CN108624505A - A kind of slow virus freezing drying protective agent and slow virus freeze-dried powder - Google Patents
A kind of slow virus freezing drying protective agent and slow virus freeze-dried powder Download PDFInfo
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- CN108624505A CN108624505A CN201810995003.1A CN201810995003A CN108624505A CN 108624505 A CN108624505 A CN 108624505A CN 201810995003 A CN201810995003 A CN 201810995003A CN 108624505 A CN108624505 A CN 108624505A
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- slow virus
- freeze
- dried powder
- protective agent
- freezing drying
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
Abstract
A kind of slow virus freezing drying protective agent of present invention offer and slow virus freeze-dried powder, the protective agent ingredient include glycerine, sucrose, mannitol, L arginine, glycine, PEG6000, gelatin and phosphate buffer（Disodium hydrogen phosphate/sodium dihydrogen phosphate）；The slow virus freeze-dried powder is lyophilized by slow virus with the protective agent in a cold or frozen state to be formed；The slow virus freeze-dried powder is preferable in subzero 80 DEG C of stability, can effectively keep lentiviral particle active.
Description
Technical field
The present invention relates to biological product technical field, especially a kind of slow virus freezing drying protective agent, that is, slow virus freeze-drying
Powder..
Background technology
Slow virus（lentivirus）One kind of human immunodeficiency virus is derived from through artificial reconstructed reverse transcription disease
Poison, by envelope protein（Such as VSVG）With capsid protein（Such as p24）It wraps up hereditary material DNA nucleic acid to constitute, DNA nucleic acid encode structures
The key molecules such as albumen gag, polymerase Pol, transport modulin Rev.The advantage of slow virus is can be logical with mediate foreign gene
It crosses LTR elements and is integrated into host genome, to which foreign gene can stablize heredity with host gene.Therefore, from exploitation
Since, slow virus is mainly used in basic research field and enters host cell as means of transport foreign gene-carrying or move
In object, the function of related gene is studied in enhancing or the expression for weakening specific purpose gene.But from American science in 2013
After family's report successfully cures 1 acute lymphatic leukemia late period infant with CART cells, slow virus is led in cellular immunotherapy
It is paid special attention in domain.
CART cells are obtained by slow virus modified human source T cell, it is characterized in that integrated antigen recognizing signal and activated downstream
Signal simultaneously plays lethal effect, avoids the dependence to auxiliary signals such as antigen presenting cells in one, Direct Recognition tumour cell.
Therefore, CART cells are more more effective than traditional T cell therapeutic effect.Currently, hundreds of clinical tests are carrying out both at home and abroad
CART cell therapies are studied, and are had 2 drugs based on CART technologies and evaluated by FDA, and hematological cancer is applied to
The treatment of patient.
Slow virus is key raw material in CART cell preparation process.The quality control of slow virus directly affects CART cells
Preparation and killing activity.CART cell production links include T cell is infected with the slow virus carrier for carrying target gene, wherein
Whether the titre of slow virus is successful very crucial to infecting.Slow virus carrier is substantially made of protein, can not be in room temperature and 4 DEG C
Under the conditions of preserve for a long time, poor to thermal stability, multigelation can reduce its titre, or even form different degrees of degradation product.
However, current, there is no ideal slow virus to freeze protection liquid, and conventional -80 DEG C preserve or are added glycerine protective agent and can also drop
Its low activity.In addition, when needing long-distance transportation, i.e., stored, may be caused due to temperature difference using larger amount of dry ice
Slow virus activity reduces.
Freeze-dried powder is that biopharmaceutical macromolecular drug commonly uses preservation form.Slow virus shell is made of multiple proteins, essence
It is protein combination body, has protein fundamental characteristics, i.e., to thermo-responsive, easy inactivation etc..But can it not stablize currently
The slow virus freeze-dried powder of preservation.
Invention content
First technical problem to be solved by this invention is to provide a kind of slow virus freezing drying protective agent.
Second technical problem to be solved by this invention is to provide a kind of based on above-mentioned protectant slow virus freeze-drying
Powder.
