CN103173493B - Preparation containing recombinant adenovirus - Google Patents
Preparation containing recombinant adenovirus Download PDFInfo
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- CN103173493B CN103173493B CN201310053079.XA CN201310053079A CN103173493B CN 103173493 B CN103173493 B CN 103173493B CN 201310053079 A CN201310053079 A CN 201310053079A CN 103173493 B CN103173493 B CN 103173493B
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Abstract
The invention relates to a preparation containing recombinant adenovirus. The preparation consists of the recombinant adenovirus, buffer liquid or a buffer system and a protective agent, wherein the buffer liquid or buffer system is any of Tris-HCl buffer liquid, disodium hydrogen phosphate-citric acid buffer liquid and arginine-hydrochloric acid, and the protective agent is selected from propylene glycol, polyethylene glycol 400 and dimethyl sulfoxide. The preparation disclosed by the invention has the outstanding advantages that the ingredients are simple, divalent cationic salts and nonionic surfactants, such as MgCl2 and tween 80, are not contained, however, the adenovirus can be excellently conserved at the temperature of 2-8 DEG C even room temperature and is enabled to have good stability, and the like.
Description
Technical field
The present invention relates to recombinant adenovirus, relate in particular to a kind of preparation that contains recombinant adenovirus.
Background technology
The fundamental principle of selecting about issuing the middle regulation auxiliary material of notice > > (No. [2008] 7, state's food medicine prison note) of pharmaceutical chemicals injection and the requirement of polycomponent Biochemical Drugs injection basic fundamental according to the < < of State Food and Drug Administration's issue: under the prerequisite of satisfying the demand, the kind of injection auxiliary material used and consumption should be the least possible.Recombinant adenovirus, as clinical medicine, can maintain under the prerequisite of its stability, requires in principle pharmaceutical adjunct kind and consumption more few better.
In the prior art of having reported, US Patent No. 2006/0205080A1 discloses a kind of Tris-HCl of comprising, MgCl
2, glycine and be selected from the composition of the aqueous cosolvent of propylene glycol, DMSO, PEG, sucrose, glycerine, tetrahydrofuran (THF), glycol ether, said preparation can be 2～8 ℃ of activity that keep preferably adenovirus, but the component in said composition is more, cause products production comparatively complicated, even strengthen the security risks of gene therapy medicament.
In addition, Chinese patent CN101163794A discloses a kind of adenovirus composition, said composition comprises adenovirus particles and can maintain pH at damping fluid and the glycerine of 8.0-9.6, has the simple advantage of component, but the stability of its long-term preservation adenovirus under 2～8 ℃ of conditions is undesirable.
Summary of the invention
The object of the present invention is to provide a kind of preparation that contains recombinant adenovirus, by comprising recombinant adenovirus, damping fluid or buffer system, prevention adenovirus generation physics or chemical transformation, the protective material of inactivation forms said preparation, and described damping fluid or buffer system are selected from any or its combination of Tris-HCl damping fluid, Lin acid hydrogen Er Na – citrate buffer solution, arginine-hydrochloric acid; Be suitable for any or its combination that protective material of the present invention is selected from propylene glycol, poly(oxyethylene glycol) 400 or dimethyl sulfoxide (DMSO).
Further, the pH of described damping fluid or buffer system is 7.4-8.6.
Further, described damping fluid or buffer system are arginine-hydrochloride buffer.
Further, described damping fluid or buffer system are Tris-HCl damping fluid.
Further, described damping fluid or buffer system are Lin acid hydrogen Er Na – citrate buffer solution.
Further, described polyol is propylene glycol, and further, in preparation of the present invention, the concentration of propylene glycol is 5-20w/v%, is preferably 10-20w/v%.
The present invention can, according to the needs of stable maintenance adenovirus activity, adjust and select required buffer concentration.In preparation of the present invention, the concentration of damping fluid is 10-100mM, and described damping fluid is selected from any of Tris-HCl damping fluid, Lin acid hydrogen Er Na – citrate buffer solution, arginine-hydrochloric acid.Such selection can obtain gratifying saving result.
