CN101912598A - Method for preparing brain protein hydrolysate for injection and preparation thereof - Google Patents
Method for preparing brain protein hydrolysate for injection and preparation thereof Download PDFInfo
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Abstract
The invention relates to a method for preparing brain protein hydrolysate for injection and a preparation thereof. The brain protein hydrolysate is prepared from pig brain tissues passing quarantine through a specific controlled enzyme hydrolysis technique, and active ingredients of the prepared brain protein hydrolysate comprise polypeptide with a molecular weight of less than 10,000 Daltons and 16 free amino acids which are hydrolyzed amino acids; each milligram of total nitrogen of the obtained brain protein hydrolysate contains 4.68 to 7.02 milligrams of amino acids; the preparation method comprises the processes of homogenization, primary enzymolysis, inactivation, secondary enzymolysis, cation exchange column adsorption, desorption, blending, ultra-filtration, preparation and the like; when the obtained brain protein hydrolysate is used for preparing the preparation, an amino acid does not need adding; and pharmacological experiment results show that the prepared preparation has good treatment effect and good safety.
Description
Technical field
The present invention relates to the biochemical pharmacology technical field, more specifically is a kind of preparation method and preparation thereof of brain protein hydrolysate for injection.
Background technology
Brain protein hydrolysate for injection is that trade name is sold with Cerebrolysin by Austrian EBEWE pharmaceutical factory the earliest.Cerebrolysin Vial be with the health pig brain through enzyme hydrolysis, purification and a kind of biochemical drug that obtains, its main component is the mixture of multiple low molecular peptide class material and several amino acids.Remarkable for treatment cerebral dysfunction disease effect.
Domesticly begin to develop Cerebrolysin Vial from the nineties in last century, to the end of the year 2009,28 of the existing disclosed patent applications that relates to Cerebrolysin Vial preparation and technology.But nearly all preparation technology adds corresponding aminoacid outside all needing when producing preparation, as patent 200610065169.0, needs the corresponding amino acid ligand of outer interpolation to make concentrated wiring liquid, obtains the stock solution of brain protein hydrolysate for injection.From angle biology, the aminoacid during Medulla sus domestica albumen is formed is completely, and being does not need to add amino acid whose, thereby existing preparation technology existing problems can only be described.
According to State Food and Drug Administration's relevant document requirement, all Cerebrolysin Vial preparation manufacturers in the whole nation all need be according to the outer aminoacid that replenishes that adds of standard.Illustrate that also the existing Cerebrolysin Vial preparation preparation technology of manufacturer also has problems.
At the problems referred to above, we have carried out the preparation method of brain protein hydrolysate for injection and the research of preparation thereof.
Summary of the invention
The present invention be with through the qualified Medulla sus domestica cerebral tissue of quarantining by specific hydrolytic degradation technology, do not prepare and need add aminoacid, to be molecular weight be the cerebroprotein hydrolysate preparation of hydrolysis amino acid less than 10000 daltonian polypeptide and 16 kinds of free amino acids to active component.
The purpose of this invention is to provide a kind of brain protein hydrolysate for injection preparation, not needing to be characterized in replenishing in addition adding aminoacid, safety is better;
Another object of the present invention provides the preparation method of a kind of brain protein hydrolysate for injection and preparation thereof.
The objective of the invention is to be achieved through the following technical solutions:
1. the preparation method of a brain protein hydrolysate for injection is characterized in that may further comprise the steps:
(1) qualified freeze or the fresh pig cerebral tissue adds 1-3 times of purified water homogenate of will quarantining,
(2) homogenate is cooled to 30-50 ℃ in 65-90 ℃ of insulation 15-45 minute, adds hydrochloric acid and transfers pH2.0-4.0, stirs the pepsin that adds Medulla sus domestica weight 1.5%-4.5%, stirs, and supernatant is collected in 30-50 ℃ of insulation 2-6 hour, precipitate centrifugal, the merging clear liquid;
(3) the supernatant concentrating under reduced pressure in 100-120 ℃ of deactivation 3-6 hour, adds water to Medulla sus domestica weight to the 1/2-1/5 of Medulla sus domestica weight behind the adjust pH, with calcic lye pH adjustment value 5.0-8.0, and the cloth bag filtration, activated carbon adsorption, decarburization gets the deactivation clear liquid;
(4) adding is by the pancreatin of Medulla sus domestica weight 1%-3% in the deactivation clear liquid, and 35-45 ℃ is incubated 1-4 hour, boils 15-30 minute, and cooling transfers pH value 3-4 to make pancreatin stop hydrolysis, leaves standstill, and filtering-depositing gets hydrolyzed solution;
(5) with treated good cation exchange column on the said hydrolyzed liquid, wash post with purified water, the reuse weak ammonia is resolved, and collects eluent, concentrate, the sodium hydroxide solution adjust pH, concentrating under reduced pressure is removed free ammonia, concentrated solution standing over night, remove precipitation, get stock solution, freeze below-20 ℃ to preserve;
(6) will freeze stock solution and thaw, the filtering precipitation, dilution adds the 0.03%-0.1% sodium sulfite, and/or adds excipient 3%-10%, is seasoning liquid;
(7) seasoning liquid is collected filtrate through holding back the ultrafiltration of 6000-10000 molecular weight ultrafilter membrane, and 0.2 μ m film aseptic filtration gets ultrafiltrate;
(8) ultrafiltrate is filled in the ampoule under the injection preparation method aseptic condition routinely, seals, sterilization detects, and packing promptly gets light yellow clear and bright injection formulation;
(9) with fill under the ultrafiltrate aseptic condition, the false add plug, put in the freeze-drier, when products temperature reaches-30 ℃--in the time of 50 ℃, continue cooling 2-4 hour, and the temperature of condenser reaches-40 ℃--in the time of 60 ℃, evacuation, when the vacuum of vacuum system was lower than 30Pa, the primary drying temperature was set at-30 ℃--and 10 ℃; The redrying temperature is set at 15 ℃-50 ℃, continues drying after reaching temperature, until up to specification, promptly gets the lyophilized formulations of off-white color or micro-yellow powder and block mixture.
A kind of brain protein hydrolysate for injection preparation comprises injection and lyophilized formulations, and its feature also is every milliliter of nitrogenous 5.4-6.8mg of injection, wherein contains free amino acid 25-50mg; Every nitrogenous 30mg of lyophilized formulations wherein contains free amino acid 150-201mg and every nitrogenous 60mg, wherein free amino acid 300-402mg; A kind of preparation method of brain protein hydrolysate for injection is characterized in that excipient is a kind of in mannitol, glucose, lactose, dextran, sucrose, gelatin hydrolysate, glycine, the Glycine sodium.
