CN1208091C - Virus deactivating method with immobilized solvent or immobilized solvent-detergent - Google Patents
Virus deactivating method with immobilized solvent or immobilized solvent-detergent Download PDFInfo
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- CN1208091C CN1208091C CN 01123658 CN01123658A CN1208091C CN 1208091 C CN1208091 C CN 1208091C CN 01123658 CN01123658 CN 01123658 CN 01123658 A CN01123658 A CN 01123658A CN 1208091 C CN1208091 C CN 1208091C
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- 239000002904 solvent Substances 0.000 title claims abstract 7
- 241000700605 Viruses Species 0.000 title claims abstract 5
- 239000003599 detergent Substances 0.000 title claims 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract 3
- 229910052799 carbon Inorganic materials 0.000 claims 1
- 230000002779 inactivation Effects 0.000 claims 1
- 150000002632 lipids Chemical class 0.000 claims 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 abstract 2
- 239000003795 chemical substances by application Substances 0.000 abstract 2
- 239000013543 active substance Substances 0.000 abstract 1
- 230000000415 inactivating effect Effects 0.000 abstract 1
- 235000012239 silicon dioxide Nutrition 0.000 abstract 1
- 239000000377 silicon dioxide Substances 0.000 abstract 1
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Abstract
The present invention relates to a method for inactivating viruses with a solvent or a solvent decontaminating agent, which is characterized in that the solvent and/or the solvent decontaminating agent is fixed to a carrier prepared from activated carbon as an active substance or from silicon dioxide. The method of the present invention has the advantages of simple and continuous operation, low cost, safety and reliability.
Description
Invention field
The present invention relates to a kind of with immobilization organic solvent (Solvant is hereinafter to be referred as S) and/or detergent (Detergent is hereinafter to be referred as D) method of inactivation of viruses in aqueous solution.
Background technology
Since nineteen seventies, owing to the viral pollution of food, medicine causes the incident of serious medical treatment ﹠ health consequence to become the problem that the public is concerned about day by day.A large amount of work has been done in order to address this problem by the science and technology circle, and various viral inactivation treatment methods develop rapidly.The viral inactivation treatment method of extensive use at present has following several.
1, the Pasteur viral method of going out
The Pasteur viral method (liquid heated 10 hours at 60 ℃) of going out is one of the most classical virus inactivating method, the inactivation of virus that utilizes viruses molecule and the difference of target product molecule on heat stable property to select.For example, one of major product of blood plasma product albumin is worldwide used the history that this method is gone out viral existing nearly half a century, and generally acknowledged good result is arranged.In the production of some other blood plasma products such as Antithrombin III, this method also is to generally acknowledge virus inactivating method efficiently.
Yet, though use this method aqueous solution is carried out effectively deactivation model virus of inactivation of virus, but the bioactive loss of some target product is also comparatively serious, needs to use heavy dose of stabilizing agent, makes the amount that a ultrafiltration operation is controlled stabilizing agent often must be set after the virus of going out.Therefore, this method can not be arranged on production line terminal (except that albumin), can not solve secondary pollution problem.At product loss of activity and being provided with specially under the situation of one destabilizing agent technology, the increase of technology cost is bigger.
2, the SD viral method of going out
It is one of most widely used virus inactivating method in the world at present that SD (Solvant-Detergent) handles, and this method is that the aqueous solution with liquid SD agent or SD agent joins and carries out the inactivation of virus reaction in the aqueous solution.This method is at first showing powerful efficient aspect the inactivation of virus of thrombin (FVIII, FIX etc.).From earlier 1990s, the SD method has been used for the production of plant-scale viral blood plasma that goes out, and this method also can be widely used in the inactivation of virus of other biological product.
Yet the virus spectrum of going out of SD method is restricted, and its deactivation to lipid-coated virus is effective, and is powerless to non-lipid-coated virus.Secondly, the application of this method must be removed technology by a SD agent and be accompanied, and can not be arranged on the production line terminal, thereby can not solve the secondary pollution problem.Be specifically designed to the technological measure of removing the SD agent owing to will set up one or two, also increased the technology cost.
3, low pH method
Utilize viruses molecule and the stability difference of goods molecule in acid medium to be prepared, make viral macromole (the viral macromole that absolute acid stability the is relatively poor) inactivation that may exist.But this method is mainly effective to lipid-coated virus, and owing to be to carry out in acid medium, this method is bigger to the side effect of other biological preparation, thereby is mainly used in the inactivation of virus of immunoglobulin.To the product of pH sensitivity, this method is inapplicable.
