CN104043099A - Cerebrolysin hydrolysate injection and preparation method thereof - Google Patents
Cerebrolysin hydrolysate injection and preparation method thereof Download PDFInfo
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- CN104043099A CN104043099A CN201410255461.3A CN201410255461A CN104043099A CN 104043099 A CN104043099 A CN 104043099A CN 201410255461 A CN201410255461 A CN 201410255461A CN 104043099 A CN104043099 A CN 104043099A
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Abstract
The invention provides a cerebrolysin hydrolysate injection and a preparation method thereof. The preparation method comprises the following steps: taking pig brain, removing impurities, and adding purified water for homogenizing; hydrolyzing homogenate through pepsin and pancreatin, and collecting a clear liquid; regulating the pH of the clear liquid to be 1.7-3, heating to 100-110 DEG C, insulating for 15-45min, then regulating pH to 8.0-9.0; conducting ultrafiltration through a 8-10kd membrane, and collecting filtrate; conducting column chromatography to the filtrate, collecting an eluent, regulating ph to 8.0-9.0, concentrating through an anion-exchange membrane, filtering through a 0.2mu m membrane to obtain a stock liquid; and freezing the stock liquid, then defreezing, adding an antioxidant, regulating the pH to be 6.9-7.5, and filtering respectively through a 8-10kd membrane and a 0.2mu m membrane. The polypeptide in the obtained cerebrolysin hydrolysate injection is similar to that of a control drug being more than or equal to 0.90, and the obtained cerebrolysin hydrolysate injection contains 16 amino acids, the nitrogen amount of total amino acids accounts for more than or equal to 85% of total nitrogen content, additional amino acids are not required, and the amino acids can be prepared completely by means of pig brain, thus being stable and safe.
Description
Technical field
The present invention relates to biological preparation field, relate in particular to a kind of brain protein hydrolysate injection and preparation method thereof.
Background technology
Brain protein hydrolysate injection product is qualified through checking, quarantining by raw material, extracts, and refining, the production technologies such as sterilizing prepare; The main component of Cerebrolysin Vial is low molecular peptide and free amino acid.Easily enter cranial nerve cell by blood brain barrier, low molecular peptide has the effect identical with naturally occurring neurotrophic factor, can induce neuron differentiation, maintain normal neurocyte function, increase generation and the synapse transmission of neurotransmitter, neuroprotective cell is avoided various infringements, regulates the metabolism of human body brain, neurotransmission and nerve growth; Can improve breakdown of glucose enzymatic activity, increase the anaerobic energy metabolism of brain to glucose, reduce the concentration of lactic acid in brain, the mitochondrion to neurocyte in the time of hypoxic-ischemic has protective effect.
Brain protein hydrolysate injection is registered by Austrian EBEWE pharmaceutical factory import last century, to the end of the year 2010, and 29 of the existing disclosed patent applications that relates to pharmaceutics of hydrolysate of brain protein and technique.
Such as Chinese invention patent document CN01130523.1 discloses a kind of process for preparing injection liquid of brain protein hydrolyzate: fresh Medulla sus domestica is removed surperficial mucosa and bloodstain etc., homogenate, double gauze filters, pepsin hydrolysis, add pancreatin hydrolysis, add at 2.5～3.0 ,-20～10 DEG C of hydrochloric acid adjust pHs and leave standstill 10～30 hours; Room temperature, double gauze filters, and adds water, and adjust pH is to neutral, filtering with microporous membrane clarification, ultrafilter membrane ultrafiltration, ultrafilter membrane concentrates, concentrated solution ebuillition of heated 15 minutes, then cooling rapidly, then microporous filter membrane and ultrafilter membrane, the ultrafiltrate obtaining, then through 121 DEG C of steam sterilizations; According to required amount of liquid medicine, calculate the aminoacid consumption that need to add by the drug standard of National Drug Administration's December in 2000 promulgation on the 28th (numbering WS1-XG-25-2000), become concentrated wiring liquid with above-mentioned solution preparation, then rare equipped pin finished product medicinal liquid, sterilizing becomes sterile preparation.And for example CN1857711A discloses the production method of a kind of Cerebrolysin Vial and lyophilized formulations thereof: by Medulla sus domestica homogenate, pepsin, pancreatin hydrolysis, upper hydroxyapatite chromatography post carries out absorption and purification, allotment peptide figure, collects object, adopts the filter membrane that is 8KD to molecular retention to carry out ultrafiltration, carry out determining nitrogen, amino acid analysis, add corresponding amino acid ligand and make concentrated wiring liquid, membrane filtration, to obtain final product.Existing preparation technology needs the corresponding aminoacid of extra interpolation just can make standard compliant Cerebrolysin Vial in the time producing preparation, and closing documentation requirements for 2008 No. 734 according to State Food and Drug Administration, Cerebrolysin Vial must not add exogenous aminoacid.Thereby we need to not add the research of the preparation method of exogenous amino acid whose Cerebrolysin Vial.