The brain protein hydrolysate for injection that a kind of preparation method of brain protein hydrolysate for injection obtains, it is characterized in that its effective ingredient is that the molecular weight that the brain hydrolysis produces is 16 kinds of free amino acids less than 10000 daltonian small-molecular peptides and generation, contain free amino acid 4.68-7.02mg in every milligram of total nitrogen of gained hydrolysate, and in every milligram of total nitrogen contained free amino acid proportion of composing and scope unit milligram meter is as follows by weight:
Aspartic Acid 0.64-0.96 glutamic acid 0.83-1.24 serine 0.25-0.46
Histidine 0.13-0.2 glycine 0.32-0.48 threonine 0.22-0.40
Alanine 0.33-0.5 arginine 0.093-0.34 valine 0.21-0.32
Methionine 0.12-0.22 tryptophan 0.03-0.07 isoleucine 0.15-0.22
Phenylalanine 0.16-0.24 leucine 0.36-0.54 lysine 0.47-0.7
Proline 0.27-0.4;
Resulting a kind of brain protein hydrolysate for injection does not need to add in addition aminoacid when the preparation preparation.
Reach the requirement that meets the brain protein hydrolysate injection standard, and do not need to replenish in addition and add aminoacid, just need improve preparation method, the control hydrolysis, make that micromolecule polypeptide and 16 kinds of free amino acid mixtures can reach best proportioning in the Cerebrolysin Vial of preparation, be implemented in the preparation Cerebrolysin Vial ejection preparation and need do not add aminoacid.
Below each step of preparation method in the technical program is analyzed:
Step 1 qualified freeze or the fresh pig cerebral tissue adds purified water homogenate of will quarantining is conventional steps.
Step 2 obtains supernatant with the homogenate enzymolysis, has played and has removed major part not by the macromole impurity effect of enzymolysis.
Step 3 supernatant concentrating under reduced pressure is to dwindle later process clear liquid volume; And by behind the adjust pH in 100-120 ℃ of deactivation 3-6 hour, play and kill the virus that may carry in the Medulla sus domestica, further remove macro-molecular protein simultaneously, for subsequent treatment creates conditions.
Step 4 adds pancreatin in the deactivation clear liquid, the insulation enzymolysis makes in the malicious clear liquid that goes out not by the small molecular protein of pepsin hydrolysis and the further hydrolysis of peptide class; Because the further remove impurity of step 3 makes easier the carrying out of enzymolysis in this step, so needs control, by boiling and transferring pH value 3-4 to make pancreatin stop hydrolysis.
Treated good cation exchange column on the hydrolyzed solution that step 5 obtains step 4 washes post with purified water, and the reuse weak ammonia is resolved, and collects eluent, concentrates, and is that the adsorption by cation exchange column is further purified the targeted activity composition; Resolving with weak ammonia can be with the complete desorbing of targeted activity composition; The sodium hydroxide solution adjust pH is an alkalescence, and concentrating under reduced pressure is removed free ammonia, further purifies the concentrated solution that will obtain; And the concentrated solution standing over night is removed precipitation, gets stock solution, plays further remove impurity effect; Freezing below-20 ℃ to preserve then is in order to remove unwanted aminoacid.It is to be noted that step 5 just is difficult to carry out if there is not the processing of step 1-4, illustrated that also technology of the present invention is complete, previous step creates conditions for next step.
Step 6 will be freezed stock solution and thaw, and the filtering precipitation is removed unwanted aminoacid and small amount of impurities, and allotment obtains seasoning liquid.
Step 7 through holding back the ultrafiltration of 6000-10000 molecular weight ultrafilter membrane, is collected filtrate with seasoning liquid, is in order further to remove the macromole polypeptide that may have in the seasoning liquid; 0.2 the aseptic filtration of μ m film obtains qualified Cerebrolysin Vial solution.
Step 8 obtains preparation by conventional injection preparation exactly
Step 9 obtains preparation by freeze drying process exactly.
By complete method of the present invention, resulting hydrolysate polypeptide molecular weight is less than 10000 dalton, contains free amino acid 4.68-7.02mg in every milligram of total nitrogen, and each seed amino acid limit is as follows in every milligram of total nitrogen:
Aspartic Acid (C
4H
7NO
4) 0.64～0.96mg glutamic acid (C
5H
9NO
4) 0.83～1.24mg
Serine (C
3H
7NO
3) 0.25～0.46mg histidine (C
6H
9N
3O
2) 0.13～0.2mg
Glycine (C
2H
5NO
2) 0.32～0.48mg threonine (C
4H
9NO
3) 0.22～0.40mg
Alanine (C
3H
7NO
2) 0.33～0.5mg arginine (C
6H
14N
4O
2) 0.093～0.34mg
Valine (C
5H
14NO
2) 0.21～0.32mg methionine (C
15H
14NO
2S) 0.12～0.22mg
Tryptophan (C
14H
12N
2O
2) 0.023～0.06mg isoleucine (C
6H
13NO
2) 0.15～0.22mg
Phenylalanine (C
9H
14NO
2) 0.16～0.24mg leucine (C
6H
13NO
2) 0.36～0.54mg
Lysine (C
6H
14N
2O
2) 0.47～0.7mg proline (C
5H
9NO
2) 0.27～0.4mg
Technology and prior art below in conjunction with the preparation method of a kind of brain protein hydrolysate for injection of the present invention and preparation thereof compare.
1. all there is not critical processes such as " concentrating under reduced pressure behind the pepsin enzymolysis; adjust pH; 100-120 ℃; 3-6 hour inactivation of virus " in the prior art. in the present invention, 100-120 ℃, 3-6 hour high temperature not only makes the virus that may be present in the porcine mesencephalic cell, particularly zoonotic virus is killed, for the safety of preparation lays the foundation, can not make simultaneously and be solidified degeneration by the high molecular weight protein of pepsin enzymolysis and other impurity, and by filter removing, for the treatment process of back has purified substrate, so it is the important ring in the whole complete process.