In order to solve the above technical problems, the technical scheme is that：
A kind of slow virus freezing drying protective agent, by glycerine, sucrose, mannitol, L-arginine, glycine, PEG6000, gelatin
And phosphate buffer（Disodium hydrogen phosphate/sodium dihydrogen phosphate）Composition, wherein
Glycerine 0.1-5.0%（V/V）
Sucrose 1-200mg/mL
Mannitol 1-200mg/mL
L-arginine 0.1-10mg/mL
Glycine 0.1-10mg/mL
PEG6000 1-10%
Gelatin 0.5-5%
Phosphate buffer 5-100mmol/L
Preferably, above-mentioned slow virus freezing drying protective agent, prescription are as follows：
Glycerine 1.0%（V/V）
Sucrose 20mg/mL
Mannitol 25mg/mL
L-arginine 1mg/mL
Glycine 2mg/mL
PEG6000 2%
Gelatin 1.5%
Phosphate buffer 50mmol/L
Preferably, above-mentioned slow virus freezing drying protective agent pH value is 7.0-7.6.
The preparation method of above-mentioned slow virus freezing drying protective agent, the specific steps are：
（1）Glycerine, sucrose, mannitol, L-arginine, glycine, PEG6000, gelatin and phosphate-buffered are weighed according to prescription
Liquid（Disodium hydrogen phosphate/sodium dihydrogen phosphate）；
（2）By glycerine, sucrose, mannitol, L-arginine, glycine, PEG6000, gelatin and phosphate buffer（Phosphoric acid hydrogen two
Sodium/phosphorus acid dihydride sodium）It is dissolved in appropriate sterile purified water, fully dissolves, it is 7.0~7.6 to adjust solution ph, is filtered with 0.22 μm
Membrane filtration degerming.
Application of the above-mentioned slow virus freezing drying protective agent in terms of preparing slow virus freeze-dried powder.
A kind of slow virus freeze-dried powder is made of slow virus and above-mentioned slow virus freezing drying protective agent.
Preferably, above-mentioned slow virus freeze-dried powder, the slow virus are derived from human immunodeficiency virus, removal virulence gene, only
Retain assembly element and infects the virus of element.
Preferably, above-mentioned slow virus freeze-dried powder, every 1 cillin bottle（0.25~0.5ml of volume after redissolution）Contain：
Slow virus 0.5~1 × 107TU
Glycerine 1.0%（V/V）
Sucrose 20mg/mL
Mannitol 25mg/mL
L-arginine 1mg/mL
Glycine 2mg/mL
PEG6000 2%
Gelatin 1.5%
Phosphate buffer 50mmol/L
Preferably, above-mentioned slow virus freeze-dried powder, pH value is 7.0~7.6 after redissolution.
Preferably, above-mentioned slow virus freeze-dried powder can be applied to scientific research CART cells, CAR-NK cells, CAR-DC cells
Preparation.
Above-mentioned slow virus method for preparing freeze-dried powder, the specific steps are：
（1）0.25~0.5ml slow virus concentrates are transferred in 1 cillin bottle with micropipette rifle；
（2）With the concentrate in vacuum freeze drying step 1, capping, in -80 DEG C of preservations.
Beneficial effects of the present invention：
Slow virus freezing drying protective agent of the present invention can effectively reduce loss of activity during slow virus freezing/redissolution,
Make up defect of the direct cryopreservation of current slow virus without effective protection agent.
Slow virus freeze-dried powder obtained by the present invention is more easily transported than the slow virus of liquid condition, does not need special ultralow temperature
Cold chain is supported, has good application value in terms of the medicine exploitation based on slow virus.
Description of the drawings
Fig. 1 slow virus freeze-dried powder appearances.
The freeze-drying of Fig. 2 slow virus is preceding and redissolves postoperative infection Activity determination.
Specific implementation mode
1 slow virus freezing drying protective agent of embodiment is prepared
（1）Appropriate disodium hydrogen phosphate and sodium dihydrogen phosphate powder are weighed, dissolving is sufficiently mixed with deionized water, with 2M sodium hydroxides
Solution adjusts pH value between 7.0~7.6, and 0.45 μm of membrane filtration obtains phosphate buffer（50mmol/L, pH7.0~
7.6）Room temperature is spare.
（2）According to following component and final concentration require to weigh glycerine, sucrose, mannitol, L-arginine, glycine,
PEG6000, gelatin are added in phosphate buffer, are sufficiently mixed dissolving, and measurement solution ph and it is adjusted to 7.0 again~
Between 7.6,0.22 μm of membrane filtration obtains slow virus freezing drying protective agent.