Summary of the invention
The object of the invention is to overcome and in existing Cerebrolysin Vial technology of preparing, need additionally to add corresponding amino acid whose defect, a kind of brain protein hydrolysate injection and preparation method thereof is provided, the brain protein hydrolysate injection small molecular peptide anti-phase peptide figure chromatographic identification and reference substance similarity >=0.90 that adopt this preparation method to prepare, add exogenous aminoacid without supplementing, rely on Medulla sus domestica hydrolysis to make completely.
First aspect of the present invention is to provide a kind of brain protein hydrolysate injection, similarity >=90% of the micromolecule polypeptide in described brain protein hydrolysate injection small molecular polypeptide and middle inspection institute Cerebrolysin Vial 140697～200602; Described brain protein hydrolysate injection contains 16 kinds of free amino acids: Aspartic Acid, glutamic acid, serine, histidine, glycine, threonine, alanine, arginine, valine, methionine, tryptophan, isoleucine, phenylalanine, leucine, lysine, proline; In described brain protein hydrolysate injection, total amino acid whose nitrogen content accounts for the more than 85% of total nitrogen of described brain protein hydrolysate injection.
Preferably, described brain protein hydrolysate injection adopts following step to be prepared from:
1) get the Medulla sus domestica that is up to the standards, roguing (removing the impurity such as blood vessel, fat), adds purified water homogenate;
2) by step 1) homogenate that obtains first after through pepsin, pancreatin hydrolysis, collect clear liquid;
3) by step 2) clear liquid that obtains regulates pH to 1.7～3, is heated to 100～110 DEG C, and insulation 15～45min (being preferably 20～40min, more preferably 25～35min), then regulates pH to 8.0～9.0;
4) cold treatment, gets clear liquid, and 8～10kd membrane ultrafiltration is collected filtrate;
5) by step 4) the upper column chromatography of the filtrate that obtains, rinse post by purified water, resolve, collect eluent, regulate pH to 8.0～9.0, anion exchange membrance concentration, 0.2 μ m membrane filtration obtains stock solution;
6) stock solution step 5 being obtained is freezed, and then thaws, and adds antioxidant, and the content of controlling antioxidant is 0.01～0.05wt%, regulates pH to 6.9～7.5,8～10kd membrane ultrafiltration, and 0.2 μ m membrane filtration, both.
Wherein, step 4) described in cold treatment, refer to below liquid cools to 10 DEG C.
Preferably, in step 1) afterwards, carry out step 2) before, homogenate heating is made to protein denaturation.
Further preferably, make protein denaturation specifically can adopt following step: to be incubated 15～30min by homogenate and 75～85 DEG C.
Preferably, step 6) described in antioxidant be sodium sulfite, sodium pyrosulfite, dibutyl phenol, sodium sulfite, sodium thiosulfate or butylated hydroxyarisol.
Preferably, step 5) in the chromatographic stuffing that uses of upper column chromatography be Amberlite IRA-200.
Preferably, step 5) described in anion exchange membrane adopt imported from America film DUS-8040C ionic membrane (aperture 0.001 μ m).
