CN104043099B - A kind of brain protein hydrolysate injection and preparation method thereof - Google Patents
A kind of brain protein hydrolysate injection and preparation method thereof Download PDFInfo
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Abstract
The invention provides a kind of brain protein hydrolysate injection and preparation method thereof, get Medulla sus domestica, roguing, add purified water homogenate; Through pepsin, pancreatin hydrolysis after homogenate is first, collect clear liquid; Clear liquid regulates pH to 1.7 ~ 3, is heated to 100 ~ 110 DEG C, and insulation 15 ~ 45min, then adjusts pH to 8.0 ~ 9.0; 8 ~ 10kd membrane ultrafiltration, collects filtrate; Filtrate Column chromatography, collects eluent, and adjust pH to 8.0 ~ 9.0, anion exchange membrance concentration, 0.2 μm of membrane filtration obtains stock solution; Stock solution is freezed, and then thaws, and adds antioxidant, adjusts pH to 6.9 ~ 7.5,8 ~ 10kd membrane ultrafiltration, 0.2 μm of membrane filtration.In gained brain protein hydrolysate injection polypeptide similar to reference substance >=0.90, containing 16 seed amino acids, the nitrogen quantity of total amino acids accounts for total nitrogen content ratio >=85%, adds aminoacid without the need to additionally supplementing, and relies on Medulla sus domestica hydrolysis obtained completely, stability and safety.
Description
Technical field
The present invention relates to field of biological, particularly relate 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, and extract, 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 break up, maintain the function of Normal neuronal cells, the generation increasing neurotransmitter and synapse transmission by Induction of neuronal, neuroprotective cell, from various infringement, regulates the metabolism of human body brain, neurotransmission and nerve growth; Breakdown of glucose enzymatic activity can be improved, increase brain to the anaerobic energy metabolism of glucose, reduce the concentration of lactic acid in brain, have protective effect when hypoxic-ischemic to the mitochondrion of neurocyte.
Brain protein hydrolysate injection is registered by the import of Austrian EBEWE pharmaceutical factory last century, to the end of the year 2010, relates to the patent application 29 of pharmaceutics of hydrolysate of brain protein and technique disclosed in existing.
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 removes surperficial mucosa and bloodstain etc., homogenate, double gauze filters, pepsin hydrolysis, add pancreatin hydrolysis, 10 ~ 30 hours are left standstill at adding hydrochloric acid adjust pH 2.5 ~ 3.0 ,-20 ~ 10 DEG C; Room temperature, double gauze filters, and adds water, and adjust pH is to neutral, filtering with microporous membrane is clarified, ultrafilter membrane ultrafiltration, and ultrafilter membrane concentrates, concentrated solution ebuillition of heated 15 minutes, then cool rapidly, then microporous filter membrane and ultrafilter membrane, the ultrafiltrate obtained, then through 121 DEG C of steam sterilizations; According to required amount of liquid medicine, the aminoacid consumption needing to add is calculated by the drug standard (numbering WS1-XG-25-2000) of National Drug Administration's December in 2000 promulgation on the 28th, 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 to molecular retention is 8KD 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 could obtain standard compliant Cerebrolysin Vial when producing preparation, and according to State Food and Drug Administration's No. 734 pass documentation requirements in 2008, Cerebrolysin Vial must not add exogenous aminoacid.Thus we need the research not adding the preparation method of exogenous amino acid whose Cerebrolysin Vial.
