CN108676085B - A kind of preparation method and its pharmaceutical composition of thymalfasin - Google Patents
A kind of preparation method and its pharmaceutical composition of thymalfasin Download PDFInfo
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Abstract
The invention belongs to pharmaceutical technology fields, and in particular to a kind of preparation method of thymalfasin and the pharmaceutical composition containing thymalfasin, the preparation method are eluted using glucan G type gel and cyanoalkysilane bonded silica gel；In thymalfasin using the method for the present invention purifying, impurity [the D-Ser1]-thymalfasin and [D-Asn28]-thymalfasin of measurement are respectively less than 0.07%；Freeze drying powder injection dissolubility made from composition of the present invention is good, stability is good, solubility is good；The freeze drying powder injection as made from the composition is easy to use, is conducive to storing, and preparation method is simple, easy to industrialized production, and production cost is low.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to the preparation method of thymalfasin and the drug containing thymalfasin
Composition.
Background technique
Thymalfasin (Thymosin α 1, also known as thymosin α1), molecular formula C129H215N33O55, it is by 28 amino acid
The polypeptide of composition, relative molecular mass 3108.28, for white or off-white powder, amino acid composition sequence is N-Ac-
Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-
Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH。
The beginning of the eighties U.S. the artificial synthesized thymalfasin in the laboratories such as Merrifield, currently, U.S. Sciclone
With the thymalfasin product of Italian Sclavo SPA, in multiple country's listings such as Italy, (day reaches trade name Zadxin
It is celestial), just enter Chinese market early in last century end.
Chinese patent CN201210052504 discloses a kind of purification process of thymalfasin, and specific steps include active carbon
Adsorption filtration, the concentration of chromatography on neutral alumina post separation, ultrafiltration membrane, ether settle centrifuge washing, drying.
Chinese patent CN201110186259 uses reverse phase C18 filler, successively uses 0.1%TFA/ acetonitrile, 0.2% phosphorus
Acid/acetonitrile and water/acetonitrile carry out first time to the thick peptide of thymalfasin respectively, purify and turn salt preparation for the second time.After product freeze-drying
Purity is up to 99% or more.
Chinese patent CN200910304817 provides a kind of thymalfasin desalinating process.Its processing step are as follows: will be acidified
Ion exchange resin is first eluted with water in Thymosin alpha 1 solution afterwards after mixing with cation exchange resin, then is eluted with alkaline solution
Exchanger resin, collects the eluent for having absorption at 230nm wavelength, and last concentrated drying obtains thymalfasin.At sample
The characteristics of reason amount is big, and desalting efficiency is good, repeats loading, is suitable for industrialized production.
During the synthesis in solid state of existing thymalfasin, since synthesis step is more, the raw material quantity of addition is more, causes
Impurity in products type is relatively more, it is known that impurity include: D-Asn28- thymalfasin, Asp28- thymalfasin, di-Ala4- chest
Gland method is new etc., and there are also many for unknown impuritie.
Thymalfasin is a kind of polypeptide, all very sensitive to pH value, temperature etc., and stability in water is poor.In order to increase
The stability for adding thymalfasin preparation is clinically prepared into freeze-dried powder, and requires to save under the conditions of 2~8 DEG C, but infuse
It penetrates with thymalfasin on production link, needs to prepare the filling quality that just can guarantee preparation using 2~8 DEG C of water for injection.
Chinese patent application 201110220652 discloses a kind of containing the Pharmaceutical composition of thymalfasin and its preparation side
Method, the pharmaceutical composition contain following component: thymalfasin, mannitol, sorbierite 0.01M NaAc_HAc buffer solution tune
PH to 3.8~4.2 is saved, but the pH value of normal human blood is 7.35~7.45, solution can be to intravascular out of this range
Chrotoplast causes to damage, and induces the chain reaction of the thrombophlebitis of platelet aggregation and excitation.
Summary of the invention
For solve it is above-mentioned the problems of in the prior art, We conducted a large amount of experimental exploration, discovery addition phosphorus
Sour disodium hydrogen-citrate buffer solution and a certain proportion of dextrosan and glycine can significantly improve thymalfasin injection system
The stability of agent.
