CN100431599C - Brain protein hydrolysate and production process of its freeze dried preparation - Google Patents
Brain protein hydrolysate and production process of its freeze dried preparation Download PDFInfo
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Images
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention relates to a brain protein hydrolysate and a preparation method for a freeze dried preparation of the brain protein hydrolysate. In the method, certificated fresh health pig brain or health pig brain stored in a froze mode is taken and homogenized by using a colloidal mill, and homogenate is collected; the homogenate is processed through hydrolysis respectively by using pepsin and pancreatin, hydrolyzed supernatant fluid is collected and filtered by using filter paper, and filtrate is collected; the filtrate is processed through adsorption separation and purification by using hydroxyapetite columns, a peptide map is prepared, and a target substance is collected and ultrafiltered by using a filter film with molecule interception of 8KD; filtrate is collected and processed through azotification and amino acid analysis, and an amino acid dosage is calculated according to a medicine standard (number: WS1-XG-25-2000) published by State Pharmaceutical Inspection Administration in December 28th in 2000; concentrated preparation solution is prepared by adding a corresponding amino acid, and processed through secondary filtration by using a filter film of 0.22 mu m to obtain brain protein hydrolysate stock solution for injection. The brain protein hydrolysate stock solution for injection can be further prepared into a brain protein hydrolysate freeze dried pharmaceutical preparation according to the prescription of a freeze dried pharmaceutical preparation. The method has the advantages of environmental protection, high yield coefficient and low cost.
Description
Technical field
The present invention relates to a kind of production method of Cerebrolysin Vial, also related to a kind of production method of Cerebrolysin Vial lyophilized formulations simultaneously, belong to pharmaceutical field.
Background technology
Cerebral tissue contains rich in protein, people study always the proteolysis of animal brain are become aminoacid and low molecular polypeptide for a long time, are used for treating diseases such as dysfunction that functional disorder, apoplexy, brain memory obstacle and cerebral trauma due to the compensatory deficiency of cerebrovascular cause, cerebral concussion sequela.
Cerebrolysin Vial claims cerebrolysin at home again, and early than early 1990s, domestic scholars is just studied its technology, quality, pharmacological effect and clinical practice, has delivered a large amount of articles.
Chinese invention patent document CN01130523.1 discloses a kind of " process for preparing injection liquid of brain protein hydrolyzate ", its content 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, 40～45 ℃ of bath temperatures, time is 6～8 hours, and the reuse sodium hydroxide is transferred pH to 7.6～7.7, adds pancreatin (pancreatin is dissolved in earlier in the calcium chloride solution of 0.02N in 4 ℃ of activation 30 minutes down) hydrolysis, 40～45 ℃ of water-bath hydrolysis 4～6 hours, obtain hydrolyzed solution, add under 2.5～3.0 ,-20～10 ℃ of the hydrochloric acid adjust pHs and left standstill 10～30 hours; At ambient temperature, filter with double gauze, the water that adds 1/2 volume again, hydro-oxidation sodium adjust pH is 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 mixed polypeptide of brain egg folding hydrolysis, 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.Its shortcoming is: (1) feed liquid repeatedly is 70,000 and 10,000 ultrafilter membrane through the microporous filter membrane in 0.5～1um aperture and the molecular weight that dams, and the ultrafiltrate that obtains makes operation more loaded down with trivial details, and the production cycle is long, is subjected to microbial contamination in the production process easily.(2) it is low to use ultrafilter membrane to carry out the ultrafiltration and concentration response rate: this process using molecular weight that dams is that 1000 ultrafilter membrane concentrates, because of the effective active composition of Cerebrolysin Vial is a micromolecule polypeptide, have seeing through and to lose in various degree in the ultrafiltration and concentration process, yield reduces greatly.