Summary of the invention
The object of the invention is to overcome in existing Cerebrolysin Vial technology of preparing needs 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 adopting this preparation method to prepare and reference substance similarity >=0.90, without the need to the exogenous aminoacid of supplementary interpolation, rely on Medulla sus domestica hydrolysis obtained 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; Amino acid whose nitrogen content total in described brain protein hydrolysate injection accounts for more than 85% of the 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, and be heated to 100 ~ 110 DEG C, insulation 15 ~ 45min (be preferably 20 ~ 40min, be more preferably 25 ~ 35min), then regulates pH to 8.0 ~ 9.0;
4) cold treatment, gets clear liquid, 8 ~ 10kd membrane ultrafiltration, collects filtrate;
5) by step 4) the filtrate Column chromatography that obtains, rinse post by purified water, resolve, collect eluent, regulate pH to 8.0 ~ 9.0, anion exchange membrance concentration, 0.2 μm of membrane filtration obtains stock solution;
6) stock solution that step 5 obtains freezed, then thaw, add antioxidant, the content controlling antioxidant is 0.01 ~ 0.05wt%, regulates pH to 6.9 ~ 7.5,8 ~ 10kd membrane ultrafiltration, 0.2 μm of membrane filtration, both.
Wherein, step 4) described in cold treatment, refer to liquid cools to less than 10 DEG C.
Preferably, in step 1) after, carry out step 2) before, homogenate heating is made protein denaturation.
Further preferably, protein denaturation is made specifically can to 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, dibutylphenol, sodium sulfite, sodium thiosulfate or butylated hydroxyarisol.
Preferably, step 5) in the chromatographic stuffing that uses of Column chromatography be AmberliteIRA-200.
Preferably, step 5) described in anion exchange membrane adopt imported from America film DUS-8040C ionic membrane (0.001 μm, aperture).
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 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 after homogenate step 1 obtained is first, collects clear liquid;
Step 3, clear liquid step 2 obtained regulates pH to 1.7 ~ 3, is heated to 100 ~ 110 DEG C, and insulation 15 ~ 45min (be preferably 20 ~ 40min, be more preferably 25 ~ 35min), then regulates pH to 8.0 ~ 9.0;
Step 4, cold treatment, gets clear liquid, 8 ~ 10kd membrane ultrafiltration, collects filtrate;
Step 5, filtrate Column chromatography step 4 obtained, rinses post by purified water, resolves, and collects eluent, and regulate pH to 8.0 ~ 9.0, anion exchange membrance concentration, 0.2 μm of membrane filtration obtains stock solution;
Step 6, stock solution step 5 obtained is freezed, and then thaws, and adds antioxidant, and the content controlling antioxidant is 0.01 ~ 0.05wt%, regulates pH to 6.9 ~ 7.5,8 ~ 10kd membrane ultrafiltration, 0.2 μm of membrane filtration.
Wherein, cold treatment described in step 4, refers to liquid cools to less than 10 DEG C.
Preferably, after step 1, before carry out step 2, homogenate heating is made protein denaturation.
Further preferably, protein denaturation is made specifically can to adopt following step: homogenate is incubated 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, dibutylphenol, sodium sulfite, sodium thiosulfate or butylated hydroxyarisol.
Preferably, the chromatographic stuffing that in step 5, Column chromatography uses is AmberliteIRA-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, collect clear liquid in step 2 and can pass through manner known in the art realizations such as filtering, centrifugal.Compared with prior art, preparation technology's tool of the present invention has the following advantages:
1, the present invention is after pepsin, pancreatin hydrolysis become the clear liquid of polypeptide and free amino acid, adjust pH to 1.7 ~ 3.0,100 ~ 110 DEG C are heated 30 minutes, again can 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 polypeptide and free amino acid Reversible binding and deactivation zoonotic virus from carrying out after double-enzyme hydrolysis, belong to end-of-pipe control, add without other biomaterial, safer;
2, adopt AmberliteIRA-200 exchange filler greatly can improve existing anion exchange and can not adsorb whole amino acid whose problem;
3, adopt anion exchange membrane, in the solution of ph8.0 ~ 9.0, amino acids negative charge, repels mutually with film surface charge, and free amino acid and polypeptide are trapped; Cation exchange membrane preferred imported from America film DUS-8040C ionic membrane, aperture is 0.001um, aperture 50d molecular weight, and free amino acid can be trapped further;
4, have employed 2 ultrafiltration in present invention process, filter for 2 times, reduce macromole sensitizer to greatest extent, ensure that particulate matter and Sterility Assurance value SAL;
5, freeze after adopting ultrafiltration in present invention process, ultrafiltration is freezed to reduce aminoacid and is separated out after removing macromole, thaw and can remove a small amount of impurity further 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; Containing 16 seed amino acids, the nitrogen quantity of total amino acids accounts for total nitrogen content ratio >=85%, containing 16 seed amino acids such as Aspartic Acid, glutamic acid, serine, histidine, glycine, threonine, alanine, arginine, valine, methionine, tryptophan, isoleucine, phenylalanine, leucine, lysine, proline.
