CN1640496A - Recombinant adenovirus lyophilized preparation and its preparing method - Google Patents
Recombinant adenovirus lyophilized preparation and its preparing method Download PDFInfo
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- CN1640496A CN1640496A CN 200410016000 CN200410016000A CN1640496A CN 1640496 A CN1640496 A CN 1640496A CN 200410016000 CN200410016000 CN 200410016000 CN 200410016000 A CN200410016000 A CN 200410016000A CN 1640496 A CN1640496 A CN 1640496A
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- recombinant adenovirus
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- protective agent
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- 238000002360 preparation method Methods 0.000 title claims abstract description 66
- 241000701161 unidentified adenovirus Species 0.000 title claims abstract description 57
- 238000000034 method Methods 0.000 title description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 38
- 239000003223 protective agent Substances 0.000 claims abstract description 24
- 150000001413 amino acids Chemical class 0.000 claims abstract description 21
- 239000003292 glue Substances 0.000 claims abstract description 11
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 8
- 239000007864 aqueous solution Substances 0.000 claims abstract description 3
- 239000000203 mixture Substances 0.000 claims description 41
- 238000009472 formulation Methods 0.000 claims description 33
- 238000004108 freeze drying Methods 0.000 claims description 31
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 24
- 235000001014 amino acid Nutrition 0.000 claims description 20
- 239000003053 toxin Substances 0.000 claims description 19
- 231100000765 toxin Toxicity 0.000 claims description 19
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 18
- 239000001963 growth medium Substances 0.000 claims description 16
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 14
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 14
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 14
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 14
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 13
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 13
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 13
- 108010010803 Gelatin Proteins 0.000 claims description 12
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 12
- 229930006000 Sucrose Natural products 0.000 claims description 12
- 239000008273 gelatin Substances 0.000 claims description 12
- 229920000159 gelatin Polymers 0.000 claims description 12
- 235000019322 gelatine Nutrition 0.000 claims description 12
- 235000011852 gelatine desserts Nutrition 0.000 claims description 12
- 239000001103 potassium chloride Substances 0.000 claims description 12
- 235000011164 potassium chloride Nutrition 0.000 claims description 12
- 239000005720 sucrose Substances 0.000 claims description 12
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 11
- 229930195725 Mannitol Natural products 0.000 claims description 11
- 241000700605 Viruses Species 0.000 claims description 11
- 239000000594 mannitol Substances 0.000 claims description 11
- 235000010355 mannitol Nutrition 0.000 claims description 11
- 210000004907 gland Anatomy 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- 235000002639 sodium chloride Nutrition 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 239000004201 L-cysteine Substances 0.000 claims description 9
- 235000013878 L-cysteine Nutrition 0.000 claims description 9
- 239000008187 granular material Substances 0.000 claims description 9
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 8
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 8
- 235000019800 disodium phosphate Nutrition 0.000 claims description 8
- 238000004821 distillation Methods 0.000 claims description 8
- 239000004202 carbamide Substances 0.000 claims description 7
- 235000013877 carbamide Nutrition 0.000 claims description 7
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 claims description 6
- 239000004223 monosodium glutamate Substances 0.000 claims description 6
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 6
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 4
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 3
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 3
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 3
- 239000003855 balanced salt solution Substances 0.000 claims description 3
- 239000008101 lactose Substances 0.000 claims description 3
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 235000011148 calcium chloride Nutrition 0.000 claims description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 2
- 239000000243 solution Substances 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 6
- 208000015181 infectious disease Diseases 0.000 description 12
- 239000007788 liquid Substances 0.000 description 6
- 238000009777 vacuum freeze-drying Methods 0.000 description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 3
- 239000013603 viral vector Substances 0.000 description 3
- 239000006184 cosolvent Substances 0.000 description 2
- 238000001415 gene therapy Methods 0.000 description 2
- PWKSKIMOESPYIA-UHFFFAOYSA-N 2-acetamido-3-sulfanylpropanoic acid Chemical compound CC(=O)NC(CS)C(O)=O PWKSKIMOESPYIA-UHFFFAOYSA-N 0.000 description 1
- 206010067268 Post procedural infection Diseases 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 229920005549 butyl rubber Polymers 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicinal Preparation (AREA)
Abstract
The present invention relates to a recombinant adenovirus freeze-dried preparation and its preparation method. The recombinant adenovirus freeze-dried preparation disclosed by said invention is the preparation which is obtained after the aqueous solution prepared by using recombinant adenovirus: protective agent (volume ratio)=1:(0.8-1.2) is frozen and dried. The above-mentioned protective agent contains animal glue, sugar, inorganic salt and amino acid culture balanced salt whose pH value is 7.2-8.0. Said recombinant adenovirus freeze-dried preparation is safe, and high in effect and good in stability.