Preferably, step 2) be specially: regulate pH to 1.7～3.0, add pepsin hydrolysis, pepsin hydrolysis carries out at 35～45 DEG C, insulation 3～5h, then regulate pH to 7.2～7.8, add pancreatin hydrolysis, pancreatin hydrolysis carries out at 35～45 DEG C, insulation 2～4h, inactivator, stops hydrolysis, collects clear liquid.
Wherein, step 2) in collect clear liquid can be by filtering, the manner known in the art such as centrifugal realizes.
Second aspect of the present invention is to provide a kind of preparation method of brain protein hydrolysate injection, comprises the following steps:
Step 1, gets the Medulla sus domestica that is up to the standards, and roguing (removing the impurity such as blood vessel, fat), adds purified water homogenate;
Step 2, through pepsin, pancreatin hydrolysis, collects clear liquid after the homogenate that step 1 is obtained is first;
Step 3, the clear liquid that step 2 is obtained regulates pH to 1.7～3, is heated to 100～110 DEG C, and insulation 15～45min (being preferably 20～40min, more preferably 25～35min), then regulates pH to 8.0～9.0;
Step 4, cold treatment, gets clear liquid, and 8～10kd membrane ultrafiltration is collected filtrate;
Step 5, the upper column chromatography of the filtrate that step 4 is obtained, rinses post by purified water, resolves, and collects eluent, regulates pH to 8.0～9.0, anion exchange membrance concentration, 0.2 μ m membrane filtration obtains stock solution;
Step 6, the stock solution that step 5 is obtained is freezed, and then thaws, and adds antioxidant, and the content of controlling antioxidant is 0.01～0.05wt%, regulates pH to 6.9～7.5,8～10kd membrane ultrafiltration, 0.2 μ m membrane filtration.
Wherein, cold treatment described in step 4, refers to below liquid cools to 10 DEG C.
Preferably, after step 1, before carry out step 2, homogenate heating is made to protein denaturation.
Further preferably, make protein denaturation specifically can adopt following step: homogenate is incubated to 15～30min at 75～85 DEG C.
Preferably, described preparation method also comprises step 7: fill, 100～120 DEG C of sterilizings, leak detection.
Preferably, the antioxidant described in step 6 is sodium sulfite, sodium pyrosulfite, dibutyl phenol, sodium sulfite, sodium thiosulfate or butylated hydroxyarisol.
Preferably, the chromatographic stuffing that in step 5, upper column chromatography uses is Amberlite IRA-200.
Preferably, anion exchange membrane described in step 5 adopts imported from America film DUS-8040C ionic membrane.
Preferably, step 2 is specially: regulate pH to 1.7～3.0, add pepsin hydrolysis, pepsin hydrolysis carries out at 35～45 DEG C, insulation 3～5h, then regulate pH to 7.2～7.8, add pancreatin hydrolysis, pancreatin hydrolysis carries out at 35～45 DEG C, insulation 2～4h, inactivator, stops hydrolysis, collects clear liquid.
Wherein, in step 2, collecting the manner known in the art such as clear liquid can pass through to filter, centrifugal realizes.Compared with prior art, preparation technology's tool of the present invention has the following advantages:
1, the present invention becomes at pepsin, pancreatin hydrolysis after the clear liquid of polypeptide and free amino acid, adjust pH to 1.7～3.0,100～110 DEG C are heated 30 minutes, can again be combined into peptide bond by precaution of hydrolysis liquid Free Amino Acids, form small peptide, simultaneously can deactivation zoonotic virus, more reasonable than existing patent, prevent that polypeptide and free amino acid Reversible binding and deactivation zoonotic virus from carrying out after double-enzyme hydrolysis, belongs to end control, add without other biomaterial, safer;
2, adopt Amberlite IRA-200 exchange filler can greatly improve existing anion exchange and can not adsorb whole amino acid whose problems;
3, adopt anion exchange membrane, in the solution of ph8.0～9.0, aminoacid is electronegative, mutually repels with film surface charge, and free amino acid and polypeptide are trapped; The preferred imported from America film of cation exchange membrane DUS-8040C ionic membrane, aperture is 0.001um, aperture 50d molecular weight, free amino acid can further be trapped;
4, in technique of the present invention, adopted 2 ultrafiltration, filtered, reduce to greatest extent macromole sensitizer, ensured particulate matter and aseptic guarantee value SAL for 2 times;
5, freeze after adopting ultrafiltration in technique of the present invention, ultrafiltration is removed and is freezed to reduce aminoacid after macromole and separate out, and thaws and can further remove a small amount of impurity after freezing;
6, similarity >=0.90 of brain protein hydrolysate injection small molecular peptide anti-phase peptide figure chromatographic identification provided by the invention and Cerebrolysin Vial 140697～200602; Contain 16 seed amino acids, the nitrogen amount of total amino acids accounts for total nitrogen content ratio >=85%, contains 16 seed amino acids such as Aspartic Acid, glutamic acid, serine, histidine, glycine, threonine, alanine, arginine, valine, methionine, tryptophan, isoleucine, phenylalanine, leucine, lysine, proline.
