CN107211576B - With the method for single aldehyde compound inactivation of viruses - Google Patents
With the method for single aldehyde compound inactivation of virusesInfo
- Publication number
- CN107211576B CN107211576B CN201010047511.0A CN201010047511A CN107211576B CN 107211576 B CN107211576 B CN 107211576B CN 201010047511 A CN201010047511 A CN 201010047511A CN 107211576 B CN107211576 B CN 107211576B
- Authority
- CN
- China
- Prior art keywords
- inactivation
- virus
- aldehyde compound
- terminator
- viruses
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 230000002779 inactivation Effects 0.000 title claims abstract description 42
- 241000700605 Viruses Species 0.000 title claims abstract description 38
- -1 aldehyde compound Chemical class 0.000 title claims abstract description 26
- 239000007788 liquid Substances 0.000 claims abstract description 8
- 150000003384 small molecules Chemical class 0.000 claims abstract description 5
- 238000000502 dialysis Methods 0.000 claims abstract description 3
- 238000003756 stirring Methods 0.000 claims description 9
- 229910000090 borane Inorganic materials 0.000 claims description 8
- UORVGPXVDQYIDP-UHFFFAOYSA-N trihydridoboron Substances B UORVGPXVDQYIDP-UHFFFAOYSA-N 0.000 claims description 8
- DHMQDGOQFOQNFH-UHFFFAOYSA-N glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 6
- 229940012952 Fibrinogen Drugs 0.000 claims description 4
- 102000008946 Fibrinogen Human genes 0.000 claims description 4
- 108010049003 Fibrinogen Proteins 0.000 claims description 4
- 229940019698 Fibrinogen containing hemostatics Drugs 0.000 claims description 4
- 239000007795 chemical reaction product Substances 0.000 claims description 4
- 239000000376 reactant Substances 0.000 claims description 4
- 239000004471 Glycine Substances 0.000 claims description 3
- GETQZCLCWQTVFV-UHFFFAOYSA-N Trimethylamine Chemical group CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 claims description 3
- PIICEJLVQHRZGT-UHFFFAOYSA-N 1,2-ethanediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 claims description 2
- 102100001249 ALB Human genes 0.000 claims description 2
- 101710027066 ALB Proteins 0.000 claims description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 2
- 239000004472 Lysine Substances 0.000 claims description 2
- 238000007792 addition Methods 0.000 claims description 2
- 235000004279 alanine Nutrition 0.000 claims description 2
- 229940050528 albumin Drugs 0.000 claims description 2
- 125000002147 dimethylamino group Chemical group [H]C([H])([H])N(*)C([H])([H])[H] 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 238000002523 gelfiltration Methods 0.000 claims description 2
- 230000014759 maintenance of location Effects 0.000 claims description 2
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 2
- YOQDYZUWIQVZSF-UHFFFAOYSA-N sodium borohydride Substances [BH4-].[Na+] YOQDYZUWIQVZSF-UHFFFAOYSA-N 0.000 claims description 2
- ODGROJYWQXFQOZ-UHFFFAOYSA-N sodium;boron(1-) Chemical compound [B-].[Na+] ODGROJYWQXFQOZ-UHFFFAOYSA-N 0.000 claims description 2
- 238000000108 ultra-filtration Methods 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 6
- 239000002994 raw material Substances 0.000 abstract description 5
- 230000000415 inactivating Effects 0.000 abstract description 4
- 208000001756 Virus Disease Diseases 0.000 abstract description 2
- 238000006116 polymerization reaction Methods 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 19
- 239000000047 product Substances 0.000 description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 6
- 210000004027 cells Anatomy 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N HCl Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- IKHGUXGNUITLKF-UHFFFAOYSA-N acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- 108091006822 Human Serum Albumin Proteins 0.000 description 2
- 238000002679 ablation Methods 0.000 description 2
- 150000001299 aldehydes Chemical class 0.000 description 2
- 239000010836 blood and blood product Substances 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000009928 pasteurization Methods 0.000 description 2
- NBBJYMSMWIIQGU-UHFFFAOYSA-N propionic aldehyde Chemical compound CCC=O NBBJYMSMWIIQGU-UHFFFAOYSA-N 0.000 description 2
- 230000003612 virological Effects 0.000 description 2
- ZTQSAGDEMFDKMZ-UHFFFAOYSA-N 1-butanal Chemical compound CCCC=O ZTQSAGDEMFDKMZ-UHFFFAOYSA-N 0.000 description 1
- KKAXNAVSOBXHTE-UHFFFAOYSA-N Boranamine Chemical class NB KKAXNAVSOBXHTE-UHFFFAOYSA-N 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 206010011409 Cross infection Diseases 0.000 description 1
- 231100000614 Poison Toxicity 0.000 description 1
- 208000009305 Pseudorabies Diseases 0.000 description 1
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 239000003633 blood substitute Substances 0.000 description 1
- QXUAMGWCVYZOLV-UHFFFAOYSA-N boride(3-) Chemical compound [B-3] QXUAMGWCVYZOLV-UHFFFAOYSA-N 0.000 description 1
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 1
- 229910052796 boron Inorganic materials 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 201000009910 diseases by infectious agent Diseases 0.000 description 1
- 229940079593 drugs Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 230000004301 light adaptation Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000001954 sterilising Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- ZMANZCXQSJIPKH-UHFFFAOYSA-N triethylamine Chemical class CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 1
Abstract
A kind of method with single aldehyde compound inactivation of viruses, it is characteristic of the invention that adding single aldehyde compound solution in feedstock solution is extracted, carries out inactivation of virus；Add terminator and terminate inactivation；Carry out changing liquid finally by dialysis or other filter methods, and remove remnants small molecule.The present invention can be by Virus inactivating present in raw material, and the ability for making product no longer have virus infection on the premise of biological products function is not influenceed, improves the security of biological products；There is advantages below compared with prior art：It is directly added into, easy to use, inactivation time is short；The polymerization between albumen will not be caused；Can be efficiently by the inactivation of virus of all categories；The present invention is applied to the inactivation of virus using the extract of people source/animal sources as the biological products preparation process of raw material.
