed solution, add hydrochloric acid and transfer pH value 2.5-3.0, left standstill 10-30 hour under-20～-10 ℃; At ambient temperature, filter with double gauze, the water that adds 1/2 volume again, hydro-oxidation sodium transfers pH value to neutral, with the aperture is the filtering with microporous membrane clarification of 0.5-1.0um, get clear liquor, be 70,000 and 10,000 ultrafilter membrane ultrafiltration with the molecular weight that dams successively, the ultrafiltrate reuse that the obtains molecular weight that dams is that 1000 ultrafilter membrane concentrates, the concentrated solution that obtains is heated to 100 ℃ of boilings 15 minutes, be cooled to below 25 ℃ then rapidly, be 70,000 and 10,000 ultrafilter membrane more successively through the microporous filter membrane in 0.5-1um aperture and the molecular weight that dams, the degerming solution that the ultrafiltrate that obtains obtained through 121 ℃ of steam sterilizations in 30 minutes again is called the proteoclastic mixed polypeptide of brain, the Freamine highly finished product.