2000, Yu Libi etc. have delivered " preparation of brain egg albumen powder and the research of enzyme solution thereof " in " aminoacid and living resources ", its content is: get bright Medulla sus domestica striping, the piece of dehematizing and clean, add normal saline homogenate by 1: 5 (w/v), add freezing acetone by 1: 10 (w/v) again and stir extraction 2 hours, filtered through gauze behind the standing demix is extracted 4 times altogether.Filter block extracts 2 times by 1: 10 (w/v) with ether, filters the filter block drying, the brain powder was by (w/v) adding distil water dissolving in 1: 20, boil 30min,, transfer pH8.0 with NaOH in 47 ± 0.5 ℃ of balances, add pancreatin hydrolysis 30h, transfer pH6.0 with HCl, boil 40min, cold slightly, filter, filtrate boils, cools off, 4 ℃ deposit and spend the night.The buchner funnel sucking filtration is collected filtrate, bathes temperature and is evaporated to 1/3 of original volume for 58～69 ℃, boil, cool off, 4 ℃ deposit 24h.Sucking filtration gets filtrate, adds no heat source water to original volume, 6% kaolin and 0.6% active carbon, transfers pH4.4～4.6, boils 1h, and sucking filtration gets filtrate while hot.Transfer pH7.0, boil, cooling, 4 ℃ are deposited more than the 24h.After use G
6Filter liquor is collected in aseptic filtration, and molecular cut off 10,000 following liquid are collected in ultrafiltration immediately, aseptic subpackaged, pyrogen testing, quality inspection.Its shortcoming is: (1) production cycle is long, and equipment investment is big, and production efficiency is low; Pancreatin hydrolysis 30h under pH8.0,47 ± 0.5 ℃ of conditions, the production cycle is long, is subjected to microbial contamination easily, is difficult to ensure drug quality; Only use pancreatin hydrolysis, hydrolysis and not exclusively, and also solid-liquid is difficult to separation, and loss is big in the production process; Use the concentrating under reduced pressure step, production efficiency is low, is unfavorable for the control to drug quality.(2), use a large amount of organic chemistry solvents, the boiling point of acetone and ether is extremely low, it is inflammable, explosive, deleterious hazardous chemical, ether also is used as anesthetis, uses a large amount of acetone and ether earlier the brain albumen precipitation to be come out to make the brain egg albumen powder, and this step is abnormally dangerous, produce in must be between hoolivan, operate loaded down with trivial details, contaminated environment, harm production operation personnel's is healthy.
Summary of the invention
The object of the present invention is to provide a kind of novel Cerebrolysin Vial production method, this method is compared with existing production method, has that production link is received less, the yield height, production cost is low and safety non-pollution.
The present invention adopts following technical scheme: a kind of production method of Cerebrolysin Vial, this method comprises the steps:
(1) the fresh or frozen healthy Medulla sus domestica of learning from else's experience and being up to the standards is used colloid mill homogenate, collects homogenate;
(2) homogenate is used pepsin, pancreatin hydrolysis respectively, collect the hydrolysis supernatant, filter, collect filter liquor;
(3) filter liquor is carried out absorption and purification with the hydroxyapatite chromatography post: the hydroxyapatite chromatography post adopts slurry type method dress post, the dress column pressure is 0.5Mpa, clean with 400mmol/L pH6.8 phosphate buffer earlier behind the dress post, reuse 20mmol/L pH6.8 phosphate buffer balance, hydroxyapatite chromatography post on the above-mentioned filter liquor is adsorbed, with 20mmol/L pH6.8 phosphate buffer eluting;
(4) collect object, adopting molecular retention is that the filter membrane of 8KD carries out ultrafiltration, collects filter liquor, carries out peptide figure, amino acid analysis check;
(5) by the WS that is numbered of National Drug Administration's December in 2000 promulgation on the 28th
1-XG-25-2000 drug standard calculates the aminoacid consumption, adds corresponding amino acid ligand and makes concentrated wiring liquid, and 0.22 μ m filter membrane carries out essence and filters, and promptly gets the stock solution of brain protein hydrolysate for injection.
