CN109821010A - A kind of freeze drying powder injection and its preparation method and application of anti-tumor protein TmSm - Google Patents
A kind of freeze drying powder injection and its preparation method and application of anti-tumor protein TmSm Download PDFInfo
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Abstract
The invention belongs to field of pharmaceutical preparations, specifically disclose the freeze drying powder injection of anti-tumor protein TmSm a kind of, include TmSm fusion protein, mannitol, glycine, trehalose, Tween 80 and phosphate buffer.The invention also discloses the method for the freeze drying powder injection for preparing anti-tumor protein TmSm and its preparing the application in the drug for treating tumour.By formula compounding pharmaceutical solution, places and be freeze-dried in freeze dryer.The product appearance being according to said method prepared is good, pharmaceutical activity is strong, has good function of tumor inhibition.Stablized using above-mentioned formula and the resulting freeze-dried powder quality of preparation method, plays the purpose of protected protein class pharmaceutical activity, operating method is easy, easy to industrialized production.
Description
Technical field
The invention belongs to the technical fields of pharmaceutical preparation, and in particular to the freeze drying powder injection of anti-tumor protein TmSm a kind of and
Preparation method and application.
Background technique
Tumour is to endanger the first big disease of human health, is always the mesh that the mankind pursue using the antitumor technology of biology
Mark.Anti-tumor protein drug is the antitumor main contents and important component of biology, for many years by anti-tumor protein
The unremitting research of drug, it has been found that the major obstacle of anti-tumor protein Drug development and research come from many aspects, wherein it
The quality of production and stable technology controlling and process be a very important aspect, it is related to the production of the anti-tumor protein drug
The feasibility of cost and industrialized production also just decides that can the drug really develop to clinic and answer to a certain extent
With.Therefore, controllable, the stable process study of anti-tumor protein drug has great importance.
Freeze-drying (freezing drying, lyophilization) full name is vacuum freeze drying (vacuum
Freeze-drying is referred to as lyophilized) refer to and water in solid, will be utilized under the conditions of low-temperature reduced-pressure by dry water-containing materials freezing
Distillation performance, make low-temperature material be dehydrated and reach a kind of drying means of drying purpose.The technology is present approximately in 1911 earliest
Year, by constantly improving and developing, have become a very important ring in many commercial pharmaceutical preparation processes at present.
Because vacuum freeze drying is to complete the removal of moisture, the equipment of the technology in the environment of low temperature, vacuum
Investment, running expense are high, and the production cycle is relatively long, relatively high very using freeze drying technology product cost produced thus
It is more, however for the drug of some thermal sensitivity, easy in inactivation especially antibody, albumen, vaccine type medication, this is so far
Optimal production method.The technology is particularly important for biological products, such as recombinant protein, antibody, vaccine.
The first protein drug Hum μ lin ratified from FDA nineteen eighty-twoTM(Genentech) it since, has had more than at present
130 protein and peptide drugs go through, and have more products to be in continual exploitation.
TmSm fusion protein is a kind of independent development, protide drug candidate with anti-tumor activity, with other eggs
White class drug is the same, necessarily faces various problems such as quality stability, transport and storage after commercialization, and freeze-dried powder system
Agent is the best approach solved these problems.Therefore, it is stable to obtain a kind of final product quality, operating method simplicity is easy to industrialize
The freeze-drying preparation process of production is formulation arts personnel's problem encountered.
Summary of the invention
In view of the deficienciess of the prior art, the first purpose of this invention is to provide a kind of anti-tumor protein TmSm's
Freeze drying powder injection has the stability for improving anti-tumor protein TmSm, and facilitates the advantages of medicament transport is with storage.
The freeze drying powder injection of anti-tumor protein TmSm of the invention includes:
The TmSm fusion protein of survival dose；
Filler；
Freeze drying protectant；
Tween 80；With
Buffer.
In embodiments of the present invention, the filler and freeze drying protectant are selected from glucose, sucrose, lactose, malt
In the group that sugar, mannitol, sorbierite, xylitol, glycine, dextran, trehalose, Tween 80, povidone and glycerol form
One or more.
