CN1640489A - Method for preparing urine polyacid peptide composition - Google Patents
Method for preparing urine polyacid peptide composition Download PDFInfo
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- CN1640489A CN1640489A CNA2004100007539A CN200410000753A CN1640489A CN 1640489 A CN1640489 A CN 1640489A CN A2004100007539 A CNA2004100007539 A CN A2004100007539A CN 200410000753 A CN200410000753 A CN 200410000753A CN 1640489 A CN1640489 A CN 1640489A
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Abstract
The present invention discloses a preparation method of urine polyacid peptide composition. Said preparation method includes the following steps: making fresh human urine undergo the processes of acidification filtration, adsorption and elution, then collecting eluent, inactivating virus by using ethyl alcohol with low pH value, making reduced pressure concentration and filtration so as to obtain the invented urine polyacid peptide composition. In said composition the molecular weighto f polypeptide is small, its bio-activity is high and toxic side effect is small, so that it can raise the action of differentiating and inducing tumor, and can reduce hypereccrisis of low-molecular metabolite of patient.
Description
Technical field
The present invention relates to a kind of preparation method of Uropoly acid-peptide composition, belong to the biological medicine technology field.
Background technology
Both at home and abroad to the history in existing several centuries of medical research of the extract of urine and urine, help agent and anti-cachexia agent owing to contain differentiating inducer, the differentiation of treatment and prophylaxis of cancer in urine and the extract thereof, so the extract of urine is applied as a kind of medicine of up-and-coming treatment cancer.
A kind of cancer-resisting substance-Uropoly acid-peptide composition that is formed through the science refining refinement by fresh Urina Hominis is disclosed such as ZL 99122051.x, wherein mainly contain hippuric acid, phenylacetylglutamine, phenylacetic acid and four kinds of organic acid of heteroauxing and a series of heterogeneity small-molecular peptides and inorganic salt, wherein four kinds of organic acid total contents are 9.81～23.58mg/ml, and total peptide content is about 20.7～26.7%; The total content of inorganic elements is 3077.24～8152.01 μ g/ml; Molecular weight is less than 10,000, and said preparation has induction, differentiation to help and anti-too much excretory synergism to tumor, and toxicity is low, the available clinically treatment of multiple late malignant tumour.
ZL 99122050.1 discloses the preparation method of Uropoly acid-peptide composition, with fresh uric acidization, removes by filter the bulky grain in the acidify urine; The bigger organic substance of molecular weight in the acidify urine is removed in ultrafiltration, the urine of the acidify after the ultrafiltration is added in the adsorption column absorption effective ingredient wherein; Be not adsorbed agent absorption with soft water washing but remain in inorganic matter in the adsorbent and hydrophilic organics hydrophilicity is adsorbed on effective ingredient on the adsorption column with pure eluting; Collect coloured effluent, after concentrating, drying obtains dry product; Be dissolved in water, adjust pH to neutral with alkali, stand at low temperature is separated out the impurity that forms in the concentration process, through removing thermal source, removing technology such as virus and be further purified.
ZL 99122049.8 discloses a kind of cancer-resisting substance---Uropoly acid-peptide composition that is formed by fresh Urina Hominis refining refinement, wherein mainly contain hippuric acid, phenylacetylglutamine, four kinds of organic acid of phenylacetic acid and heteroauxing and heterogeneity small-molecular peptides and inorganic salt, said composition has induction to tumor, differentiation helps and anti-too much excretory synergism, and toxicity is low, can promote cancerous cell to move towards normal differentiation or make cancer cell-apoptosis, can be used for the treatment of multiple late malignant tumour clinically and prevent cancer return, the result shows that this medicine has therapeutic effect and avirulence to kinds of tumors, have very high selectivity, improved curative effect greatly cancer.
Though, above-mentioned patent disclosure urine extract composition, content, extraction process and therapeutic effect thereof, but continuous development along with biotechnology, in order further to reduce impurity and the virus in the extract, the assurance effective ingredient can be given full play to drug effect, also need checking, active component and the structure of extraction process, inactivation of viruses technology and doing further research aspect the treatment the urine extract, such as how further removing the macromole impurity in the extract, harmful virus, reduce pyrogen, determine the molecular weight ranges and the sequence thereof of micromolecule polypeptide.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of Uropoly acid-peptide composition, this method has overcome deficiency of the prior art, select the inactivation of viruses technology of a suitable extraction process and science, virus and macromole impurity have been removed effectively, the molecular weight of polypeptide is little in the compositions of preparation, the biological activity height, and toxic and side effects is little, tumor induction, differentiation are helped that better effect is arranged, and lowered the excessively low molecule metabolites matter of drainage of patient.
In order to realize the object of the invention, the technical scheme of employing is:
The method of a kind of Uropoly acid-peptide composition of the present invention comprises the steps:
Fresh Urina Hominis through acidify, is filtered, and absorption behind the eluting, is characterized in that, with the eluent collected through low pH value ethanol inactivation of virus after, again through concentrating under reduced pressure, filter Uropoly acid-peptide composition.
Specifically, comprise the steps:
1) the Freshman urine of collecting is regulated acidity to pH between 2～3, remove by filter diameter in the acidify urine greater than 20 microns impurity, hold back macromole impurity through ultrafiltration again, filtrate;
2) then with filtrate after adsorption column absorption, in purified water washing adsorption column, impurity again and use the ethanol elution adsorption column, collect eluent;
3) described eluent hangs down pH value ethanol inactivation of virus;
4), get Uropoly acid-peptide composition after the filtration again through concentrating under reduced pressure.
Wherein, regulate urine pH in the step 1) between 2～3, regulate the acidity of urine and can use hydrochloric acid, sulphuric acid and be fit to other acid of the present invention.
The present invention is in order to keep the stability of effective ingredient in the fresh urine, to suppress the breeding of antibacterial with acidification of urine.Research worker is through discovering that the pH value of urine preferably is controlled between 2.0～3.0 simultaneously, and the organic acid in the urine all is ionic condition like this, and what be easier to adsorb in the leaching process finishes.
Described filtration can be adopted the filter in 20 μ m apertures, with in the filtering acidify urine greater than 20 μ m granule foreigns.
Described ultrafiltration is in order to remove the bigger organic substance of molecular weight in the acidify urine, and molecular cut off often adopts ultrafilter membrane greater than 10,000 organic impurities, and the pressure of ultrafiltration is controlled to be 5～15Mpa, such as tubular membrane and field of medicaments ultrafiltration apparatus commonly used.
Step 2) adsorption column absorption is adopted in the absorption described in, and the adsorbent in the adsorption column can be selected C18 post, XAD-7, XAD-8 or XAD-16 for use and be fit to other adsorbents of the present invention, preferred XAD-16 post and XAD-7, most preferably XAD-16.
Before adsorption column uses, wash adsorbent respectively with pure and mild purified water, the dress adsorption column adds in the adsorption column with the acidify urine of certain flow velocity after with ultrafiltration, uses the effective ingredient in the adsorbents adsorb filtrate; In the acidify urine a large amount of inorganic ions and Organic substance are arranged, what be not adsorbed after entering adsorbent lodges in the post, is not adsorbed with the soft water washing but remains in inorganic matter and hydrophilic organics hydrophilicity in the adsorbent, the purification effective ingredient; Be adsorbed on effective ingredient on the adsorbent with pure eluting; Collect eluent.Through the absorb-elute process, just make the effective ingredient desorption that is adsorbed like this, effective ingredient enters eluent.
With respect to 20 kilograms of freshly voided urines, the consumption of adsorbent is 5～20 kilograms, is preferably 10～20 kilograms; The consumption of the alcohol of above-mentioned immersion adsorbent is 15～25 liters, and soak time is 5～20 minutes; Preferable amount is 18～25 liters, and soak time is 10～20 minutes; The consumption of the pure and mild purified water of washing adsorbent is 15～25 liters, and alcohol is 1～2: 2～4 with the ratio of deionized water; The flow velocity that acidify urine adds adsorption column is 0.1～2L/min, is preferably 1～2L/min; Washing is 5～30L with the consumption of purified water, is preferably 15～30L; Elution speed is 0.1～3L/min, is preferably 0.1～2L/min; The consumption of eluent alcohol is 5～20L, is preferably 10～20L; Elution speed is 0.1～3L/min, is preferably 0.5～2L/min; Above-mentioned eluting can use ethanol, methanol, propanol and be fit to other alcohols of the present invention, preferred alcohol.
Low pH value ethanol inactivation of virus is in the step 3): concentration of alcohol is 70%～80% in the adjusting eluent, and adjust pH to 4.0～5.5, under 20 ℃～27 ℃, places 4～8 hours.
Used ethanol is good with 95%～99% medicinal alcohol, and adjust pH can and be fit to other acid of the present invention with 1～2mol/L hydrochloric acid, sulphuric acid, is preferably hydrochloric acid.
Because Uropoly acid-peptide composition of the present invention is the multi-component material that extracts from urine, therefore must carry out inactivation of virus removes various viruses, but must consider to guarantee not influence the activity of compositions when removing virus.Uropoly acid-peptide composition of the present invention adopts low pH value ethanol inactivation of virus to remove lipid-coated virus (comprising HIV (human immunodeficiency virus), hepatitis B virus, hepatitis C virus, Sindbis virus and rabies virus etc.), so not only guarantee the prescription of biological product, and enlarged the acquisition range of urine.
Research worker of the present invention is analyzed and researched and the contrast experiment from the method for existing inactivation of viruses, gropes to be fit to ablation method of the present invention.The method of five kinds of inactivation of viruses commonly used is respectively: 1. Pasteurization (pasteurization): 60 ℃, 10 hours; 2. organic solvent/detergent (S/D) facture; 3. low pH is incubated the method for putting; 4. dry heating method: 80 ℃ of heating 72 hours; 5. membrane filtering method.At first, 80 ℃ of heating were not suitable for this product in 72 hours, can destroy this product activity, and with the method for S/D, but that organic solvent has is residual, influenced product quality; Select for use the viral method of going out in classical 60 ℃, 10 hours to remove virus, but the inactivation of viruses method was higher to equipment requirements in 60 ℃, 10 hours, the difficult control of temperature, behind the mass production, the shuttle of 50 tons of urines palpuses of every batch processing super large, time-consuming, and excessive cycle, cost is too high, and has potential safety hazard, the most important thing is product activity influential, so the method for existing inactivation of viruses is all undesirable.
At above situation, research worker of the present invention is found out a kind of low pH value ethanol inactivation of viruses method, and its method is: adding ethanol to solution concentration of alcohol is 70%～80%, and adjust pH to 4.0～5.5, under 20 ℃～27 ℃, places 4～8 hours.And low pH value ethanol disinfection method and pasteurization have been carried out the contrast test of active anticancer influence, draw low pH value ethanol disinfection method and be better than pasteurization, and easy saving time, reduce cost, do not destroy the activity of organic acid and polypeptide, can also effectively remove lipid-coated virus simultaneously.
Concentrating under reduced pressure described in the step 4) is: with sodium hydroxide, sodium carbonate, sodium bicarbonate and the suitable adjusting PH with base value 5.5～6.5 of the present invention of 1～3mol/L, used alkali is preferably sodium hydroxide earlier; Evacuation heats up then, and vacuum degree control is 0.08～0.1MPa, and temperature is no more than 40 ℃, and be 8～10 hours heat time heating time, and being concentrated into the solution solids total amount is 200～300mg/ml.
Described filtration can be adopted the method for membrane filtration, and described filter membrane employing aperture is 0.45～5 micron a filter membrane.
Need to adjust pH value after filtering or before filtering, the hydrochloric acid of the sodium hydroxide of available 1～3mol/L, sodium carbonate, sodium bicarbonate or 1～3mol/L, sulphuric acid and suitable other alkali of the present invention or sour adjust pH 5.5～7.5.
After filtering, promptly get Uropoly acid-peptide composition.