Preparation of the present invention is comprised of recombinant adenovirus, 10-50mM damping fluid and 10% propylene glycol, and pH is 7.8; Described damping fluid is selected from any of Tris-HCl damping fluid, Lin acid hydrogen Er Na – citrate buffer solution, arginine-hydrochloric acid.
Lin acid hydrogen Er Na – citric acid of the present invention and arginine-hydrochloride buffer not only have good shock absorption, also adenovirus are had to provide protection to a certain degree.
It is simple that preparation of the present invention has component, do not contain divalent cation salt and nonionogenic tenside, as MgCl
2with tween-80 etc., also can be 2～8 ℃ of fine preservation adenovirus of room temperature even, and make it have the outstanding advantages such as satisfactory stability.
Preparation of the present invention is mainly used in the recombinant adenovirus of preserving gene therapy, also can be applicable to preserve the recombinant adenovirus of any wild-type adenovirus or other purposes.Specifically, comprise all replication-defective adenoviral or replication competent adenovirus that utilize recombinant DNA technology to build, and the wild-type adenovirus of all people or animal or its sudden change adenovirus.
Adopt the virus titer value of international TCID50 method calibrating and the ratio (being specific activity) of the virion number that ultraviolet method is examined and determine to evaluate the stability of recombinant adenovirus of the present invention.
The present invention adopts TCID50 method to measure recombinant adenovirus titre, its principle is: the viral sample through serial dilution is infected to 293 cells, the cell hole of some amount that each extent of dilution infects is cultivated 10 days, examine under a microscope the cell situation in each cell hole, statistics sick cell hole, utilize TCID50 calculation formula, calculate virus titer.
TCID50/ml=10
-[-L-d(s-0.5)]+b
Wherein, L: be minimum dilution logarithm; D: be poor between logarithm of dilution; S: be the positive boring ratio rate of pathology sum; B: be virus inoculation amount, be scaled the logarithm of the multiple of every ml volumes.
The present invention adopts the granule number of spectrophotometry adenovirus, and its principle is: in theory, the composition of wild-type 5 type adenovirus is 87% protein and 13%DNA.Therefore, the concentration of adenovirus particles can directly be measured with ultraviolet spectrophotometer.The tryptophane of protein and tyrosine residues have uv-absorbing at 277nm place, and double-stranded linear DNA has obtained the maximum absorption at 260nm.This law adopts and to contain 0.1%SDS(sodium lauryl sulphate) preparation damping fluid, viral capsid is resolved into protein and DNA, recycling double-stranded linear DNA has obtained the maximum absorption at 260nm place, when having washing agent, granule density and 260nm light absorption value are proportional, with spectrophotometer, in 260nm place, measure the absorbance of trial-product, adopt formula (1AU=1.1 * 10
12vP/mL) calculate virion number.The scope of application of Test Virus granule number is trial-product virion number>=2 * 10
11vP/mL.
The final judge index of recombinant adenovirus stability of the present invention is, according to the viral specific activity that detects gained virus titer and granule number calculating gained, i.e. viral specific activity=virus titer/virion number * 100%.
National drug food Surveillance Authority (SFDA) treats in research and quality of the pharmaceutical preparations control techniques governing principle > > and stipulates at < < people's gene, and clinical recombinant adenovirus specific activity should be higher than 3.3%; (the < < Chinese Medical Journal > > such as Zhang Xiaozhi, 2004, the 84th the 10th phase of volume), in " quality controls of recombinant adenovirus clinical grade gene therapy goods ", mention, while evaluating the stability of adenovirus preparation, in preparation, the specific activity of adenovirus should be greater than 3.3%.”
Except as otherwise noted, percentage composition of the present invention is bulking value percentage composition.
The preparation method of recombinant adenovirus of the present invention can adopt this area common recombinant adenovirus preparation method gained, in general comprises that cell recovery, seed cell preparation, virus infection, amplification, lysis, clarification filtration, the concentrated liquid, stoste of changing the process such as prepares
The preparation method of recombinant adenovirus toxin preparation of the present invention can adopt this area conventionally to prepare the method for recombinant adenovirus, in general comprise the steps: by stoste change liquid, detectable level, with the pharmaceutical adjunct of the good concentration of proportioning dilute, degerming, packing obtain finished product.
Another object of the present invention is to provide recombinant adenovirus toxin preparation for the preparation of the application in gene therapy medicament.