Present invention process is simple, obtained brain protein hydrolysate injection small molecular polypeptide and 16 kinds of free aminoacid contents constant, in gained brain protein hydrolysate injection polypeptide similar to reference substance >=0.90, containing 16 seed amino acids, the nitrogen quantity of total amino acids accounts for total nitrogen content ratio >=85%, adds aminoacid without the need to additionally supplementing, and relies on Medulla sus domestica hydrolysis obtained completely, efficacy stability, safer; Injection is finally can sterile injectable preparation, and SAL guarantee value is higher, and ampoule encapsulates, and more seals, and improves drug safety.
Accompanying drawing explanation
The finger printing of the brain protein hydrolysate injection that Fig. 1 provides for the embodiment of the present invention 1 and middle inspection institute Cerebrolysin Vial 140697 ~ 200602;
Fig. 2 is 16 seed amino acid HPLC-UV detection of middle inspection institute Cerebrolysin Vial 140697 ~ 200602;
The free amino acid HPLC-UV detection of the brain protein hydrolysate injection that Fig. 3 provides for the embodiment of the present invention 1;
The total amino acids HPLC-UV detection of the brain protein hydrolysate injection that Fig. 4 provides for the embodiment of the present invention 1;
The finger printing of the brain protein hydrolysate injection that Fig. 5 provides for the embodiment of the present invention 2 and middle inspection institute Cerebrolysin Vial 140697 ~ 200602;
The free amino acid HPLC-UV detection of the brain protein hydrolysate injection that Fig. 6 provides for the embodiment of the present invention 2;
The total amino acids HPLC-UV detection 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 the present invention better.
Embodiment 1
Cerebral tissue (quarantining qualified) gets 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, and cooling; Hydrochloric acid adjusts pH1.8, pepsin hydrolysis, and 42 DEG C are incubated 3 hours, and sodium hydroxide adjusts pH7.6, pancreatin hydrolysis, and 42 DEG C are incubated 2 hours, collected by centrifugation clear liquid; Hydrochloric acid adjusts pH2, is incubated 30 minutes at 105 DEG C, and then sodium hydroxide adjusts pH8.8; Cold treatment, extracts supernatant, and 10kd membrane ultrafiltration removes macromole, collects filtrate 100L; Column chromatography, rinses post by purified water, resolves, and collects eluent, and regulate pH to 8.0 ~ 9.0, be concentrated into 50L with DUS-8040C ionic membrane, 0.2 μm of membrane filtration, obtains stock solution, less than-15 DEG C Cryopreservations; Stock solution is thawed, and allotment, adds sodium sulfite 0.01wt%, regulates pH6.9 ~ 7.5,8 ~ 10kd membrane ultrafiltration, 0.2 μm of membrane filtration, fill; 115 DEG C, 30 minutes, leak detection etc., prepared 5ml specification injection with small volume.
Similarity measures:
Reference substance is middle inspection institute Cerebrolysin Vial 140697 ~ 200602.Take octadecylsilane chemically bonded silica as filler (250mm × 4mm, particle diameter 5 μm); With 0.1% trifluoroacetic acid solution for mobile phase A; With 0.085% trifluoracetic acid acetonitrile solution--0.1% trifluoroacetic acid solution (80:20), for Mobile phase B, carries out gradient elution by table 1; Flow velocity is 0.8ml per minute; Determined wavelength is 276nm.Column temperature 40 DEG C; Calculate by similarity evaluation, result is as shown in Fig. 1 and table 2.