Description
Technical field:
The invention belongs to biological preparation and preparation field, be specifically related to recombinant adenovirus lyophilized formulations and preparation method.
Background technology:
Along with going deep into gradually of gene therapy research, the development of advancing by leaps and bounds has been arranged as its basic viral vector research.Adopt the gene transfer technique treatment human diseases of viral vector not only to study, and entered into clinical experimental stage in the chamber of experimentizing.Adenovirus is pathogenic little and do not induce canceration to the people because of it, and host cell is wide, breeding titre height, and struck capacity is big, and becomes the focus of viral vector research in recent years.Even if but the liquid recombinant adenovirus toxin preparation that contains stabilizing agent is also comparatively responsive to temperature, bring big difficulty for transportation, preservation, this has limited its application clinically to a great extent.Up to the present, Shang Weiyou can be in the lyophilizing recombinant adenovirus toxin preparation listing of 4-8 ℃ of long preservation.
Summary of the invention:
Technical problem to be solved by this invention is to solve existing recombinant adenovirus stability of formulation, provides a kind of safe, efficient, good stability, effect duration long lyophilization recombinant adenovirus toxin preparation.
Recombinant adenovirus lyophilized formulations disclosed by the invention is to be recombinant adenovirus by volume ratio: protective agent=1: the preparation that the aqueous solution that (0.8-1.2) is mixed with obtains after lyophilization, wherein to comprise animal glue, sugar, inorganic salt and pH be that 7.2-8.0 aminoacid is cultivated balanced salt to protective agent.
Recombinant adenovirus of the present invention is meant genetic engineering modified adenovirus.
Animal glue of the present invention is selected from gelatin, osseocolla or hide glue etc.;
Described sugar is selected from one or more in sucrose, mannitol, trehalose, lactose, the maltose;
Described inorganic salt is selected from one or more in sodium chloride, potassium chloride, potassium dihydrogen phosphate, sodium hydrogen phosphate, calcium chloride, the magnesium chloride;
Described aminoacid culture medium balanced salt is selected from commercially available basic aminoacid culture medium (Dulbecco ' s Modified Eagle Medium, be called for short D-MEM) balanced salt.
The animal glue and the sugar that adopt in the lyophilized formulations protective agent of the present invention can play the support effect, if add the heat stability that trehalose can improve the live virus preparation; The inorganic salt that adopts is for the pH value of regulating preparation and wait and ooze a little, to satisfy physiological adaption requirement and safety of medicine, stable, effective requirement; The pH value that adopts is that the aminoacid culture medium balanced salt of 7.2-8.0 is a cosolvent, to increase the dissolubility of adenovirus, enhanced stability.
In protective agent of the present invention, also can add other compositions: cosolvent such as carbamide, monosodium glutamate etc.; Antioxidant synergist such as L-cysteine etc.; With further raising stability of formulation.
The preferred final weight volume of protective agent each component of the present invention (w/v) concentration is animal glue 0.5-2.0%, sugar 1.5～21.5%, and inorganic salt 0.1～12%, aminoacid is cultivated balanced salt 0.5～2%.
Protective agent of the present invention is preferably filled a prescription and is consisted of (w/v) sucrose 1.5-5.5%; mannitol 4.5-10.5%; gelatin 0.5-2.0%; L-cysteine 0.03-0.15%; trehalose 1.5-5.5%; potassium chloride 0.1-0.3%; potassium dihydrogen phosphate 0.1-0.3%; pH value is basic aminoacid culture medium (D-MEM) balanced salt 0.5～2% of 7.2-8.0; sodium chloride 3.5～8.5%, sodium hydrogen phosphate 1.2～2.9%, carbamide 0.4～1.2%; monosodium glutamate 0.4～1.2%, all the other are the pure water of resistivity=18.2M Ω/cm.