Compared with prior art, recombinant adenovirus toxin preparation of the present invention has following advantage:
Recombinant adenovirus toxin preparation of the present invention not only can fine preservation recombinant adenovirus activity, effectively prevent gathering or the division of adenovirus, and component is simple, do not contain divalent cation salt, as MgCl
2, be beneficial to production and the clinical use of recombinant adenovirus.Thereby recombinant adenovirus toxin preparation of the present invention not only can well be preserved recombinant adenovirus and other adenovirus thereof, also has the production control of being easy to and industrialization, low cost of manufacture, the higher advantage such as better of preparation security.Preparation of the present invention has outstanding advantage in the preservation of recombinant adenovirus.
Embodiment
Below with reference to embodiment, illustrate the present invention, embodiments of the invention, only for technical scheme of the present invention is described, are not to further restriction of the present invention.
The impact of embodiment 1pH value on recombinant adenovirus stability in preparation
In order to study the impact of pH value on recombinant adenovirus stability in preparation, under 25 ℃ of conditions, we have studied in the scope of pH6.8～8.6, and pH value is respectively 6.8,7.2,7.4,7.6,7.8,8.2,8.6 o'clock, the impact of pH value on recombinant adenovirus stability in preparation.
Contriver, by detecting virus titer (TCID50/mL) and the specific activity of recombinant adenovirus in preparation, has studied the impact of pH value on recombinant adenovirus stability, the results are shown in Table 1.
The impact of table 1pH value on recombinant adenovirus stability in preparation
Note: preparation consist of 10mMTris+10% propylene glycol (w/v), with HCl, regulate pH.
From table 1, at pH, lower than 7.4 o'clock, the recombinant adenovirus in preparation was more unstable, and pH is too high, and pungency is larger.Therefore, preparation selection pH of the present invention is 7.4～8.6.Wherein pH value is 7.8 o'clock, and the stability of adenovirus is fine, and close to the pH value of human body, adverse drug reaction is less.
Embodiment 2MgCl
2impact on recombinant adenovirus stability in preparation
In order to study MgCl
2impact on recombinant adenovirus stability in preparation, under 25 ℃ of conditions, the present invention adopts simultaneous test, has studied and in preparation, has had MgCl
2the whether impact on recombinant adenovirus stability.Wherein, experimental group preparation is recombinant adenovirus toxin preparation of the present invention, comprises recombinant adenovirus, Tris-HCl damping fluid and is selected from the protective materials such as propylene glycol, poly(oxyethylene glycol) 400, dimethyl sulfoxide (DMSO), does not contain MgCl in preparation
2; Control group preparation, except the recombinant adenovirus toxin preparation of the present invention that contains same composition and same amount, also comprises MgCl in preparation
2.
By detecting virus titer (TCID50/mL) and the specific activity of recombinant adenovirus in preparation, studied MgCl
2impact on recombinant adenovirus stability, the composition of preparation and result of study are in Table 2.
Table 2MgCl
2impact on recombinant adenovirus stability in preparation
Note: Tris concentration is 50mM, regulates pH78 with HCl; Each protectant percentage is w/v%.
From table 2, recombinant adenovirus toxin preparation of the present invention with added MgCl
2control formulation compare, under 25 ℃ of conditions, the two is to keeping the stability of recombinant adenovirus substantially there is no difference.As everyone knows, as medicine for treatment thing, the supplementary product kind adding in preparation and consumption are more few better.Visible, simplification preparation of the present invention compared with prior art has advantage in the preservation of recombinant adenovirus.
Stability study under 25 ℃ of conditions of embodiment 3 recombinant adenovirus toxin preparation of the present invention
Under 25 ℃ of conditions, studied the stability of recombinant adenovirus in recombinant adenovirus toxin preparation of the present invention and prior art preparation (being the disclosed preparation of CN101163794A1).Wherein, experimental group preparation is recombinant adenovirus toxin preparation of the present invention, comprises recombinant adenovirus, Tris-HCl damping fluid and is selected from the protective materials such as propylene glycol, poly(oxyethylene glycol) 400, dimethyl sulfoxide (DMSO), does not contain MgCl
2; Control formulation is the disclosed preparation of CN101163794A1.
