CN107661355A - A kind of method for the inactivation of virus being used in Uropoly acid-peptide semi-finished product preparation process - Google Patents
A kind of method for the inactivation of virus being used in Uropoly acid-peptide semi-finished product preparation process Download PDFInfo
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- CN107661355A CN107661355A CN201710910345.4A CN201710910345A CN107661355A CN 107661355 A CN107661355 A CN 107661355A CN 201710910345 A CN201710910345 A CN 201710910345A CN 107661355 A CN107661355 A CN 107661355A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/22—Urine; Urinary tract, e.g. kidney or bladder; Intraglomerular mesangial cells; Renal mesenchymal cells; Adrenal gland
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
- A61L2/18—Liquid substances or solutions comprising solids or dissolved gases
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2202/00—Aspects relating to methods or apparatus for disinfecting or sterilising materials or objects
- A61L2202/20—Targets to be treated
- A61L2202/21—Pharmaceuticals, e.g. medicaments, artificial body parts
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Abstract
The invention discloses a kind of method for the inactivation of virus being used in Uropoly acid-peptide semi-finished product preparation process, comprise the following steps：A. urine is collected, carries out acidizing pretreatment；B. by pretreated acidifying urine ultrafiltration membrance filter；C. resin adsorption, washing, elution are carried out after the filtrate after ultrafiltration being carried out into pH value adjustment and collects eluent；D. the eluent of collection is adjusted into pH value, concentration of alcohol and temperature and carries out inactivation of virus；E. pH value is adjusted after inactivation terminates to 6.0~6.5, carries out being concentrated to give concentrate；F. the concentrate adjustment pH value is filtered, and obtains Uropoly acid-peptide semi-finished product.The present invention by preparing Uropoly acid-peptide semi-finished product during with organic solvent carries out inactivation of virus, it is simple and feasible, avoid the risk that the virus of Uropoly acid-peptide product infects.
Description
Technical field
The invention belongs to Uropoly acid-peptide fabricating technology field, and in particular to one kind is used for Uropoly acid-peptide semi-finished product and prepared
During carry out inactivation of virus method.
Background technology
Uropoly acid-peptide is to be less than 6000D polypeptides containing a variety of organic acids and molecular weight made from extraction purification from Male urine
Deng composition, for its main component by hippuric acid, phenylacetylglutamine and polypeptide etc., its character is dark-brown clear liquid, is had
Off-odor.
Uropoly acid-peptide injection is mainly used in the treatment of advanced breast cancer, non-tiny born of the same parents' patients with lung cancer.
CN 1052333248A disclose application of the Uropoly acid-peptide in scar medicine is treated.
Uropoly acid-peptide is the active ingredient extracted from Male urine, is to belong to multicomponent biochemical drug, state food medicine
Product supervision and management general bureau constantly pays close attention to the security of biochemical drug, puts into effect《Biological tissue extracted product and eukaryotic cell expression
The Viral safety assessment technique of product evaluates rule》、《Blood product removal/inactivation of viruses technical method and checking refer to
Lead principle》With《Multicomponent biochemistry medicine injection main technique requirements》A series of safety requirements, process of the Uropoly acid-peptide in preparation
In should have specific remove or the method for inactivation of viruses.Traditional method on inactivation of virus has not including as follows at present
It is several.
Dry thermoinactivation：(80 DEG C of 72h or 100 DEG C of 30min) its principle by the selection to temperature and action time, makes disease
Malicious structural damage, so that virus loses infection activity.
S/D factures：(1%Ttitonx100+0.3%TNBP more than 4 hours or 1%Ttiton80+0.3%TNBP24
DEG C more than 6 hours) its principle is that organic solvent can make lipoid be come off from virus surface, make virus structure destroyed so as to lose
Infection activity.
Membrane filter method：Nano-film filtration virus removal technology is that product is filtered using the filter membrane between 15-45nm,
Using principle is sieved, in nano-film filtration, the viral or other pathogen more than filter sizes is trapped within film, chi
Very little less protein product can then continue to retain in the product by filter membrane.
Irradiation：Pass through alpha ray and ultraviolet treatment with irradiation.This physical method handles multicomponent Biochemical Drugs system
Product will not remain the material endangered now in product.