In embodiments of the present invention, the buffer is selected from potassium phosphate buffer, sodium phosphate buffer, lemon
One of acid sodium-salt buffer, HEPES buffer solution.
In embodiments of the present invention, filler be mannitol and glycine, freeze drying protectant be trehalose and tween,
Buffer is sodium phosphate buffer.
In embodiments of the present invention, the concentration of anti-tumor protein TmSm is 2~6mg/ml, and the concentration of mannitol is 10
~30mg/ml, the concentration of glycine are 2~8mg/ml, and the concentration of trehalose is 20~60mg/ml, and the concentration of Tween 80 is 0.5
~1.5mg/ml, the sodium phosphate buffer of sodium phosphate buffer 50mM, pH 6.5.
In embodiments of the present invention, the concentration of anti-tumor protein TmSm is 5mg/ml, and the concentration of mannitol is 20mg/
Ml, the concentration of glycine are 5mg/ml, and the concentration of trehalose is 30mg/ml, and the concentration of Tween 80 is 0.8mg/ml.
Second object of the present invention is to provide a kind of side of freeze drying powder injection for preparing above-mentioned anti-tumor protein TmSm
Method, method includes the following steps:
1) it according to recipe quantity compounding pharmaceutical solution, and is divided in freeze-drying container；
2) product is placed in freeze dryer, fast cooling carries out pre-freeze to product；
3) freeze dryer baffle temperature is set, a lyophilization is carried out under the conditions of -35 DEG C~-25 DEG C；
4) freeze dryer baffle temperature is set, carries out secondary parsing-desiccation under the conditions of 10 DEG C~20 DEG C.
In embodiments of the present invention, in step 2), the condition of pre-freeze is -80 DEG C of freezings 3h, -8 DEG C of annealing 2h, so
- 45 DEG C of freezing 3h afterwards.
In embodiments of the present invention, in step 3), the condition of a lyophilization is the shelf at -25 DEG C
At a temperature of lyophilization 20h.
In embodiments of the present invention, in step 4), the condition of the secondary parsing-desiccation is 20 DEG C of parsing-desiccations
8h。
Third object of the present invention is to provide the freeze drying powder injection of above-mentioned anti-tumor protein TmSm in preparation for controlling
Treat the application in the drug of tumour.
In embodiments of the present invention, tumour is breast cancer, cancer of pancreas and lung cancer.
The anti tumor activity in vitro of TmSm freeze drying powder injection evaluates display, to breast cancer B-Cap-37, breast cancer MCF-
7/s, cancer of pancreas SW1990 and tetra- kinds of cancer cells of lung cancer A549 have anti-tumor activity, and wherein lung cancer A549 cell is most sensitive.
Technical solution of the present invention has following advantageous effects:
First, it establishes a kind of for the quality stabilization of anti-tumor protein drug, controllable dosage form and preparation method, guarantee
The activity and curative effect of protein drug, and realize circulation and long term storage.
Second, preparation method of the invention is simple, has the feasibility of commercialized development, is suitable for large-scale continuous life
It produces.
Third, anti-tumor protein TmSm freeze drying powder injection product appearance prepared by the present invention is good, and pharmaceutical activity is strong, and property is steady
Fixed, interior bacteriotoxin residual is few, and purity is high has immunologic competence, and stability is good, has very strong anti tumor activity in vitro,
To breast cancer cell, it is ideal anticancer drug that pancreatic cancer cell and lung carcinoma cell, which have rush apoptosis of tumor cells effect,.
Detailed description of the invention
Fig. 1 is the result figure that TmSm fusion protein is identified with Western Blot method, can prove that the TmSm albumen has
Immunologic competence.
Fig. 2 is growth inhibition curve of the TmSm albumen to B-Cap-37 breast cancer cell.
Fig. 3 is the unit of activity measurement and Rate activity calculated result of TmSm powder-injection.
Fig. 4 is the ultra-violet absorption spectrum scanning figure of TmSm fusion protein, a length of 278nm of maximum absorption wave.
Fig. 5 is outside drawing after TmSm powder-injection redissolves, and medical fluid is transparent, clarity is high, without insoluble matter after display is redissolved.