Description
Technical field
The invention belongs to biological product technical field, and in particular to (especially contain albumen in biological products
Product) production in virus inactivating method.
Background technology
The extract (such as albumin, fibrinogen, cell factor) originated in people or animal body
When preparing the biological products such as blood product, protein drug, biological dressing, to prevent the cross-infection of virus,
Its production technology must possess the ability for having removal/inactivation of viruses, should there is specific in process of production
The method of removal/inactivation of viruses.Because many blood products belong to national defence war preparedness medicine, therefore this hair
The method of bright inactivation of virus has Practical significance to national defence material for preparedness against war.
Due to the material of inhomogeneity biological source, the species that it has Virus Pollution is different, selected virus
The emphasis of removal/ablation method is also different, so have the method for a variety of removal/inactivations.Conventional
Method has：(1) pasteurization (pasteurization), (2) dry heating method (mainly for freeze-dried products), (3)
Organic solvent/detergent (S/D) facture, (4) membrane filter method, (5) incubation at low pH value, (6) chemical ablation
Method.Various methods have certain use condition, and the species of removal/inactivation of viruses also has certain model
Enclose, some methods can not be used alone, need to be used in combination with other method.And for the disease in extract
Poison inactivation, usual processing time is longer, treating capacity is limited, inactivation of virus is not thorough enough, and cost is higher.
Many aldehyde compounds, as conventional virus-inactivating agent, are a kind of very effective using glutaraldehyde
Virus inactivating method, but this method applies in general to the surface sterilization of environment or vessel, and application method is spray
The mode spilt or soaked.Because many aldehyde compounds can cause protein-crosslinking, change the property of protein.
United States Patent (USP) US6914127B2, US5895810, US5905141 etc. are used as crosslinking agent using glutaraldehyde
Blood substitute is prepared, because the addition speed of glutaraldehyde is very slow and consumption is smaller, glutaraldehyde can be quick
React and be consumed with the amido of protein surface, make the concentration of free glutaraldehyde in solution very small, therefore
Do not possess the ability of inactivation of virus；Glutaraldehyde is had no for inactivation of virus and had been reported that, other use are also had no
Aldehyde radical molecule carries out the report of inactivation of virus to biological products.
The content of the invention
It is an object of the invention to provide a kind of method with single aldehyde compound inactivation of viruses, it is allowed to be applied to
The inactivation of virus of biological products is prepared as raw material using people/animal origin extract, will can be existed in raw material
Virus inactivating, make product no longer have virus infection ability, do not influenceing biological products function
On the premise of effect, the security of biological products is improved.
Method with single aldehyde compound inactivation of viruses proposed by the invention, specific steps include：
Step 1: the extraction feedstock solution adjustment pH that the human or animal for needing inactivation of virus is originated is big
In 6.0；Single aldehyde compound solution is added, the final concentration of free list aldehyde compound in mixed liquor is existed
0.1%~3%, maintain inactivation 0.2~3 hour；Its concentration and inactivation time are the differences according to viral species
It is adjusted, described single aldehyde compound is any for formaldehyde or acetaldehyde or propionic aldehyde or butyraldehyde；
Step 2: being stirred according to the mol ratio of single aldehyde compound and terminator for 1 to 2~100 amount
Terminator is added, stirring terminates inactivation；Described terminator is the boride with reduction, can be with
Select any of dimethylamino borine or trimethylamine groups borine or triethyamino borine or sodium borohydride
Kind；To make termination more abundant, before terminator is added, according to single aldehyde compound and amine compound
Mol ratio adds amine compound for 1 to 5~100 amount stirring, adds terminator and terminates inactivation；
Amine compound is any of glycine or lysine or alanine or ethylenediamine；
Step 3: the mixed liquor after termination is changed with the method for dialysis or ultrafiltration or gel filtration
Liquid, removes remnants small molecule reactant and reaction product, is replaced into and retention and next step process phase
The solution of adaptation.