By detecting virus titer (TCID50/mL) and the specific activity of recombinant adenovirus in preparation, studied the stability under 25 ℃ of conditions of recombinant adenovirus toxin preparation of the present invention, the composition of preparation and detected result are in Table 3.
Stability study under 25 ℃ of conditions of table 3 recombinant adenovirus toxin preparation of the present invention
Note: Tris concentration is 50mM, regulates pH78 with HCl, each protectant percentage is w/v%.
From table 3, compare with control group preparation, recombinant adenovirus toxin preparation of the present invention has more excellent stability, under 25 ℃ of conditions, can preserve recombinant adenovirus 3 months, and specific activity is all more than 3.3%, and the specific activity of control group preparation is only 2.48%, lower than criterion of acceptability (specific activity of gene therapy recombinant adenovirus should be not less than 3.3%).
The impact of embodiment 4 buffer systems on recombinant adenovirus stability in preparation
Under 25 ℃ of conditions, the present invention has studied three kinds of buffer system (citric acid-Na
2hPO
4, Arg-HCl, Tris-HCl) impact on recombinant adenovirus stability in preparation.
By detecting virus titer (TCID50/mL) and the specific activity of recombinant adenovirus in preparation, studied the impact of buffer system on recombinant adenovirus preparation stability in preparation, the composition of preparation and result of study are in Table 4.
From table 4, under 25 ℃ of conditions, Tris-HCl, citric acid-Na
2hPO
4, Arg-HCl buffer system can better keep the stability of recombinant adenovirus in preparation.
The impact of table 4 buffer system on recombinant adenovirus stability in preparation
Note: in Tris-HCl damping fluid, Tris concentration is 50mM, regulates pH78 with HCl; Citric acid-Na
2hPO
4in damping fluid, citric acid concentration is 50mM, uses Na
2hPO
4regulate pH78; In Arg-HCl damping fluid, arginine concentration is 50mM, with HCl, regulates pH78; Each protectant percentage is w/v%.
Stability study under 4 ℃ of conditions of embodiment 5 recombinant adenovirus toxin preparation of the present invention
Under 4 ℃ of conditions, studied the stability of recombinant adenovirus in recombinant adenovirus toxin preparation of the present invention and prior art preparation (being the disclosed preparation of CN101163794A).Wherein, experimental group preparation is recombinant adenovirus toxin preparation of the present invention, and control group preparation is prior art preparation.
By detecting virus titer (TCID50/mL) and the specific activity of recombinant adenovirus in preparation, studied the stability of recombinant adenovirus toxin preparation of the present invention under 4 ℃ of conditions, the composition of preparation and result of study are in Table 5.
As shown in Table 5, compare with control group preparation, under 4 ℃ of conditions, in recombinant adenovirus toxin preparation of the present invention, the stability of recombinant adenovirus is far above the stability of adenovirus in prior art.
In addition, table 5 data also show, in same buffer system, different protective materials are also variant to the stability influence of recombinant adenovirus, and protective material preferred sequence is propylene glycol, poly(oxyethylene glycol) 400, methyl-sulphoxide, and poor is glycerol.
The stability study of table 5 recombinant adenovirus toxin preparation of the present invention
Note: in Tris-HCl damping fluid, Tris concentration is 50mM, regulates pH78 with HCl; Citric acid-Na
2hPO
4in damping fluid, citric acid concentration is 50mM, uses Na
2hPO
4regulate pH78; In Arg-HCl damping fluid, arginine concentration is 50mM, with HCl, regulates pH78; In Histidine-HCl damping fluid, histidine concentrations is 50mM, with HCl, regulates pH78; Each protectant percentage is w/v%.
The impact of embodiment 6 propylene glycol concentration on recombinant adenovirus stability in preparation
Under 25 ℃ of conditions, the present invention has studied the impact of propylene glycol concentration (5%, 10%, 15%, 20%) on recombinant adenovirus stability in preparation.By detecting virus titer (TCID50/mL) and the specific activity of recombinant adenovirus in preparation, studied the impact of propylene glycol concentration on recombinant adenovirus preparation stability in preparation, the composition of preparation and result of study are in Table 6.