Another technical problem to be solved by this invention is to provide the preparation method of above-mentioned recombinant adenovirus lyophilized formulations.
The preparation of recombinant adenovirus lyophilized formulations disclosed by the invention comprises the following steps:
By above-mentioned protective agent prescription, take by weighing each component, add pure water to prescribed volume and fully dissolve, standby; Again recombinant adenovirus is pressed interior gland-containing virus amount 1.0 * 10
12The concentration of granule number/ml is with 1: (0.8-1.2) join in above-mentioned protectant solution; fill; preparation is put into freeze drying box; pre-freeze is to-30 ℃--40 ℃, kept evacuation 3-5 hour; distillation; dry again, make formulation temperature by 0 ℃-35 ℃, promptly make lyophilization recombinant adenovirus toxin preparation.
Recombinant adenovirus lyophilized formulations with prescription of the present invention and method prepare detects evidence through infection titer, and preparation has kept the original activity of recombinant adenovirus; And have good stability through 30 months stability test proof.Compared with prior art, technology of the present invention is reasonable, safety and good stability, and effect duration is long, provides good prospects for application for adopting recombinant adenovirus as the clinical experiment of the gene therapy of carrier.
Description of drawings:
The stable correlation curve of lyophilized formulations and liquid preparation under Fig. 1 4-8 ℃ condition
The specific embodiment:
Embodiment 1
To contain freeze drying protectant the recombinant adenovirus toxin preparation, by interior gland-containing virus amount 0.5 * 10
12The concentration packing of granule number/ml, the 1ml/ bottle, a part jump a queue 4 ℃ of preservations of gland as the preparation lyophilizing before infection titer detect usefulness, another part preparation adds butyl rubber and plug adds to 1/3 place, carries out lyophilization again.3 different lot numbers contain the recombinant adenovirus toxin preparation of freeze drying protectant after lyophilization; infection titer detects proof: by the lyophilizing recombinant adenovirus toxin preparation that the inventive method is made, preparation lyophilization postoperative infection titre only descends at 0.03-0.05logTCID before than lyophilization
50Between/the ml.(table 1)
Table 1: infection titer is measured before and after the lyophilizing of recombinant adenovirus toxin preparation
| The preparation lot number | ??????????????logTCID 50/ml | ||
| Before the lyophilizing | After the lyophilizing | Front and back are poor | |
| ????20001001 | ????10.51 | ????10.48 | ????0.03 |
| ????20001201 | ????10.69 | ????10.79 | ????-0.1 |
| ????20001202 | ????10.56 | ????10.51 | ????0.05 |
Embodiment 2
Same lot number, contain sucrose, mannitol, gelatin, the L-cysteine, trehalose, potassium chloride, potassium dihydrogen phosphate, basis aminoacid culture medium (Dulbecco ' s ModifiedEagle Medium, be called for short D-MEM) the recombinant adenovirus lyophilized formulations of balanced salt and without freeze dried liquid control formulation, leave in the same 4-8 ℃ refrigerator, regularly randomization is measured the preparation infection titer, the result proves: the lyophilizing recombinant adenovirus toxin preparation of making by the inventive method, has good stable, deposited 30 months under 4-8 ℃ of condition, the preparation infection titer descends at 0.5logTCID
50In/the ml, obviously descend, reach 3logTCID and deposit 30 months infection titers at 4-8 ℃ without cryodesiccated liquid control formulation
50/ ml.This result proves: deposit the infection titer fall that 30 months infection titer and lyophilized formulations deposit 30 months without cryodesiccated liquid control formulation under 4-8 ℃ of condition and differ greatly.(table 2, Fig. 1)
Table 2: 4-8 ℃ of preservation of infections titer determination of lyophilizing recombinant adenovirus toxin preparation
| The preparation lot number | ?????????????????????????logTCID 50/ml | ||||||
| 0 month | March | June | December | 18 months | 24 months | 30 months | |
| Liquid preparation | 10.56 | ?10.39 | ?10.31 | ?9.98 | ?8.94 | ?8.08 | ?7.50 |