Specifically, the present invention provides:
A kind of purification process of thymalfasin, comprising the following steps:
Step 1: thymalfasin crude product being dissolved in disodium hydrogen phosphate-citrate buffer solution buffer, with glucan G type
Gel is stationary phase, and disodium hydrogen phosphate-citrate buffer solution is mobile phase, collects purpose peak and obtains the first eluent, freeze-drying；
Step 2: the freeze-dried powder of the first eluent is dissolved in mobile phase A, using cyanoalkysilane bonded silica gel as stationary phase,
It is A phase with 1% acetonitrile solution that mass concentration is 0.05~0.1% perchloric acid, mass concentration is 0.05~0.1% perchloric acid
95% acetonitrile solution be B phase, gradient is 40%B → 70%B, carries out second and purifies, and linear gradient elution collects elution
Liquid, by eluent membrane filtration, concentration；
Step 3: being slowly added to ether into concentrate, stir, after standing 10-12 hours at 4-6 DEG C, thymus gland method is precipitated
Newly.
The pH of the disodium hydrogen phosphate-citrate buffer solution is 2.0~4.0.
A kind of pharmaceutical composition containing thymalfasin, comprising thymalfasin, excipient, buffering pair, feature is described
Excipient be mixture that dextrose and glycine form.
The composition includes the ingredient of following parts by weight: 1.5~2 parts by weight thymalfasins, 40-60 parts by weight figuration
Agent, buffering are to appropriate.
Dextrosan is spray drying dextrosan in the excipient.
The weight ratio of dextrosan and glycine is (3~5) in the excipient: 1.
Buffering is to for pH=7.0 disodium hydrogen phosphate-citrate buffer solution used in described pharmaceutical composition.
The pH value of the composition is 6.6~7.5.
A method of the pharmaceutical composition containing thymalfasin prepares freeze-dried powder, method includes the following steps:
It takes excipient to be dissolved in the water for injection of 2/3 recipe quantity, stirs evenly；
Recipe quantity thymalfasin is added, surveys pH value, adjusts pH to 6.6~7.5 with disodium hydrogen phosphate-citrate buffer solution,
Water for injection is added, freeze-dried powder is lyophilized to obtain with miillpore filter refined filtration to full dose.
Compared with the prior art, the present invention has the following advantages and good effect:
1, in the thymalfasin using the method for the present invention purifying, the impurity [D-Ser of measurement1]-thymalfasin and [D-
Asn28]-thymalfasin is respectively less than 0.07%.
2, the freeze drying powder injection as made from the composition has good stability, and solubility is good；It is lyophilized as made from the composition
Powder-injection is easy to use, is conducive to storing, and preparation method is simple, easy to industrialized production, and production cost is low.
Specific embodiment
The following describes the present invention further through the description of specific embodiments, but it is to limit of the invention that this, which is not,
System, those skilled in the art's basic thought according to the present invention can make various modifications or improvements, but without departing from this
The basic thought of invention, is all within the scope of the present invention.
By thymalfasin crude product prepared by the method for CN1390853A, being detected its HPLC purity is 94.52%.
Embodiment 1
Step 1: thymalfasin crude product 5g pH2.6 disodium hydrogen phosphate-citrate buffer solution being dissolved, concentration is made
The solution of 200mg/ml, using SephadexG-15 as stationary phase, using pH2.6 disodium hydrogen phosphate-citrate buffer solution as mobile phase,
Elution speed is 2ml/min, elutes 20min, Detection wavelength 210nm, collects purpose peak and obtains the first eluent, freeze-drying；
Step 2: the freeze-dried powder 4.2g of the first eluent is dissolved in mobile phase A, using YMC-Pack CN as stationary phase,
It is A phase with the acetonitrile solution A (volume ratio is 99% water/1% acetonitrile) that mass concentration is 0.05% perchloric acid, mass concentration is
The acetonitrile solution B (volume ratio is 5% water/95% acetonitrile) of 0.05% perchloric acid is B phase, flow velocity 60ml/min, elution
30min, gradient are 40%B → 70%B, carry out second and purify, and Detection wavelength 210nm collects purity and is greater than 95% or more
Eluent, by 0.22 μm of membrane filtration of eluent, concentration；
Step 3: it is slowly added to 8ml ether into this concentrate, stirs, after standing 10-12 hours at a temperature of 4-6 DEG C,
The thymalfasin of precipitation is dry, the thymalfasin 2.82g that purity is 99.93% is obtained, purifying total recovery is 56.4%.