2. column chromatography of the prior art is not to adopt the cation exchange filler, is not to use the ammonia eluting. the available technology adopting glucose gel, and its practical function and ultrafiltration are similar; Also have and adopt anion exchange in the technology, yet anion exchange can not be adsorbed whole aminoacid, if adopt the hydrochloride buffer eluting, the DEAE-SephodexA-25 anionite is exactly a sieve chromatography so, with NF membrane be principle of uniformity, and NF membrane sees through the dalton that the molecular weight of particle is generally 300-1000, according to 16 seed amino acid molecular weight of standard (WS1-XG-25-2000) all less than 300 dalton, therefore pass through nanofiltration technique, free amino acid is all fallen by sieve in theory, in the actual production practice then is that most free amino acids are dropped, and therefore will reach standard to amino acid whose requirement, just can only replenish in addition and add free amino acid.
The present invention adopts cation exchange column, can adsorb whole aminoacid and small-molecular peptides, with the ammonia eluting can eluting all aminoacid and the small-molecular peptides of absorption, so kept enough aminoacid in the Cerebrolysin Vial that obtains of the present invention, do not need the outer aminoacid that adds.
3. compared with the prior art, following processing step all is the technology that prior art does not have in the technology of the present invention.
A. concentrating under reduced pressure behind the pepsin enzymolysis is transferred pH value, and 110 ℃, deactivation in 6 hours; B. use calcium hydroxide to regulate pH 6.5-7.5 after the deactivation; C. use activated carbon decolorizing after the deactivation; D. through deactivation, the clear liquid reuse pancreatin enzymolysis after the decolouring; E. transfer pH value 3.0 with oxalic acid behind the pancreatin enzymolysis; F. with cation seperation column absorption, ammonia is resolved;
G.. transfer the pH value concentrating under reduced pressure to take off free ammonia with sodium hydroxide; H. every mgN contains the ratio and the scope thereof of 16 seed amino acids.
Therefore major advantage of the present invention is:
1. the proteinic degree of hydrolysis of control hydrolysis brain makes its protein 75%-85% be hydrolyzed to free amino acid, and 15%-25% is hydrolyzed to the mixture less than 10000 dalton's micromolecule polypeptides, does not add aminoacid, does not contain the protein of macromolecule.
2. by the adjust pH high-temperature inactivation, stop the risk of zoonotic virus, medication is safer.
3. with cation exchange column method purification micromolecule polypeptide and 16 kinds of free amino acids, ensured the purity of Cerebrolysin Vial.
4. freeze-dried powder dosage form, preparation stabilization, material is malleable not, but long preservation reduces adverse reaction rate, improves drug safety.
5. it is simple to have technology, micromolecule polypeptide and 16 kinds of free amino acid proportions constant, and contain aminoacid 4.68-7.02mg in every milligram of total nitrogen in the gained Cerebrolysin Vial, efficacy stability, the characteristics of safety.
6. gained Cerebrolysin Vial preparation efficacy stability, safety is good.
Test is by adopting different preparation methoies relatively process character and effect below.
Method 1: prior art for preparation method.
Technological process is: healthy Medulla sus domestica is cleaned, add 1.5 times of waters for injection, homogenate → pepsin hydrolysis, 42 ± 2 ℃ of hydrolysis temperatures, pH3.0, enzyme dosage is a Medulla sus domestica weight 3%, enzymolysis 6 hours, the enzymolysis solution freeze overnight → enzymolysis solution absorption supernatant that thaws adds Medulla sus domestica weight 0.3% pancreatin, calcium chloride activation 1 hour, 42 ± 2 ℃ of following hydrolysis 4 hours are boiled and were ended enzymolysis in 20 minutes, cool off supernatant → supernatant ultrafiltration (crossing 10,000 dalton), getting the ultrafiltration liquid measure is that 2 times → ultrafiltrate of Medulla sus domestica weight is through nanofiltration (crossing 300-1000 dalton), keep the liquid measure of damming and be the Medulla sus domestica weight → liquid that dams through ultrafiltration (crossing 10,000 dalton), ultrafiltrate → sampling and measuring aminoacid and peptide figure, add antioxidant (sodium sulfite 0.85g/L), get seasoning liquid → seasoning liquid through ultrafiltration (crossing 10,000 dalton), get ultrafiltrate.
Method 2:, get hydrolyzed solution by the technology of the present invention preparation method.
All feed intake 5 kilograms of Medulla sus domestica of 2 kinds of methods, 10 kilograms of hydrolyzed solutions, repeat 3 times.
Aminoacid and peptide figure measure all and carry out according to standard (WS1-XG-25-2000).
The different preparation methoies of table 1 are process character and effect relatively
| Project | Unit | Method 1 | Method 1 |
| Single batch of complete process time | h? | 75±5? | 50±5? |
| Total nitrogen content | mg/ml? | 2±0.5? | 6.2±0.2? |
| Amino acid content | mg/ml? | 10±2? | 35±5? |
| Amino acid contained kind | Kind | 10? | 16? |
[0071]Table 1 is the result show: process of the present invention has significant difference compared with the prior art.
Various amino acid contents and standard WS1-GX-25-2001 require relatively (unit: mg/ml) of content in 2 kinds of method gained of table 2 hydrolyzed solution
| Aminoacid | Method 1 | Method 2 | Standard |
| Aspartic Acid | 0.70-0.91? | 3.84-5.76? | 2.40-3.60? |
| Glutamic acid | 1.01-1.05? | 4.98-7.44? | 3.20-4.80? |
| Serine | Do not measure | 1.50-2.76? | 0.21-0.39? |
| Histidine | Do not measure | 0.78-1.20? | 1.04-1.56? |
| Glycine | 0.66-0.93? | 1.92-2.88? | 1.20-1.80? |
| Threonine | Do not measure | 1.32-2.40? | 0.21-0.39? |
| Alanine | 1.34-1.42? | 1.98-3.00? | 2.40-3.60? |
| Arginine | Do not measure | 0.56-2.04? | 0.30-1.10? |
| Valine | 0.71-0.94? | 1.26-1.92? | 1.60-2.40? |
| Methionine | Do not measure | 0.72-1.32? | 0.35-0.65? |
| Tryptophan | Do not measure | 0.18-0.42? | 0.35-0.65? |
| Isoleucine | 0.42-0.48? | 0.90-1.32? | 1.60-2.40? |
| Phenylalanine | 0.21-0.31? | 0.96-1.44? | 1.60-2.40? |
| Leucine | 2.05-2.63? | 2.16-3.24? | 4.80-7.20? |
| Lysine | 2.06-2.25? | 2.82-4.02? | 4.80-7.20? |
| Proline | 0.84-1.08? | 1.60-2.40? | 1.60-2.40? |
| Total amount | 10-12? | 28.08-42.12? | 28.08-42.14? |
Table 2 is the result show: technology of the present invention and prior art are having significant difference aspect resulting aminoacid and the content thereof; Require content relatively with standard WS1-GX-25 2001, aspect amino acid contained kind and total amount, be consistent, but be inconsistent aspect the amino acid whose content out of the ordinary.