Fig. 6 is the electrophoretogram for the purity of protein that non-reduced SDS-PAGE method detects TmSm freeze drying powder injection, shows freeze-dried powder
The purity of TmSm fusion protein in injection does not occur degrading and polymerizeing situation still 98% or more.
Fig. 7 is the chromatogram for the purity of protein that RP-HPLC method detects TmSm freeze drying powder injection, according to areas of peak normalization method
Measuring TmSm purity of protein in freeze drying powder injection is 98.1%.
Fig. 8 is that the hemolytic of TmSm freeze drying powder injection detects, and as a result negative control pipe and medicine group do not find that haemolysis is existing
As.
Fig. 9 is the Detection of Stability of TmSm freeze drying powder injection results, it can be seen that temperature is higher, and 6 months activity are damaged
Mistake is bigger, and 6 months loss of activity are saved at 4 DEG C and are lower than 4%.
Figure 10 is the evaluation of TmSm freeze drying powder injection anti tumor activity in vitro, it can be seen that microscope observes different tumours
Cell after TmSm drug effect for 24 hours after, compared with blank control, cell morphologically has occurred apparent variation, occurs thin
Born of the same parents' apoptosis early stage phenomenon.
Figure 11 is TmSm albumen to the inhibiting effect of four kinds of growth of tumour cell, is had to four kinds of tumour cells antitumor
Effect, wherein most sensitive to TmSm albumen is A549 cell.
Figure 12 is that Annexin V-FITC/PI method analyzes Apoptosis results, it can be seen that TmSm fusion protein is added
Afterwards, apparent apoptosis phenomenon has occurred in breast cancer B-Cap-37 cell.Have this further demonstrates TmSm fusion protein and promotees to swell
Apoptosis of tumor effect.
Figure 13 is the freeze-drying process curve graph of TmSm fusion protein.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following implementation will be helpful to this field
Technical staff further understands the present invention, but the invention is not limited in any way.Unless otherwise specified, make in following embodiments
It is commercially available conventional reagent that reagent is tested in room.
Embodiment one: the present embodiment for preparing of the freeze drying powder injection of anti-tumor protein TmSm is prepared for by the group of following content
The anti-tumor protein TmSm freeze drying powder injection being grouped as:
The TmSm fusion protein of 5mg/ml, the mannitol of 20mg/ml, the trehalose of the glycine of 5mg/ml, 30mg/ml,
The Tween 80 of 0.8mg/ml, the sodium phosphate buffer that 50mM, pH are 6.5, filling specification are 2ml/ branch.
The sodium phosphate buffer that 50mM, pH are 6.5 is prepared: being taken sodium phosphate about 8.2g, is set in 1000ml water, ultrasound is molten
Solution, and use phosphorus acid for adjusting pH to 6.5, filtering, it is ultrasonic to obtain the final product.
The composition of the freeze drying powder injection of the anti-tumor protein TmSm of the invention of table 1
The preparation of anti-tumor protein TmSm: being prepared by genetic engineering, has 90% homology with Survivin,
Survivin mutant protein (abbreviation TmSm) includes 20,34,84,117 site simple point mutations, multipoint mutation, groups
Close mutation, and the above site simultaneous mutation.
The freeze drying powder injection of anti-tumor protein TmSm of the invention the preparation method is as follows:
Active constituent TmSm fusion protein 10mg, the mannitol 40mg, glycine 10mg, trehalose of survival dose are weighed according to table 1
Then 60mg, Tween 80 1.6mg use 50mM, the sodium phosphate buffer dissolved dilution that pH is 6.5 to 2ml, packing is extremely
It is lyophilized in glass tube vial；Or equal proportion is amplified to 1000 amounts, sample solution is per bottled amount 2ml；Sample is placed into freeze dryer
In, carry out pre-freeze, fast cooling to -80 DEG C of freezing 3h, after be warming up to -8 DEG C of annealing 2h, be then cooled to -45 DEG C of freezing 3h；One
Baffle temperature is risen to -25 DEG C, lyophilization 20h by secondary drying；Baffle temperature is risen to 20 DEG C, parsing-desiccation by redrying
8h.Obtain anti-tumor protein TmSm freeze drying powder injection of the invention.