Several ablation methods above, are not particularly suited for Uropoly acid-peptide semi-finished product and apply in process.
Patent application 99122050.1 discloses a kind of preparation method of Uropoly acid-peptide composition, by fresh uric acid, mistake
Filter out the bulky grain in acidifying urine；Ultrafiltration removes the larger organic substance of acidifying urine middle-molecular-weihydroxyethyl, and the acidifying after ultrafiltration is urinated
Add in adsorption column, adsorb active ingredient therein；The not to be adsorbed dose of nothing adsorbed but remained in adsorbent is washed with soft water
The active ingredient of machine thing and hydrophilic organics hydrophilicity with alcohol elution absorption on adsorption column；Coloured efflux is collected, after concentration, is dried
Obtain dry product；Be dissolved in water, pH adjusted to neutrality with alkali, stand at low temperature separates out the impurity formed in concentration process, through except thermal source,
Except the techniques such as virus are further purified.
Patent application 200410000753.9 discloses a kind of preparation method：By fresh human urine it is acidified, filtering, resin column
Absorption, washing.After de-, inactivate, then be concentrated under reduced pressure through low ph value ethanol, be filtrated to get Uropoly acid-peptide composition.
The preparation process of Uropoly acid-peptide is aforementioned patents disclosed, but it is not specific to Viral inactivation techniques in preparation process
Research with openly.Not yet find at present about application of the Uropoly acid-peptide in preparation process about Viral inactivation techniques and report
Road.
The content of the invention
It is an object of the invention to provide a kind of method for the inactivation of virus being used in Uropoly acid-peptide semi-finished product preparation process, solution
Uropoly acid-peptide semi-finished product of having determined the problem of inactivation of virus, avoid the wind of the virus infection of Uropoly acid-peptide product in preparation process
Danger.
For achieving the above object, present invention employs following technical scheme：
A kind of method for the inactivation of virus being used in Uropoly acid-peptide semi-finished product preparation process, it is characterised in that
Comprise the following steps：
A. urine is collected, carries out acidizing pretreatment；
B. by pretreated acidifying urine ultrafiltration membrance filter；
C. resin adsorption, washing, elution are carried out after the filtrate after ultrafiltration being carried out into pH value adjustment and collects eluent；
D. the eluent of collection is adjusted into pH value, concentration of alcohol and temperature and carries out inactivation of virus；
E. pH value is adjusted after inactivation terminates to 6.0~6.5, carries out being concentrated to give concentrate；
F. the concentrate adjustment pH value is filtered, and obtains Uropoly acid-peptide semi-finished product.
Preferably, urine is Male urine in the step a.
Preferably, acidizing pretreatment refers to hydrochloric acid adjustment urine ph values to 2.0~4.0 in the step a, then carries out sheet frame
The impurity for removing more than 20um is filtered, and Mm filter is used to remove more than 5um impurity.
Preferably, the aperture of milipore filter can retain 10KD molecular weight in the step b.
Preferably, the filtrate progress pH value in the step c after ultrafiltration is adjusted to 2.0~3.0；The resin is macropore tree
Fat adsorbent, model XAD-16N, adsorbent volume are 1 with acidifying urine volume ratio:16.
Preferably, the absorption uses Pyrex post, and diameter is with high than being 1:5；Acidifying urine in extraction process, purifying
The volume ratio of water and ethanol is 4:4:1；Adsorption flow rate：Wash flow velocity：Elution flow rate is 3:3:1；Purified water is carried out after absorption to wash
Wash and carry out ethanol elution after being less than 120us/cm to electrical conductivity and collect eluent.
Preferably, the eluent of collection is adjusted into pH value to 4.5~5.0 with 2M sodium hydroxides in the step d；Use alcohol
Concentration of alcohol in meter measurement known volume eluent, adds more than 95% ethanol or the amount of purified water, adjusts the second of eluent
Determining alcohol is to 73%-75%；Temperature is set as 23 DEG C -27 DEG C, and soaking time is 6 hours.
Preferably, the extract solution after inactivation is adjusted into pH value to 6.0-6.5 with 2M sodium hydroxides in the step e；Then
By extract solution using being concentrated in vacuo, density is concentrated into 1.100-1.120.