We experimentize according to " GLP " " chemicals acute toxicity test technological guidance principle ", " chemicals zest, anaphylaxis and hemolytic investigative technique guideline ", " chemicals long term toxicity test technological guidance principle " related request, result of study shows: the Cerebrolysin Vial preparation of the present invention's preparation, to haemolysis, irritated, toxicity, half causes death and waits critical index to estimate, and all meets injection product regulation; More unexpectedly acute toxicity testing 6mg/mlN injects 0.8ml (the pharmacopeia injection volume is 0.5ml) non-toxic reaction (according to the evaluation regulation injection 0.48ml of 120 times of using dosages); Do not measure half lethal dose, and with all making half lethal dose in the commercially available prod of concentration, this is complete unexpected safe result.Pharmacodynamic study demonstrates, and under therapeutic dose the therapeutic effect of animal pattern preparation of the present invention is better than commercially available similar preparation, but there was no significant difference.
Embodiment
Below by embodiment technical solution of the present invention is described further, but the invention is not restricted to following embodiment.
Embodiment 1:
The fresh pig cerebral tissue 20kg that (1) will quarantine qualified adds 2 times of purified water homogenate,
(2) homogenate is cooled to 40 ℃ in 80 ℃ of insulations 20 minutes, adds hydrochloric acid and transfers pH3.0, stirs to add the 600g pepsin, stirs, and supernatant is collected in 30-50 ℃ of insulation 4 hours, precipitate centrifugal, the merging clear liquid;
(3) the supernatant concentrating under reduced pressure in 110 ℃ of deactivations 6 hours, gets deactivation clear liquid 4kg to 1/5 of Medulla sus domestica weight behind the adjust pH, adds water to Medulla sus domestica weight, with Ca (OH)
2Liquid adjust pH 7.0, the cloth bag filtration, charcoal is taken off in activated carbon adsorption, gets clear liquid.
(4) add pancreatin 400g in the deactivation clear liquid, 40 ℃ are incubated 3 hours, boil 30 minutes, and pH3.0 is transferred in cooling, leave standstill, and filtering-depositing gets hydrolyzed solution;
(5) with treated good cation exchange column on the said hydrolyzed liquid, wash post with purified water, reuse 8-10% ammonia is resolved, and collects eluent, concentrates, the sodium hydroxide solution adjust pH is 10, concentrating under reduced pressure is removed free ammonia, and concentrated solution is removed precipitation in 0-3 ℃ of standing over night, get stock solution 10kg, freeze below-20 ℃ to preserve; The calibrating of stock solution: character: be light yellow liquid.
Differentiate: need testing solution chromatogram and reference substance solution chromatogram basically identical.
Every milligram of total nitrogen content, aminoacid kind and total amount conformance with standard (WS1-XG-25-2000), (every milligram of total nitrogen contains amount of amino acid 4.68-7.02mg, 16 kinds of free amino acids of aminoacid kind conformance with standard regulation).
(6) will freeze stock solution and thaw, the filtering precipitation, dilution adds seasoning liquid total amount 0.05% sodium sulfite, is seasoning liquid;
(7) seasoning liquid is collected ultrafiltrate by the ultrafilter membrane ultrafiltration of 6000-10000 molecular weight, and 0.2 μ m film aseptic filtration gets ultrafiltrate;
(8) ultrafiltrate is filled in the ampoule under the injection preparation method aseptic condition routinely, seals, sterilization, detect, packing promptly gets every and contains 3200 of the light yellow clear and bright injection formulations of total nitrogen 30mg, the nitrogenous 5.4-6.8mg of the every ml of injection, wherein free amino acid 25-50mg.
Embodiment 2:
The fresh pig cerebral tissue 40kg that (1) will quarantine qualified adds 2 times of purified water homogenate,
(2) homogenate is cooled to 40 ℃ in 80 ℃ of insulations 20 minutes, adds hydrochloric acid and transfers pH3.0, stirs to add the 1200g pepsin, stirs, and supernatant is collected in 42 ± 2 ℃ of insulations 4 hours, precipitate centrifugal, the merging clear liquid;
(3) the supernatant concentrating under reduced pressure in 110 ℃ of deactivations 6 hours, gets deactivation clear liquid 8kg to 1/5 of Medulla sus domestica weight behind the adjust pH, adds water to Medulla sus domestica weight, with Ca (OH)
2Liquid adjust pH 7.0, the cloth bag filtration, charcoal is taken off in activated carbon adsorption, gets clear liquid.
(4) add pancreatin 800g in the deactivation clear liquid, 40 ± 2 ℃ are incubated 3 hours, boil 30 minutes, and pH3.0 is transferred in cooling, leave standstill, and filtering-depositing gets hydrolyzed solution;
(5) with treated good cation exchange column on the said hydrolyzed liquid, wash post with purified water, reuse 8%-9% ammonia is resolved, and collects eluent, concentrates, the sodium hydroxide solution adjust pH is pH10, concentrating under reduced pressure is removed free ammonia, and concentrated solution is removed precipitation in 04 ℃ of standing over night, get stock solution 20kg, freeze below-20 ℃ to preserve; The calibrating of stock solution: character: be light yellow liquid.
Differentiate: need testing solution chromatogram and reference substance solution chromatogram basically identical.
Every milligram of total nitrogen content, aminoacid kind and total amount conformance with standard (WS1-XG-25-2000).
(6) will freeze stock solution and thaw, the filtering precipitation, dilution adds seasoning liquid total amount 0.04% sodium sulfite, is seasoning liquid;
(7) seasoning liquid is collected ultrafiltrate through holding back the ultrafiltration of 6000-10000 molecular weight ultrafilter membrane, and 0.2 μ m film aseptic filtration gets ultrafiltrate;
(8) ultrafiltrate is filled in the ampoule under the injection preparation method aseptic condition routinely, seals, sterilization detects, and packing promptly gets every and contains 1600 of the light yellow clear and bright injection formulations of total nitrogen 60mg.