In above-mentioned preparation process, the dosage of active constituent and auxiliary material is weighed according to the component proportion of table 1, wherein
Quality/end solution volume of W/V expression component.
Make the freeze-drying process curve of TmSm fusion protein, the result is shown in Figure 13.
Embodiment two: identification the present embodiment of anti-tumor protein TmSm freeze drying powder injection is identified with Western Blot method
TmSm fusion protein.
After the dissolution of 20ml water for injection is added in TmSm freeze drying powder injection sample prepared by embodiment one, polyacrylamide is used
Gel electrophoresis (SDS-PAGE) is separated, and is identified by fusion protein obtained by more purification and renaturation.
Western Blot step and reagent: 20ml injection is added in TmSm freeze drying powder injection sample prepared by embodiment one
After being dissolved with water, separated with polyacrylamide gel electrophoresis (SDS-PAGE), when this electrophoresis need on gel bilateral symmetry
Loading, centre are standard molecular weight albumen Marker.After electrophoresis, glue is separated from the middle, half glue coomassie of the first from left is bright
Indigo plant dyeing, right half glue are used for transferring film, carry out transferring film using transfer method is partly done.Pvdf membrane (Thermo company) is first soaked with methanol
1-2min is steeped, filter paper impregnates with transfering buffering liquid, and graphite spreads 3 layers of filter paper of same size respectively from bottom to top, pvdf membrane, then
3 layers of filter paper, bubble of rushing.Transferring film electric current is 25mA, and the time is about 50min.Confining liquid is then added (containing 5% skimmed milk power
TPBS) protein binding in close membrane is used distilled water flushing film after completely shaking 1h on shaking table, then is washed 4 times with TPBS, and each shaking table is fast
Shake 5min；The rabbit-anti people survivin primary antibody (Bioworld company) that 4ml dilutes 500 times through PBS is added, shakes 1h slowly；It uses again
TPBS is washed 4 times, shakes 5min fastly every time；The goat antirabbit secondary antibody that PBS dilutes 1000 times of horseradish peroxidase-labeled is added
(Bioworld company), shakes 1h slowly；It is washed 3 times with TPBS, shakes 5min fastly every time, then washed 1 time with PBS, shake 5min fastly；By pvdf membrane
Albumen is spread out on preservative film up, is added on film after taking each 1ml mixing of reagent A and B, is reacted 2min.By pvdf membrane face
It is spread out on preservative film, folds downward.In darkroom, film is put on film and is exposed 1min, press film is first put
Enter developer solution, followed by water, place into fixing solution, then water, finally film is taken out it can be seen that melanoprotein band.Again will
Whether the developed band on pvdf membrane is compared consistent with the destination protein band of the other half SDS-PAGE electrophoresis.
Testing result: as shown in Figure 1, being detected albumen can specifically be immunized with rabbit-anti people's survivin primary antibody
Reaction.Therefore it can prove that the TmSm albumen has immunologic competence.
Embodiment three: the appearance and solubility of anti-tumor protein TmSm freeze drying powder injection detect the present embodiment and have detected implementation
The appearance of anti-tumor protein TmSm freeze drying powder injection prepared by example one mainly from color, whether atrophy disintegration, crack etc. into
Row evaluation, solubility are then that the sterile water for injection of 2ml is added into the cillin bottle each containing TmSm powder needle and immediately begins to
Timing can record dissolution time and solution character in the process with slight wobble bottle body.
As a result, it has been found that all samples can obtain in the 20s after the water for injection that 2ml is added, transparent, clarity is high, nothing
The medical fluid of insoluble matter, is shown in Fig. 5.