Technique of the present invention is simple, the brain protein hydrolysate injection small molecular polypeptide making and 16 kinds of free aminoacid contents are constant, in gained brain protein hydrolysate injection polypeptide similar to reference substance >=0.90, contain 16 seed amino acids, the nitrogen amount of total amino acids accounts for total nitrogen content ratio >=85%, adds aminoacid without extra supplementing, and relies on Medulla sus domestica hydrolysis to make completely, efficacy stability, safer; Injection is for finally can sterilizing injection, and SAL guarantee value is higher, ampoule encapsulation, and more sealing, improves drug safety.
Brief description of the drawings
The brain protein hydrolysate injection that Fig. 1 provides for the embodiment of the present invention 1 and the finger printing of middle inspection institute Cerebrolysin Vial 140697～200602;
Fig. 2 is 16 seed amino acid high performance liquid chromatogram collection of illustrative plates of middle inspection institute Cerebrolysin Vial 140697～200602;
The free amino acid high performance liquid chromatogram collection of illustrative plates of the brain protein hydrolysate injection that Fig. 3 provides for the embodiment of the present invention 1;
The total amino acids high performance liquid chromatogram collection of illustrative plates of the brain protein hydrolysate injection that Fig. 4 provides for the embodiment of the present invention 1;
The brain protein hydrolysate injection that Fig. 5 provides for the embodiment of the present invention 2 and the finger printing of middle inspection institute Cerebrolysin Vial 140697～200602;
The free amino acid high performance liquid chromatogram collection of illustrative plates of the brain protein hydrolysate injection that Fig. 6 provides for the embodiment of the present invention 2;
The total amino acids high performance liquid chromatogram collection of illustrative plates of the brain protein hydrolysate injection that Fig. 7 provides for the embodiment of the present invention 2.
Detailed description of the invention
With reference to the accompanying drawings, in conjunction with concrete embodiment, the present invention will be further described, to understand better the present invention.
Embodiment 1
Cerebral tissue (quarantining qualified) is got 100kg after removing the impurity such as blood vessel, fat, adds the homogenate of 1 times of volume purified water, in 82 DEG C of insulations 15 minutes, cooling; Hydrochloric acid is adjusted pH1.8, pepsin hydrolysis, and 42 DEG C are incubated 3 hours, and sodium hydroxide is adjusted pH7.6, pancreatin hydrolysis, 42 DEG C are incubated 2 hours, centrifugal collection clear liquid; Hydrochloric acid is adjusted pH2, is incubated 30 minutes at 105 DEG C, and then sodium hydroxide is adjusted pH8.8; Cold treatment, extracts supernatant, and 10kd membrane ultrafiltration is removed macromole, collects filtrate 100L; Upper column chromatography, rinses post by purified water, resolves, and collects eluent, regulates pH to 8.0～9.0, is concentrated into 50L with DUS-8040C ionic membrane, and 0.2 μ m membrane filtration, obtains stock solution ,-15 DEG C of Cryopreservations below; Stock solution is thawed, and allotment, adds sodium sulfite 0.01wt%, regulates pH6.9～7.5,8～10kd membrane ultrafiltration, 0.2 μ m membrane filtration, fill; 115 DEG C, 30 minutes, leak detection etc., prepared 5ml specification injection with small volume.