The present invention also provides a kind of production method of Cerebrolysin Vial lyophilized formulations, and this method is:
(1) the fresh or frozen healthy Medulla sus domestica of learning from else's experience and being up to the standards is used colloid mill homogenate, collects homogenate;
(2) homogenate is used pepsin, pancreatin hydrolysis respectively, collect the hydrolysis supernatant, filter, collect filter liquor;
(3) filter liquor is carried out absorption and purification with the hydroxyapatite chromatography post: the hydroxyapatite chromatography post adopts slurry type method dress post, the dress column pressure is 0.5Mpa, clean with 400mmol/L pH6.8 phosphate buffer earlier behind the dress post, reuse 20mmol/L pH6.8 phosphate buffer balance, hydroxyapatite chromatography post on the above-mentioned filter liquor is adsorbed, with 20mmol/L pH6.8 phosphate buffer eluting;
(4) collect object, adopting molecular retention is that the filter membrane of 8KD carries out ultrafiltration, collects filter liquor, carries out peptide figure, amino acid analysis check;
(5) by the WS that is numbered of National Drug Administration's December in 2000 promulgation on the 28th
1-XG-25-2000 drug standard calculates the aminoacid consumption, adds corresponding amino acid ligand and makes concentrated wiring liquid, and 0.22 μ m filter membrane carries out essence and filters, and gets the stock solution of brain protein hydrolysate for injection;
(6) brain protein hydrolysate for injection stock solution and excipient and solvent are mixed, through coarse filtration and fine straining, divide in the cillin bottle of packing into decolorizing with activated carbon;
(7) handle through lyophilizing, get lyophilized formulations.
The production method of this Cerebrolysin Vial of the present invention has following advantage:
1, do not use Hazardous Chemical Substances such as acetone, ether to prepare the brain egg albumen powder, fresh or frozen Medulla sus domestica directly extracts Cerebrolysin Vial through production technologies such as two enzyme enzymolysis, the tool safety non-pollution, less put into production equipment, reduce advantages such as production link.
2, double-enzyme hydrolysis brain tissue homogenate liquid, adopt extraction, the centrifugal acquisition supernatant of residue, after filtration, go up hydroxyapatite chromatography post absorption and purification, production technologies such as ultrafiltration, hydrolysis is complete, can remove foreigh protein removing and macromole impurity, make the HPLC impurity peak area, reach the separating effect of existing technology less than 5%.
3, adopt two enzyme enzymolysis, extraction, the centrifugal acquisition supernatant of residue, after filtration, go up hydroxyapatite chromatography post absorption and purification, production technologies such as ultrafiltration, advantage be shorten greatly in production stage, cycle, yield improves, production cost reduces, production environment requires to reduce, easy and simple to handle, reduced medicine and be subjected to microbial contamination, rotten chance.This process stabilizing is reliable, GMP compatible.
Description of drawings
Fig. 1 is the brain protein hydrolysate for injection freeze-drying curve.
Fig. 2 differentiates collection of illustrative plates for brain protein hydrolysate injection.
Fig. 3 differentiates collection of illustrative plates for brain protein hydrolysate for injection.
The specific embodiment
Be described further below in conjunction with the production method of the drawings and specific embodiments Cerebrolysin Vial of the present invention.