Example IV: the water content detection of anti-tumor protein TmSm freeze drying powder injection: referring to Chinese Pharmacopoeia Karl Fischer
Moisture titration is determined the residual moisture of six TmSm freeze drying powder injection samples, is computed and obtains TmSm powder-injection
Residual moisture be 1.8 ± 0.1%.
| Sample | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 |
| Residual moisture (%) | 1.8 | 1.81 | 1.81 | 1.80 | 1.79 | 1.80 |
Example IV one: detection of bacterial endotoxin the present embodiment of anti-tumor protein TmSm freeze drying powder injection is to embodiment one
Bacteria endotoxin content is detected in the freeze drying powder injection of preparation.2015 editions Chinese Pharmacopoeia four<1143>methods 1 of reference,
The endotoxin limit < 5EU/ml of protein drug, and sensitivity of the limulus reagent experiment is 0.25EU/ml, sensitivity meets the requirements, this
Method and result are effective.
Testing result: test solution is negative, and test sample positive control is positive, and positive control is the positive, negative right
According to be it is negative, meet the requirements.
Embodiment five: the purity of protein of anti-tumor protein TmSm freeze drying powder injection detects the present embodiment with a variety of methods to reality
Apply the purity of protein of the freeze drying powder injection of the preparation of example one.
1. non-reduced SDS-PAGE electrophoretic determination uses non-reduced SDS-PAGE electrophoresis technique determining, the isomery of protein
Body, oligomer will embody different swimming degree in electrophoresis.Content can be detected down to 0.2 μ g using Coomassie brilliant blue dyeing
Single protein band.10 μ g applied sample amounts are selected in this experiment, so that micro existing impurity protein can show that, if
The band of impurity protein is not shown, then there is no the single contaminant albumen that content is equal to or more than 0.2ug for explanation, that is to say, bright sample
Without the single contaminant albumen for being more than 2% in product.Therefore, non-reduced SDS-PAGE can not only delicately reflect different molecular weight
Protein different distributions, moreover it is possible to reflect the isomers and oligomer of same protein, measure target protein purity.For this
Literary grace detects the purity of TmSm fusion protein in freeze-dried powder with this method.
According to the concentration for the protein solution that Lowry method measures, loading is carried out with 10 μ g, 5 μ g, 1 μ g, 0.2 μ g respectively,
As a result see Fig. 6.Image analysis system through gel imaging carries out gray scale scanning, and non-reduced SDS-PAGE method can be examined as the result is shown
Measure the protein content of 0.2 μ g.It can be seen that the purity of the TmSm fusion protein in freeze drying powder injection does not go out still 98% or more
Now degradation and polymerization situation.
The purity of protein of 2.RP-HPLC method detection powder-injection
Instrument: Waters, Symmetry 300
Chromatographic column: C4 column, 5um
Mobile phase: Tris-HCl: normal propyl alcohol=820:285
Column temperature: 45 DEG C
Detection wavelength: 220nm
Detect flow velocity: 0.5ml/min
Applied sample amount: 20 μ l
Purity calculates: calculating by area normalization method, accounts for the percentage of total mark area as purity using the peak area of target peak
Data.
Testing result: target protein appearance in about 5min effectively separates target peak with other miscellaneous peaks.Peak
It is 98.1%, i.e. TmSm fusion protein that area normalization method, which measures TmSm albumen in freeze drying powder injection and accounts for the percentage of total protein,
Purity is 98.1%, as shown in Figure 7.
Embodiment six: the hemolytic test rat vein blood sampling of anti-tumor protein TmSm freeze drying powder injection is in glass strain
In sterile chamber, fully shake.Centrifuge 1000rpm 5min, is collected by centrifugation cell, is washed 2 times with PBS, then with 0.9%
Normal saline at 2% red cell suspension.One bottle of powder-injection of the albumen of TmSm containing 10mg is taken, ultrapure water is added, is made
The TmSm albumen of 1mg/ml final concentration.Taken shown according to the form below different volumes TmSm albumen (1mg/ml), 2% red cell suspension,
Physiological saline and distilled water mixing, prepare test sample, negative control and positive control.All test tube warm bath one are more than hour,
Whether there is or not haemolysis or agglutinating reactions for observation.Without haemolysis or agglutinating reaction in tested property management three hours, it is considered as qualification.