Adopt a kind of Uropoly acid-peptide composition of preparation method gained of the present invention, its effective ingredient mainly comprises hippuric acid, phenylacetylglutamine, 4-hydroxyl phenylacetic acid, 5-hydroxyindoleacetic acid and 20～30 kinds of micromolecule polypeptide active component, and the molecular weight of micromolecule polypeptide active component is 400～6000D.
Wherein preferred micromolecule polypeptide active component molecular weight is between 400～2800D.
By analysis, a word symbol of the sequence of two main peptides is in the wherein said polypeptide active composition:
AVEGPSSALGPLGP
Alanine-valine-glutamic acid-glycine-proline-serine-serine-alanine-leucine-glycine-proline-leucine-glycine-proline, 14 peptides;
LGPSTPGPPPNGGA
Leucine-glycine-proline-serine-threonine-proline-glycine-proline-proline-proline-asparagine-glycine-glycine-alanine, 14 peptides.
The sequence that research worker of the present invention determines main two peptides in the Uropoly acid-peptide composition is to the medical research of urine extracting solution and its artificial synthetic polypeptide effective ingredient is laid the foundation.
Uropoly acid-peptide composition of the present invention is 100ml by compositions, and wherein the content of polypeptide is 100mg～800mg; Hippuric acid content is 200mg～500mg, and phenylacetyl glutamy content is 250mg～600mg, and 4-hydroxyl phenylacetic acid content is 30mg～80mg, and 5-hydroxyindoleacetic acid content is 5mg～50mg.
Wherein be: hippuric acid 180, phenylacetic acid glutamine 265,4-hydroxyl phenylacetic acid 152,5-hydroxyindoleacetic acid 193 with four kinds of organic acid molecular weight of ES mass spectrometric determination.
The nitrogen content of Uropoly acid-peptide composition of the present invention is 14～45mg/ml.
The pH value of described Uropoly acid-peptide composition is 5.5～7.5; Be preferably 5.5～6.5.
Uropoly acid-peptide composition of the present invention can add different adjuvant according to the method on the pharmaceutics to be processed, and makes different dosage form products.
Uropoly acid-peptide composition of the present invention can be made into injection, powder ampoule agent for injection, tablet or capsule etc., is preferably injection.
Can adopt the preparation method of injection such as injection, earlier the urine extract be diluted with water for injection, and adjust pH to 6.5～7.5, again through removing thermal source, degerming, filtration, removing virus and promptly get the Uropoly acid-peptide injection.
Specifically, be diluted to the solution that contains total solid 30～50mg/ml with water for injection,, remove thermal source with removing the thermal source film then with sodium hydroxide or the hydrochloric acid adjust pH 6.5～7.5 of 1～3mol/L, and, promptly get the Uropoly acid-peptide injection at last through the sterilization of degerming filter membrane with except that viral membrane filtration.
Described remove the thermal source film be Millipore remove the thermal source film, it is 0.20～0.30 micron filter membrane that described filter membrane adopts the aperture, removes the filter membrane that viral filter membrane can adopt 20～50 nanometer NFP Filters.
The Uropoly acid-peptide injection of being made by Uropoly acid-peptide composition of the present invention has adopted the deactivation mode twice, promptly behind eluting, adopt low pH value ethanol deactivation method to remove lipid-coated virus, and employing removes parvovirus except that viromembrane before the preparation finished product, the biological activity that has kept compositions so effectively, and can thoroughly remove virus in the urine, to satisfy the requirement of biological product.
Other dosage forms of the present invention can adopt those skilled in the art's common technology to be prepared, and can adopt lyophilization or spray-dired method such as powder ampoule agent for injection.
The main effective ingredient of Uropoly acid-peptide composition treatment of the present invention and prophylaxis of cancer comprises: differentiating inducer, and differentiation helps agent and anti-cachexia agent, and its pharmacology analysis is open in ZL 99122049.8.
Differentiating inducer is the special antagonist of unusual Methyl transporters compound enzyme, and the most important effective ingredient of Uropoly acid-peptide composition is micromolecule polypeptide and organic acid, is to have the very cancer-resisting substance of high selectivity.
It is the inhibitor of each member's enzyme of Methyl transporters compound enzyme that differentiation helps agent, the inducing action that very strong enhancement differentiating inducer is arranged, 4-hydroxyl phenylacetic acid, 5-hydroxyindoleacetic acid and hippuric acid are the competitive inhibitors of MAT in the Uropoly acid-peptide composition, in differentiation therapy indispensable role, differentiation helps agent can promote differentiation on the one hand, can prevent again that on the other hand the cell damage that differentiating inducer causes from finish differentiation smoothly, recurrence just can obviously reduce; And can lower the demand of differentiating inducer.
Wherein the phenylacetic acid glutamine can be protected the ability of chemistry supervision; generation with prophylaxis of cancer; improve cancer patient simultaneously in addition and excessively drain the function of low molecule metabolite; Uropoly acid-peptide composition also proves the curative effect that can significantly increase chemotherapy in the result of dying patient's clinical practice; and alleviate side effect, improve patient's quality of life.The effect that Uropoly acid-peptide composition has obvious anticancer to shift, itself does not have toxicity again, prevents the ideal medicament that recurs beyond doubt after the operation.
Uropoly acid-peptide composition of the present invention balances out the cancer factor of unusual Methyl transporters compound enzyme, makes the cancerous cell can't normal differentiation, can only carry out end Mo differentiation as normal cell.
The present invention has adopted the new preparation method of effective component extracting from human urine, adopt new employing to hang down pH value ethanol virus inactivating method, the activity that has not only kept extract, virus (lipid-coated virus and other parvovirus) and thermal source have also been eliminated effectively, removed macromole impurity, reduced the toxic and side effects of said composition, improved the tumor induction, broken up the effect of help, and lowered the patient and excessively drained low molecule metabolites matter, promoting the normal differentiation of end eventually of cancerous cell, apoptosis, is ideal antitumor drug.The Uropoly acid-peptide composition of the process of the present invention simultaneously extraction gained has been controlled the molecular weight ranges of micromolecule polypeptide, can obtain the polypeptide of the following molecular weight of 6000D, has determined sequence, the molecular weight of four kinds of organic acid compositions and each components contents of two kinds of main peptides.
Through carrying out drug effect, toxicity, study of pharmacy such as clinical, show Uropoly acid-peptide composition of the present invention because by adopting new preparation extraction process, remove macromole impurity and various virus, safer, the avirulence of product, and hepatocarcinoma, pulmonary carcinoma, the isocellular growth of leukemia there is obvious inhibitory action, but the activity of enzyme in the anticancer effectively shifts in the anticancer and recurrence, is a kind of effective induction preparation; Find that by relative analysis the clinical safety reliability of Uropoly acid-peptide composition of the present invention is higher, biological activity, active anticancer and tumour inhibiting rate have improved 5-10% than ZL 99122049.8 disclosed compositionss.
Description of drawings
Fig. 1 is for concerning between purified water washing throwing amount and the pH value
Inner bag vacuum and time graph when Fig. 2 is ethanol evaporation
Inner bag temperature and time curve when Fig. 3 is ethanol evaporation
Fig. 4 is hippuric acid, phenylacetylglutamine reference substance HPLC chromatogram
Fig. 5 is hippuric acid, phenylacetylglutamine test sample HPLC chromatogram
Fig. 6 is 4-hydroxyl phenylacetic acid, 5-hydroxyindoleacetic acid reference substance HPLC chromatogram
Fig. 7 is 4-hydroxyl phenylacetic acid, 5-hydroxyindoleacetic acid test sample HPLC chromatogram
Fig. 8 is that three kinds of organic acid molecular weight in the injection are measured in liquid-matter coupling
Fig. 9 is the molecular weight that 5-hydroxyindoleacetic acid in the injection is measured in liquid-matter coupling
Figure 10 is the chromatography of ions figure [20022611 Sm (SG, 2 * 6)] of sample
Figure 11 is total mass spectrum [20,022,611 408 (16.707) Cm (372:428)] in I district
Figure 12 is chromatography of ions figure [20,022,611 408 (16.707) M3]
Figure 13-Figure 17 is chromatography of ions figure [20,022,611 408 (16.707) partial enlarged drawings
Figure 18 is total mass spectrum [20,022,611 864 (32.201) Cm (832:1161)] in II district
Figure 19 is collection of illustrative plates [20,022,611 864 (32.201) M3]
Figure 20 is collection of illustrative plates [20,022,611 1438 (51.806) M3]
Figure 21 is total mass spectrum [20,022,611 1438 (51.806) Cm (1324:1444)] in I II district
Figure 22 is the sequencing result of m/z 626.36
Figure 23 is m/z 626.36 peptide sequences
Figure 24 is the sequencing result of m/z 609.84
Figure 25 is the peptide sequence of m/z 609.84
Figure 26 is the blank sample ultraviolet absorpting spectrum
Figure 27 is the standard sample ultraviolet absorpting spectrum
Figure 28 is the trap curve
Figure 29 is the ultraviolet absorpting spectrum of this sample
Figure 30 contains the spectral absorption curve of 75% ethanol sample
Figure 31 removes the spectral absorption curve of ethanol sample
The specific embodiment
Following embodiment further describes the present invention, but described embodiment only is used to illustrate the present invention rather than restriction the present invention.
Experimental example 1
This experimental example relates to the test of adsorption conditions of the present invention.
1. the urine pH value determines
Characteristic according to adsorbent: the substance-adsorbing to the nonionic state is strong, again because the main component of our product is four kinds of organic acid and small peptide material.According to four kinds of organic acid pKa values, the pH value of urine was transferred to 2.0～3.0 o'clock, make organic acid all be ionic condition, be easy to absorption.
2. the rate of charge of acidify urine
Use soak with ethanol 24 hours before the test of XAD-16 adsorbent, dressing up 3 sectional areas then is 0.018m
2The adsorption column of ( 20cm, high 60cm), column volume is about 5L.
Acidify urine lot number is respectively 990301,990302,990303, and inventory is 5L, 10L, 20L, 30L, 40L, 50L.
The soft water washing amount is 30L.
The 95% ethanol elution 20L that feeds intake.
Collect ethanol elution, observe, the color of record eluent, and survey the absorption value at A430 place, draw following data with ultraviolet spectrophotometry.
The influence of different inventorys to 95% ethanol elution A430 trap urinated in table 1 acidify
| Acidify urine inventory (L) | The color of three batches of ethanol elution and A430 trap | |||||
| ????990301 | ????990302 | ??990303 | ||||
| ????5 | Colourless | 0.083 | Colourless | 0.057 | Colourless | ?0.074 |
| ????10 | Colourless | 0.214 | Colourless | 0.195 | Colourless | ?0.201 |
| ????20 | Colourless | 0.296 | Colourless | 0.289 | Colourless | ?0.312 |
| ????30 | Faint yellow | 0.365 | Faint yellow | 0.353 | Faint yellow | ?0.378 |
| ????40 | Faint yellow | 0.374 | Faint yellow | 0.366 | Faint yellow | ?0.381 |
| ????50 | Faint yellow | 0.382 | Faint yellow | 0.368 | Faint yellow | ?0.389 |
To above-mentioned data analysis: when acidify urine volume was higher than 30L, absorption value changed minimum, and the XAD-16 absorption that reached capacity be described when adding acidify and urinate 30L, therefore, can reach a conclusion, acidify urine inventory and XAD-16 adsorbent volume ratio be 6: 1 be the best.
3. purified water rate of charge
The rate of charge of purified water is determined the clean result of impurity according to purified water.We reflect its clean result, and have designed following experimental program to measure amount and the pH value variation tendency thereof that contains " chloride " in the washing relief liquor:
After choosing 3 XAD-16 posts pretreatment of stating test, add 30L acidify urine earlier, select purified water 10L, 20L, 30L, 40L, five kinds of inventorys of 50L to wash adsorption column respectively then, respectively get final washing relief liquor, by " purified water quality standard " Shen " chloride " detection method, check the amount of " chloride " in the relief liquor.Test 3 batches altogether, each batch test 3 times the results are shown in Table 2.