The production of embodiment 1, brain protein hydrolysate for injection (stock solution)
Fresh or the frozen healthy Medulla sus domestica 100.0kg that learns from else's experience and be up to the standards, remove non-cerebral tissue things such as connective tissue and broken bone, clean with water for injection, drain, earlier be twisted into fragment with meat grinder, scale to 200 purpose colloid mill is levigate with regulating, and collects homogenate, and the water for injection that adds 150.0kg (1.5 times of weights) stirs evenly.Serosity is heated to 42 ℃ ± 2 ℃, transfer pH to 1.5～1.7 with 4.5% hydrochloric acid, add the pepsic ratio of 10g (1%) by every kg serosity, adding 2.5kg pepsin is kept the condition hydrolysis 8 hours of pH1.5～1.7,42 ℃ ± 2 ℃.With 5M NaOH the pepsin hydrolysis serosity is transferred pH to 7.1～7.3, the ratio (1%) that adds the 10g pancreatin in every kg serosity, add the 2.5kg pancreatin, after stirring evenly, maintain 7.5～7.7,42 ℃ ± 2 ℃ hydrolysis of pH 4 hours, enzymolysis finishes post-heating and is warming up to 100 ℃, is incubated after 15 minutes, is cooled to below 30 ℃.Transfer pH2.8～3.0, standing over night with 4.5% hydrochloric acid.Sucking filtration gets supernatant 176.0kg, and residue is centrifugal with centrifuge, collects the 53.0kg of supernatant, merges supernatant, and 229.0kg carries out coarse filtration altogether, collects filter liquor and gets 227.0kg, transfers pH to 6.8～7.0 with 5M NaOH, and is standby.With hydroxyapatite furnishing pasty state, adopt slurry type method dress post with water for injection, dress column pressure 0.5Mpa.Clean with the 400mmol/LpH6.8 phosphate buffer earlier behind the dress post, reuse 20mmol/L pH6.8 phosphate buffer balance is adsorbed hydroxyapatite chromatography post on the above-mentioned filter liquor, with 20mmol/L pH6.8 phosphate buffer eluting.Collect object and get 285.0kg, using molecular retention is that the filter membrane of 8K carries out ultrafiltration, collects filter liquor and gets 274.0kg, carries out peptide figure, aminoacid check.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, filter 0.22 μ m filter membrane carries out essence, promptly get the stock solution 292.0kg of brain protein hydrolysate for injection.This stock solution can be by lyophilized formulations prescription preparation finished product.
Under 100Kg inventory situation, the Cerebrolysin Vial stock solution total nitrogen that obtains is 5069.12g, is higher than prior art 2636.48g, shows that yield has improved about 93% approximately.
The production method of embodiment 2, brain protein hydrolysate for injection lyophilized preparation
(1) preparation of supplementary material
In 10000 grades of clean areas, add in the Agitation Tank by recipe quantity examining qualified Cerebrolysin Vial stock solution and mannitol entirely, add water for injection to scale, fully stirring and evenly mixing is pressed amount of liquid 0.2% and is added activated carbon, stir decolouring 20 minutes, 0.45um the film coarse filtration through 0.22um film fine straining, is surveyed its content again, send between 100 grades of packing, stand-by.
Prescription is: Cerebrolysin Vial stock solution is equivalent to total nitrogen 30.0g approximately
Mannitol 240g
Water for injection adds to 4000.0ml
Be distributed into 1000 bottles
In the prescription, Cerebrolysin Vial stock solution is principal agent, and mannitol is excipient, and water for injection is solvent.
(2) preparation of packaging material
1. the preparation of cillin bottle
The 7ml cillin bottle is after ultrasonic waves for cleaning, and then respectively with deionized water, water for injection washing, the reuse clean compressed air dries up, and reaches 280-300 ℃ tunnel baking oven sterilization and removes pyrogen by temperature in 0.5 hour at last, and is stand-by.
2. the preparation of butyl rubber plug
Butyl rubber plug is cleaned with deionized water respectively earlier, then with water for injection rinsing repeatedly, and moist heat sterilization 20 minutes under 121 ℃ of conditions then, afterwards, vacuum is taken out most steam, cools, and is stand-by.
3. the preparation of aluminium lid
After the purified water of aluminium lid cleans, under 120 ℃ of conditions xeothermic 150 minutes, stand-by.
(3) packing, half tamponade
After the packing debugging machine is normal, will send between packing, determine the packing volume, carry out packing by injection packing rules, half tamponade according to content through the empty bottle that clean drying and sterilizing cools.
(4) lyophilizing
The 7ml cillin bottle that branch is installed sample is sent in the freeze drying box, carries out the lyophilizing operation by the specific freeze-drying curve of brain protein hydrolysate for injection shown in Figure 1, and lyophilizing is pressed into plug in the cillin bottle after finishing fully.