Testing result: negative control pipe and medicine group do not find haemolysis, illustrate the frozen-dried protective agent prescription being added
And freeze-drying process does not cause the great variety of solution osmotic pressure, this lyophilized protein product meets zoopery requirement, can be used for
Animal internal injection.See Fig. 8.
Embodiment seven: the Detection of Stability of anti-tumor protein TmSm freeze drying powder injection puts the freeze drying powder injection of TmSm albumen
It is placed under the conditions of 4 DEG C, 25 DEG C and 37 DEG C, was sampled respectively at the 0th, 1,2,3,4,5,6 month and measures its antitumor vigor
Unit.
Testing result: temperature is higher, and its 6 months loss of activity is bigger.In addition, 4 DEG C of preservations, 6 months loss of activity most
It is small, it is lower than 4%.See Fig. 9.
The anti tumor activity in vitro of eight: TmSm freeze drying powder injection of embodiment detects
TmSm fusion protein anti tumor activity in vitro standard detecting method:
1. B-Cap-37 cell (ATCC) is with 1 × 105The amount of a/ml is inoculated in 25cm2Tissue Culture Flask.Logarithmic growth phase
Cell, with 0.25% pancreatin digestion after, be added by DMEM complete medium (10% newborn bovine serum, 100IU/L penicillin,
100mg/L streptomysin, pH7.2) and cell is blown and beaten, make to form cell suspension.It is 1 × 10 with blood counting chamber meter cell number5A/
ml.Every 100 μ l of hole is inoculated in 96 porocyte culture plates, in 37 DEG C, 5%CO216h is cultivated in incubator, observes cell in hole
It covers with to 70~80%.
2. TmSm fusion protein (preparation method is shown in embodiment one) is diluted to different concentration with DMEM complete medium.
3. discarding culture medium old in 96 orifice plates, PBS is added in an outermost circle, is detailed in following table, the 1st is classified as not celliferous sky
200 μ l DMEM complete mediums are added in white control, every hole；Sample to be detected can be added in 2-9 column；10th column are added 200
The hole μ l/ buffer makees cell negative control.
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
| 1 | Blank | S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | Neg |
| 2 | Blank | S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | Neg |
| 3 | Blank | S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | Neg |
| 4 | Blank | S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | Neg |
| 5 | Blank | S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | Neg |
| 6 | Blank | S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | Neg |
Note: Blank-blank control, Neg- negative control, S1-S8 are various concentration albumen
4. the sample to be tested of 200 μ l difference dilutions is then added into 2-9 column cell hole, each dilution makees 6
Multiple holes (n=6).
5. 96 orifice plates of sample will have been added to set the CO of 37 DEG C, 5%2Continue to cultivate 48h in incubator, microscopically observation is thin
Born of the same parents' metamorphosis, preliminary judging result.
6. MTT and DMSO is added referring to 3.2.3 method, and the OD value at every hole 490nm is measured, then calculates drug to swollen
The inhibiting rate of oncocyte.
The TmSm fusion protein of process purifying and renaturation to various concentration is under the conditions of 37 DEG C of temperature, to B- after 48 hours
The inhibiting rate curve of Cap-37 breast cancer cell is studied, and as a result sees Fig. 2.With being made according to part in a linear relationship in figure
Equation is computed and obtains activity value.Figure it is seen that inhibiting rate is about in two sections 5-30% and 30-68%,
Concentration is approximate with inhibiting rate in a linear relationship.Because the concentration variation range and inhibiting rate value in first section are smaller, thus
Using it as TmSm active unit interval of definition, sample is easy in dilution beyond the range of linearity, to cause sample
The deviation of anti-tumor activity detection.Therefore the higher linearly interval of concentration is selected here.Regression equation: y=0.6495x+
0.1634, R2=0.998.
Testing result: it is 1 list that every 48h, which inhibits the TmSm fusion protein amount of 50% breast cancer B-Cap-37 cell growth,
Position.It is calculated according to the TmSm albumen in experiment, 1 unit is equivalent to the TmSm albumen of 15.35 ± 0.13 μ g.
The anti tumor activity in vitro of the TmSm freeze drying powder injection of nine: TmSm freeze drying powder injection of embodiment detects test sample:
It one bottle of TmSm freeze drying powder injection, is diluted with culture medium, prepares obtain 1.3,2.6,5.2,10.4 and 15.6units/ml not respectively
With the TmSm sample solution of concentration.