Alternative is selected purified water washing throwing amount when being 50L, and every discharging 2.5L measures its pH value, tests 3 batches, the results are shown in Figure 1.
" chloride " check result in the purified water washing relief liquor of table 2 different volumes
| Purified water volume (L) | ???????????990304 | ???????????990305 | ???????????990306 | ||||||
| The 1st time | The 2nd time | The 3rd time | The 1st time | The 2nd time | The 3rd time | The 1st time | The 2nd time | The 3rd time | |
| ????10 | Muddy | Muddy | Muddy | Muddy | Muddy | Muddy | Muddy | Muddy | Muddy |
| ????20 | Little apparent muddiness | Little apparent muddiness | Little apparent muddiness | Little apparent muddiness | Little apparent muddiness | Little apparent muddiness | Little apparent muddiness | Little apparent muddiness | Little apparent muddiness |
| ????30 | Clarification | Clarification | Clarification | Clarification | Clarification | Clarification | Clarification | Clarification | Clarification |
| ????40 | Clarification | Clarification | Clarification | Clarification | Clarification | Clarification | Clarification | Clarification | Clarification |
| ????50 | Clarification | Clarification | Clarification | Clarification | Clarification | Clarification | Clarification | Clarification | Clarification |
Analyze above-mentioned data as can be known, when the washing relief liquor reached 25.0L, its pH value variation tendency was quite mild, and the impurity hexyl originally washes entirely.And when the purified water inventory reached 30L, the impurity such as chloride that washed out were quite low, thus with the inventory of purified water and XAD-16 adsorption column volume ratio be 6: 1 for best.
4. ethanol rate of charge
The ethanol rate of charge, what of total weight of solids in the Uropoly acid-peptide concentrated solution during with different inventory ethanol elution, and take all factors into consideration two indexs of ethanol consumption and determine.Select XAD-16 adsorption column in the above-mentioned test for use, after the pretreatment, add 30L acidify urine, earlier with 30L purified water washing XAD-16 adsorption column, drop into 95% medicinal alcohol 5.0L, 7.5L, 10.0L, 12.5L, 15.0L more respectively, the effective ingredient that eluting is adsorbed is collected extracting solution and is carried out concentrating under reduced pressure, measure the total solid and the volume of concentrated solution, calculate the total weight of solids in the concentrated solution.Test 3 batches altogether, each batch carries out 3 times, after the meter average, draws following data, sees Table 3.
Table 3 different volumes ethanol elution is to the influence of total weight of solids in the Uropoly acid-peptide concentrated solution
| Ethanol consumption (L) | Total weight of solids (g) in three batches of Uropoly acid-peptide solution | ||
| ????990401 | ????990402 | ????990403 | |
| ????5.0 | ????20.6 | ????20.2 | ????21.9 |
| ????7.5 | ????25.1 | ????24.8 | ????24.2 |
| ????10. | ????26.4 | ????26.2 | ????25.9 |
| ????12.5 | ????26.7 | ????26.5 | ????26.3 |
| ????15.0 | ????26.9 | ????26.6 | ????26.6 |
Analyze above-mentioned data as can be known, when the ethanol consumption is about the twice of adsorbent volume, be enough to the active substance that eluting is adsorbed.After the ethanol inventory surpassed 7.5L, total weight of solids increased not obviously in the concentrated solution, and therefore alcoholic acid inventory is 1.5: 1st with the ratio of XAD-16 adsorption column volume, and is best.
5.XAD-16 adsorbent is reused number of times
We have carried out following test to XAD-16 adsorbent access times, after the XAD-16 sorbent treatment is good, add acidify urine 30L, with purified water 30L washing, reuse 95% medicinal alcohol 7.5L eluting.Select 3 groups of adsorbents, carry out 300 tests respectively, collect the 50th, 100,150,200,250,300 times eluent is evaporated to equal volume.Measure each time and make the total solid of concentrated solution and the HBL-100 cell mass is suppressed percentage ratio, draw following data.
Table 4 XAD-16 adsorbent is reused the influence of number of times to several indexs
| Project | Total solid mg/ml | The HBL-100 cell mass is suppressed % | ||||
| The adsorbent numbering | ????01 | ????02 | ????03 | ????01 | ????02 | ????03 |
| The adsorbent access times | ????1 | ????281 | ????269 | ????277 | ????100 | ????98 | ????99 |
| ????50 | ????286 | ????282 | ????283 | ????99 | ????99 | ????99 | |
| ????100 | ????279 | ????279 | ????269 | ????99 | ????100 | ????100 | |
| ????150 | ????282 | ????268 | ????252 | ????100 | ????99 | ????98 | |
| ????200 | ????256 | ????247 | ????248 | ????98 | ????100 | ????100 | |
| ????220 | ????219 | ????210 | ????210 | ????88 | ????90 | ????90 | |
| ????250 | ????183 | ????171 | ????183 | ????84 | ????80 | ????82 |
His-and-hers watches 4 are analyzed as can be known, and when XAD-16 is setting under the technology, when using 200 times, the total solid of concentrated solution is more or less the same when using 1,50,150 time, and after 200 times, active anticancer descends to some extent, and total solid lowers greatly.This shows that under the regulation process conditions, the access times of XAD-16 are decided to be 200 times.Can eliminate after using 200 times.
6. total solid is urinated yield to acidify
Calculate the yield of total solid, investigate ten batches continuously, the results are shown in Table 5 acidify urine.
Table 5 total solid total amount is urinated yield table to acidify
| Lot number | Acidify urine inventory L | Concentrated solution volume L | Total solid mg/ml | Yield % (g/l) |
| ????020401 | ????7680 | ????24.4 | ????209 | ????66.4 |
| ????020402 | ????7920 | ????21.7 | ????229 | ????62.7 |
| ????020103 | ????7200 | ????20.8 | ????222 | ????64.1 |
| ????020404 | ????7200 | ????21.6 | ????203 | ????60.9 |
| ????020405 | ????7680 | ????22.8 | ????210 | ????62.3 |
| ????020406 | ????7680 | ????24.8 | ????203 | ????65.6 |
| ????020407 | ????7680 | ????23.6 | ????200 | ????61.5 |
| ????020408 | ????7200 | ????18.6 | ????256 | ????66.1 |
| ????020409 | ????7200 | ????22.5 | ????206 | ????64.4 |
| ????020410 | ????7200 | ????17.7 | ????260 | ????63.9 |
| Meansigma methods | ????7464 | ????21.85 | ????219.8 | ????64.3 |
Sum up: his-and-hers watches 5 are analyzed as can be known, and under regulation technology, the total solid total amount is 64.3% (g/L) to the yield meansigma methods of acidify urine, wherein is up to 66.4% (g/L), and minimum is 60.9% (g/L).
Therefore the yield that total solid is urinated acidify is decided to be 60.0～67.0% (g/L).
7. the regeneration of adsorbent
When sorbent circulation was used, the organic solvent that eluting is used can be washed the material on the post to the greatest extent, and when water washed to chlorine (CL-) feminine gender then, pH value and purified water were near getting final product.After sorbent circulation is used a period of time, when finding that residue pollutes adsorbent for yellowish-brown, then need to handle with acid-alkali regeneration.After soaking 12h with 0.5MNaOH earlier, be washed to neutrality, reuse 0.5MHCl is washed to neutrality after soaking 12h.
Experimental example 2
This experimental example relates to the research of removing viral method.
Because of Uropoly acid-peptide solution is many component substance of extracting from urine, so inactivation of virus is the pith of our technical study, the method for at present general inactivation of viruses has five kinds:
(1) Pasteurization (pasteurization): 60 ℃, 10 hours;
(2) organic solvent/detergent (S/D) facture;
(3) low pH is incubated the method for putting;
(4) dry heating method: 80 ℃ were heated 72 hours;
(5) membrane filtering method.
Research worker of the present invention is analyzed and researched and the contrast experiment to the method for five kinds of inactivation of viruses.At first 80 ℃ of heating were not suitable for this product in 72 hours, can destroy this product activity, and with the method for S/D, but that organic solvent has is residual, influenced product quality; Select for use the viral method of going out in classical 60 ℃, 10 hours to remove virus, but the inactivation of viruses method was higher to equipment requirements in 60 ℃, 10 hours, the difficult control of temperature, behind the mass production, the shuttle of 50 tons of urines palpuses of every batch processing super large, time-consuming, and excessive cycle, cost is too high and have a potential safety hazard; By analysis, the method for the existing inactivation of viruses of discovery is all undesirable.At this situation and by checking repeatedly, research worker of the present invention is found out a kind of low pH value ethanol disinfection method, and its method is: adding ethanol to solution concentration of alcohol is 70%～80%, and adjust pH to 4.0～5.5, under 20 ℃～27 ℃, placed 4～8 hours.And low pH value ethanol disinfection method and pasteurization carried out the contrast test of active anticancer influence, draw low pH value ethanol disinfection method and be better than pasteurization, and easy saving time, reduce cost, do not destroy the activity of organic acid and polypeptide.
A technology: extracting solution adjust pH 4.0-5.5, concentration of alcohol are 70-80%, under 20 ℃～27 ℃, place inactivation of viruses 4～8 hours.
B technology: the extracting solution adjust pH was 6.0-6.5, in 60 ℃, 10 hours inactivation of viruses.
Two kinds of inactivation of viruses technologies are investigated, and investigate by " Uropoly acid-peptide solution quality draft standard ", test 10 batches altogether, the results are shown in Table 6.
Two kinds of virus inactivation technologies of table 6 are to anticancer active influence
| Project | Body suppression ratio to human breast cancer cell strain HBL-100 | Body outer suppressioning test IC50 (mg/ml) to people's hepatocarcinoma Smmu7721 | |||
| Standard code | Must not be less than 90% | Should be less than 2.0 | |||
| Carry out technology | A technology | B technology | A technology | B technology | |
| Lot number | ????020901 | ????98％ | ????96％ | ????1.7 | ????1.8 |
| ????020902 | ????97％ | ????99％ | ????1.6 | ????1.6 | |
| ????020903 | ????99％ | ????96％ | ????1.7 | ????1.5 | |
| ????020904 | ????100％ | ????94％ | ????1.4 | ????1.7 | |
| ????020905 | ????95％ | ????85％ | ????1.9 | ????1.8 | |
| ????020906 | ????99％ | ????94％ | ????1.8 | ????1.6 | |
| ????021001 | ????96％ | ????96％ | ????1.5 | ????1.6 | |
| ????021002 | ????96％ | ????87％ | ????1.8 | ????1.8 | |
| ????021003 | ????100％ | ????100％ | ????1.7 | ????1.5 | |
| ????021004 | ????100％ | ????97％ | ????1.6 | ????1.9 | |
From table 6 analytical data as can be known, A technology is measured 10 batches qualification rate equal 100% to two kinds of active anticancers.It only is 80% that B technology has two batches of defective (85%, 87%, regulation should less than 90%) qualification rates to the active anticancer of human breast cancer cell strain HBL-100; To in 10 batches of the human liver cancer cell Smmu7721 active anticancers 1 batch defective (2.1) are arranged, qualification rate is 90%.This shows that A technology is better than B technology.
Uropoly acid-peptide injection with the production of A inactivation technology, carry out HbsAg, HCV five of Chinese Military Medical Science Institutes and nine respectively, HIV-1, HIV-2 check, the result is all negative, carry out HbsAg, HCV antibody, HIV-1, HIV-2 antibody and the antibody test of syphilis spiral respectively with domestic reagent box and import reagent box, all negative.
Uropoly acid-peptide injection of the present invention adopts inactivation technology twice, earlier with low pH value ethanol inactivation of viruses method, adopts the filter membrane that removes virus to carry out inactivation treatment again before fill.This ethanol of 70%～80% and remove two step of viromembrane inactivation technology in five of Chinese Military Medical Science Institutes and nine checkings of carrying out inactivation technology, HIV-1 is added to and contains in 70%～80% alcoholic acid Uropoly acid-peptide extracting solution as a result, soak after 4～8 hours, titre 5.23～5.25 log that descend, and cytopathy does not appear through cell culture blind passage three generations; The inactivating efficacy of Pseudorabies virus 〉=5.38～6.00logTCID50; The inactivating efficacy of Sindbis virus 〉=5.63～5.88logTCID50.75% second ferment is handled 6 hours three batch samples, and cytopathy does not appear in the blind passage three generations.