| Lyophilized formulations | 10.51 | ?10.37 | ?10.19 | ?10.12 | ?10.12 | ?10.04 | ?10.04 |
Embodiment 3:
To contain sucrose; mannitol; gelatin; the L-cysteine; trehalose; potassium chloride; the freeze drying protectant of potassium dihydrogen phosphate and basic aminoacid culture medium (Dulbecco ' s ModifiedEagle Medium; be called for short D-MEM) the recombinant adenovirus toxin preparation of balanced salt and do not contain sucrose; mannitol; gelatin; the L-cysteine; trehalose; potassium chloride; the freeze drying protectant of potassium dihydrogen phosphate and basic aminoacid culture medium (Dulbecco ' s Modified EagleMedium; be called for short D-MEM) balanced salt solution; recombinant adenovirus toxin preparation with the PBS dilution; carry out lyophilization under same vacuum lyophilization condition, randomization is measured the preparation infection titer then.Result's proof does not add protectant preparation after lyophilization, and virus activity is lost substantially.(table 3)
Table 3: freeze drying protectant is to the influence of recombinant adenovirus lyophilized formulations infection titer
| The preparation lot number | Protective agent | ?????logTCID 50/ml | |
| Before the lyophilizing | After the lyophilizing | ||
| ??20000201 | ????- | ????10.48 | ????＜7.5 |
| ????10.51 | ????＜7.5 | ||
| ??20000201 | ????+ | ????10.88 | ????10.75 |
| ????10.69 | ????10.56 | ||
Embodiment 4
Protective agent prescription: sucrose 1.5g; mannitol 5g, gelatin 1.6g, L-cysteine 0.08g; trehalose 1.5g; potassium chloride 0.2g, potassium dihydrogen phosphate 0.2g, pH value are basic aminoacid culture medium (D-MEM) the balanced salt 2g (available from GIBCO company) of 7.2-8.0; sodium chloride 8.0g; sodium hydrogen phosphate 2.9g, carbamide 1.2g, monosodium glutamate 0.5g
Preparation:
By prescription each component is mixed, add pure water, again recombinant adenovirus is pressed interior gland-containing virus amount 1.0 * 10 to 100ml
12The concentration of granule number/ml joined in the above-mentioned protective agent with 1: 0.8; mix homogeneously carries out fill; adopt vacuum freeze-drying method, preparation is put into freeze drying box, pre-freeze is to-38 ℃; kept 4 hours; evacuation, distillation, dry again; make formulation temperature by 0 ℃-35 ℃, promptly make lyophilization recombinant adenovirus toxin preparation.
Embodiment 5
Protective agent prescription: sucrose 5.5g; mannitol 10g, gelatin 1.2g, L-cysteine 0.03g; trehalose 5.5g; potassium chloride 0.3g, potassium dihydrogen phosphate 0.1g, pH value are basic aminoacid culture medium (D-MEM) the balanced salt 0.5g (available from GIBCO company) of 7.2-8.0; sodium chloride 4.0g; sodium hydrogen phosphate 1.3g, carbamide 0.8g, monosodium glutamate 0.8g
Preparation:
By prescription each component is mixed, add pure water, again recombinant adenovirus is pressed interior gland-containing virus amount 1.0 * 10 to 100ml
12The concentration of granule number/ml joined in the above-mentioned protective agent with 1: 0.9; mix homogeneously carries out fill; adopt vacuum freeze-drying method, preparation is put into freeze drying box, pre-freeze is to-30 ℃; kept 3 hours; evacuation, distillation, dry again; make formulation temperature by 0 ℃-35 ℃, promptly make lyophilization recombinant adenovirus toxin preparation.
Embodiment 6
Protective agent prescription prescription: sucrose 4.0g; mannitol 7.5g, gelatin 0.8g, L-cysteine 0.15g; trehalose 4.0g; potassium chloride 0.1g, potassium dihydrogen phosphate 0.3g, pH value are basic aminoacid culture medium (D-MEM) the balanced salt 1g (available from GIBCO company) of 7.2-8.0; sodium chloride 6.0g; sodium hydrogen phosphate 2.0g, carbamide 0.5g, monosodium glutamate 1.2g
Preparation:
By prescription each component is mixed, add pure water, again recombinant adenovirus is pressed interior gland-containing virus amount 1.0 * 10 to 100ml
12The concentration of granule number/ml joined in the above-mentioned protective agent with 1: 1.1; mix homogeneously carries out fill; adopt vacuum freeze-drying method, preparation is put into freeze drying box, pre-freeze is to-35 ℃; kept 3.5 hours; evacuation, distillation, dry again; make formulation temperature by 0 ℃-35 ℃, promptly make lyophilization recombinant adenovirus toxin preparation.