Embodiment 3:
The fresh pig cerebral tissue 20kg that (1) will quarantine qualified adds 2 times of purified water homogenate,
(2) homogenate is cooled to 40 ℃ in 80 ℃ of insulations 20 minutes, adds hydrochloric acid and transfers pH3.0, stirs to add the 600g pepsin, stirs, and supernatant is collected in 30-50 ℃ of insulation 4 hours, precipitate centrifugal, the merging clear liquid;
(3) the supernatant concentrating under reduced pressure in 110 ℃ of deactivations 6 hours, gets deactivation clear liquid 4kg to 1/5 of Medulla sus domestica weight behind the adjust pH, adds water to Medulla sus domestica weight, with Ca (OH)
2Liquid adjust pH 7.0, the cloth bag filtration, charcoal is taken off in activated carbon adsorption, gets clear liquid.
(4) add pancreatin 400g in the deactivation clear liquid, 40 ℃ are incubated 3 hours, boil 30 minutes, and pH3.0 is transferred in cooling, leave standstill, and filtering-depositing gets hydrolyzed solution;
(5) with treated good cation exchange column on the said hydrolyzed liquid, wash post with purified water, reuse 8-10% ammonia is resolved, and collects eluent, concentrates, the sodium hydroxide solution adjust pH is 10, concentrating under reduced pressure is removed free ammonia, and concentrated solution is removed precipitation in 03 ℃ of standing over night, get stock solution 10kg, freeze below-20 ℃ to preserve; The calibrating of stock solution: character: be light yellow liquid.
Differentiate: need testing solution chromatogram and reference substance solution chromatogram basically identical.
Every milligram of total nitrogen content, aminoacid kind and total amount conformance with standard (WS1-XG-25-2000).
(6) will freeze stock solution and thaw, the filtering precipitation, dilution adds seasoning liquid total amount 0.06% sodium sulfite, is seasoning liquid;
(7) ultrafiltrate is collected in the ultrafilter membrane ultrafiltration of seasoning liquid through holding back the 6000-10000 molecular weight, and 0.2 μ m film aseptic filtration gets ultrafiltrate;
(8) with fill under the ultrafiltrate aseptic condition, the false add plug, put in the freeze-drier, when products temperature reaches-30 ℃--in the time of 50 ℃, continue cooling 2-4 hour, and the temperature of condenser reaches-40 ℃--in the time of 60 ℃, evacuation, when the vacuum of vacuum system was lower than 30Pa, the primary drying temperature was set at-30 ℃--and 10 ℃; The redrying temperature is set at 15 ℃-50 ℃, continues dryly after reaching temperature, until up to specification, promptly gets 3200 in the preparation of every off-white color that contains total nitrogen 30mg or micro-yellow powder and block.
Embodiment 4:
The fresh pig cerebral tissue 40kg that (1) will quarantine qualified adds 2 times of purified water homogenate,
(2) homogenate is cooled to 40 ℃ in 80 ℃ of insulations 20 minutes, adds hydrochloric acid and transfers pH3.0, stirs to add the 1200g pepsin, stirs, and supernatant is collected in 42 ± 2 ℃ of insulations 4 hours, precipitate centrifugal, the merging clear liquid;
(3) the supernatant concentrating under reduced pressure in 110 ℃ of deactivations 6 hours, gets deactivation clear liquid 8kg to 1/5 of Medulla sus domestica weight behind the adjust pH, adds water to Medulla sus domestica weight, with Ca (OH)
2Liquid adjust pH 7.0, the cloth bag filtration, charcoal is taken off in activated carbon adsorption, gets clear liquid.
(4) add pancreatin 800g in the deactivation clear liquid, 40 ± 2 ℃ are incubated 3 hours, boil 30 minutes, and pH3.0 is transferred in cooling, leave standstill, and filtering-depositing gets hydrolyzed solution;
(5) with treated good cation exchange column on the said hydrolyzed liquid, wash post with purified water, reuse 8%-9% ammonia is resolved, and collects eluent, concentrates, the sodium hydroxide solution adjust pH is pH10, concentrating under reduced pressure is removed free ammonia, and concentrated solution is removed precipitation in 0-4 ℃ of standing over night, get stock solution 20kg, freeze below-20 ℃ to preserve; The calibrating of stock solution: character: be light yellow liquid.
Differentiate: need testing solution chromatogram and reference substance solution chromatogram basically identical.
Every milligram of total nitrogen content, aminoacid kind and total amount conformance with standard (WS1-XG-25-2000).
(6) will freeze stock solution and thaw, the filtering precipitation, dilution adds seasoning liquid total amount 0.07% sodium sulfite, adds seasoning liquid total amount 5% dextran simultaneously, is seasoning liquid;
(7) seasoning liquid is collected ultrafiltrate through holding back the ultrafiltration of 6000-10000 molecular weight ultrafilter membrane, and 0.2 μ m film aseptic filtration gets ultrafiltrate;
(8) with fill under the ultrafiltrate aseptic condition, the false add plug, put in the freeze-drier, when products temperature reaches-30 ℃--in the time of 50 ℃, continue cooling 2-4 hour, and the temperature of condenser reaches-40 ℃--in the time of 60 ℃, evacuation, when the vacuum of vacuum system was lower than 30Pa, the primary drying temperature was set at-30 ℃--and 10 ℃; The redrying temperature is set at 15 ℃-50 ℃, continues dryly after reaching temperature, until up to specification, promptly gets 3200 in the preparation of every off-white color that contains total nitrogen 60mg or micro-yellow powder and block.
Embodiment 5:
The fresh pig cerebral tissue 20kg that (1) will quarantine qualified adds 2 times of purified water homogenate,
(2) homogenate is cooled to 38 ℃ in 85 ℃ of insulations 18 minutes, adds hydrochloric acid and transfers pH3.2, stirs to add the 500g pepsin, stirs, and supernatant is collected in 40 ± 2 ℃ of insulations 5 hours, precipitate centrifugal, the merging clear liquid;
(3) the supernatant concentrating under reduced pressure in 120 ℃ of deactivations 3 hours, gets deactivation clear liquid 5kg to 1/4 of Medulla sus domestica weight behind the adjust pH, adds water to Medulla sus domestica weight, with Ca (OH)
2Liquid adjust pH 7.0, the cloth bag filtration, charcoal is taken off in activated carbon adsorption, gets clear liquid.