Similarity is measured:
Reference substance is middle inspection institute Cerebrolysin Vial 140697～200602.Taking octadecylsilane chemically bonded silica as filler (250mm × 4mm, particle diameter 5 μ are m); Taking 0.1% trifluoroacetic acid solution as mobile phase A; Taking 0.085% trifluoracetic acid acetonitrile solution--0.1% trifluoroacetic acid solution (80:20), as Mobile phase B, carries out gradient elution by table 1; Flow velocity is 0.8ml per minute; Detection wavelength is 276nm.40 DEG C of column temperatures; Press similarity evaluation and calculate, result is as shown in Fig. 1 and table 2.
Gradient elution table in table 1 similarity mensuration
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 40 | 50 | 50 |
| 45 | 0 | 100 |
| 53 | 0 | 100 |
| 55 | 100 | 0 |
| 65 | 100 | 0 |
Table 2 embodiment 1 fingerprint similarity evaluation result
| ? | S1 | S2 | Reference fingerprint |
| S1 | 1 | 0.951 | 0.971 |
| S2 | 0.951 | 1 | 0.997 |
| Reference fingerprint | 0.971 | 0.997 | 1 |
From Fig. 1 and table 2, the similarity of the micromolecule polypeptide in the 5ml specification injection with small volume that the present embodiment provides and middle inspection institute Cerebrolysin Vial 140697～200602 is 0.951, meets quality control requirement (>=0.90).
Determined amino acid:
Reference substance is middle inspection institute Cerebrolysin Vial 140697～200602.AccQ Tag method, 4umNora～Pak
tMc
18(3.9 × 150mm) post, mobile phase A is pH5.0,0.1mol/L, acetic acid-phosphate buffer; Mobile phase B is 60%HPLC level acetonitrile, carries out gradient elution by table 3; Use AccQFluor derivating agent column front derivation, it is 248nm that UV detects wavelength, and flow velocity is 1ml per minute, and column temperature is 37 DEG C.Testing result is as shown in Fig. 2,3 and 4.
Gradient elution table in table 3 determined amino acid
| Time (min) | Flow velocity (ml/min) | Mobile phase A (%) | Mobile phase B (%) | Curve |
| Initial | 1.0 | 100 | 0 | * |
| 0.5 | 1.0 | 98 | 2 | 6 |
| 15.0 | 1.0 | 93 | 7 | 6 |
| 19.0 | 1.0 | 90 | 10 | 6 |
| 32.0 | 1.0 | 67 | 33 | 6 |
| 33.0 | 1.0 | 67 | 33 | 6 |
| 34.0 | 1.0 | 0 | 100 | 6 |
| 37.0 | 1.0 | 0 | 100 | 6 |
| 38.0 | 1.0 | 100 | 0 | 6 |
| 42.0 | 1.0 | 100 | 0 | 6 |
From Fig. 2 and Fig. 3, in the 5ml specification injection with small volume that the present embodiment provides, amino acid whose retention time and middle inspection institute Cerebrolysin Vial 140697～200602 is consistent, contains 16 seed amino acids (Aspartic Acid, glutamic acid, serine, histidine, glycine, threonine, alanine, arginine, valine, methionine, tryptophan, isoleucine, phenylalanine, leucine, lysine, proline).
As shown in Figure 4, in the 5ml specification injection with small volume that the present embodiment provides, the nitrogen content of total amino acids accounts for 88.3% of total nitrogen.
Embodiment 2
Cerebral tissue (check, quarantine and be qualified) is got 100kg after removing the impurity such as blood vessel, add the homogenate of 2 times of volume purified water, in 85 DEG C of insulations 20 minutes, cooling, hydrochloric acid was adjusted pH2.0, pepsin hydrolysis, 42 DEG C are incubated 4 hours, and sodium hydroxide is adjusted pH7.8, pancreatin hydrolysis, 42 DEG C are incubated 2 hours, centrifugal collection clear liquid; Hydrochloric acid is adjusted pH2, and 110 DEG C are incubated 30 minutes, and sodium hydroxide is adjusted pH9.0; Cold treatment, extracts supernatant, and 10kd membrane ultrafiltration is removed macromole, collects filtrate 150L; Upper column chromatography, rinses post by purified water, resolves, and collects eluent, regulates pH to 8.0～9.0, and with the concentrated 50L of DUS-040C ionic membrane, 0.2 μ m membrane filtration, obtains stock solution ,-15 DEG C of following Cryopreservations; Stock solution is thawed, and allotment, adds sodium sulfite 0.02wt%, regulates pH to 6.9～7.5,8～10kd membrane ultrafiltration, 0.2 μ m membrane filtration, fill; 115 DEG C, 30 minutes, leak detection etc., prepared 10ml specification injection with small volume.