For removing parvovirus (B19 etc.), increase and use Viresolve in addition
TMThe Millipore filter membrane of NFP Filters model is removed the parvovirus of 18～24nm.The filter membrane that world health organisation recommendations is removed virus should be 35nm or littler.Remove the process certification result of virus with the film of Viresolve NFP model, the titre of HIV 4.50～5.23 log that descend, and cytopathy does not appear through cell culture blind passage three generations; This can remove 4.8～5.6logTCID50 poliovirus except that viromembrane empirical tests.
Experimental example 3
This experimental example relates to the experimental study to vacuum concentration.
Add the 500L ethanol extract in double conic rotary vacuum dryer, open vacuum and hot water circulating pump, begin heating, control vacuum is 0.08～0.1MPa, and the external jacket water temperature is no more than 62 ℃, about 10 hours of heat time heating time.Inner bag temperature of per 30 minutes records, pressure, when reaching the evaporation terminal point, the inner bag temperature descends fast, concentrates and finishes.Carry out 3 batches altogether.The results are shown in Figure 2 and Fig. 3.
More than 3 batches of vacuum concentration (lot number is respectively: 020401,020402,020403) analytical data as can be known: control vacuum is 0.08～0.1MPa, and the external jacket water temperature is no more than 62 ℃, can guarantee that the inner bag temperature is no more than 40 ℃.When the inner bag temperature descends fast, concentrate and finish, be 8-10 hour heat time heating time.
Experimental example 4
This experimental example relates to the investigation that the Uropoly acid-peptide injection filters front and back active anticancer, content of peptides and organic acid content.
1. active anticancer was investigated before and after the Uropoly acid-peptide injection filtered
Get the Uropoly acid-peptide injection after three batches of filtrates after the aseptic filtration and these three batches remove virus, the method under measuring according to " Uropoly acid-peptide injection quality standard draft " active anticancer is measured, and the results are shown in Table 7.
Table 7 removes the comparison of virus filtration front and back active anticancer
| Lot number 030301 | Lot number 030302 | Lot number 030303 | |||
| Mg/ml before filtering | Filter back mg/ml | Mg/ml before filtering | Filter back mg/ml | Mg/ml before filtering | Filter back mg/ml |
| ??0.76 | ??0.78 | ??1.32 | ??1.36 | ??1.35 | ??1.40 |
As seen from Table 7, remove the three batches of active anticancer there was no significant differences in virus filtration front and back, the Viresolve of the Millpore company that uses in the technology is described
TMThe viromembrane that removes of NFP Filters model does not influence the injection active anticancer.
2. content of peptides was investigated before and after three batches of Uropoly acid-peptide injection filtered
Get the Uropoly acid-peptide injection after three batches of filtrates after the aseptic filtration and these three batches remove virus, measure, the results are shown in Table 8 according to the assay method of polypeptide under " Uropoly acid-peptide injection quality standard draft " assay item.
Table 8 removes the comparison of virus filtration front and back content of peptides
| Lot number 030301 | Lot number 030302 | Lot number 030303 | |||
| Mg/ml before filtering | Filter back mg/ml | Mg/ml before filtering | Filter back mg/ml | Mg/ml before filtering | Filter back mg/ml |
| ??1.83 | ??1.85 | ??2.08 | ??2.06 | ??2.52 | ??2.46 |
It can be seen from the table, remove the three batches of content of peptides there was no significant differences in virus filtration front and back, the Vireso lve of the Millipore company that uses in the technology is described
TMThe viromembrane that removes of NFP Filters model does not influence content of peptides in the injection.
3. four kinds of organic acid contents were investigated before and after three batches of Uropoly acid-peptide injection filtered
Get the Uropoly acid-peptide injection after three batches of filtrates after the aseptic filtration and these three batches remove virus, drink assay method according to the following four kinds of organic acid of " Uropoly acid-peptide injection quality standard draft " assay item and measure, the results are shown in Table 9.
Table 9 removes the comparison of the four kinds of organic acid contents in virus filtration front and back
| Four kinds of organic acid titles | Lot number 030301 | Lot number 030302 | Lot number 030303 | |||
| Mg/ml before filtering | Filter back mg/ml | Mg/ml before filtering | Filter back mg/ml | Mg/ml before filtering | Filter back mg/ml | |
| Hippuric acid | ????2.44 | ????2.44 | ????2.84 | ????2.84 | ????2.73 | ????2.74 |
| The acid of phenylacetyl glutamy | ????2.28 | ????2.26 | ????2.80 | ????2.72 | ????2.60 | ????2.55 |
| Phenylacetic acid | ????2.48 | ????2.50 | ????2.74 | ????2.74 | ????2.96 | ????2.93 |
| Heteroauxing | ????0.05 | ????0.06 | ????0.06 | ????0.06 | ????0.08 | ????0.08 |
As seen from Table 9, remove the three batches of four kinds of organic acid assays in virus filtration front and back basically identical as a result, the Viresolve of the Millipore company that uses in the technology is described
TMThe viral filter membrane that removes of NFP Filters model does not influence four kinds of organic acid content.
Experimental example 5
This experimental example relates to the structural identification of each composition in the frequent micturition acid phthalein compositions.
Frequent micturition of the present invention acid phthalein compositions contains four kinds of organic acid (hippuric acid, phenylacetylglutamine, 4-hydroxyl phenylacetic acid, 5-hydroxyindoleacetic acid) and a series of small-molecular peptides isoreactivity composition for what extract from the healthy human urine, the structural identification test is mainly proved conclusively above-mentioned two big class materials.Four kinds of organic acid are tested with high performance liquid chromatography, adopt application of sample to confirm and separation determination and mass spectrometric determination molecular weight; Small-molecular peptides adopts liquid-matter coupling, measures the molecular weight ranges of peptide, and has determined the wherein sequence of two main peptides.
1. four kinds of organic acid structural identifications (HPLC method) in the frequent micturition acid phthalein
1.1 Instrumental Analysis type HPLC, day island proper Tianjin LC-10ATVP of company, detector, SPD-10AVP.Chromatographic column, DikMA company, Luna 5 μ 18 (Z), 250 * 4.6mn (dm).
1.2 reference substance 4-hydroxyl phenylacetic acid; The 5-hydroxyindoleacetic acid; Hippuric acid; Phenylacetylglutamines etc. are available from Sigma company.
1.3 chromatographic condition mobile phase: methanol: water: glacial acetic acid=250: 750: 5 flow velocitys: 1ml/min; Sample size: 20 μ l; Detect wavelength: 260nm.
1.4 the preparation of reference substance solution
Precision takes by weighing 5-hydroxyindoleacetic acid reference substance 20mg, puts in the 50ml measuring bottle, adds the about 40ml of water, just dissolving in ultrasonic 30 minutes, and thin up is to scale, as storing solution.
Precision takes by weighing hippuric acid reference substance 45mg, phenylacetyl paddy amine acyl reference substance 60mg, 4-hydroxyl phenylacetic acid reference substance 45mg, put in the same 50ml measuring bottle, add the about 40ml of water, made dissolving in ultrasonic 30 minutes, precision is measured 5-hydroxyindoleacetic acid reference substance storing solution 5.0ml again, put in the above-mentioned 50ml measuring bottle, thin up is to scale, mixing.
1.5 the preparation of need testing solution
It is an amount of to get this product, and thin up is made the solution that contains 8mg among every 1ml, promptly.
1.6 algoscopy
Get each 20 μ l injecting chromatograph of above-mentioned reference substance solution and need testing solution, the record chromatogram uses external standard method with 4 kinds of organic acid content of calculated by peak area.
1.7 measurement result sees Table 10 and Fig. 4～Fig. 7.
Four kinds of organic acid of table 10 retention time in the HPLC chromatogram compares
| Reference substance Rt (min) | Hippuric acid 12.398 | Phenylacetylglutamine 13.265 | This acetic acid of 4-hydroxyl 41.465 | 5-hydroxyindoleacetic acid 51.065 |
| Uropoly acid-peptide injection Rt (min) | 12.398 | ????13.398 | ????41.598 | ????50.132 |
Find out each organic acid retention time basically identical from Fig. 4～Fig. 7, provable this product contains above four kinds of organic acid.
2. four kinds of organic acid structural identifications of Uropoly acid-peptide (mass spectrography)
With four kinds of organic acid molecular weight of ES mass spectrometric determination, see Fig. 8, Fig. 9.
From Fig. 8, Fig. 9, obtain four kinds of organic acid molecular weight:
3. electrospray mass spectrometer is measured peptide composition sequence and molecular weight
Testing conditions cation detection mode, capillary voltage 3000V, Cone voltage 50V, Collision voltage 30V, Mep detector voltage 2000V.
Peptide preface example analysis software is the Biolynx of Micromass company.
Analytical method electrospray mass spectrometer analytical method
Test result
Find out from total mass spectrum in I district, contain 20 polypeptide approximately, about 5 of relative amount the higher person [seeing 2022611408 (16.707) M3] wherein, their molecular ion peak [M+H]+be respectively 1218.69,1240.67,1251.69,1322.74,1452.81 (molecular weight=molecular ion peaks-1.0078).
Find out from total mass spectrum in II district: o'clock only find a tangible quasi-molecular ions [M+H] +=1251.73 in m/z＞700.
In 5 larger peptides in I district, select two bigger ions of intensity to carry out the tandem mass spectrum analysis, measure its peptide sequence.
Conclusion: contain the 20-30 peptide species in the Uropoly acid-peptide, their molecular weight 400～6000D, preferred molecular weight is 400-2800D.Wherein the sequence of two main peptides is
AVEGPSSALGPLGP
Alanine-valine-glutamic acid-glycine-proline-serine-serine-alanine-leucine-glycine-proline-leucine-glycine-proline, 14 peptides
LGPSTPGPPPNGGA
Leucine-glycine-proline-serine-threonine-proline-glycine-proline-proline-proline-asparagine-glycine-glycine-alanine, 14 peptides
4. conclusion
4.1 proved conclusively in the Uropoly acid-peptide existence of four kinds of organic acid and peptide composition with HPLC method and two kinds of methods of liquid one matter coupling chromatography, and the molecular weight 400～6000D of peptide composition, preferred molecular weight is 400-2800 D.See Figure 10.
Four kinds of organic acid structural identifications the results are shown in Table 11.
Four kinds of organic acid reference substances of table 11 and sample Mw contrast
| Theoretical value Mw | Hippuric acid 179.2 | Phenylacetylglutamine 264 | 4-hydroxyl phenylacetic acid 152.2 | 5-hydroxyindoleacetic acid 193.3 |
| Measured value Mw | ???180 | ????265 | ?????152 | ??????193 |
4.2 electron spray mass spectrometry has been proved conclusively peptide composition in the Uropoly acid-peptide, peptide species surplus this product contains 20 altogether, and its molecular weight 400～6000D, preferred molecular weight is 400～2800D, and has measured wherein with tandem mass spectrum that the sequence of two main polypeptide fractions is:
AVEGPSSALGPLGP
Alanine-valine-glutamic acid-glycine-proline-serine-serine-alanine-leucine-glycine-proline-leucine-glycine-proline, 14 peptides
LGPSTPGPPPNGGA
Leucine-glycine-proline-serine-threonine-proline-glycine-proline-proline-proline-asparagine-glycine-glycine-alanine, 14 peptides.
Table 12 is measured the peptide content result with Folin-phenol method
| Test number (TN) | ????1 | ????2 | ????3 |
| Total solid (mg) | ????417 | ????586.8 | ????665.4 |
| Total peptide amount (mg) | ????17.56 | ????28.29 | ????30.07 |
| Content of peptides (%) | ????4.21 | ????4.82 | ????4.52 |
The polypeptide average content is 4.52% in every as seen from Table 12 100mg Uropoly acid-peptide composition.