Gradient elution table during table 1 similarity measures
| 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.AccQTag 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, UV determined wavelength is 248nm, 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, containing 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 provides of the present embodiment, the nitrogen content of total amino acids accounts for 88.3% of total nitrogen.
Embodiment 2
Cerebral tissue (check, quarantine qualified) gets 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 adjusts pH2.0, pepsin hydrolysis, 42 DEG C are incubated 4 hours, and sodium hydroxide adjusts pH7.8, pancreatin hydrolysis, 42 DEG C are incubated 2 hours, collected by centrifugation clear liquid; Hydrochloric acid adjusts pH2, and 110 DEG C are incubated 30 minutes, and sodium hydroxide adjusts pH9.0; Cold treatment, extracts supernatant, and 10kd membrane ultrafiltration removes macromole, collects filtrate 150L; Column chromatography, rinses post by purified water, resolves, and collects eluent, and regulate pH to 8.0 ~ 9.0, concentrate 50L with DUS-040C ionic membrane, 0.2 μm of membrane filtration, obtains stock solution, less than-15 DEG C 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 μm of 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 result
| 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, containing 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 provides of the present embodiment, the nitrogen content of total amino acids accounts for 86.8% of total nitrogen.
Embodiment 3
Cerebral tissue (check, quarantine qualified) gets 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 adjusts pH3.0, pepsin hydrolysis, 35 DEG C are incubated 5 hours, and sodium hydroxide adjusts pH7.2, pancreatin hydrolysis, 45 DEG C are incubated 3 hours, collected by centrifugation clear liquid; Hydrochloric acid adjusts pH3.0, and 110 DEG C are incubated 30 minutes, and sodium hydroxide adjusts pH8.0; Cold treatment, extracts supernatant, and 10kd membrane ultrafiltration removes macromole, collects filtrate 150L; Column chromatography, rinses post by purified water, resolves, and collects eluent, and regulate pH to 8.0 ~ 9.0, concentrate 50L with DUS-040C ionic membrane, 0.2 μm of membrane filtration, obtains stock solution, less than-15 DEG C 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 μm of membrane filtration, fill; 115 DEG C, 30 minutes, leak detection etc., prepared 5ml specification injection with small volume.
Embodiment 4
Cerebral tissue (check, quarantine qualified) gets 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 adjusts pH1.7, pepsin hydrolysis, 40 DEG C are incubated 5 hours, and sodium hydroxide adjusts pH7.2, pancreatin hydrolysis, 45 DEG C are incubated 3 hours, collected by centrifugation clear liquid; Hydrochloric acid adjusts pH2.8, and 110 DEG C are incubated 45 minutes, and sodium hydroxide adjusts pH8.0; Cold treatment, extracts supernatant, and 10kd membrane ultrafiltration removes macromole, collects filtrate 150L; Column chromatography, rinses post by purified water, resolves, and collects eluent, and regulate pH to 8.0 ~ 9.0, concentrate 50L with DUS-040C ionic membrane, 0.2 μm of membrane filtration, obtains stock solution, less than-15 DEG C 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 μm of membrane filtration, fill; 115 DEG C, 30 minutes, leak detection etc., prepared 10ml specification injection with small volume.
Embodiment 5
Cerebral tissue (check, quarantine qualified) gets 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 adjusts pH1.7, pepsin hydrolysis, 40 DEG C are incubated 5 hours, and sodium hydroxide adjusts pH7.2, pancreatin hydrolysis, 45 DEG C are incubated 3 hours, collected by centrifugation clear liquid; Hydrochloric acid adjusts pH1.7., and 110 DEG C are incubated 15 minutes, and sodium hydroxide adjusts pH8.0; Cold treatment, extracts supernatant, and 10kd membrane ultrafiltration removes macromole, collects filtrate 150L; Column chromatography, rinses post by purified water, resolves, and collects eluent, and regulate pH to 8.0 ~ 9.0, concentrate 50L with DUS-040C ionic membrane, 0.2 μm of membrane filtration, obtains stock solution, less than-15 DEG C 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 μm of membrane filtration, fill; 115 DEG C, 30 minutes, leak detection etc., prepared 5ml specification injection with small volume.