Freeze-drying process is: 1, after the packing, be cooled to-38 ℃, keep 3h; 2, evacuation, reach 14Pa after, begin to be warming up to 5 ℃, keep 10h; 3, keep vacuum 14Pa, be warming up to 15 ℃, keep 15h; 4, keep vacuum 14Pa, be warming up to 25 ℃, keep 3h; 5, tamponade recovers atmospheric pressure, outlet.6, lyophilizing overall process needs 34h approximately.
(5) roll lid
The semi-finished product that lyophilizing is finished roll lid, simultaneously the full review of sampling.
(6) visual inspection
When rolling lid, carry out visual inspection, reject the outward appearance defective work.
(7) labeling
The semi-finished product that visual inspection is qualified are pasted label by the labeling rule of operation.
(8) outer package
Labeling finishes, product examine entirely qualified after, dress capsule, middle box, big case, warehouse-in then.
(9) differentiate collection of illustrative plates
Adopting the instrument model is SPD-10Avp LC-10Atvp, and detector type is a ultraviolet, and the detection wavelength is 280nm, and the post model is TSK-GEL G2000SWxl, and column temperature is 30 ℃, and sample size is 10ul, and flow is 0.5ml/min.The discriminating atlas analysis collection of illustrative plates of standard substance brain protein hydrolysate injection (Cerebrolysin) is seen Fig. 1, and analysis result sees Table 1
Table 1 standard substance brain protein hydrolysate injection analysis result
The discriminating collection of illustrative plates of brain protein hydrolysate for injection lyophilized formulations as shown in Figure 2, analysis result sees Table 2
Table 2 brain protein hydrolysate for injection lyophilized formulations analysis result
By Fig. 1 and Fig. 2 as can be known, to table 1 when and table 2 contrast the brain protein hydrolysate for injection lyophilized formulations with the chromatogram basically identical of standard sample, conformance with standard.
Claims (4)
1, a kind of production method of Cerebrolysin Vial, this method comprises the steps:
(1) the fresh or frozen healthy Medulla sus domestica of learning from else's experience and being up to the standards is used colloid mill homogenate, collects homogenate;
(2) homogenate is used pepsin, pancreatin hydrolysis respectively, collect the hydrolysis supernatant, filter, collect filter liquor;
(3) filter liquor is carried out absorption and purification with the hydroxyapatite chromatography post: the hydroxyapatite chromatography post adopts slurry type method dress post, the dress column pressure is 0.5Mpa, clean with 400mmol/L pH6.8 phosphate buffer earlier behind the dress post, reuse 20mmol/L pH6.8 phosphate buffer balance, hydroxyapatite chromatography post on the above-mentioned filter liquor is adsorbed, with 20mmol/L pH6.8 phosphate buffer eluting;
(4) collect object, adopting molecular retention is that the filter membrane of 8KD carries out ultrafiltration, collects filter liquor, carries out peptide figure, amino acid analysis check;
(5) by the WS that is numbered of National Drug Administration's December in 2000 promulgation on the 28th
1-XG-25-2000 drug standard calculates the aminoacid consumption, adds corresponding amino acid ligand and makes concentrated wiring liquid, and 0.22 μ m filter membrane carries out essence and filters, and promptly gets the stock solution of brain protein hydrolysate for injection.