Experimental example 6
This experimental example relates to the sequencing of peptide composition in the frequent micturition acid phthalein compositions and partial peptide thereof.
Sample title: Uropoly acid-peptide composition (lot number 20020801)
Sample state: coffee-like lyophilized powder
Test item: seek peptide composition and measure the sequence of partial peptide
Examination criteria and other specification requirement:
(1) electrospray mass spectrometer analytical method detailed rules and regulations.
(2) testing conditions: cation detection mode.Capillary voltage 3000V, cone voltage 50V, Collision voltage 30V.MCP detector voltage 2200V.
(3) peptide sequence analysis software is the Biolynx of Micromass company.
Testing environment: temperature: 22 ℃
Detecting instrument title and model: Q-TOF2 ESI-MS/MS (Micromass)
The apparatus measures error is 0.01%.
Analytical method and test result:
One, analytical method
(1) The pretreatment:
1. take by weighing the about 10.0mg of test sample (Uropoly acid-peptide composition, coffee-like lyophilized powder) in 10ml tool plug scale test tube.
2. add the 5.0ml chloroform, fully concussion and supersound extraction 3 minutes (KQ-100 ultrasonic cleaner, Kunshan, Jiangsu Dianshan Lake detecting instrument factory).Left standstill 10 minutes, and the careful sucking-off of three formylmerphalan alkane was discarded with clean suction pipe.
3. add the 5.0ml petroleum ether, all the other steps are with 2.
4. repeating step 2 and step 3.
5. add the 5.0ml dehydrated alcohol, all the other steps are with 2.Repeat 3 times.Residue dries naturally stand-by (about 30 minutes).
(2) searching of peptide components and identification
The sample dissolution of above-mentioned processing is got 1 μ L after centrifugal and is carried out " capillary high performance liquid chromatography is received and risen electron spray-level Four bar flight time tandem mass spectrum " automated analysis in 5ml 1% formic acid deionized water.
(3) sequencing of partial peptide composition
The sample dissolution of above-mentioned processing is got 5 μ L after centrifugal and is carried out " receive and rise electron spray-quadrupole rod flight time tandem mass spectrum " and analyze in 5ml 1% formic acid deionized water.Select 1-2 main peak to carry out sequencing.
Two, result
1. the chromatography of ions figure of the sample of above-mentioned processing sees Figure 10 [20022611 Sm (SG, 2 * 6)], and it can be divided into three zones:
The retention time in I district is 14min～19min
The retention time in II district is 30min～45min;
The retention time in III district is 45min～55min.
2.I total mass spectrum in district is seen Figure 11 [20,022,611 408 (16.707) Cm (372:428)] (top, the upper right corner are corresponding chromatogram),
In m/z＞900 o'clock, do not find tangible quasi-molecular ions.See Figure 12 [20022611408 (16.707) M3] (bottom) with the collection of illustrative plates after the specific software MaxEnt3 conversion.In m/z=900～2300 scopes, find tangible quasi-molecular ions, and mainly concentrate in m/z=900～1500 scopes; And in m/z=300～900 scopes, still having the part ion peak, they are to be with unicharged molecule, wherein may comprise partial peptide.Relatively the mass spectrum before and after the conversion as can be seen: contain more than 20 peptide approximately in the chromatography of ions I district of test sample, about 5 of relative amount the higher person (seeing 5 of partial enlarged drawings such as [20,022,611 408 (16.707) M3]) (seeing Figure 13-17) wherein, their molecular ion peak [M+H]+be respectively 1218.69,1240.67,1251.69,1322.74,1452.81 (molecular weight=molecular ion peaks-1.0078).
3.II total mass spectrum in district is seen Figure 18 [20,022,611 864 (32.201) Cm (832:1161)] (top, the upper right corner are corresponding chromatogram),
In m/z＞700 o'clock, tangible quasi-molecular ions is found at the end.See Figure 19 [20022611864 (32.201) M3] P0 (bottom) with the collection of illustrative plates after the specific software MaxEnt3 conversion.In m/z＞700 o'clock, only find a tangible quasi-molecular ions [M+H] +=1251.73; And in m/z＜700 scopes quasi-molecular ions with the conversion before basically identical, they mainly are to be with unicharged organic molecule.Relatively the mass spectrum before and after the conversion as can be seen: peptide composition seldom in the chromatography of ions II district of test sample.
4.III total mass spectrum in district is seen Figure 20 [20,022,611 1438 (51.806) Cm (1324:1444)] (bottom, the upper right corner be correspondence chromatogram), in m/z＞700 o'clock, does not find tangible quasi-molecular ions.See Figure 21 [20,022,611 1438 (51.806) M3] (top) with the collection of illustrative plates after the specific software MaxEnt3 conversion.Mass spectrum before and after the conversion is almost consistent, and therefore, peptide composition seldom in the chromatography of ions III district of test sample.
5. select two bigger ions of intensity to carry out the tandem mass spectrum analysis:
The sequencing of m/z 626.36 the results are shown in Figure 22 20022615a, and Figure 23 20022615a-wkh-1 is seen in the sequence report, and sequence is:
AVEGPSSALGPLCGP。Single isotopic mass is 1250.7043Da, and value of calculation is 1250.6506Da, and deviation is 0.05Da.The sequencing of m/z 609.84 the results are shown in Figure 24 20022615b, and Figure 25 20022615b-wkh-1 is seen in the sequence report, and sequence is:
LGPSTPGPPPNGGA。Single isotopic mass is 1217.6644Da, and value of calculation is 1217.6040Da, and deviation is 0.06Da.
Three, conclusion
Contain the 20-30 peptide species in the Uropoly acid-peptide composition of the present invention, their molecular weight 400～6000D, wherein the sequence of two main peptides is:
AVEGPSSALGPLCGP
LGPSTPGPPPNGGA
Experimental example 7
This experimental example relates to the mensuration of total peptide content.
Sample state: dark-brown powder
Test item: total peptide content
Examination criteria and other specification requirement: ultraviolet and visible absorption spectra method general rule
Testing environment: temperature: 16 ℃, humidity: 31%
Detecting instrument title and model: HP-8453 ultraviolet-uisible spectrophotometer, DAD diode array detector
Wavelength accuracy: ± 0.6nm, absorbance accuracy: ± 0.05AU, resolution: 1nm
The analytical test result:
The pre-treatment of sample
1.1 precision takes by weighing 3 parts of an amount of semi-finished product, places the centrifuge tube of band stopper respectively, adds the 10mL carbon trichloride, ultrasonic 10 minutes, treat standing demix after, remove liquid with careful suction of suction pipe.
1.2 in above-mentioned centrifuge tube, add the mL petroleum ether, ultrasonic 10 minutes, with the 2000rpm rotating speed, centrifugal 2 minutes, abandoning supernatant.
1.3 add the 10mL absolute methanol again, ultrasonic 10 minutes, with the 2000rpm rotating speed, centrifugal 2 minutes, abandoning supernatant.
1.4 the operation under 1.3 of the triplicates.
1.5 dry up sample in the centrifuge tube with nitrogen.
The preparation of 2 standard solution
Take by weighing 10.6mg bovine serum albumin (Sigma company), water is settled to the 50mL volumetric flask, makes standard solution, and its concentration is 0.212mg.mL.
3 reagent preparation
3.1 4% sodium carbonate liquor: weighing sodium carbonate 2.0g, water are settled to the 50mL volumetric flask, shake up.
3.2 1% curpic carbonate solution: take by weighing sodium sulfate 20.0mg, water is settled to the 2mL volumetric flask, shakes up.
3.3 0.2molL
-1Sodium hydroxide solution: take by weighing the 0.40g sodium hydroxide, water is settled to the 50mL volumetric flask, shakes up.
3.4 2% potassium sodium tartrate solution: take by weighing sodium potassium tartrate tetrahydrate 40.0mg, water is settled to the 2mL volumetric flask, shakes up.
3.5 alkaline copper test solution: face and use preceding preparation.Get 4% sodium carbonate liquor and 0.2molL
-1Each 50mL of sodium hydroxide solution, 1% copper-bath and each 1mL of 2% potassium sodium tartrate solution shake up.
3.6 Folin-phenol test solution: 2.0molL
-1, purchase in Beijing through company of section, face with before being diluted with water to 1.0molL
-1
The preparation of 4 standard curves
Draw standard solution 0.00,0.20,0.40,0.60,0.80,1.00mL places 10 test tubes respectively, adds water to 1.00mL, adds alkaline copper test solution 5.00mL more respectively, shakes up, and room temperature was placed 10 minutes.Add phenol test solution 0.05mL successively, shake up.Put in 30 ℃ of water-baths and be incubated 30 minutes, taking-up is put and is chilled to room temperature, makes blank to add the standard solution pipe, measures trap at 750nm wavelength place, with protein concentration C (mgmL
-1) be abscissa, trap A is vertical coordinate production standard line taking (seeing Figure 26-28): C=0.3209 * A, r=0.999.
Half-finished mensuration
With 1.5 samples of handling down, water is settled to the 2.00mL volumetric flask, takes out 0.50mL and is diluted with water to 2.00mL, takes out 0.30mL again and adds water to 1.00mL, and all the other operations are with 4.Provide the concentration C value by the HP-8453 work station, see Figure 29 (04003065).
6 results calculate
C wherein: concentration (mgmL
-1), volume (6.50mL) when V1:Folin-phenol reacts, V2: the liquor capacity that from the volumetric flask of standardize solution, takes out (0.50mL), V3: the volume behind the compositions standardize solution (2.00mL), V4: with the liquor capacity (0.50mL) after the V2 dilution, V5: with the liquor capacity (0.30mL) that takes out among the V4
Table 13
| Numbering | ????1 | ????2 | ????3 |
| Trap A concentration is from C (mgmL -1) total peptide weight (mg) composition weight (mg) content % average content % standard deviation in the compositions | ????0.31562 ????0.10128 ????17.555 ????417.0 ????4.21 | ????0.50861 ????0.16321 ????28.289 ????586.8 ????4.82 ????4.52 ????0.31 | ????0.54066 ????0.17349 ????30.07 ????665.4 ????4.52 |
Experimental example 8
This experimental example relates to the quality control of Uropoly acid-peptide composition.
This experimental example Uropoly acid-peptide composition is from concentrating under reduced pressure, with the concentrated solution that obtains after the filtration of 1.2 μ m filter membranes.The content of polypeptide is 100mg～800mg among every 100ml; Hippuric acid content is 200mg～500mg, and phenylacetyl glutamy content is 250mg～600mg, and 4-hydroxyl phenylacetic acid content is 30mg～80mg, and 5-hydroxyindoleacetic acid content is 5mg～50mg.
[character] this product is the dark-brown clear liquid.
In the chromatogram that [discriminating] writes down under the assay item, the retention time at four kinds of organic acid peaks should be with the retention time at reference substance peak be consistent accordingly separately.
[inspection] pH value is got this product, measures (two appendix VI of Chinese Pharmacopoeia version in 2000 H) in accordance with the law, and pH value should be 6.5～7.5.
It is an amount of that the clarity of solution and color are got this product, 100 times of dilute with waters, and solution should be clarified.The apparent color of institute is measured at 430nm wavelength place according to spectrophotography (two appendix IV of Chinese Pharmacopoeia version in 2000 A), and the trap value must not cross 0.6.
The total solid precision is measured this product 2ml, places the evaporating dish of constant weight, behind the water bath method, is dried to constant weight at 105 ℃, and its total solid should be 200-300mg/ml.
The nitrogen content precision is measured this product 1ml, measures (two appendix VII of Chinese Pharmacopoeia version in 2000 D) in accordance with the law, and the nitrogen content of this product should be not less than 14mg/ml.