Negative control group is the buffer in maximum concentration sample to be detected.B-Cap-37, MCF-7/s and SW1990 are thin
The drug effect group of born of the same parents' (Shanghai Inst. of Life Science, CAS) is the DMEM that drug containing is added according to the final concentration of setting
Complete medium；The drug effect group of A549 cell is that the RMIP1640 that drug containing is added according to the final concentration of setting is cultivated completely
Base.6 multiple holes (n=6) of each concentration, in 37 DEG C after dosing, 5%CO2It is cultivated in incubator.
Several tumour cells are in 37 DEG C after dosing, 5%CO2It is cultivated 24 hours in incubator, it is aobvious to take out placement inverted light
Micro- microscopic observation cellular morphology variation.
The TmSm sample of different vigor gradients is to breast cancer B-Cap-37 cell, breast cancer MCF-7/s, cancer of pancreas SW1990
The detection time of the inhibiting rate of cell and lung cancer A549 cell is culture 48h.
Testing result: after microscope observes different tumour cells after TmSm drug effect for 24 hours, with blank control phase
Than apparent variation morphologically has occurred in cell.Cell volume at this time obviously becomes smaller, and gap occurs in iuntercellular, and cell goes out
Now shrink；Cell membrane is complete but there is vesicle sample protrusion on surface, there is cells float.These are all Apoptosis early stage phenomenons.See figure
10。
In general, when inhibiting rate of the drug to tumour cell reaches 30%, it is certain can just to illustrate that this drug has
Anti-tumor activity.TmSm powder-injection has antitumor effect to tetra- kinds of tumour cells of B-Cap-37, MCF-7/S, SW1990 and A549
Fruit.Wherein most sensitive is A549 cell, is secondly SW1990, least sensitive tumour cell is MCF-7/S.See Figure 11.
The tumour of TmSm freeze drying powder injection of the invention is added in the bis- dye method detections of ten: Annexin V-FITC/PI of embodiment
Phenomena of apoptosis experimental procedure:
1. B-Cap-37 cell culture is carried out by the cell culture processes of embodiment eight, when cell is paved with 80% cell bottle, with 1
×105A cell/ml is seeded to 6 orifice plates.Dosing after culture 16h.
2. test sample is prepared: one bottle of TmSm freeze drying powder injection, a certain amount of culture medium dilution is added, it is single that preparation obtains 10
Position/ml TmSm sample solution.Negative control group is the buffer in sample to be detected.
3. after 48h dosing, cell is collected, is washed twice with PBS, the digestion of 0.25% pancreatin.
4. culture medium is added, digestion is terminated, centrifuge tube is collected；1000rpm is centrifuged 5min, discards supernatant liquid.
5. PBS (4 DEG C) is washed 1 time, centrifuge 1000rpm is centrifuged 5min, discards supernatant liquid, and gently piping and druming makes into individual cells
Suspension.
7. handling cell about 1~5 × 105/ ml is cleaned 2 times with PBS, be added 500 μ l 1 × combine Buffer (Shanghai beauty
Season Bioisystech Co., Ltd).
8. being separately added into 5 μ l Annexin V-FITC (Shanghai Mei Ji Bioisystech Co., Ltd) and 5 μ lPI (Shanghai beauty
Season Bioisystech Co., Ltd) (50 μ g/ml).
9. room temperature is protected from light 5min.
9. being detected on flow cytometer.Annexin V-FITC (Ex=488nm, Em=530nm), with FITC signal
(FL1) and PI signal.
Data are read on two-parameter point diagram, apoptotic cell is Annexin V+/PI-, the 4th section cell ratio of the upper right corner in figure
Example is that the ratio of apoptotic cell occurs.