Embodiment 7
The protective agent prescription: lactose 1.5g, mannitol 5g, gelatin 1.6g, trehalose 5.5g, potassium dihydrogen phosphate 0.2g, pH value are basic aminoacid culture medium (D-MEM) the balanced salt 2g (available from GIBCO company) of 7.2-8.0, sodium chloride 8.0g, sodium hydrogen phosphate 2.9g
Preparation:
By prescription each component is mixed, add pure water, again recombinant adenovirus is pressed interior gland-containing virus amount 1.0 * 10 to 100ml
12The concentration of granule number/ml joined in the above-mentioned protective agent with 1: 1.2; mix homogeneously carries out fill; adopt vacuum freeze-drying method, preparation is put into freeze drying box, pre-freeze is to-37 ℃; kept 3.5 hours; evacuation, distillation, dry again; make formulation temperature by 0 ℃-35 ℃, promptly make lyophilization recombinant adenovirus toxin preparation.
Embodiment 8
Prescription: maltose 1.5g, sucrose 5g, gelatin 1.2g, L-cysteine 0.08g, trehalose 1.5g, potassium chloride 0.2g, potassium dihydrogen phosphate 0.2g, pH value is basic aminoacid culture medium (D-MEM) the balanced salt 2g (available from GIBCO company) of 7.2-8.0, sodium chloride 8.0g, carbamide 1.2g
Preparation:
By prescription each component is mixed, add pure water, again recombinant adenovirus is pressed interior gland-containing virus amount 1.0 * 10 to 100ml
12The concentration of granule number/ml joined in the above-mentioned protective agent with 1: 1.0; mix homogeneously carries out fill; adopt vacuum freeze-drying method, preparation is put into freeze drying box, pre-freeze is to-36 ℃; kept 5 hours; evacuation, distillation, dry again; make formulation temperature by 0 ℃-35 ℃, promptly make lyophilization recombinant adenovirus toxin preparation.
Embodiment 9
Prescription: sucrose 1.5g, hide glue 1.6g, trehalose 1.5g, potassium chloride 0.2g, potassium dihydrogen phosphate 0.2g, pH value are basic aminoacid culture medium (D-MEM) the balanced salt 2g (available from GIBCO company) of 7.2-8.0, sodium chloride 8.0g
Preparation:
By prescription each component is mixed, add pure water, again recombinant adenovirus is pressed interior gland-containing virus amount 1.0 * 10 to 100ml
12The concentration of granule number/ml joined in the above-mentioned protective agent with 1: 1.1; mix homogeneously carries out fill; adopt vacuum freeze-drying method, preparation is put into freeze drying box, pre-freeze is to-39 ℃; kept 4 hours; evacuation, distillation, dry again; make formulation temperature by 0 ℃-35 ℃, promptly make lyophilization recombinant adenovirus toxin preparation.
Claims (8)
1, recombinant adenovirus lyophilized formulations; it is characterized in that said preparation is is recombinant adenovirus by volume ratio: protective agent=1: the preparation that the aqueous solution that (0.8-1.2) is mixed with obtains after lyophilization, wherein to comprise animal glue, sugar, inorganic salt and pH be that 7.2-8.0 aminoacid is cultivated balanced salt to protective agent.
2, recombinant adenovirus lyophilized formulations according to claim 1; the final weight volumetric concentration that it is characterized in that the contained each component of wherein said protective agent is animal glue 0.5-2.0%; sugar 1.5～21.5%, inorganic salt 0.1～12%, aminoacid is cultivated balanced salt 0.5～2%.
3, recombinant adenovirus lyophilized formulations according to claim 1 and 2 is characterized in that wherein said animal glue is selected from gelatin, osseocolla or hide glue.
4, recombinant adenovirus lyophilized formulations according to claim 1 and 2 is characterized in that wherein said sugar is selected from one or more in sucrose, mannitol, trehalose, lactose, the maltose.
5, recombinant adenovirus lyophilized formulations according to claim 1 and 2 is characterized in that wherein said inorganic salt is selected from one or more in sodium chloride, potassium chloride, potassium dihydrogen phosphate, sodium hydrogen phosphate, calcium chloride, the magnesium chloride.