Polypeptide fractions is measured according to efficient liquid-liquid chromatography (two appendix V of Chinese Pharmacopoeia version in 2000 D) and is measured.The relative percentage composition of 3 main peptides should be not less than 50.0% in the gained chromatogram.
Particulate matter is got 1 bottle of this product, checks (two appendix IX of Chinese Pharmacopoeia version in 2000 C) in accordance with the law, should be up to specification.
Bacterial endotoxin is got this product, checks (Shen state pharmacopeia version appendix in 2000 XI E) in accordance with the law, and containing the endotoxin amount among every 1ml should be less than 0.5EU (sensitivity of the limulus reagent be 0.0625EU/ml).
[assay] measured according to high performance liquid chromatography (two appendix VD of Chinese Pharmacopoeia version in 2000).
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Methanol-water-glacial acetic acid (250: 750: 5) is a mobile phase; Flow velocity 1ml/min; The detection wavelength is 260nm.Number of theoretical plate calculates with the hippuric acid peak should be not less than 2500.
The preparation of reference substance solution
Precision takes by weighing 5-hydroxyindoleacetic acid reference substance 20mg, puts in the 50ml measuring bottle, with the dissolving of the hydroxide sodium solution of 0.05mol/L and be diluted to scale, as stock solution.
Precision takes by weighing hippuric acid reference substance 45mg, phenylacetylglutamine reference substance 60mg, 4-hydroxyl phenylacetic acid reference substance 45mg, put in the same 50ml measuring bottle, add the about 40ml dissolving of sodium hydroxide solution of 0.05mol/L, precision is measured heteroauxing reference substance stock solution 5.0ml again, put in the same 50ml measuring bottle, be diluted to scale with method, mixing, product solution in contrast.
The preparation of need testing solution
It is an amount of to get this product, and the solution that contains 8mg among every 1ml is made in the sodium hydroxide solution dilution that adds 0.05mol/L, promptly.
Algoscopy is got above-mentioned reference substance solution and each 20 μ l injecting chromatograph of need testing solution, and the record chromatogram uses external standard method with four kinds of organic acid content of calculated by peak area.
[determination of activity] following two kinds of cell strains are chosen any one kind of them to measure and are got final product.
Algoscopy: Hep-G2 hepatoma cell strain or CL.1-0 lung cancer cell line
Press Uropoly acid-peptide injection quality standard draft method and measure, IC50 answers≤2.0mg/ml.
Experimental example 9
This experimental example relates to HIV deactivation checking.
1. material
1.1 Uropoly acid-peptide composition (containing 73-75% ethanol): lot number 021001,021101,021102.
1.2 human immunodeficiency virus: HIV-1 IIIB strain
1.3 MT4 cell
1.4 cell culture fluid: RPMI 1640 adds 10% newborn calf serum
1.5 96 porocyte culture plates
2. method
Get the Uropoly acid-peptide composition 0.9ml that contains 73-75% ethanol, add HIV-1 (TCID50=7.25) 0.1ml, respectively at 0,1/4,1,2,6 hour sampling 0.1ml, do 10 times of serial dilutions with cell culture fluid at once, be added in 96 orifice plates that contain the MT4 suspension, adsorb and change liquid after 1 hour, sucking-off 0.1ml supernatant replenishes the fresh cell culture fluid of 0.1ml.If blank (sample adds virus), positive control (culture fluid adds virus), negative control (sample adds culture fluid).With the titre of MT4 cell micro culture method mensuration HIV, with the index of cytopathy (CPE) as the existence of differentiation virus.Use cell culture blind passage three generations to cytopathic sample not occurring, continue the situation of observation of cell pathological changes.
3. result
021001 batch sample is carried out duplicate detection three times, and 021101 and 021102 batch sample is respectively carried out one-time detection, the results are shown in Table 14, to the continuous blind passage three generations of the sample that pathological changes do not occur, the results are shown in Table 15.
The result of table 14 three batch sample titration of virus
| The sample lot number | The checking number of times | HIV titre (TCID 50) | ||||
| ????0hr | ????1/4hr | ????1hr | ????2hr | ????6hr | ||
| ????021001 | For the first time | ????5.55 | ????＜2.00 | ????＜2.00 | ????＜2.00 | ????＜2.00 |
| For the second time | ????5.23 | ????＜2.00 | ????＜2.00 | ????＜2.00 | ????＜2.00 | |
| For the third time | ????5.00 | ????＜2.00 | ????＜2.00 | ????＜2.00 | ????＜2.00 | |
| ????021101 | For the first time | ????5.55 | ????＜2.00 | ????＜2.00 | ????＜2.00 | ????＜2.00 |
| ????021102 | For the first time | ????5.23 | ????＜2.00 | ????＜2.00 | ????＜2.00 | ????＜2.00 |
| Positive control | ????7.25 | ????7.23 | ????7.25 | ????7.23 | ????7.25 | |
The triple-substituted result of table 15 blind passage (CPE)
| The sample lot number | The checking number of times | Sample time | The first generation | The second filial generation | The third generation | |||
| ??10 2 | ?10 3 | ??10 2 | ??10 3 | ??10 2 | ??10 3 | |||
| ??021001 | For the first time | ??1/4hr | ???- | ??- | ???- | ???- | ???- | ???- |
| ??1hr | ???- | ??- | ???- | ???- | ???- | ???- | ||
| ??2hr | ???- | ??- | ???- | ???- | ???- | ???- | ||
| ??6hr | ???- | ??- | ???- | ???- | ???- | ???- | ||
| For the second time | ??1/4hr | ???- | ??- | ???- | ???- | ???- | ???- | |
| ??1hr | ???- | ??- | ???- | ???- | ???- | ???- | ||
| ??2hr | ???- | ??- | ???- | ???- | ???- | ???- | ||
| ??6hr | ???- | ??- | ???- | ???- | ???- | ???- | ||
| For the third time | ??1/4hr | ???- | ??- | ???- | ???- | ???- | ???- | |
| ??1hr | ???- | ??- | ???- | ???- | ???- | ???- | ||
| ??2hr | ???- | ??- | ???- | ???- | ???- | ???- | ||
| ??6hr | ???- | ??- | ???- | ???- | ???- | ???- | ||
| ??021101 | For the first time | ??1/4hr | - | - | - | - | - | - |
| ??1hr | - | - | - | - | - | - | ||
| ??2hr | - | - | - | - | - | - | ||
| ??6hr | - | - | - | - | - | - | ||
| ??021102 | For the first time | ??1/4hr | - | - | - | - | - | - |
| ??1hr | - | - | - | - | - | - | ||
| ??2hr | - | - | - | - | - | - | ||
| ??6hr | - | - | - | - | - | - |
Annotate :-expression cell does not have pathological changes.
4. conclusion
From The above results as can be seen, HIV-1 is added in the Uropoly acid-peptide composition that contains 73-75% ethanol, soak after 6 hours, and 5.23-5.25 Log of titre decline, and cytopathy does not appear through cell culture blind passage three generations.
Experimental example 10
This experimental example relates to the effect assessment that ethanol inactivation of viruses and membrane filtration in the Uropoly acid-peptide extraction process remove virus
1. purpose:
Be to estimate the effect that 75% ethanol is removed poliovirus (plan HAV) to the inactivating efficacy and the membrane filtration of Sindbis (intending HCV) and Pseudorabies virus (intending HBV) in the Uropoly acid-peptide production technology.
2. experiment material:
(1) indicator virus: with Sindbis virus and Pseudorabies virus indicator virus, the indicator virus of the no lipid-coated virus of gray nucleus virus conduct as lipid-coated virus.Above-mentioned virus is the indicator virus of defined in " the inactivation of virus rules " that Chinese Drug Administration 2002.5 issues.
(2) cell strain: Vero cell, BHK21 cell.They are by the MEM culture medium culturing that contains 10% hyclone.
(3) contain 75% ethanol Uropoly acid-peptide sample and the Uropoly acid-peptide semi-finished product that are used for membrane filtration: attached through alcohol desorption, the ethanol content in the Uropoly acid-peptide sample detects with gas chromatography, according to measurement result concentration of alcohol is adjusted to 75%, and PH is 4.5.The Uropoly acid-peptide semi-finished product that are used for membrane filtration are the semi-finished product through filtering with microporous membrane of the present invention.Above sample provides by producer.
(4) dehydrated alcohol (analytical pure): chemical reagent factory in Beijing produces.
(5) filter membrane: the Viresolve NFP column filter membrane that Millipore company produces, connect various terminal and sebific duct the previous day in experiment, rise the distilled water flushing filter membrane by producer's requirement with 5-10.It is standby to pack autoclave sterilization then.Because this kind filter membrane costs an arm and a leg, in order to reduce cost, membrane filtration three batch samples are adopted in this experiment.Method is after having filtered a batch sample, soaks this filter membrane respectively 2 hours with 0.2NNaOH and 0.2N HCl, and is extremely neutral with distilled water flushing respectively again.Dry the back and repack, disinfection with high pressure steam is used further to handle another batch sample.
(6) peristaltic pump: the BT00-100M peristaltic pump that Lange company in Baoding produces, be furnished with YZ 1515W pump head, filter pressure is 1-2bar.
3. experimental technique:
(1) control sample
Get 500ml and contain the alcoholic acid Uropoly acid-peptide sample in 75% left and right sides.Boil off ethanol (volume about remaining 15-20%) through placing vaporizer, return to original volume with the injection sterile saline.Sodium bicarbonate solution with 10% during experiment is transferred PH4.5-5.0 (being the pH value in the production technology), with the filtration sterilization of syringe needle filter.This boils off Uropoly acid-peptide sample that ethanol returns to original volume and to contain 75% left and right sides alcoholic acid Uropoly acid-peptide sample spectra absorption curve consistent.Get this and boil off Uropoly acid-peptide (PH4.5-5.0) the sample 9ml+1ml indicator virus (titre 7-7.5log) that ethanol returns to original volume, mixing is got 0.2ml+1.8ml culture fluid (containing 10% hyclone) immediately and is done dilution in 1: 10, is contrast in 0 o'clock.All the other samples are put in 25 ℃ ± 1 ℃ the incubator and are hatched 6h, get 0.2ml speed+1.8ml culture fluid (containing 10% hyclone) then and contrast as 6h.Sample adds in 96 well culture plates of inoculating cell after dilution immediately, cultivates observation of cell pathological changes situation behind the 72h.Do three batches of repetitions.
(2) handle sample:
Get and contain 75% left and right sides alcoholic acid Uropoly acid-peptide sample 6ml+1ml indicator virus+3ml dehydrated alcohol mixing (the ethanol final concentration is 75%), put in 25 ℃ ± 1 ℃ the incubator, respectively at 5,15,30,60,120,240,360min sampling 0.2ml+1.8ml culture fluid (containing 10% hyclone) does dilution in 1: 10, to stop alcoholic acid continuous action.Sample adds in 96 well culture plates of inoculating cell after dilution immediately, cultivates observation of cell pathological changes situation behind the 72h.Do three batches of repetitions.
(3) stop contrast:
Get 2ml and contain in the alcoholic acid Uropoly acid-peptide sample adding 18ml culture fluid in 75% left and right sides (containing 10% tire Niu Yinqing), do dilution in 1: 10, be stop buffer.With 1: 100 times of virus dilution of this stop buffer (identical) and put in 25 ℃ ± 1 ℃ incubator and hatch, in 96 orifice plates that directly add inoculating cell after 0h and the 6h sampling, survey the virus titer that stops contrast with the viral dilution degree of sample treatment group.Every batch of experiment is all done simultaneously and is stopped contrast.
(4) membrane filter method: get the Uropoly acid-peptide semi-finished product 900ml that producer provides, add 100ml poliovirus (7.5LgTCID
50), do power with peristaltic pump behind the mixing and under 1-2bar pressure, promote sample and remove virus by the Viresolve NFP column membrane filtration that Millipore company produces.Sampling is done dilution in 1: 10 with the culture fluid that contains 10% hyclone and is inoculated in 96 well culture plates that cover with cell monolayer immediately, observation of cell pathological changes situation after 72h cultivates respectively before and after sample filtering.Do three batches of repetitions.