Protective agent component and content：
Glycerine 1.0%（V/V）
Sucrose 20mg/mL
Mannitol 25mg/mL
L-arginine 1mg/mL
Glycine 2mg/mL
PEG6000 2%
Gelatin 1.5%
Phosphate buffer 50mmol/L
It is prepared by 2 slow virus freeze-dried powder of embodiment
（1）The present invention conventionally prepares RCR deficiency slow virus stostes：Prepare auxiliary packaging plasmid（pspax2）, packet
Film quality grain（pMD）, the albumen containing GFP expression plasmid（pCDHGFP）And 293T cells；By 3 plasmids of preparation according to certain
Ratio mixes（pspax2：pMD：pCDHGFP=2：1：3）And 293T cells are entered with the transfection of PEI reagents；Fresh training is replaced after 12h
Foster base continues to cultivate, and collecting supernatant in 48h, 72h respectively obtains stoste containing slow virus and after 0.45 μm of membrane filtration in 4 DEG C of guarantors
It deposits spare.
（2）The present invention purifies slow virus stoste in anion-exchange chromatography method：Appropriate tris alkali is weighed, with deionized water
Equilibration buffer A is prepared respectively（25mM Tris, pH8.0）And elution buffer B（1M Tris, pH8.0）, take appropriate A liquid and B
Liquid mixes, and obtains washing buffer C（200mM Tris, pH8.0）；Slow virus stoste is adjusted to 25mM with appropriate tris alkali, and
It is 8.0 to adjust pH value；Then in sequence（A liquid balance-slow virus stoste crosses the elution of column-A liquid washing-C liquid washing-B liquid）Cross Q
Sepharose chromatographic columns collect B liquid elution fractions, obtain slow virus refined solution.
（3）The present invention replaces slow virus refined solution with ultrafiltration：The super filter tube for taking molecular cut off 60kDa, in ultra-clean work
Make that slow virus refined solution is moved into super filter tube in platform, with 3000rpm, 4 DEG C of centrifugation 10min；Throw aside in bottom pipe liquid and with slow
Viral freezing drying protective agent is added into super filter tube upper layer casing, is repeated and is centrifuged 3-4 times, obtains the pre- frozen stock solution of slow virus.
（4）Packing freeze-drying obtains slow virus freeze-dried powder：The pre- frozen stock solution of slow virus is dispensed according to 0.5ml to 1 west
It in woods bottle, through vacuum freeze drying, seals and obtains slow virus freeze-dried powder, in -80 DEG C of preservations.
3 slow virus freeze-dried powder expression activitiy of embodiment is analyzed
The slow virus freeze-dried powder of 3 batches is produced with production technology described in embodiment 2 according to formula described in embodiment 1, and
To carrying out transduction activity measurement before and after freeze-drying（TU/ML）, testing result is as follows：
The front and back transduction activity measurement result of 1 slow virus freeze-dried powder of table freeze-drying
Claims (8)
1. a kind of slow virus freezing drying protective agent, it is characterised in that：By glycerine, sucrose, mannitol, L-arginine, glycine,
PEG6000, gelatin and phosphate buffer（Disodium hydrogen phosphate/sodium dihydrogen phosphate）Composition, wherein
Glycerine 0.1-5.0%（V/V）
Sucrose 1-200mg/mL
Mannitol 1-200mg/mL
L-arginine 0.1-10mg/mL
Glycine 0.1-10mg/mL
PEG6000 1-10%
Gelatin 0.5-5%
Phosphate buffer 5-100mmol/L.
2. slow virus freezing drying protective agent according to claim 1, it is characterised in that：
Constituent content is as follows：
Glycerine 1.0%（V/V）
Sucrose 20mg/mL
Mannitol 25mg/mL
L-arginine 1mg/mL
Glycine 2mg/mL
PEG6000 2%
Gelatin 1.5%
Phosphate buffer 50mmol/L
Solution ph is 7.0-7.6.
3. slow virus freezing drying protective agent preparation method according to claim 1, it is characterised in that：
The specific steps are：
（1）Glycerine, sucrose, mannitol, L-arginine, glycine, PEG6000, gelatin and phosphate are weighed according to constituent content
Buffer solution；
（2）Glycerine, sucrose, mannitol, L-arginine, glycine, PEG6000, gelatin are added separately to phosphate buffer
In, it fully dissolves, with 0.22 μm of membrane filtration, is placed in 4 DEG C of preservations.