2, a kind of production method of Cerebrolysin Vial lyophilized formulations, this method is:
(1) the fresh or frozen healthy Medulla sus domestica of learning from else's experience and being up to the standards is used colloid mill homogenate, collects homogenate;
(2) homogenate is used pepsin, pancreatin hydrolysis respectively, collect the hydrolysis supernatant, filter, collect filter liquor;
(3) filter liquor is carried out absorption and purification with the hydroxyapatite chromatography post: the hydroxyapatite chromatography post adopts slurry type method dress post, the dress column pressure is 0.5Mpa, clean with 400mmol/L pH6.8 phosphate buffer earlier behind the dress post, reuse 20mmol/L pH6.8 phosphate buffer balance, hydroxyapatite chromatography post on the above-mentioned filter liquor is adsorbed, with 20mmol/L pH6.8 phosphate buffer eluting;
(4) collect object, adopting molecular retention is that the filter membrane of 8KD carries out ultrafiltration, collects filter liquor, carries out peptide figure, amino acid analysis check;
(5) by the WS that is numbered of National Drug Administration's December in 2000 promulgation on the 28th
1-XG-25-2000 drug standard calculates the aminoacid consumption, adds corresponding amino acid ligand and makes concentrated wiring liquid, and 0.22 μ m filter membrane carries out essence and filters, and gets the stock solution of brain protein hydrolysate for injection;
(6) brain protein hydrolysate for injection stock solution and excipient and solvent are mixed, through coarse filtration and fine straining, divide in the cillin bottle of packing into decolorizing with activated carbon;
(7) handle through lyophilizing, get lyophilized formulations.
3, the production method of Cerebrolysin Vial lyophilized formulations as claimed in claim 2, it is characterized in that: described excipient is a mannitol, solvent is a water for injection, adds 240g mannitol with the brain protein hydrolysate for injection stock solution of total nitrogen 30g, adds the injection water again to 4000ml.
4, as the production method of claim 2 or 3 described Cerebrolysin Vial lyophilized formulations, it is characterized in that: described lyophilizing processing procedure is kept 3h for being cooled to-38 ℃ earlier, evacuation then, reach 14Pa after, begin to be warming up to 5 ℃, keep 10h; Keep vacuum 14Pa again, be warming up to 15 ℃, keep 15h; Further keep vacuum 14Pa, be warming up to 25 ℃, keep 3h; Tamponade recovers atmospheric pressure, outlet.
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| CN104225572A (en) * | 2014-08-19 | 2014-12-24 | 北华大学 | Cerebroprotein hydrolysate and preparation method thereof |
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| CN101637457B (en) * | 2009-09-01 | 2011-06-08 | 广州市隆赋药业有限公司 | Preparation method for cerebroprotein hydrolysate freeze-dried injection for injection |
| CN102579584A (en) * | 2011-01-06 | 2012-07-18 | 赵能 | Health care pills for dredging channels and collaterals and treating facial paralysis |
| CN103142991B (en) * | 2013-03-27 | 2015-01-21 | 山西普德药业股份有限公司 | Cerebroprotein hydrolysate and lyophilized powder thereof for injection |
| CN105039482A (en) * | 2015-08-31 | 2015-11-11 | 刘冬明 | Method for preparing active pig brain polypeptide |
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| CN111363771B (en) * | 2020-03-18 | 2023-03-14 | 湖北瑞邦生物科技有限公司 | Production process of pig brain extract with strong oxidation resistance and pig brain extract |
| CN113908174B (en) * | 2021-10-29 | 2022-06-24 | 北京四环制药有限公司 | Efficient and safe preparation method and application of cerebroprotein hydrolysate |
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| CN1666777A (en) * | 2004-03-08 | 2005-09-14 | 王玫 | Medicinal preparation of brain protolysate for injection and preparation method thereof |
| CN1228082C (en) * | 2004-03-19 | 2005-11-23 | 严家定 | Method for preparing pharmaceutics of hydrolysate of brain protein |
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| CN1356141A (en) * | 2001-11-24 | 2002-07-03 | 海南斯达制药有限公司 | Process for preparing injection liquid of brain protein hydrolyzate |
| CN1666777A (en) * | 2004-03-08 | 2005-09-14 | 王玫 | Medicinal preparation of brain protolysate for injection and preparation method thereof |
| CN1228082C (en) * | 2004-03-19 | 2005-11-23 | 严家定 | Method for preparing pharmaceutics of hydrolysate of brain protein |
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| CN104225572A (en) * | 2014-08-19 | 2014-12-24 | 北华大学 | Cerebroprotein hydrolysate and preparation method thereof |
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