From table 6, propylene glycol concentration and recombinant adenovirus stability are linear dependence substantially, and propylene glycol concentration is higher, and in preparation, the stability of recombinant adenovirus is better.And, when the concentration of propylene glycol is not less than 10w/v%, get final product the stability of recombinant adenovirus in fine preservation preparation.
The impact of table 6 propylene glycol concentration on recombinant adenovirus stability in preparation
Note: in Tris-HCl damping fluid, Tris concentration is 50mM, regulates pH78 with HCl; Citric acid-Na
2hPO
4in damping fluid, citric acid concentration is 50mM, uses Na
2hPO
4regulate pH78; In Arg-HCl damping fluid, arginine concentration is 50mM, with HCl, regulates pH78; The percentage of propylene glycol is w/v%.
The impact of embodiment 7 buffer salinities on recombinant adenovirus stability in preparation
Under 25 ℃ of conditions, the present invention has studied the impact of buffer salinity (10mM, 50mM, 100mM) on recombinant adenovirus stability in preparation in buffer system.
By detecting virus titer (TCID50/mL) and the specific activity of recombinant adenovirus in preparation, studied the impact of buffer salinity on recombinant adenovirus preparation stability in preparation, the composition of preparation and result of study are in Table 7.
The impact of table 7 buffer salinity on recombinant adenovirus stability in preparation
Note: pH78, the percentage of propylene glycol is w/v%.
From table 7, in buffer system, in citric acid concentration and preparation, the stability of recombinant adenovirus is obvious linear relationship, the concentration of citric acid is higher, in preparation, the stability of recombinant adenovirus is better, when the concentration of citric acid is not less than 50mM in buffer system, get final product the stability of recombinant adenovirus in fine preservation preparation; In buffer system, in arginine concentration and preparation, the stability of recombinant adenovirus is obvious linear relationship, arginic concentration is higher, in preparation, the stability of recombinant adenovirus is better, when arginic concentration is not less than 50mM in buffer system, get final product the stability of recombinant adenovirus in fine preservation preparation; In buffer system, in the concentration of Tris and preparation, the stability dependency of recombinant adenovirus is not strong, and when in buffer system, the concentration of Tris is not less than 10mM, preparation of the present invention is under different Tris concentration, and in preparation, the stability difference of recombinant adenovirus is little.
Embodiment 8 preparation of the present invention is preserved the impact of high density recombinant adenovirus stability
For test preparation of the present invention is preserved greater concn recombinant adenovirus stability, designed 2,4,6,8, six recombinant adenovirus concentration gradients of 10E12vp/mL, after sample preparation in 25 ℃ of preservations, respectively at sampling in 0,1,2,3 month, detect virus titer and granule number, calculate specific activity, the results are shown in Table 8.
Table 8 preparation of the present invention is preserved the impact of high density recombinant adenovirus stability
Note: pH78, the percentage of propylene glycol is w/v%.
From table 8, in three kinds of preparation damping fluids of the present invention, the stability of adenovirus and its concentration are all significant linear relationship, preparation of the present invention is preserved the following recombinant adenovirus of 2～6E12vp/mL concentration good stability, and the recombinant adenovirus of preserving 8～10E12vp/mL concentration also can maintain the stability of 1～2 month 25 ℃ of conditions.
Claims (1)
1. a preparation that contains recombinant adenovirus, described preparation is comprised of recombinant adenovirus, 50mM damping fluid and 10% dimethyl sulfoxide (DMSO), pH is 7.8, and wherein, described damping fluid is selected from any of Tris-HCl damping fluid, Sodium phosphate dibasic-citrate buffer solution, arginine-hydrochloride buffer.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1347450A (en) * | 1999-04-09 | 2002-05-01 | 阿文蒂斯药物股份有限公司 | Composition for preservation of infections recombinant adenoviruses |
| US20040033239A1 (en) * | 2001-03-06 | 2004-02-19 | Evans Robert K | Adenovirus formulations |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1347450A (en) * | 1999-04-09 | 2002-05-01 | 阿文蒂斯药物股份有限公司 | Composition for preservation of infections recombinant adenoviruses |
| US20040033239A1 (en) * | 2001-03-06 | 2004-02-19 | Evans Robert K | Adenovirus formulations |
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