(4) add pancreatin 500g in the deactivation clear liquid, 40 ± 2 ℃ are incubated 3.5 hours, boil 28 minutes, and pH3.0 is transferred in cooling, leave standstill, and filtering-depositing gets hydrolyzed solution;
(5) with treated good cation exchange column on the said hydrolyzed liquid, wash post with purified water, reuse 8% ammonia is resolved, and collects eluent, concentrates, the sodium hydroxide solution adjust pH is pH10, concentrating under reduced pressure is removed free ammonia, and concentrated solution is removed precipitation in 0-3 ℃ of standing over night, get stock solution 10kg, freeze below-20 ℃ to preserve;
The calibrating of stock solution: character: be light yellow liquid.
Differentiate: need testing solution chromatogram and reference substance solution chromatogram basically identical.
Every milligram of total nitrogen content, aminoacid kind and total amount conformance with standard (WS1-XG-25-2000).
(6) will freeze stock solution and thaw, the filtering precipitation, dilution adds seasoning liquid total amount 0.06% sodium sulfite, adds seasoning liquid total amount 6% mannitol simultaneously, is seasoning liquid;
(7) ultrafiltrate is collected in the ultrafilter membrane ultrafiltration of seasoning liquid through holding back the 6000-10000 molecular weight, and 0.2 μ m film aseptic filtration gets ultrafiltrate;
(8) with fill under the ultrafiltrate aseptic condition, the false add plug, put in the freeze-drier, when products temperature reaches-30 ℃--in the time of 50 ℃, continue cooling 2-4 hour, and the temperature of condenser reaches-40 ℃--in the time of 60 ℃, evacuation, when the vacuum of vacuum system was lower than 30Pa, the primary drying temperature was set at-30 ℃--and 10 ℃; The redrying temperature is set at 15 ℃-50 ℃, continues dryly after reaching temperature, until up to specification, promptly gets 1600 in the preparation of every off-white color that contains total nitrogen 60mg or micro-yellow powder and block.
Embodiment 6:
The fresh pig cerebral tissue 20kg that (1) will quarantine qualified adds 3 times of purified water homogenate,
(2) homogenate is cooled to 35 ℃ in 90 ℃ of insulations 15 minutes, adds hydrochloric acid and transfers pH2.8, stirs to add the 300g pepsin, stirs, and supernatant is collected in 38 ± 2 ℃ of insulations 6 hours, precipitate centrifugal, the merging clear liquid;
(3) the supernatant concentrating under reduced pressure in 115 ℃ of deactivations 5 hours, gets deactivation clear liquid 7kg to 1/3 of Medulla sus domestica weight behind the adjust pH, adds water to Medulla sus domestica weight, with Ca (OH)
2Liquid adjust pH 7.0, the cloth bag filtration, charcoal is taken off in activated carbon adsorption, gets clear liquid.
(4) add pancreatin 300g in the deactivation clear liquid, 42 ± 2 ℃ are incubated 4 hours, boil 30 minutes, and pH3.5 is transferred in cooling, leave standstill, and filtering-depositing gets hydrolyzed solution;
(5) with treated good cation exchange column on the said hydrolyzed liquid, wash post with purified water, reuse 8.5% ammonia is resolved, and collects eluent, concentrates, the sodium hydroxide solution adjust pH is pH10, concentrating under reduced pressure is removed free ammonia, and concentrated solution is removed precipitation in 0-4 ℃ of standing over night, get stock solution 10kg, freeze below-20 ℃ to preserve; The calibrating of stock solution: character: be light yellow liquid.
Differentiate: need testing solution chromatogram and reference substance solution chromatogram basically identical.
Every milligram of total nitrogen content, aminoacid kind and total amount conformance with standard (WS1-XG-25-2000).
(6) will freeze stock solution and thaw, the filtering precipitation, dilution adds seasoning liquid total amount 0.08% sodium sulfite, adds seasoning liquid total amount 8% dextran simultaneously, is seasoning liquid;
(7) ultrafiltrate is collected in the ultrafilter membrane ultrafiltration of seasoning liquid through holding back the 6000-10000 molecular weight, and 0.2 μ m film aseptic filtration gets ultrafiltrate;
(8) with fill under the ultrafiltrate aseptic condition, the false add plug, put in the freeze-drier, when products temperature reaches-30 ℃--in the time of 50 ℃, continue cooling 2-4 hour, and the temperature of condenser reaches-40 ℃--in the time of 60 ℃, evacuation, when the vacuum of vacuum system was lower than 30Pa, the primary drying temperature was set at-30 ℃--and 10 ℃; The redrying temperature is set at 15 ℃-50 ℃, continues dryly after reaching temperature, until up to specification, promptly gets 3300 in the preparation of every off-white color that contains total nitrogen 30mg or micro-yellow powder and block.
Embodiment 7:
The fresh pig cerebral tissue 15kg that (1) will quarantine qualified adds 2 times of purified water homogenate,
(2) homogenate is cooled to 42 ℃ in 80 ± 5 ℃ of insulations 30 minutes, adds hydrochloric acid and transfers pH2.5, stirs to add the 270g pepsin, stirs, and supernatant is collected in 45 ± 3 ℃ of insulations 3 hours, precipitate centrifugal, the merging clear liquid;
(3) the supernatant concentrating under reduced pressure in 105 ℃ of deactivations 6 hours, gets deactivation clear liquid 10kg to 1/2 of Medulla sus domestica weight behind the adjust pH, adds water to Medulla sus domestica weight, with Ca (OH)
2Liquid adjust pH 7.0, the cloth bag filtration, charcoal is taken off in activated carbon adsorption, gets clear liquid.
(4) add pancreatin 150g in the deactivation clear liquid, 43 ± 2 ℃ are incubated 3 hours, boil 25 minutes, and pH3.2 is transferred in cooling, leave standstill, and filtering-depositing gets hydrolyzed solution;
(5) with treated good cation exchange column on the said hydrolyzed liquid, wash post with purified water, reuse 8.5-9.5% ammonia is resolved, and collects eluent, concentrates, the sodium hydroxide solution adjust pH is pH10, concentrating under reduced pressure is removed free ammonia, and concentrated solution is removed precipitation in 0-4 ℃ of standing over night, get stock solution 15kg, freeze below-20 ℃ to preserve; The calibrating of stock solution: character: be light yellow liquid.
Differentiate: need testing solution chromatogram and reference substance solution chromatogram basically identical.
Every milligram of total nitrogen content, aminoacid kind and total amount conformance with standard (WS1-XG-25-2000).