(5) blind passage: all be cell blind passage three generations through 75% Ethanol Treatment sample and the sample behind membrane filtration for every batch.
(6) titration of virus:
Adopt micromethod.The sample got of different time in above-mentioned each group behind 10 times of serial dilutions, is added in 96 orifice plates of inoculating cell immediately, put 5%CO
2In the incubator, hatch 72h for 37 ℃, observation of cell pathological changes (CPE) situation under the mirror.Use 1% violet staining cell plates simultaneously, saving result.Virus titer is pressed the KarberShi method and is calculated.
Annotate: first dilution factor of sample is owing to contain Uropoly acid-peptide and ethanol respectively before and after control sample, processing sample, termination contrast and the membrane filtration, must dilute with the culture fluid that contains 10% hyclone, to guarantee that adding cell plates has enough nutrition later on for three days need of cell growth, otherwise the dysgonic phenomenon of cell can occur, remaining dilution factor gets final product with keeping liquid.
4. result
(1) 75% ethanol is to the inactivating efficacy (table 16 and table 17) of Pseudorabies virus in the Uropoly acid-peptide:
Table 16 75% ethanol is to the inactivating efficacy of Pseudorabies virus in the Uropoly acid-peptide
| Group | Incubation time (min) | ?????????????????Lg?TCID 50 | ||
| Sample 1 (021101) | Sample 2 (021102) | Sample 3 (021103) | ||
| Processed group | ????5 | ????＜-0.5 | ????＜-0.5 | ????＜-0.5 |
| Processed group | ????15 | ????＜-0.5 | ????＜-0.5 | ????＜-0.5 |
| Processed group | ????30 | ????＜-0.5 | ????＜-0.5 | ????＜-0.5 |
| Processed group | ????60 | ????＜-0.5 | ????＜-0.5 | ????＜-0.5 |
| Processed group | ????120 | ????＜-0.5 | ????＜-0.5 | ????＜-0.5 |
| Processed group | ????240 | ????＜-0.5 | ????＜-0.5 | ????＜-0.5 |
| Processed group | ????360 | ????＜-0.5 | ????＜-0.5 | ????＜-0.5 |
| The sample contrast | ????0 | ????5.50 | ????5.13 | ????4.88 |
| The sample contrast | ????360 | ????4.13 | ????0.88 | ????1.13 |
| Stop contrast | ????0 | ????5.63 | ????5.25 | ????5.25 |
| Stop contrast | ????360 | ????5.13 | ????4.25 | ????4.13 |
Annotate: sample contrast 360min virus titer is on the low side to be because sample contrast pH value is adjusted due to the error.This shows that Pseudorabies virus is relatively more responsive to acid (low PH).
The blind passage result of table 17 75% ethanol after to Pseudorabies virus deactivation 360min in the Uropoly acid-peptide
| The sample lot number | A blind passage generation (4 days) | Blind passage secondary (8 days) | Blind passage three generations (12 days) |
| ????021101 | ????(-) | ????(-) | ????(-) |
| ????021102 | ????(-) | ????(-) | ????(-) |
| ????021103 | ????(-) | ????(-) | ????(-) |
| The cell contrast | ????(-) | ????(-) | ????(-) |
(2) 75% ethanol are to the inactivating efficacy (table 18 and table 19) of Sindbis virus in the Uropoly acid-peptide:
Table 18 75% ethanol is to the inactivating efficacy of Sindbis virus in the Uropoly acid-peptide
| Group | Incubation time (min) | ?????????????????????Lg?TCID 50 | ||
| Sample 1 (021101) | Sample 2 (021102) | Sample 3 (021103) | ||
| Processed | 5 | ＜-0.5 | ＜-0.5 | ＜-0.5 |
| | 15 | ＜-0.5 | ＜-0.5 | ＜-0.5 |
| | 30 | ＜-0.5 | ＜-0.5 | ＜-0.5 |
| | 60 | ＜-0.5 | ＜-0.5 | ＜-0.5 |
| | 120 | ＜-0.5 | ＜-0.5 | ＜-0.5 |
| | 240 | ＜-0.5 | ＜-0.5 | ＜-0.5 |
| | 360 | ＜-0.5 | ＜-0.5 | ＜-0.5 |
| The | 0 | 5.38 | 5.13 | 5.25 |
| The | 360 | 5.00 | 4.75 | 4.75 |
| Stop contrast | ??0 | ??5.63 | ??5.00 | ??5.13 |
| Stop contrast | ??360 | ??5.13 | ??4.88 | ??4.88 |
The blind passage result of table 19 75% ethanol after to Sindbis inactivation of virus 360min in the Uropoly acid-peptide
| The sample lot number | A blind passage generation (4 days) | Blind passage secondary (8 days) | Blind passage three generations (12 days) |
| ????021101 | ????(-) | ????(-) | ????(-) |
| ????021102 | ????(-) | ????(-) | ????(-) |
| ????021103 | ????(-) | ????(-) | ????(-) |
| The cell contrast | ????(-) | ????(-) | ????(-) |
(3) membrane filter method removes viral effect:
Table 20 membrane filter method is removed the effect of poliovirus
| The sample lot number | Lg TCID before the filter 50 | Filter back | 1 generation of cell blind passage |
| ????021101 | ????5.50 | ????-0.13 | ????(+) |
| ????021102 | ????5.63 | ????0.25 | ????(+) |
| ????021103 | ????5.63 | ????0.75 | ????(+) |
Annotate: the cell contrast is all negative in the blind passage experiment.
5. conclusion
(1) but 75% ethanol deactivation is tested in the Uropoly acid-peptide production technology Sindbis and pseudorabies indicator virus.Under 25 ℃ ± 1 ℃ condition, indicator virus more than 75% ethanol effect 5min, the inactivating efficacy 〉=5.38-6.00LgTCID of Pseudorabies virus
50, the inactivating efficacy of Sindbis virus 〉=5.63-5.88 LgTCID
50,, there is no cytopathic effect (CPE) with each batch sample blind passage three generations of 6 hours of 75% Ethanol Treatment.The termination control experiment shows that 75% ethanol no longer has deactivation to the indicator virus that is added after 10 times of dilutions.
(2) observed Viresolve NFP filter membrane can be removed 4.8-5.6 LgTCID in the test
50Poliovirus, cell blind passage all have cytopathy (CPE) to occur after 1 generation, can remove a certain amount of virus though membrane filter method is described, but can not remove the indicator virus that is added fully.
In addition, the result of table 20 also shows, new filter membrane uses for the first time that to remove viral result better, after handling through twice NaOH and HCl, filter membrane is used further to remove virus filtration, the effect of then removing virus slightly descends, and removes 4.88 LgTCID but still can reach
50Poliovirus.Therefore suggestion:, remove viral filter membrane and can only must not reuse with once in process of production for guaranteeing to remove viral effect.
The about 500ml of 1mol/L hydrochloric acid solution is added in the 20kg Freshman urine, and regulating pH is 3, with the filter cloth of 1 square metre of sheet frame filter and 20 μ m, in the filtering acidify urine greater than 20 μ m granule foreigns; With the tubular membrane ultrafiltration of molecular cut off 10,000, controlled pressure is 5Mpa, removes molecular weight in the acidify urine greater than 10,000 organic impurities, collects low molecule filtrate; Use XAD-16 as adsorbent then, consumption is 10 kilograms, earlier with 20 liters of soak with ethanol adsorbents 10 minutes, make adsorption column, reuse deionized water flush away ethanol is used 15 liters of ethanol and 25 liters of deionized water wash adsorption columns once more successively, will hang down molecule filtrate subsequently with in 1L/ minute the flow velocity adding post, added in about 20 minutes, with the flow velocity washing XAD-16 adsorption column of 20L purified water with 1L/ minute; The ethanol of reuse 10L carries out eluting with 1L/ minute flow velocity, makes the effective ingredient desorption that is adsorbed, and collects eluent;
Regulate concentration of alcohol to 73% with 95% medicinal alcohol then,, placed 6 hours for 2 ℃ 25 ℃ of scholars, with inactivation of viruses with 1mol/L hydrochloric acid adjust pH 4.0; Use the sodium hydroxide adjust pH 6.5 of 2mol/L subsequently, pump in the enamel pot, the beginning evacuation heats up, and makes the inner bag temperature be no more than 40 ℃, keeps being concentrated into 200mg/ml in about 8 hours; With the sodium hydroxide of 2mol/L fine setting pH value 7.0, be that the filter membrane filtration under diminished pressure of 1.2 μ m is removed impurity with the aperture, filtered solution through quality standard check Uropoly acid-peptide composition.
By analysis, the present embodiment Uropoly acid-peptide composition, effective ingredient comprises hippuric acid, phenylacetylglutamine, 4-hydroxyl phenylacetic acid, 5-hydroxyindoleacetic acid and a series of micromolecule polypeptide active component, wherein in the micromolecule polypeptide active component 26 peptide species are arranged, its maximum molecular weight is about 2800D, and minimum molecular weight is about 800D; Two main peptide sequence symbols are respectively: AVEGPSSALGPLGP (alavalgluglyproserseralaleuglyproleuglypro), 14 peptides; LGPSTPGPPPNGGA (leuglyproserthrproglyproproproasnglyglyala), L4 peptide.
By the urine extract is l00mg, wherein, is 100ml by the urine extract, and wherein the content of polypeptide is 600mg; Hippuric acid content is 300mg, and phenylacetyl glutamy content is 250mg, and 4-hydroxyl phenylacetic acid content is 80mg, and 5-hydroxyindoleacetic acid content is 30mg.Wherein be: hippuric acid 180, phenylacetic acid glutamine 265,4-hydroxyl phenylacetic acid 152,5-hydroxyindoleacetic acid 193 with four kinds of organic acid molecular weight of ES mass spectrometric determination.
With embodiment 1, different is again Uropoly acid-peptide composition to be prepared into injection; The Uropoly acid-peptide composition that is about to gained is diluted to the solution that contains total solid 40mg/ml with water for injection, with the hydrochloric acid adjust pH 7.5 of 2mol/L; The thermal source film that removes of reuse Millipore removes thermal source; With the aperture is the filter membrane of 0.22 μ m, aseptic filtration; With the filter membrane of 30nm Millipore, remove virus removal; Be packed as the Uropoly acid-peptide injection of 100ml/ bottle at last with filling and sealing machine.
The about 550ml of 1mol/L hydrochloric acid solution is added in the 20kg Freshman urine, and regulating pH is 2.5, with the filter cloth of 1 square metre of sheet frame filter and 20 μ m, in the filtering acidify urine greater than 20 μ m granule foreigns; Use tubular membrane ultrafiltration, controlled pressure is 8Mpa, removes the organic impurities of macromolecule in the acidify urine, collects filtrate; Use XAD-16 as adsorbent then, consumption is 15 kilograms, earlier with 25 liters of soak with ethanol adsorbents 15 minutes, make adsorption column, reuse deionized water flush away ethanol is used 20 liters of ethanol and 20 liters of deionized water wash adsorption columns once more successively, will hang down molecule filtrate subsequently with in 1.5L/ minute the flow velocity adding post, added in about 20 minutes, with the flow velocity washing XAD-16 adsorption column of 20L purified water with 1.5L/ minute; The ethanol of reuse 15L carries out eluting with 1L/ minute flow velocity, makes the effective ingredient desorption that is adsorbed, and collects eluent;
Regulate concentration of alcohol to 75% with 95% medicinal alcohol then,, placed 8 hours for 2 ℃ 25 ℃ of scholars, with inactivation of viruses with 1mol/L hydrochloric acid adjust pH 4.5; Use the hydrochloric acid adjust pH 6.0 of 2mol/L subsequently, pump in the enamel pot, the beginning evacuation heats up, and makes the inner bag temperature be no more than 40 ℃, keeps being concentrated into 250mg/ml in about 10 hours; With the sodium hydroxide of 2mol/L fine setting pH value 6.5, be that the filter membrane filtration under diminished pressure of 1.2 μ m is removed impurity with the aperture, filtered solution through quality standard check Uropoly acid-peptide composition.