Preferably, concentrate carries out microfiltration membrane with 2M sodium hydroxides adjustment pH value to 6.0~8.0 in the step f
Filtering, obtains Uropoly acid-peptide semi-finished product
The present invention is through Bio-pharmaceutical Technology Co., Ltd. Suzhou Auspicious's (numbering：LCJS/BG201605-TLSW-001) examine
Fixed, addition Pseudorabies virus (PRV), Xin Debishi viral (Sindbis), encephalomyocarditis virus (EMCV) are verified, inactivate
Significant effect, fat coating and non-lipid-coated virus can be inactivated.
Invention advantage：
The present invention by preparing Uropoly acid-peptide semi-finished product during with organic solvent carry out inactivation of virus, it is simple and feasible,
Avoid the risk of the virus infection of Uropoly acid-peptide product.
Brief description of the drawings
Fig. 1 is PRV titration curve figures under the conditions of organic solvent 74% (± 1%) ethanol acidity；
Fig. 2 is PRV titration curve figures under organic solvent 74% (± 1%) ethanol neutrallty condition；
Fig. 3 is Sindbis titration curve figures under the conditions of organic solvent 74% (± 1%) ethanol acidity；
Fig. 4 is Sindbis titration curve figures under organic solvent 74% (± 1%) ethanol neutrallty condition；
Fig. 5 is EMCV titration curve figures under the conditions of organic solvent 74% (± 1%) ethanol acidity；
Fig. 6 is EMCV titration curve figures under organic solvent 74% (± 1%) ethanol neutrallty condition.
Embodiment
Technical scheme is further described below in conjunction with preferred embodiment,
Inactivation of virus and the specific implementation step verified are carried out using the method for the invention：
Adjust urine extracting liquid pH value pH5.0 ± 0.1 (selecting the pH5.0 condition poor to inactivation of virus) and neutral urine
Concentration of alcohol is to 74% ± 1% in extract solution pH7.0 ± 0.1, in 23 DEG C of (selecting the temperature conditionss poor to inactivation of virus) water
Bath inactivation 6h, is measured by sampling virus titer in zero point, 0.25h, 0.5h, 1h, 2h, 4h and 6h respectively.
For further prove organic solvent ethanol to virus deactivation, while centering urine extract solution (7.0 ±
0.1) the ethanol inactivation of viruses under the conditions of is studied.
In acid (pH5.0 ± 0.1) and three kinds of indicator virus titre mean decreases of neutral extract solution (pH7.0 ± 0.1)
It see the table below 1-6；
PRV titre mean decreases in three batch samples after 23 DEG C of inactivations under the conditions of table 1 74% (± 1%) ethanol acidity
PRV titre mean decreases in three batch samples after the lower 23 DEG C of inactivations of table 2 74% (± 1%) ethanol neutrallty condition
Under the conditions of table 3 74% (± 1%) ethanol acidity after 23 DEG C of inactivations in three batch samples Sindbis titres averagely under
Depreciation
After the lower 23 DEG C of inactivations of table 4 74% (± 1%) ethanol neutrallty condition in three batch samples Sindbis titres averagely under
Depreciation
EMCV titre mean decreases in three batch samples after 23 DEG C of inactivations under the conditions of table 5 74% (± 1%) ethanol acidity
EMCV titre mean decreases in three batch samples after the lower 23 DEG C of inactivations of table 6 74% (± 1%) ethanol neutrallty condition
Three kinds of indicator virus population mean drop-out values are shown in Table 7 in three batch organic solvents processing sample
Three kinds of indicator virus titre population mean drop-out values in the batch organic solvent of table 7 three processing sample
It may indicate that from table 1 to table 7：
The 23 DEG C of zero points samplings under acid and neutrallty condition respectively of organic solvent 74% (± 1%) ethanol, PRV and
Sindbis virus titers are down to below test limit, in acid and neutral extract solution PRV virus titers population mean decline >=
4.3logs；Sindbis titres population mean declines >=5.0logs；
Organic solvent 74% (± 1%) ethanol in acid condition 23 DEG C inactivation 0.5h, EMCV viruses be down to test limit with
Under, titre population mean declines >=6.0logs；23 DEG C of inactivation 1h, EMCV viruses are down to below test limit in neutral conditions,
Titre population mean declines >=6.0logs；Illustrate that acid condition contributes to EMCV viruses faster to inactivate.