(6) will freeze stock solution and thaw, the filtering precipitation, dilution adds seasoning liquid total amount 0.03% sodium sulfite, is seasoning liquid;
(7) ultrafiltrate is collected in the ultrafilter membrane ultrafiltration of seasoning liquid through holding back the 6000-10000 molecular weight, and 0.2 μ m film aseptic filtration gets ultrafiltrate;
(8) with fill under the ultrafiltrate aseptic condition, the false add plug, put in the freeze-drier, when products temperature reaches-30 ℃--in the time of 50 ℃, continue cooling 2-4 hour, and the temperature of condenser reaches-40 ℃--in the time of 60 ℃, evacuation, when the vacuum of vacuum system was lower than 30Pa, the primary drying temperature was set at-30 ℃--and 10 ℃; The redrying temperature is not decided to be 15 ℃-50 ℃, continues dryly after reaching temperature, until up to specification, promptly gets 2600 in the preparation of every off-white color that contains total nitrogen 30mg or micro-yellow powder and block.
Embodiment 8:
The fresh pig cerebral tissue 30kg that (1) will quarantine qualified adds 3 times of purified water homogenate,
(2) homogenate is cooled to 40 ℃ in 80 ± 2 ℃ of insulations 30 minutes, adds hydrochloric acid and transfers pH3.0, stirs to add the 600g pepsin, stirs, and supernatant is collected in 40 ± 2 ℃ of insulations 6 hours, precipitate centrifugal, the merging clear liquid;
(3) the supernatant concentrating under reduced pressure in 110 ℃ of deactivations 6 hours, gets deactivation clear liquid 7.5kg to 1/4 of Medulla sus domestica weight behind the adjust pH, adds water to Medulla sus domestica weight, with Ca (OH)
2Liquid adjust pH 7.0, the cloth bag filtration, charcoal is taken off in activated carbon adsorption, gets clear liquid.
(4) add pancreatin 600g in the deactivation clear liquid, 40 ± 2 ℃ are incubated 4 hours, boil 30 minutes, and pH3.0 is transferred in cooling, leave standstill, and filtering-depositing gets hydrolyzed solution;
(5) with treated good cation exchange column on the said hydrolyzed liquid, wash post with purified water, reuse 8% ammonia is resolved, and collects eluent, concentrates, the sodium hydroxide solution adjust pH is pH10, concentrating under reduced pressure is removed free ammonia, and concentrated solution is removed precipitation in 0-4 ℃ of standing over night, get stock solution 20kg, freeze below-20 ℃ to preserve;
The calibrating of stock solution
Character: be light yellow liquid.
Differentiate: need testing solution chromatogram and reference substance solution chromatogram basically identical.
Every milligram of total nitrogen content, aminoacid kind and total amount conformance with standard (WS1-XG-25-2000).
(6) will freeze stock solution and thaw, the filtering precipitation, dilution adds seasoning liquid total amount 0.09% sodium sulfite, adds seasoning liquid total amount 8% Glycine sodium simultaneously, is seasoning liquid;
(7) ultrafiltrate is collected in the ultrafilter membrane ultrafiltration of seasoning liquid through holding back the 6000-10000 molecular weight, and 0.2 μ m film aseptic filtration gets ultrafiltrate;
(8) with fill under the ultrafiltrate aseptic condition, the false add plug, put in the freeze-drier, when products temperature reaches-30 ℃--in the time of 50 ℃, continue cooling 2-4 hour, and the temperature of condenser reaches-40 ℃--in the time of 60 ℃, evacuation, when the vacuum of vacuum system was lower than 30Pa, the primary drying temperature was set at-30 ℃--and 10 ℃; The redrying temperature is set at 15 ℃-50 ℃, continues dryly after reaching temperature, until up to specification, promptly gets 2500 in the preparation of every off-white color that contains total nitrogen 60mg or micro-yellow powder and block.
Claims (5)
1. the preparation method of a brain protein hydrolysate for injection is characterized in that may further comprise the steps:
(1) qualified freeze or the fresh pig cerebral tissue adds 1-3 times of purified water homogenate of will quarantining,
(2) homogenate is cooled to 30-50 ℃ in 65-90 ℃ of insulation 15-45 minute, adds hydrochloric acid and transfers pH2.0-4.0, stirs the pepsin that adds Medulla sus domestica weight 1.5%-4.5%, stirs, and supernatant is collected in 30-50 ℃ of insulation 2-6 hour, precipitate centrifugal, the merging clear liquid;
(3) the supernatant concentrating under reduced pressure in 100-120 ℃ of deactivation 3-6 hour, adds water to Medulla sus domestica weight to the 1/2-1/5 of Medulla sus domestica weight behind the adjust pH, with calcic lye pH adjustment value 5.0-8.0, and the cloth bag filtration, activated carbon adsorption, decarburization gets the deactivation clear liquid;
(4) adding is by the pancreatin of Medulla sus domestica weight 1%-3% in the deactivation clear liquid, and 35-45 ℃ is incubated 1-4 hour, boils 15-30 minute, and cooling transfers pH value 3-4 to make pancreatin stop hydrolysis, leaves standstill, and filtering-depositing gets hydrolyzed solution;
(5) with treated good cation exchange column on the said hydrolyzed liquid, wash post with purified water, the reuse weak ammonia is resolved, and collects eluent, concentrate, the sodium hydroxide solution adjust pH, concentrating under reduced pressure is removed free ammonia, concentrated solution standing over night, remove precipitation, get stock solution, freeze below-20 ℃ to preserve;
(6) will freeze stock solution and thaw, the filtering precipitation, dilution adds the 0.03%-0.1% sodium sulfite, and/or adds excipient 3%-10%, is seasoning liquid;
(7) seasoning liquid is collected filtrate with the ultrafiltration of 6000-10000 molecular weight ultrafilter membrane, and 0.2 μ m film aseptic filtration gets ultrafiltrate;
(8) ultrafiltrate is filled in the ampoule under the injection preparation method aseptic condition routinely, seals, sterilization detects, and packing promptly gets light yellow clear and bright injection formulation;
(9) with fill under the ultrafiltrate aseptic condition, the false add plug, put in the freeze-drier, when products temperature reaches-30 ℃--in the time of 50 ℃, continue cooling 2-4 hour, and the temperature of condenser reaches-40 ℃--in the time of 60 ℃, evacuation, when the vacuum of vacuum system was lower than 30Pa, the primary drying temperature was set at-30 ℃--and 10 ℃; The redrying temperature is set at 15 ℃-50 ℃, continues drying after reaching temperature, until up to specification, promptly gets the lyophilized formulations of off-white color or micro-yellow powder and block mixture.