4. application of the slow virus freezing drying protective agent described in claim 1 in terms of slow virus freeze-dried powder preparation.
5. a kind of slow virus freeze-dried powder, is made of lentiviral particle and slow virus freezing drying protective agent described in claim 1.
6. slow virus freeze-dried powder according to claim 5, it is characterised in that：The slow virus is autonomous replication deficiency disease
Poison does not have RCR activity.
7. slow virus freeze-dried powder according to claim 5, it is characterised in that：Volume 0.5mL after each cillin bottle redissolves, contains
Have：
RCR deficiencies slow virus 0.5~1 × 107 TU
Glycerine 1.0%（V/V）
Sucrose 20mg/mL
Mannitol 25mg/mL
L-arginine 1mg/mL
Glycine 2mg/mL
PEG6000 2%
Gelatin 1.5%
Phosphate buffer 50mmol/L
PH value is 7.0-7.6 after redissolution.
8. the preparation method of slow virus freeze-dried powder according to claim 5, it is characterised in that：
The specific steps are：
（1）Slow virus stoste is centrifuged into 10min through 4000rpm ultrafiltration, adds appropriate slow virus freezing drying protective agent, repeat from
The heart 3 times, and it is 0.5mL to adjust final volume；
（2）By slow virus displacement liquid through 0.22 μm of membrane filtration degerming, it is added to the cillin bottle of aseptic process, it is dry through vacuum refrigeration
Dry, sealing bottleneck is up to slow virus freeze-dried powder.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810995003.1A CN108624505A (en) | 2018-08-29 | 2018-08-29 | A kind of slow virus freezing drying protective agent and slow virus freeze-dried powder |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810995003.1A CN108624505A (en) | 2018-08-29 | 2018-08-29 | A kind of slow virus freezing drying protective agent and slow virus freeze-dried powder |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108624505A true CN108624505A (en) | 2018-10-09 |
Family
ID=63708999
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810995003.1A Pending CN108624505A (en) | 2018-08-29 | 2018-08-29 | A kind of slow virus freezing drying protective agent and slow virus freeze-dried powder |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109929814A (en) * | 2019-04-12 | 2019-06-25 | 南京科佰生物科技有限公司 | Slow virus stabilizer and its application method |
| CN112080524A (en) * | 2019-06-13 | 2020-12-15 | 南京艾德免疫治疗研究院有限公司 | Preparation method of lentivirus vector cryopreservation protective solution |
| CN112831525A (en) * | 2020-10-21 | 2021-05-25 | 东莞清实生物科技有限公司 | Simple and efficient lentivirus cryopreservation liquid and application thereof |
Citations (3)
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| WO2002011540A1 (en) * | 2000-08-03 | 2002-02-14 | Merck & Co., Inc. | Rotavirus vaccine formulations |
| WO2003086443A1 (en) * | 2002-04-11 | 2003-10-23 | Medimmune Vaccines, Inc. | Spray freeze dry of compositions for intranasal administration |
| 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 |
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2018
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002011540A1 (en) * | 2000-08-03 | 2002-02-14 | Merck & Co., Inc. | Rotavirus vaccine formulations |
| WO2003086443A1 (en) * | 2002-04-11 | 2003-10-23 | Medimmune Vaccines, Inc. | Spray freeze dry of compositions for intranasal administration |
| 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 |
Non-Patent Citations (2)
| Title |
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| 朱敖兰等: "生物制品冻干保护剂及其保护机理的研究进展", 《喀什师范学院学报》 * |
| 韦晓玲等: "冻干对BMP-2 基因重组慢病毒载体生物学效应的影响", 《上海口腔医学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109929814A (en) * | 2019-04-12 | 2019-06-25 | 南京科佰生物科技有限公司 | Slow virus stabilizer and its application method |
| CN112080524A (en) * | 2019-06-13 | 2020-12-15 | 南京艾德免疫治疗研究院有限公司 | Preparation method of lentivirus vector cryopreservation protective solution |
| CN112831525A (en) * | 2020-10-21 | 2021-05-25 | 东莞清实生物科技有限公司 | Simple and efficient lentivirus cryopreservation liquid and application thereof |
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Application publication date: 20181009 |