Three batch sample organic solvents in acid and neutral virus titer change curve as shown in figs. 1 to 6.
Three batch sample organic solvents are respectively from left to right to carry from X-axis in acid and neutral virus titer change curve
Take in liquid plus sampling (control be used as initial titer) during virus 0, (± 1%) ethanol of organic solvent 74% acidity (pH5.0 ±
0.1) sampled with neutral (pH7.0 ± 0.1) 23 DEG C of inactivation zero points, 0.25h, 0.5h, 1h, 2h, 4h and 6h；Three batch experiment data
Close, three curves essentially coincide.
From Fig. 1 to Fig. 6, three batch samples inactivation dynamic curve may indicate that：Organic solvent can inactivate at 23 DEG C zero
Below PRV and Sindbis viruses to the test limit of acid and neutral extract solution；0.5h is handled under acid condition, EMCV can be inactivated
Below virus to test limit；1h is inactivated under neutrallty condition, can be inactivated below EMCV viruses to test limit.
Pseudorabies virus (PRV), Xin Debishi viral (Sindbis), encephalomyocarditis virus are separately added into extract solution
(EMCV) pH value, is adjusted to 5.0 ± 0.1 with 2M sodium hydroxides, and regulation concentration of alcohol 74% (± 1%), temperature is at 23 DEG C, the time
6h, its organic solvent virus is inactivated, the as shown by data of checking.
Through 74% (± 1%) ethanol, 23 DEG C of zero point samplings, acidic extraction liquid (pH5.0 ± 0.1) and neutral extract solution
PRV titres are down to below test limit in (pH7.0 ± 0.1), titre population mean drop-out value >=4.3logs；
Through 74% (± 1%) ethanol, 23 DEG C of zero point samplings, acidic extraction liquid (pH5.0 ± 0.1) and neutral extract solution
Sindbis titres are down to below test limit in (pH7.0 ± 0.1), titre population mean drop-out value >=5.0logs；
Through 74% (± 1%) ethanol, 23 DEG C of zero point samplings, acidic extraction liquid (pH5.0 ± 0.1) and neutral extract solution
EMEC titres are down to below test limit in (pH7.0 ± 0.1), titre population mean drop-out value >=6.0logs；
Through 74% (± 1%) ethanol, 23 DEG C of inactivation 1h, EMEC titres are down to inspection in neutral extract solution (pH7.0 ± 0.1)
It is following to survey limit, titre population mean drop-out value >=6.0logs；
Three indicator viruses can be gone out completely in i.e. acid (pH5.0 ± 0.1) and neutral extract solution (pH7.0 ± 0.1)
It is living, it was demonstrated that Uropoly acid-peptide preparation technology organic solvent ethanol plays a leading role to inactivation of virus.
Organic solvent virus is inactivated in Uropoly acid-peptide preparation process, and inactivation of virus is imitated using in manufacturing condition
The worst condition of fruit, it is not optimal embodiment.Inactivated in its Uropoly acid-peptide preparation technology with organic solvent virus
Technique pH value 4.5-5.0；Concentration of alcohol 73%-75%；23 DEG C -27 DEG C of temperature, 6h is in practical range for insulation, its inactivation of virus
It is effective.
It is pointed out that as described above is only to explain the preferred embodiments of the invention, it is right according to this to be not intended to
The present invention makees any formal limitation, therefore all have any modification for making the relevant present invention under identical spirit
Or change, it should all be included in that the invention is intended to the category protected.
Claims (9)
- A kind of 1. method for the inactivation of virus being used in Uropoly acid-peptide semi-finished product preparation process, it is characterised in thatComprise the following steps：A. urine is collected, carries out acidizing pretreatment；B. by pretreated acidifying urine ultrafiltration membrance filter；C. resin adsorption, washing, elution are carried out after the filtrate after ultrafiltration being carried out into pH value adjustment and collects eluent；D. the eluent of collection is adjusted into pH value, concentration of alcohol and temperature and carries out inactivation of virus, inactivation adjusted after terminating pH value to 6.0~6.5, carry out being concentrated to give concentrate；F. the concentrate adjustment pH value is filtered, and obtains Uropoly acid-peptide semi-finished product.