Similarity method for measuring is with reference to embodiment 1, and result is as shown in Fig. 5 and table 4.
Table 4 embodiment 2 fingerprint similarity evaluation results
| ? | S1 | S2 | Reference fingerprint |
| S1 | 1 | 0.956 | 0.997 |
| S2 | 0.956 | 1 | 0.976 |
| Reference fingerprint | 0.997 | 0.976 | 1 |
From Fig. 5 and table 4, the similarity of the micromolecule polypeptide in the 10ml specification injection with small volume that the present embodiment provides and middle inspection institute Cerebrolysin Vial 140697～200602 is 0.956, meets quality control requirement (>=0.90).
The method of determined amino acid is with reference to embodiment 1, and result is as shown in Fig. 2, Fig. 6 and Fig. 7.From Fig. 2 and Fig. 6, in the 10ml specification injection with small volume that the present embodiment provides, amino acid whose retention time and middle inspection institute Cerebrolysin Vial 140697～200602 is consistent, contains 16 seed amino acids (Aspartic Acid, glutamic acid, serine, histidine, glycine, threonine, alanine, arginine, valine, methionine, tryptophan, isoleucine, phenylalanine, leucine, lysine, proline).As shown in Figure 7, in the 10ml specification injection with small volume that the present embodiment provides, the nitrogen content of total amino acids accounts for 86.8% of total nitrogen.
Embodiment 3
Cerebral tissue (check, quarantine and be qualified) is got 100kg after removing the impurity such as blood vessel, add the homogenate of 2 times of volume purified water, in 85 DEG C of insulations 30 minutes, cooling, hydrochloric acid was adjusted pH3.0, pepsin hydrolysis, 35 DEG C are incubated 5 hours, and sodium hydroxide is adjusted pH7.2, pancreatin hydrolysis, 45 DEG C are incubated 3 hours, centrifugal collection clear liquid; Hydrochloric acid is adjusted pH3.0, and 110 DEG C are incubated 30 minutes, and sodium hydroxide is adjusted pH8.0; Cold treatment, extracts supernatant, and 10kd membrane ultrafiltration is removed macromole, collects filtrate 150L; Upper column chromatography, rinses post by purified water, resolves, and collects eluent, regulates pH to 8.0～9.0, and with the concentrated 50L of DUS-040C ionic membrane, 0.2 μ m membrane filtration, obtains stock solution ,-15 DEG C of following Cryopreservations; Stock solution is thawed, and allotment, adds sodium pyrosulfite 0.02wt%, regulates pH to 6.9～7.5,8～10kd membrane ultrafiltration, 0.2 μ m membrane filtration, fill; 115 DEG C, 30 minutes, leak detection etc., prepared 5ml specification injection with small volume.
Embodiment 4
Cerebral tissue (check, quarantine and be qualified) is got 100kg after removing the impurity such as blood vessel, add the homogenate of 2 times of volume purified water, in 75 DEG C of insulations 20 minutes, cooling, hydrochloric acid was adjusted pH1.7, pepsin hydrolysis, 40 DEG C are incubated 5 hours, and sodium hydroxide is adjusted pH7.2, pancreatin hydrolysis, 45 DEG C are incubated 3 hours, centrifugal collection clear liquid; Hydrochloric acid is adjusted pH2.8, and 110 DEG C are incubated 45 minutes, and sodium hydroxide is adjusted pH8.0; Cold treatment, extracts supernatant, and 10kd membrane ultrafiltration is removed macromole, collects filtrate 150L; Upper column chromatography, rinses post by purified water, resolves, and collects eluent, regulates pH to 8.0～9.0, and with the concentrated 50L of DUS-040C ionic membrane, 0.2 μ m membrane filtration, obtains stock solution ,-15 DEG C of following Cryopreservations; Stock solution is thawed, and allotment, adds sodium pyrosulfite 0.05wt%, regulates pH to 6.9～7.5,8～10kd membrane ultrafiltration, 0.2 μ m membrane filtration, fill; 115 DEG C, 30 minutes, leak detection etc., prepared 10ml specification injection with small volume.