4, membrane filter method
Utilize viruses molecule and the difference of goods molecule on the hydrodynamics size to be prepared, the viral macromole that may exist is separated from goods.But this method be used to strengthen the virus safe of the less protein formulation of molecular dimension, and production cost is very high mainly as a kind of viral inactivation treatment method of complementarity.
5, photodynamics method
With the photodynamics method of antozone, be one of the method for studying at most in the viral blood plasma production of going out at present as active basis.Because this method can be used as the production terminal, might solve secondary pollution.In the photodynamic inactivation virus treated, (patent publication No.: disclose a kind of method of inactivation of virus CN1224657A), be the single water solublity naphthalene class peroxide of 2-200mM or their mixture with concentration wherein contacts 5～200 minutes at 15～80 ℃ times and pending biological preparation to one of the present inventor at a Chinese patent of application in 1998.This method needs special light-source system, and its extensive use is restricted.
Under present condition, develop a kind of simple to operate, continuous, with low cost, safe and reliable virus inactivating method, be still the target that science and technology is pursued.
Summary of the invention
Therefore, the object of the present invention is to provide a kind of virus inactivating method with the above advantage.
According to the invention provides a kind of method, it is characterized in that described solvent or solvent-detergent are fixed on the carrier of being made by active carbon, and described virus is selected from lipid-coated virus with solvent or solvent-detergent inactivation of viruses.
The active substance of described carrier is active carbon or silicon dioxide, and it can be graininess, fibrous, felted or filter plate shape.And solvent or solvent-detergent are fixed on the described carrier by physics or chemical action.
Method of the present invention is used for by injection or the oral and final aqueous solution that contacts with human body.This aqueous solution includes but not limited to blood plasma and blood plasma derivant, biological product, natural plants goods.
The specific embodiment
By the SD virus inactivating method of USA New York Blood Center invention, and, for example, only add the shared virus inactivating method of viral method of going out of S and S and ethanol etc., all be referred to as the SD method in the present invention afterwards through the deriving method of other people exploitation.The SD method is liquid virus inactivating agent (S or SD) directly to be dispersed in carry out inactivation of virus in the aqueous solution.The method needs the long inactivation of virus response time, is generally at ambient temperature more than 6 hours, but also needs the special step of removing the SD agent.
In a Chinese patent (exercise question is " as the surfactant and the active carbon desorption method thereof of new-type desorbent ", and number of patent application is 00120625.7) of our application, we find that active carbon has adsorption to detergent.(exercise question is " a kind of method of removing as the organic solvent and/or the detergent of virus inactivating agent " to another Chinese patent of having applied at us, number of patent application is 01104343.1) in, we have invented and have utilized the active substance that contains active carbon to carry out the method that the SD agent is removed.Although make that therefore the technology cost that removes the SD agent greatly descends, viral reaction does not touch itself to going out.Along with going deep into of our research, we find, use the immobilization SD virus treated of going out, and can reduce the virus response time of going out effectively, improve the seriality of production process, reduce the technology input of removing the SD agent.
Though do not want to be limited to the restriction of any theory, but we think when passing through in the reactor of fluid media (medium) by the solid phase carrier that contains SD that may contain fat after birth virus, viruses molecule and SD molecule react, and make the viruses molecule structural damage, and make the viruses molecule inactivation thus.If the SD molecular concentration from the effusive fluid media (medium) of reaction medium is above standard, also can in reactor, add the solid phase carrier of absorption SD agent, to remove the SD agent of q.s, satisfy the residue criterion of SD agent.
In the method for the invention, but be fixed on suitable carriers with being used for the SD agent physics of inactivation of viruses or chemical, preferably fix by physical method.The active substance of described carrier includes but not limited to active carbon and silicon dioxide.Wherein active carbon for example is the form of activated carbon powder, active carbon filter plate, NACF or NACF felt, and silicon dioxide for example can be Aerosil or the form that contains the filter plate of Aerosil.
For the organic solvent S among the present invention, any solvent well known by persons skilled in the art all is suitable, and it includes but not limited to TNbP (three normal-butyl phosphate esters) commonly used in the aqueous solution inactivation of virus.
For the detergent D among the present invention, any detergent well known by persons skilled in the art all is suitable, and it includes but not limited to Tweens detergent (for example Tween-80) and Triton class detergent (for example Triton * 100) commonly used in the aqueous solution inactivation of virus.
The virus of available method deactivation of the present invention generally is meant lipid-coated virus, and it includes but not limited to HIV (human immunodeficiency virus), hepatitis B virus, hepatitis C virus and other fat peplos hepatitis virus well known by persons skilled in the art etc.