2. the preparation method of a kind of brain protein hydrolysate for injection according to claim 1 is characterized in that comprising injection and lyophilized formulations with the ejection preparation of this hydrolysate preparation; Its feature also is every milliliter of nitrogenous 5.4-6.8mg of injection, contains free amino acid 25-50mg; Every nitrogenous 30mg of lyophilized formulations contains free amino acid 150-201mg and every nitrogenous 60mg, contains free amino acid 300-402mg.
3. the preparation method of a kind of brain protein hydrolysate for injection according to claim 1 is characterized in that excipient is a kind of in mannitol, glucose, lactose, dextran, sucrose, gelatin hydrolysate, glycine, the Glycine sodium.
4. the brain protein hydrolysate for injection that preparation method as claimed in claim 1 obtains, it is characterized in that its effective ingredient is that the molecular weight that the brain hydrolysis produces is 16 kinds of free amino acids less than 10000 daltonian small-molecular peptides and generation, contain free amino acid 4.68-7.02mg in every milligram of total nitrogen of gained hydrolysate, and in every milligram of total nitrogen contained free amino acid proportion of composing and scope unit milligram meter is as follows by weight:
Aspartic Acid 0.64-0.96 glutamic acid 0.83-1.24 serine 0.25-0.46
Histidine 0.13-0.2 glycine 0.32-0.48 threonine 0.22-0.40
Alanine 0.33-0.5 arginine 0.093-0.34 valine 0.21-0.32
Methionine 0.12-0.22 tryptophan 0.03-0.07 isoleucine 0.15-0.22
Phenylalanine 0.16-0.24 leucine 0.36-0.54 lysine 0.47-0.7
Proline 0.27-0.4.
5. brain protein hydrolysate for injection according to claim 4, its feature do not need to add in addition aminoacid when also being with the ejection preparation of this hydrolysate preparation.
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| CN102166200A (en) * | 2011-04-12 | 2011-08-31 | 罗诚 | Freeze-drying composition containing cerebroprotein hydrolysates and preparation method of freeze-drying composition |
| CN103191403A (en) * | 2013-04-02 | 2013-07-10 | 黑龙江迪龙制药有限公司 | Method for preparing brain protein hydrolyzate |
| CN103919807A (en) * | 2014-05-06 | 2014-07-16 | 云南盟生药业有限公司 | Preparation method of cerebrolysin vial with high and stable nitrogen content |
| CN104043099A (en) * | 2014-06-10 | 2014-09-17 | 广州一品红制药有限公司 | Cerebrolysin hydrolysate injection and preparation method thereof |
| CN104096214A (en) * | 2014-06-10 | 2014-10-15 | 广州一品红制药有限公司 | Cerebroprotein hydrolysate freeze-dried powder injection and preparation method thereof |
| CN104096215A (en) * | 2014-07-07 | 2014-10-15 | 湖南利诺生物药业有限公司 | Pure natural cerebroprotein hydrolysate raw material preparation method |
| CN104127439A (en) * | 2014-07-16 | 2014-11-05 | 湖南利诺生物药业有限公司 | Natural composite amino acid raw material preparation method and natural composite amino acid raw material |
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| CN104587444A (en) * | 2014-12-24 | 2015-05-06 | 内蒙古天奇生物科技有限公司 | Preparation method of high-protein-content cerebroprotein hydrolysate |
| CN108478777A (en) * | 2018-05-22 | 2018-09-04 | 广东隆赋药业股份有限公司 | A kind of brain protein hydrolysate for injection（Ⅱ）The preparation method of preparation |
| CN113908174A (en) * | 2021-10-29 | 2022-01-11 | 北京四环制药有限公司 | Efficient and safe preparation method and application of cerebroprotein hydrolysate |
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| CN102166200A (en) * | 2011-04-12 | 2011-08-31 | 罗诚 | Freeze-drying composition containing cerebroprotein hydrolysates and preparation method of freeze-drying composition |
| CN103191403A (en) * | 2013-04-02 | 2013-07-10 | 黑龙江迪龙制药有限公司 | Method for preparing brain protein hydrolyzate |
| CN103919807A (en) * | 2014-05-06 | 2014-07-16 | 云南盟生药业有限公司 | Preparation method of cerebrolysin vial with high and stable nitrogen content |
| CN104096214B (en) * | 2014-06-10 | 2015-12-30 | 广州一品红制药有限公司 | A kind of Cerebrolysin Vial lyophilized injectable powder and preparation method thereof |
| CN104096214A (en) * | 2014-06-10 | 2014-10-15 | 广州一品红制药有限公司 | Cerebroprotein hydrolysate freeze-dried powder injection and preparation method thereof |
| CN104043099A (en) * | 2014-06-10 | 2014-09-17 | 广州一品红制药有限公司 | Cerebrolysin hydrolysate injection and preparation method thereof |
| CN104043099B (en) * | 2014-06-10 | 2016-04-06 | 广州一品红制药有限公司 | A kind of brain protein hydrolysate injection and preparation method thereof |
| CN104096215A (en) * | 2014-07-07 | 2014-10-15 | 湖南利诺生物药业有限公司 | Pure natural cerebroprotein hydrolysate raw material preparation method |
| CN104127439A (en) * | 2014-07-16 | 2014-11-05 | 湖南利诺生物药业有限公司 | Natural composite amino acid raw material preparation method and natural composite amino acid raw material |
| CN104189007A (en) * | 2014-09-09 | 2014-12-10 | 湖南利诺生物药业有限公司 | Method for preparing natural compound amino acid raw materials |
| CN104189007B (en) * | 2014-09-09 | 2017-09-29 | 湖南利诺生物药业有限公司 | The preparation method of natural compound amino-acid raw material |
| CN104587444A (en) * | 2014-12-24 | 2015-05-06 | 内蒙古天奇生物科技有限公司 | Preparation method of high-protein-content cerebroprotein hydrolysate |
| CN104587444B (en) * | 2014-12-24 | 2018-04-03 | 内蒙古天奇生物科技有限公司 | A kind of preparation method of high protein content Cerebrolysin Vial |
| CN108478777A (en) * | 2018-05-22 | 2018-09-04 | 广东隆赋药业股份有限公司 | A kind of brain protein hydrolysate for injection（Ⅱ）The preparation method of preparation |
| CN113908174A (en) * | 2021-10-29 | 2022-01-11 | 北京四环制药有限公司 | Efficient and safe preparation method and application of cerebroprotein hydrolysate |
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