Embodiment 5
Cerebral tissue (check, quarantine and be qualified) is got 150kg after removing the impurity such as blood vessel, add the homogenate of 1 times of volume purified water, in 80 DEG C of insulations 15 minutes, cooling, hydrochloric acid was adjusted pH1.7, pepsin hydrolysis, 40 DEG C are incubated 5 hours, and sodium hydroxide is adjusted pH7.2, pancreatin hydrolysis, 45 DEG C are incubated 3 hours, centrifugal collection clear liquid; Hydrochloric acid is adjusted pH1.7., and 110 DEG C are incubated 15 minutes, and sodium hydroxide is adjusted pH8.0; Cold treatment, extracts supernatant, and 10kd membrane ultrafiltration is removed macromole, collects filtrate 150L; Upper column chromatography, rinses post by purified water, resolves, and collects eluent, regulates pH to 8.0～9.0, and with the concentrated 50L of DUS-040C ionic membrane, 0.2 μ m membrane filtration, obtains stock solution ,-15 DEG C of following Cryopreservations; Stock solution is thawed, and allotment, adds sodium pyrosulfite 0.05wt%, regulates pH to 6.9～7.5,8～10kd membrane ultrafiltration, 0.2 μ m membrane filtration, fill; 115 DEG C, 30 minutes, leak detection etc., prepared 5ml specification injection with small volume.
The injection that embodiment 3-5 is provided carries out similarity mensuration, and assay method is with reference to embodiment 1.The similarity of the injection small molecular polypeptide that embodiment 3-5 provides and middle inspection institute Cerebrolysin Vial 140697～200602 is respectively 0.957,0.952,0.954, all meets quality control requirement (>=0.90).
The injection that embodiment 3-5 is provided carries out determined amino acid, and assay method is with reference to embodiment 1.The injection Free Amino Acids that embodiment 3-5 provides is consistent with reference substance retention time, contains 16 seed amino acids, and the ratio that in the injection that embodiment 3-5 provides, the nitrogen content of total amino acids accounts for total nitrogen is respectively 87.5%, 86.9%, 88.2%.
Above specific embodiments of the invention be have been described in detail, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and alternative also all among category of the present invention.Therefore, equalization conversion and the amendment done without departing from the spirit and scope of the invention, all should contain within the scope of the invention.
Claims (10)
1. a brain protein hydrolysate injection, is characterized in that, similarity >=90% of the micromolecule polypeptide in described brain protein hydrolysate injection small molecular polypeptide and middle inspection institute Cerebrolysin Vial 140697～200602; Described brain protein hydrolysate injection contains 16 kinds of free amino acids: Aspartic Acid, glutamic acid, serine, histidine, glycine, threonine, alanine, arginine, valine, methionine, tryptophan, isoleucine, phenylalanine, leucine, lysine, proline; In described brain protein hydrolysate injection, total amino acid whose nitrogen content accounts for the more than 85% of total nitrogen of described brain protein hydrolysate injection.
2. brain protein hydrolysate injection according to claim 1, is characterized in that, described brain protein hydrolysate injection adopts following step to be prepared from:
1) get the Medulla sus domestica that is up to the standards, roguing, adds purified water homogenate;
2) by step 1) homogenate that obtains first after through pepsin, pancreatin hydrolysis, collect clear liquid;
3) by step 2) clear liquid that obtains regulates pH to 1.7～3, is heated to 100～110 DEG C, and insulation 15～45min, then regulates pH to 8.0～9.0;
4) cold treatment, gets clear liquid, and 8～10kd membrane ultrafiltration is collected filtrate;
5) by step 4) the upper column chromatography of the filtrate that obtains, rinse post by purified water, resolve, collect eluent, regulate pH to 8.0～9.0, anion exchange membrance concentration, 0.2 μ m membrane filtration obtains stock solution;
6) stock solution step 5 being obtained is freezed, and then thaws, and adds antioxidant, and the content of controlling antioxidant is 0.01～0.05wt%, regulates pH to 6.9～7.5,8～10kd membrane ultrafiltration, and 0.2 μ m membrane filtration, both.