The aqueous solution that available method of the present invention is handled includes but not limited to water, blood plasma and blood plasma derivant, biological product, medicine, health protection articles for use etc.
The implementation condition of method of the present invention does not have concrete restriction, but pH is 2.0-12.0 generally speaking, and temperature is-5 to 60 ℃, and fluid dynamics condition (flow velocity, pressure) then can be determined according to the inactivation of virus confirmatory experiment.
Embodiment 1: the preparation of immobilization SD
Carry out the enforcement of present embodiment with following ready made material: 1) active carbon filter plate (for example AKS4 or AKS5, Germany Seitz Schenk company), 2) activated carbon felt (ZC-1200A for example, China's purple river charcoal fiber company limited) and 3) contain the grease removal filter plate (for example Delipid, U.S. CUNO company) of Aerosil.These materials at first are formed into the filter of diameter 6cm, thickness 4.2mm.TNbP/Triton * 100 are deployed into the aqueous solution of 1% (V/V).Make this mobile phase flow velocity with 5ml/min below room temperature pass through the said fixing phase respectively then, and circulated repeatedly under the same conditions 40 minutes.S or the SD immobilization amount on different materials can be controlled (another part exercise question with reference to us is the invention of " as the surfactant and the active carbon desorption method thereof of new-type desorbent ", and its number of patent application is 00120625.7) by the dosage of regulating the surfactant that is used as desorbent that adds this aqueous solution.Concrete experiment condition and the results are shown in shown in the following table 1.
The absorption that table 1:SD agent is gone up mutually at immobilization carrier
| Immobile phase | The SD agent solution | Final filtered solution | Immobilization SD | |||||
| S (V/V) | D (V/V) | Ethanol (V/V) | Volume | S (V/V) | D (V/V) | S (V/V) | D (V/V | |
| AKS4 | 1％ | 1％ | 10％ | 25ml | 0.15％ | 0.21％ | 1.79％ | 1.66％ |
| ZC- 1200A | 1％ | 1％ | 25％ | 25ml | 0.18％ | 0.23％ | 1.73％ | 1.62％ |
| DELIPID | 1％ | 1％ | 0 | 25ml | 0.25％ | 0.16％ | 1.58％ | 1.77％ |
| AKS4 | 1％ | 0 | 15％ | 25ml | 0.14％ | 0 | 1.81％ | 0 |
Embodiment 2: the preparation of immobilization SD agent
In the process of preparation active carbon slab, activated fibre felt and Delipid filter plate, SD agent soon joins in the mixture of raw material and mixes before the molding oven dry.In the case, the SD agent will be adsorbed on the materials such as activated carbon powder or Aerosil granule quantitatively, again through the normal preparation technology of filter plate, fiber felt, promptly obtain immobilization SD agent.Experiment parameter that it is crucial and experimental result are seen shown in the following table 2:
The preparation condition and the result of table 2:SD immobile phase
| The SD immobile phase | The filter plate mixture | SD amount in the filter plate | ||
| The TNbP amount | Trtion×100 | S | D | |
| The active carbon slab of SD | 1％(w/w) | 1％(w/w) | 1％(v/v) | 1％(v/v) |
| SD grease removal filter plate | 1％(w/w) | 1％(w/w) | 0.3％(v/v) | 1％(v/v) |
Annotate: all meterings all are to be standard with every cubic metre of filter plate amount contained or that disengage
Embodiment 3: the inactivation of virus of human gamma globulin (HGG)
150ml contains model virus, and (the HIV initial titer is 10
4.2, VSV initial titer 10
4.8, sindbis virus initial titer 10
5.1) human gamma globulin (HGG) (protein content 8%, pH4.5) solution are 100-300Lm at flow velocity
-2, pressure≤1bar condition under pass through respectively: the active carbon filter plate (thickness: 4.2mm, the diameter: 6cm) that A) contain 1.81% (V/V) TNbP; B) contain grease removal filter plate (thickness: 4.2mm, the diameter: 6cm) of 1.58% (V/V) TNbP and 1.77% (V/V) Triton * 100 (V/V); C) (the mixture weight ratio is 50/50, and reactor thickness is 4.2mm, diameter: 6cm) to contain the active carbon/kieselguhr mixture of 1.79% (V/V) TNBP and 1.66% (V/V) Triton * 100.Connect an active carbon filter (thickness 4.2mm, diameter: 6cm, AKS4, moral Seitz Schenk company) behind each immobilization SD device.Do behind the filtrate collection of gained further to analyze, it the results are shown in Table shown in 3 and 4.