By analysis, the present embodiment Uropoly acid-peptide composition, effective ingredient comprises hippuric acid, phenylacetylglutamine, 4-hydroxyl phenylacetic acid, 5-hydroxyindoleacetic acid and a series of micromolecule polypeptide active component, wherein in the micromolecule polypeptide active component 30 peptide species are arranged, its maximum molecular weight is about 4000D, and minimum molecular weight is about 400D;
By the urine extract is 100mg, wherein, is 100ml by the urine extract, and wherein the content of polypeptide is 800mg; Hippuric acid content is 200mg, and phenylacetyl glutamy content is 400mg, and 4-hydroxyl phenylacetic acid content is 50mg, and 5-hydroxyindoleacetic acid content is 5mg.
With embodiment 3, different is, again Uropoly acid-peptide composition is prepared into lyophilized injectable powder, the Uropoly acid-peptide composition of gained is diluted to the solution that contains total solid 30mg/ml with water for injection, with the hydrochloric acid adjust pH 7.0 of 3mol/L; The thermal source film that removes of reuse Millipore removes thermal source; With the aperture is the filter membrane of 0.22 μ m, aseptic filtration; With the filter membrane of 20nm Millipore, remove virus removal; Last fill (loading amount 2.5ml/ props up), Uropoly acid-peptide lyophilized injectable powder product is made in lyophilizing behind the false add plug.
The about 500ml of 1mol/L hydrochloric acid solution is added in the 20kg Freshman urine, and regulating pH is 2, with the filter cloth of 1 square metre of sheet frame filter and 20 μ m, in the filtering acidify urine greater than 20 μ m granule foreigns; With the tubular membrane ultrafiltration of molecular cut off 10,000, controlled pressure is 15Mpa, removes molecular weight in the acidify urine greater than 10,000 organic impurities, collects molecular weight and is lower than 10000 low molecule filtrate; Use XAD-16 as adsorbent then, consumption is 20 kilograms, earlier with 20 liters of soak with ethanol adsorbents 20 minutes, make adsorption column, reuse deionized water flush away ethanol is used 15 liters of ethanol and 25 liters of deionized water wash adsorption columns once more successively, will hang down molecule filtrate subsequently with in 11/ minute the flow velocity adding post, added in about 20 minutes, with the flow velocity washing XAD-16 adsorption column of 20L purified water with 1L/ minute; The ethanol of reuse 10L carries out eluting with 1L/ minute flow velocity, makes the effective ingredient desorption that is adsorbed, and collects eluent;
Regulate concentration of alcohol to 73% with 95% medicinal alcohol then,, placed 10 hours for 2 ℃ 25 ℃ of scholars, with inactivation of viruses with 1mol/L hydrochloric acid adjust pH 5.0; Use the sodium hydroxide adjust pH 6.3 of 2mol/L subsequently, pump in the enamel pot, the beginning evacuation heats up, and makes the inner bag temperature be no more than 40 ℃, keeps being concentrated into 200mg/ml in about 8 hours; With specification is 101 filter paper, and filtration under diminished pressure is removed impurity, with the sodium hydroxide fine setting pH value 5.5 of 2mol/L, is that the filter membrane filtration under diminished pressure of 1.2 μ m is removed impurity with the aperture, filtered solution through quality standard check Uropoly acid-peptide composition.
By analysis, the present embodiment Uropoly acid-peptide composition, effective ingredient comprises hippuric acid, phenylacetylglutamine, 4-hydroxyl phenylacetic acid, 5-hydroxyindoleacetic acid and a series of micromolecule polypeptide active component, wherein in the micromolecule polypeptide active component 20 peptide species are arranged, its maximum molecular weight is about 6000D, and minimum molecular weight is about 1000D;
By the urine extract is 100mg, wherein, is 100ml by the urine extract, and wherein the content of polypeptide is 100mg; Hippuric acid content is 500mg, and phenylacetyl glutamy content is 600mg, and 4-hydroxyl phenylacetic acid content is 30mg, and 5-hydroxyindoleacetic acid content is 50mg.
With 30 gram Uropoly acid-peptide semi-finished product and 80 gram starch mixed pelletizations, cross 20 mesh sieve granulate, drying adds 2 gram magnesium stearate compactings in flakes, promptly gets the Uropoly acid-peptide composition tablet.
More than given embodiment be in order to further specify the specific embodiments of the invention scheme, feature of the present invention, advantage and other details are not to be used for limiting protection scope of the present invention; Any variation of being done in the present invention's design all should be among scope of the present invention.
Claims (10)
1. the preparation method of a Uropoly acid-peptide composition comprises the steps:
Fresh Urina Hominis through acidify, is filtered, and absorption behind the eluting, is characterized in that, with the eluent collected through low pH value ethanol inactivation of virus after, again through concentrating under reduced pressure, filter Uropoly acid-peptide composition.
2. the preparation method of a kind of Uropoly acid-peptide composition according to claim 1 is characterized in that, comprises the steps:
1) the Freshman urine of collecting is regulated acidity to pH between 2～3, remove by filter diameter in the acidify urine greater than 20 microns impurity, hold back macromole impurity through ultrafiltration again, filtrate;
2) then with filtrate after adsorption column absorption, in purified water washing adsorption column, impurity again and use the ethanol elution adsorption column, collect eluent;
3) described eluent hangs down pH value ethanol inactivation of virus;
4), get Uropoly acid-peptide composition after the filtration again through concentrating under reduced pressure.
3. the preparation method of a kind of Uropoly acid-peptide composition according to claim 2 is characterized in that, wherein regulates urine pH in the step 1) between 2～3, regulates the acidity of urine and can use hydrochloric acid, sulphuric acid and be fit to other acid of the present invention; Described filtration can be adopted the filter in 20 μ m apertures, with in the filtering acidify urine greater than 20 μ m granule foreigns; Ultrafilter membrane is adopted in described ultrafiltration, and the pressure of ultrafiltration is controlled to be 5～15Mpa.
4. the preparation method of a kind of Uropoly acid-peptide composition according to claim 2, it is characterized in that, step 2) adsorption column absorption is adopted in the absorption described in, adsorbent in the adsorption column can be selected C18 post, XAD-7, XAD-8 or XAD-16 for use and be fit to other adsorbents of the present invention, preferred XAD-16 post and XAD-7.
5. according to the preparation method of claim 2 or 4 described a kind of Uropoly acid-peptide compositions, it is characterized in that, step 2) concrete steps are: before adsorption column uses, wash adsorbent respectively with pure and mild deionized water, the dress adsorption column, add in the adsorption column with the acidify urine of certain flow velocity after, with the effective ingredient in the adsorbents adsorb filtrate ultrafiltration; In the acidify urine a large amount of inorganic ions and Organic substance are arranged, what be not adsorbed after entering adsorbent lodges in the post, is not adsorbed with the soft water washing but remains in inorganic matter and hydrophilic organics hydrophilicity in the adsorbent, the purification effective ingredient; Be adsorbed on effective ingredient on the adsorbent with pure eluting; Collect eluent.
6. the preparation method of a kind of Uropoly acid-peptide composition according to claim 5 is characterized in that, with respect to 20 kilograms of freshly voided urines, the consumption of adsorbent is 5～20 kilograms, is preferably 10～20 kilograms; The consumption of the alcohol of above-mentioned immersion adsorbent is 15～25 liters, and soak time is 5～20 minutes; Preferable amount is 18～25 liters, and soak time is 10～20 minutes; The consumption of the pure and mild deionized water of washing adsorbent is 15～25 liters, and alcohol is 1～2: 2～4 with the ratio of purified water; The flow velocity that acidify urine adds adsorption column is 0.1～2L/min, is preferably 1～2L/min; Washing is 5～30L with the consumption of purified water, is preferably 15～30L; Elution speed is 0.1～3L/min, is preferably 1～2L/min; The consumption of eluent alcohol is 5～20L, is preferably 10～20L; Elution speed is 0.1～3L/min, is preferably 0.5～2L/min; Above-mentioned eluting can use ethanol, methanol, propanol and be fit to other alcohols of the present invention, preferred alcohol.
7. the preparation method of a kind of Uropoly acid-peptide composition according to claim 2, it is characterized in that low pH value ethanol inactivation of virus is in the step 3): concentration of alcohol is 70%～80% in the adjusting eluent, and adjust pH to 4.0～5.5, under 20 ℃～27 ℃, placed 4～8 hours.
8. the preparation method of a kind of Uropoly acid-peptide composition according to claim 7, it is characterized in that, used ethanol is good with 95%～99% medicinal alcohol, and adjust pH can and be fit to other acid of the present invention with 1～2mol/L hydrochloric acid, sulphuric acid, is preferably hydrochloric acid.
9. the preparation method of a kind of Uropoly acid-peptide composition according to claim 2, it is characterized in that, concentrating under reduced pressure described in the step 4) is: with sodium hydroxide, sodium carbonate, sodium bicarbonate and the suitable adjusting PH with base value 5.5～6.5 of the present invention of 1～3mol/L, used alkali is preferably sodium hydroxide earlier; Evacuation heats up then, and vacuum degree control is 0.08～0.1MPa, and temperature is no more than 40 ℃, and be 8～10 hours heat time heating time, and being concentrated into the solution solids total amount is 200～300mg/ml.
10. the preparation method of a kind of Uropoly acid-peptide composition according to claim 2 is characterized in that, described Uropoly acid-peptide composition can be made into injection, powder ampoule agent for injection, tablet or capsule etc., is preferably injection; The preparation method of injection is: be diluted to the solution that contains total solid 30～50mg/ml with water for injection, sodium hydroxide or hydrochloric acid adjust pH 6.5～7.5 with 1～3mol/L, remove thermal source with removing the thermal source film then, and, promptly get the Uropoly acid-peptide injection at last through the degerming membrane filtration with except that viral filter membrane sterilization; Described remove the thermal source film be Millipore remove the thermal source film, it is 0.20～0.30 micron filter membrane that described degerming filter membrane adopts the aperture, removes the filter membrane that viral filter membrane can adopt 20～50 nanometer NFP Filters.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2004100007539A CN1640489A (en) | 2004-01-16 | 2004-01-16 | Method for preparing urine polyacid peptide composition |
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| CNA2004100007539A CN1640489A (en) | 2004-01-16 | 2004-01-16 | Method for preparing urine polyacid peptide composition |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101991601B (en) * | 2009-08-10 | 2012-09-26 | 张义兴 | Method for preparing uroacitide composition |
| CN107661355A (en) * | 2017-09-29 | 2018-02-06 | 泰凌生物制药江苏有限公司 | A kind of method for the inactivation of virus being used in Uropoly acid-peptide semi-finished product preparation process |
| CN107693543A (en) * | 2017-09-29 | 2018-02-16 | 泰凌生物制药江苏有限公司 | A kind of method of inactivation of virus for Uropoly acid-peptide preparation technology |
| CN113041285A (en) * | 2019-12-27 | 2021-06-29 | 国药集团北京华邈药业有限公司 | Method for rapidly processing qualified nux vomica powder product |
-
2004
- 2004-01-16 CN CNA2004100007539A patent/CN1640489A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101991601B (en) * | 2009-08-10 | 2012-09-26 | 张义兴 | Method for preparing uroacitide composition |
| CN107661355A (en) * | 2017-09-29 | 2018-02-06 | 泰凌生物制药江苏有限公司 | A kind of method for the inactivation of virus being used in Uropoly acid-peptide semi-finished product preparation process |
| CN107693543A (en) * | 2017-09-29 | 2018-02-16 | 泰凌生物制药江苏有限公司 | A kind of method of inactivation of virus for Uropoly acid-peptide preparation technology |
| CN113041285A (en) * | 2019-12-27 | 2021-06-29 | 国药集团北京华邈药业有限公司 | Method for rapidly processing qualified nux vomica powder product |
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