The inactivation that the present invention is used for biological products using single aldehyde compound will not cause the crosslinking of protein,
And the mechanism that single aldehyde compound kills virus is unclear, it may be possible to by aldehyde radical and cell surface or inside
The amino of protein reacts with reference to caused by, causes the inactivation of virus.
The present invention has advantages below compared with prior art：1. it is directly added into, easy to use, inactivation
Time is short, after aldehydes molecule is added, and can all be inactivated viral typically within 30min；2. not
The polymerization between albumen can be caused；3. single aldehyde compound in higher concentrations, can efficiently will be entirely
The inactivation of virus of portion's species；4. in the basic conditions, the deactivation of aldehydes molecule can be stronger；5. pass through
Cross the crystalloid fluid changed in liquid process, solution and be replaced into the solution being adapted with next step process.The present invention
Suitable for the inactivation of virus using the extract of people source/animal sources as the biological products preparation process of raw material.
Embodiment
Illustrate the embodiment of technical solution of the present invention below in conjunction with instantiation.
Inactivation of virus is tested：
Step 1: taking the PINPROL 90mi that concentration is 10mg/ml, 10ml porcine pseudorabies are added
Malicious (PRV)；PH to 8 is adjusted with sodium hydroxide (or hydrochloric acid) solution；Sample 20ml；
Step 2: the formalin 10ml for taking concentration to be 1%, is added in above-mentioned solution, stirs
Afterwards in 20min, 40min, the separately sampled 20ml of 60min；It is separately added into 1mol/L diformazans
Aminoboranes 2ml, stirring reaction 0.3 hour；
Step 3: above-mentioned each sample is dialysed with 2L physiological saline respectively, liquid one is changed within every 4 hours
It is secondary, liquid is changed altogether 4 times, obtain detecting sample；Detect that virus is remaining in each sample respectively with cell infection method
Titre, cell be Syria hamster kidney cell line (BHK-21)；Assay is shown, before inactivation
Sample outside, in other all samples virus titer drop be more than 4log values, by blind passage, three generations tests,
Do not find that cell is abnormal, virus-free toxicity yet.
Example 1,
Step 1: the pig fibrinogen 9 0ml that concentration is 10mg/ml is taken, with sodium hydroxide (or salt
Acid) solution tune pH to 8；
Step 2: the acetaldehyde solution 10ml for taking concentration to be 1%, is added in above-mentioned solution, stirs
Maintain 40 minutes afterwards；Add 1mol/L trimethylamine groups borine 10ml, stirring reaction 0.5 hour；
Step 3: the mixed liquor after termination is carried out with 30kDa milipore filters with physiological saline to change liquid, remove
Remaining small molecule reactant and reaction product, until Boron contents are less than 1ug/mL, obtain inactivation of virus
Fibrinogen solution.
Example 2,
Step 1: the human serum albumin 90ml that concentration is 40mg/ml is taken, with sodium hydroxide (or hydrochloric acid)
Solution adjusts pH to 9；
Step 2: the propionic aldehyde solution 10ml for taking concentration to be 1.5%, is added in above-mentioned solution, stirring is equal
Maintained 60 minutes after even；0.5mol/L glycine solution 10ml are added, 1mol/L triethylamines are added
Base borine 15ml stirring reactions 1 hour；
Step 3: the mixed liquor after termination is loaded into bag filter, carried out with physiological saline as dialyzate
Analysis, removes remnants small molecule reactant and reaction product, liquid is changed once per 4h, carry out 4 times altogether,
Resulting solution is the human albumin solution after inactivation of virus.
Claims (3)
1. a kind of method with single aldehyde compound inactivation of viruses, it is characterised in that specific steps include：
Step 1: the extraction feedstock solution adjustment pH that the human or animal for needing inactivation of virus is originated is big
In 6.0；Single aldehyde compound solution is added, the final concentration of free list aldehyde compound in mixed liquor is existed
0.1%~3%, maintain inactivation 0.2~3 hour, wherein, extract be albumin, fibrinogen or
Cell factor；
Step 2: adding by the mol ratio of single aldehyde compound and terminator for 1 to 2~100 amount stirring
Enter terminator, stirring terminates inactivation；Described terminator be dimethylamino borine or trimethylamine groups borine,
Or triethyamino borine or sodium borohydride is any；
Step 3: the mixed liquor after termination is carried out with the method for dialysis or ultrafiltration or gel filtration
Liquid is changed, remnants small molecule reactant and reaction product is removed, is replaced into and retention and next step work
The adaptable solution of skill.
2. inactivation of viruses method according to claim 1, it is characterised in that in the step 2
It is 1 to 5~100 by the mol ratio of single aldehyde compound and amine compound before middle addition terminator
Amount stirring adds amine compound, adds terminator and terminates inactivation.
3. inactivation of viruses method according to claim 2, it is characterised in that described is aminated
Compound is any of glycine or lysine or alanine or ethylenediamine.
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107211576B true CN107211576B (en) | 2014-10-22 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| GR03 | Grant of secret patent right | ||
| DC01 | Secret patent status has been lifted |