Table 3: the deactivation of model virus
| A | B | C | |
| HIV | ≥10 4.1 | ≥10 4.1 | ≥10 4.1 |
| VSV | ≥10 4.7 | ≥10 4.7 | ≥10 4.7 |
| Sindbis virus | ≥10 5.0 | ≥10 5.0 | ≥10 5.0 |
Table 4: the analysis of human gamma globulin (HGG)
| Index | Before immobilization SD handles | After immobilization SD handles | ||
| A | B | C | ||
| Anti-HBV activity (IU/ml) | 53 | 49 * | 48 * | 45 * |
| Antibody activity (IU/ml) | 24 | 18.5 * | 19 * | 17.3 * |
| Monomer+dimer | 100 | 100 | 100 | 100 |
| Polymer | 0 | 0 | 0 | 0 |
*: dilution effect
In addition, in three kinds of reactor filtrate the content of TNBP all less than 5ppm.
Embodiment 4: the inactivation of virus of whole plasm
150ml contains people's whole plasm of the listed model virus of following table 5, and (albumen contains 5.7%, pH7.85), is 100-300LM at flow velocity
-2Under the condition of pressure≤1bar successively by through equilibrated active carbon filter plate (with embodiment 3) and another active carbon filter plate (the thickness 4.2mm that does not contain the SD agent that contains 1.79% (V/V) TNbP and 1.66% (V/V) Triton * 100 of normal saline, diameter 6cm, AKS4, Germany Seitz Schenk company), gained filtrate is done further to analyze after collection, and it the results are shown in Table shown in 5 and 6.
Table 5: the deactivation of model virus
| Virus | Initial titer | Detect titre | Deactivation |
| HIV | 10 4.2 | ≤10 0.1 | ≥10 4.1 |
| VSV | 10 4.8 | ≤10 0.1 | ≥10 4.7 |
| Sendai virus | 10 5.1 | ≤10 0.1 | ≥10 5.0 |
Table 6: the analysis result of blood plasma after filtration
| Analyze | The result |
| Protein recovery | ≥95％ |
| TNBP | 5ppm |
| Triton×100 | ＜10ppm |
| The acetate fiber electrophoretic analysis | Indistinction before and after immobilization SD handles |
| Blood coagulation eight factor response rate | ≥70％ |
Embodiment 5: the inactivation of virus of engineered protein reaction
Reorganization Alpha interferon (interferon concentration 1,150,000 IU/ml that contain the listed model virus of following table 7, pH7.0) be the step that repeats embodiment 4 under the 1ml/min condition at flow velocity, but only use the active carbon filter plate that is fixed with the SD agent, supernatant is collected the back and is done further to analyze, and it the results are shown in shown in the following table 7.
Table 7: the inactivation of virus in the recombinant interferon
| Virus | Initial titer | Detect titre | Deactivation |
| HIV | 10 4.2 | ≤10 0.1 | ≥10 4.1 |
| VSV | 10 4.8 | ≤10 0.1 | ≥10 4.7 |
| Sendai virus | 10 5.1 | ≤10 0.1 | ≥10 5.0 |
Claims (2)
1,, it is characterized in that described solvent or solvent-detergent are fixed on the carrier of being made by active carbon, and described virus is selected from lipid-coated virus with the method for solvent or solvent-detergent inactivation of viruses.
2, the method for claim 1, wherein said carrier are graininess, fibrous, felted or filter plate shape.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01123658 CN1208091C (en) | 2001-08-30 | 2001-08-30 | Virus deactivating method with immobilized solvent or immobilized solvent-detergent |
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|---|---|---|---|
| CN 01123658 CN1208091C (en) | 2001-08-30 | 2001-08-30 | Virus deactivating method with immobilized solvent or immobilized solvent-detergent |
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| CN1402974A CN1402974A (en) | 2003-03-19 |
| CN1208091C true CN1208091C (en) | 2005-06-29 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US12258540B2 (en) | 2017-10-30 | 2025-03-25 | Takeda Pharmaceutical Company Limited | Environmentally compatible detergents for inactivation of lipid-enveloped viruses |
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| WO2006116899A1 (en) * | 2005-04-29 | 2006-11-09 | Chengdu Kuachang Medical Industrial Limited | The system, solid phase medium, device and method for bio-fluid treatment |
| CN1820786B (en) * | 2005-04-29 | 2012-05-23 | 成都夸常医学工业有限公司 | Method, system and device for effectively reducing pathogen hazard |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US12258540B2 (en) | 2017-10-30 | 2025-03-25 | Takeda Pharmaceutical Company Limited | Environmentally compatible detergents for inactivation of lipid-enveloped viruses |
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