Testing result: cellular control unit is nearly all in living cells quadrant, only a few cell apoptosis and death.Dosing
Afterwards, compared with the control, on the two-parameter point diagram of FS/SS, living cells quadrant points are significantly reduced, and the point of apoptotic cell quadrant and dead
The point for dying cell quadrant increased significantly.It can thus be seen that breast cancer B-Cap-37 cell occurs after TmSm fusion protein is added
Apparent apoptosis phenomenon.Have this further demonstrates TmSm fusion protein powder-injection and promote apoptosis of tumor cells effect, sees figure
12。
This specific embodiment is only explanation of the invention, is not limitation of the present invention, those skilled in the art
Member can according to need the modification that not creative contribution is made to the present embodiment after reading this specification, but as long as at this
All by the protection of Patent Law in the scope of the claims of invention.
Claims (12)
1. a kind of freeze drying powder injection of anti-tumor protein TmSm, characterized by comprising:
The TmSm fusion protein of survival dose；
Filler；
Freeze drying protectant；
Tween 80；With
Buffer.
2. the freeze drying powder injection of anti-tumor protein TmSm according to claim 1, which is characterized in that the filler and jelly
Dry protective agent is selected from glucose, sucrose, lactose, maltose, mannitol, sorbierite, xylitol, glycine, dextran, seaweed
One or more of sugar, Tween 80, povidone and group of glycerol composition.
3. the freeze drying powder injection of anti-tumor protein TmSm according to claim 2, which is characterized in that the buffer is selected from
One of potassium phosphate buffer, sodium phosphate buffer, sodium citrate salt buffer, HEPES buffer solution.
4. the freeze drying powder injection of anti-tumor protein TmSm according to claim 3, which is characterized in that filler is mannitol
And glycine, freeze drying protectant are trehalose and tween, buffer is sodium phosphate buffer.
5. the freeze drying powder injection of anti-tumor protein TmSm according to claim 4, which is characterized in that anti-tumor protein TmSm
Concentration be 2 ~ 6 mg/ml, the concentration of mannitol is 10 ~ 30mg/ml, and the concentration of glycine is 2 ~ 8mg/ml, trehalose it is dense
Degree is 20 ~ 60mg/ml, and the concentration of Tween 80 is 0.5 ~ 1.5mg/ml, the sodium phosphate of sodium phosphate buffer 50mM, pH 6.5
Salt buffer.
6. the freeze drying powder injection of anti-tumor protein TmSm according to claim 5, which is characterized in that anti-tumor protein TmSm
Concentration be 5mg/ml, the concentration of mannitol is 20mg/ml, and the concentration of glycine is 5mg/ml, and the concentration of trehalose is 30mg/
Ml, the concentration of Tween 80 are 0.8mg/ml.
7. a kind of method for preparing the freeze drying powder injection of anti-tumor protein TmSm described in any one of -6 according to claim 1,
It is characterized in that, comprising the following steps:
1) it according to recipe quantity compounding pharmaceutical solution, and is divided in freeze-drying container；
2) product is placed in freeze dryer, fast cooling carries out pre-freeze to product；
3) freeze dryer baffle temperature is set, a lyophilization is carried out under the conditions of -35 DEG C ~ -25 DEG C；
4) freeze dryer baffle temperature is set, carries out secondary parsing-desiccation under the conditions of 10 DEG C ~ 20 DEG C.
8. the freeze drying powder injection preparation method of anti-tumor protein TmSm according to claim 7, which is characterized in that in step 2
In, the condition of pre-freeze is -80 DEG C of freezings 3h, -8 DEG C of annealing 2h, then -45 DEG C of freezing 3h.
9. the freeze drying powder injection preparation method of anti-tumor protein TmSm according to claim 7, which is characterized in that in step 3)
In, the condition of a lyophilization is the lyophilization 20h under -25 DEG C of shelf temperature.
10. the freeze drying powder injection preparation method of anti-tumor protein TmSm according to claim 7, which is characterized in that in step
4) in, the condition of the secondary parsing-desiccation is 20 DEG C of parsing-desiccation 8h.
11. the freeze drying powder injection of anti-tumor protein TmSm according to claim 1 is preparing the drug for treating tumour
In application.
12. application according to claim 11, which is characterized in that the tumour is breast cancer, cancer of pancreas and lung cancer.
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Application publication date: 20190531 |