6, recombinant adenovirus lyophilized formulations according to claim 1 and 2 is characterized in that wherein said aminoacid culture medium balanced salt is selected from basic aminoacid culture medium balanced salt solution.
7; recombinant adenovirus lyophilized formulations according to claim 1 and 2; it is characterized in that wherein said protectant bulking value percentage composition is sucrose 1.5-5.5%; mannitol 4.5-10.5%; gelatin 0.5-2.0%; L-cysteine 0.03-0.15%; trehalose 1.5-5.5%; potassium chloride 0.1-0.3%; potassium dihydrogen phosphate 0.1-0.3%, pH value are the basic aminoacid culture medium balanced salt solution 0.5～2% of 7.2-8.0, sodium chloride 3.5～8.5%; sodium hydrogen phosphate 1.2～2.9%; carbamide 0.4～1.2%, monosodium glutamate 0.4～1.2%, all the other are the pure water of resistivity=18.2M Ω/cm.
8, the preparation method of recombinant adenovirus lyophilized formulations according to claim 1 is characterized in that the preparation of said preparation comprises the following steps:
Press the protective agent prescription, take by weighing each component, add pure water to prescribed volume and fully dissolve, standby; Again recombinant adenovirus is pressed interior gland-containing virus amount 1.0 * 10
12The concentration of granule number/ml is with 1: (0.8-1.2) join in above-mentioned protectant solution; fill; preparation is put into freeze drying box; pre-freeze is to-30 ℃--40 ℃, kept evacuation 3-5 hour; distillation; dry again, make formulation temperature by 0 ℃-35 ℃, promptly make lyophilization recombinant adenovirus toxin preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100160007A CN100475268C (en) | 2004-01-17 | 2004-01-17 | Recombinant adenovirus lyophilized preparation and preparing method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100160007A CN100475268C (en) | 2004-01-17 | 2004-01-17 | Recombinant adenovirus lyophilized preparation and preparing method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1640496A true CN1640496A (en) | 2005-07-20 |
| CN100475268C CN100475268C (en) | 2009-04-08 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004100160007A Expired - Lifetime CN100475268C (en) | 2004-01-17 | 2004-01-17 | Recombinant adenovirus lyophilized preparation and preparing method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104797242A (en) * | 2012-10-02 | 2015-07-22 | 特兰斯吉恩股份有限公司 | Virus-containing formulation and use thereof |
| CN106492213A (en) * | 2016-12-05 | 2017-03-15 | 天津康希诺生物技术有限公司 | A kind of adenoviruss lyophilization additive and adenoviruss lyophilized formulations |
| CN110960484A (en) * | 2019-11-05 | 2020-04-07 | 深圳市天达康基因工程有限公司 | Recombinant adenovirus sustained-release preparation, preparation method and application thereof |
| WO2021244120A1 (en) * | 2020-06-01 | 2021-12-09 | 康希诺生物股份公司 | Sars-cov-2 vaccine |
-
2004
- 2004-01-17 CN CNB2004100160007A patent/CN100475268C/en not_active Expired - Lifetime
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104797242A (en) * | 2012-10-02 | 2015-07-22 | 特兰斯吉恩股份有限公司 | Virus-containing formulation and use thereof |
| US10111947B2 (en) | 2012-10-02 | 2018-10-30 | Transgene S.A. | Virus-containing formulation and use thereof |
| CN106492213A (en) * | 2016-12-05 | 2017-03-15 | 天津康希诺生物技术有限公司 | A kind of adenoviruss lyophilization additive and adenoviruss lyophilized formulations |
| WO2018103601A1 (en) * | 2016-12-05 | 2018-06-14 | 康希诺生物股份公司 | Freeze-drying additive for adenovirus and freeze-dried preparation of adenovirus |
| CN110960484A (en) * | 2019-11-05 | 2020-04-07 | 深圳市天达康基因工程有限公司 | Recombinant adenovirus sustained-release preparation, preparation method and application thereof |
| WO2021244120A1 (en) * | 2020-06-01 | 2021-12-09 | 康希诺生物股份公司 | Sars-cov-2 vaccine |
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| Publication number | Publication date |
|---|---|
| CN100475268C (en) | 2009-04-08 |
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