- 2. the method for the inactivation of virus being used for according to claim 1 in Uropoly acid-peptide semi-finished product preparation process, its feature exist In urine is Male urine in the step a.
- 3. the method for the inactivation of virus being used for according to claim 1 in Uropoly acid-peptide semi-finished product preparation process, its feature exist In acidizing pretreatment refers in the step a adjusts urine ph values to 2.0~4.0 with hydrochloric acid, then carries out plate-frame filtering and be used to remove More than 20um impurity, and Mm filter are used to remove more than 5um impurity.
- 4. the method for the inactivation of virus being used for according to claim 1 in Uropoly acid-peptide semi-finished product preparation process, its feature exist In the aperture of milipore filter can retain 10KD molecular weight in the step b.
- 5. the method for the inactivation of virus being used for according to claim 1 in Uropoly acid-peptide semi-finished product preparation process, its feature exist In the filtrate in the step c after ultrafiltration carries out pH value and adjusted to 2.0~3.0；The resin is macroporous resin adsorption agent, type Number it is XAD-16N, adsorbent volume is 1 with acidifying urine volume ratio:16.
- 6. the method for the inactivation of virus being used for according to claim 5 in Uropoly acid-peptide semi-finished product preparation process, its feature exist In the absorption uses Pyrex post, and diameter is with high than being 1:5；Acidifying urine, purified water and ethanol in extraction process Volume ratio is 4:4:1；Adsorption flow rate：Wash flow velocity：Elution flow rate is 3:3:1；Purified water is carried out after absorption to wash to electrical conductivity Less than progress ethanol elution after 120us/cm and collect eluent.
- 7. the method for the inactivation of virus being used for according to claim 1 in Uropoly acid-peptide semi-finished product preparation process, its feature exist In by the eluent of collection 2M sodium hydroxides adjustment pH value to 4.5~5.0 in the step d；The body known to alcohol meter measures Concentration of alcohol in product eluent, more than 95% ethanol or the amount of purified water are added, adjust the concentration of alcohol of eluent to 73%- 75%；Temperature is set as 23 DEG C -27 DEG C, and soaking time is 6 hours.
- 8. the method for the inactivation of virus being used for according to claim 1 in Uropoly acid-peptide semi-finished product preparation process, its feature exist In by the 2M sodium hydroxides adjustment pH value to 6.0-6.5 of the extract solution after inactivation in the step e；Then extract solution is used It is concentrated in vacuo, is concentrated into density in 1.100-1.120.
- 9. the method for the inactivation of virus being used for according to claim 1 in Uropoly acid-peptide semi-finished product preparation process, its feature exist In concentrate carries out microfiltration membrane filtering with 2M sodium hydroxides adjustment pH value to 6.0~8.0 in the step f, and it is more to obtain urine Sour peptide semi-finished product.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1640489A (en) * | 2004-01-16 | 2005-07-20 | 合肥永生制药有限公司 | Method for preparing urine polyacid peptide composition |
| CN101991601A (en) * | 2009-08-10 | 2011-03-30 | 张义兴 | Method for preparing uroacitide composition |
| CN105233248A (en) * | 2015-09-08 | 2016-01-13 | 泰凌(中国)投资有限公司 | Application of uroacitides to preparation of cicatrix treatment medicines |
-
2017
- 2017-09-29 CN CN201710910345.4A patent/CN107661355A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1640489A (en) * | 2004-01-16 | 2005-07-20 | 合肥永生制药有限公司 | Method for preparing urine polyacid peptide composition |
| CN101991601A (en) * | 2009-08-10 | 2011-03-30 | 张义兴 | Method for preparing uroacitide composition |
| CN105233248A (en) * | 2015-09-08 | 2016-01-13 | 泰凌(中国)投资有限公司 | Application of uroacitides to preparation of cicatrix treatment medicines |
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Application publication date: 20180206 |