3. brain protein hydrolysate injection according to claim 2, is characterized in that, in step 1) afterwards, carry out step 2) before, homogenate heating is made to protein denaturation.
4. brain protein hydrolysate injection according to claim 2, is characterized in that step 6) described in antioxidant be sodium sulfite, sodium pyrosulfite, dibutyl phenol, sodium sulfite, sodium thiosulfate or butylated hydroxyarisol.
5. brain protein hydrolysate injection according to claim 2, is characterized in that step 5) in the chromatographic stuffing that uses of upper column chromatography be Amberlite IRA-200, described anion exchange membrane adopts imported from America film DUS-8040C ionic membrane.
6. a preparation method for brain protein hydrolysate injection, is characterized in that, comprises the following steps:
Step 1, gets the Medulla sus domestica that is up to the standards, and roguing adds purified water homogenate;
Step 2, through pepsin, pancreatin hydrolysis, collects clear liquid after the homogenate that step 1 is obtained is first;
Step 3, the clear liquid that step 2 is obtained regulates pH to 1.7～3, is heated to 100～110 DEG C, and insulation 15～45min, then regulates pH to 8.0～9.0;
Step 4, cold treatment, gets clear liquid, and 8～10kd membrane ultrafiltration is collected filtrate;
Step 5, the upper column chromatography of the filtrate that step 4 is obtained, rinses post by purified water, resolves, and collects eluent, regulates pH to 8.0～9.0, anion exchange membrance concentration, 0.2 μ m membrane filtration obtains stock solution;
Step 6, the stock solution that step 5 is obtained is freezed, and then thaws, and adds antioxidant, and the content of controlling antioxidant is 0.01～0.05wt%, regulates pH to 6.9～7.5,8～10kd membrane ultrafiltration, 0.2 μ m membrane filtration.
7. preparation method according to claim 6, is characterized in that, after step 1, before carry out step 2, homogenate heating is made to protein denaturation.
8. preparation method according to claim 6, is characterized in that, also comprises step 7: fill, 100～120 DEG C of sterilizings, leak detection.
9. preparation method according to claim 6, is characterized in that, the antioxidant described in step 6 is sodium sulfite, sodium pyrosulfite, dibutyl phenol, sodium sulfite, sodium thiosulfate or butylated hydroxyarisol.
10. preparation method according to claim 6, is characterized in that, the chromatographic stuffing that in step 5, upper column chromatography uses is Amberlite IRA-200, and anion exchange membrane adopts imported from America film DUS-8040C ionic membrane.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109371081A (en) * | 2018-03-06 | 2019-02-22 | 江西康宝医药生物科技有限公司 | A kind of preparation method and products thereof of activity pig brain polypeptide |
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101912598A (en) * | 2010-06-02 | 2010-12-15 | 刘燎原 | Method for preparing brain protein hydrolysate for injection and preparation thereof |
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| CN101912598A (en) * | 2010-06-02 | 2010-12-15 | 刘燎原 | Method for preparing brain protein hydrolysate for injection and preparation thereof |
Non-Patent Citations (2)
| Title |
|---|
| 国家药典委员会: "注射用脑蛋白水解物质量标准", 《国药典化发2012[2012]400号》 * |
| 国家药典委员会: "注射用脑蛋白水解物质量标准", 《国药典化发2012[2012]400号》, 20 July 2012 (2012-07-20) * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109371081A (en) * | 2018-03-06 | 2019-02-22 | 江西康宝医药生物科技有限公司 | A kind of preparation method and products thereof of activity pig brain polypeptide |
| 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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