Carry out similarity mensuration to the injection that embodiment 3-5 provides, 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).
Carry out determined amino acid to the injection that embodiment 3-5 provides, 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, and containing 16 seed amino acids, 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%.
Be described in detail specific embodiments of the invention above, 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 substituting also all among category of the present invention.Therefore, equalization conversion done without departing from the spirit and scope of the invention and amendment, all should contain within the scope of the invention.
Claims (9)
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; Amino acid whose nitrogen content total in described brain protein hydrolysate injection accounts for more than 85% of the total nitrogen of described brain protein hydrolysate injection;
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, add purified water homogenate;
2) through pepsin, pancreatin hydrolysis after homogenate step 1) obtained is first, clear liquid is collected;
3) by step 2) clear liquid that obtains regulates pH to 1.7 ~ 3, and be heated to 100 ~ 110 DEG C, insulation 15 ~ 45min, then regulates pH to 8.0 ~ 9.0;
4) cold treatment, gets clear liquid, 8 ~ 10kd membrane ultrafiltration, collects filtrate;
5) filtrate Column chromatography step 4) obtained, rinses post by purified water, resolves, and collects eluent, and regulate pH to 8.0 ~ 9.0, anion exchange membrance concentration, 0.2 μm of membrane filtration obtains stock solution;
6) stock solution that step 5 obtains freezed, then thaw, add antioxidant, the content controlling antioxidant is 0.01 ~ 0.05wt%, regulates pH to 6.9 ~ 7.5,8 ~ 10kd membrane ultrafiltration, 0.2 μm of membrane filtration, both.
2. brain protein hydrolysate injection according to claim 1, is characterized in that, after step 1), carry out step 2) before, homogenate heating is made protein denaturation.
3. brain protein hydrolysate injection according to claim 2, is characterized in that, the antioxidant described in step 6) is sodium sulfite, sodium pyrosulfite, dibutylphenol, sodium sulfite, sodium thiosulfate or butylated hydroxyarisol.
4. brain protein hydrolysate injection according to claim 2, is characterized in that, the chromatographic stuffing that in step 5), Column chromatography uses is AmberliteIRA-200, and described anion exchange membrane adopts imported from America film DUS-8040C ionic membrane.
5. 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, roguing, adds purified water homogenate;
Step 2, through pepsin, pancreatin hydrolysis after homogenate step 1 obtained is first, collects clear liquid;
Step 3, clear liquid step 2 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, 8 ~ 10kd membrane ultrafiltration, collects filtrate;
Step 5, filtrate Column chromatography step 4 obtained, rinses post by purified water, resolves, and collects eluent, and regulate pH to 8.0 ~ 9.0, anion exchange membrance concentration, 0.2 μm of membrane filtration obtains stock solution;
Step 6, stock solution step 5 obtained is freezed, and then thaws, and adds antioxidant, and the content controlling antioxidant is 0.01 ~ 0.05wt%, regulates pH to 6.9 ~ 7.5,8 ~ 10kd membrane ultrafiltration, 0.2 μm of membrane filtration.
6. preparation method according to claim 5, is characterized in that, after step 1, before carry out step 2, homogenate heating is made protein denaturation.
7. preparation method according to claim 5, is characterized in that, also comprises step 7: fill, 100 ~ 120 DEG C of sterilizings, leak detection.
8. preparation method according to claim 5, is characterized in that, the antioxidant described in step 6 is sodium sulfite, sodium pyrosulfite, dibutylphenol, sodium sulfite, sodium thiosulfate or butylated hydroxyarisol.
9. preparation method according to claim 5, is characterized in that, the chromatographic stuffing that in step 5, Column chromatography uses is AmberliteIRA-200, and anion exchange membrane adopts imported from America film DUS-8040C ionic membrane.
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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 |
| 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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| 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 |
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| Title |
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| 注射用脑蛋白水解物质量标准;国家药典委员会;《国药典化发2012[2012]400号》;20120720;第1页 * |
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