Embodiment 2
Step 1: thymalfasin crude product 8g being dissolved in pH2.6 disodium hydrogen phosphate-citrate buffer solution and is dissolved, concentration is made
The solution of 250mg/ml, using SephadexG-25 as stationary phase, pH2.6 disodium hydrogen phosphate-citrate buffer solution is mobile phase, is washed
De- speed is 5ml/min, elutes 20min, Detection wavelength 210nm, collects purpose peak and obtains the first eluent, freeze-drying；
Step 2: the freeze-dried powder 7.1g of the first eluent is dissolved in mobile phase A, using YMC-Pack CN as stationary phase,
It is A phase with the acetonitrile solution A (volume ratio is 99% water/1% acetonitrile) that mass concentration is 0.1% perchloric acid, mass concentration is
The acetonitrile solution B (volume ratio is 5% water/95% acetonitrile) of 0.1% perchloric acid is B phase, flow velocity 75ml/min, elution
25min, gradient are 40%B → 70%B, carry out second and purify, and Detection wavelength 210nm collects purity and is greater than 95% or more
Eluent, by 0.22 μm of membrane filtration of eluent, concentration；
Step 3: it is slowly added to 10ml ether into this concentrate, stirs, after standing 10-12 hours at a temperature of 4-6 DEG C,
The thymalfasin of precipitation is dry, the thymalfasin 4.87g that purity is 99.81% is obtained, purifying total recovery is 60.9%.
Embodiment 3
Step 1: thymalfasin crude product 50g is dissolved in pH2.6 disodium hydrogen phosphate-citrate buffer solution, with
SephadexG-15 is stationary phase, using pH2.6 disodium hydrogen phosphate-citrate buffer solution as mobile phase, elution speed 25ml/
Min elutes 50min, Detection wavelength 210nm, collects purpose peak and obtains the first eluent, freeze-drying；
Step 2: the freeze-dried powder 41.3 of the first eluent is dissolved in mobile phase A, using YMC-Pack CN as stationary phase,
It is A phase with the acetonitrile solution A (volume ratio is 99% water/1% acetonitrile) that mass concentration is 0.08% perchloric acid, mass concentration is
The acetonitrile solution B (volume ratio is 5% water/95% acetonitrile) of 0.08% perchloric acid is B phase, flow velocity 75ml/min, elution
25min, gradient are 40%B → 70%B, carry out second and purify, and Detection wavelength 210nm collects purity and is greater than 95% or more
Eluent, by 0.22 μm of membrane filtration of eluent, concentration；
Step 3: being slowly added to 300ml ether into this concentrate, stir, stand 10-12 hours at a temperature of 4-6 DEG C
Afterwards, the thymalfasin of precipitation is dry, the thymalfasin 34.38g that purity is 99.82% is obtained, purifying total recovery is 68.76%.
Test case 1:
Related substance takes this product appropriate, accurately weighed, and 0.02mol/L (pH7.0) phosphate buffer is added to dissolve and dilute
The solution of the thymalfasin containing 0.5mg/ml is made as test solution；Precision measures 2ml, is placed in 100ml volumetric flask, adds
0.02mol/L (pH 7.0) phosphate buffer is diluted to scale, shakes up, and as contrast solution, precision measures test solution
HPLC is injected with each 20 μ l of reference substance solution, records chromatogram, if any impurity peaks in the chromatogram of test solution, removes solvent peak
Outside acetic acid peak, [D-Asn28]-thymalfasin is not greater than 2.0%, [D-Ser1]-thymalfasin is not greater than 1.5%, other
Single impurity peak area is not greater than contrast solution main peak area (2.0%), and the sum of each impurity peaks peak area is not greater than control
2 times (4.0%) of the peak area of main peak in liquid chromatography figure
Assay is measured according to high performance liquid chromatography (general rule 0512).
Chromatographic condition and system suitability are filler with octadecylsilane chemically bonded silica；With ammonium sulphate buffer
(ammonium sulfate 26.4g, phosphoric acid 25ml is taken to be dissolved in water and be diluted to 2000ml)-acetonitrile (90:10) is mobile phase A, with ammonium sulfate
Buffer-acetonitrile (50:50) is Mobile phase B；Column temperature is 50 DEG C；Detection wavelength is 210nm；According to the form below carries out gradient elution.It takes
Thymalfasin reference substance and [D-Ser1]-thymalfasin and [D-Asn28]-thymalfasin reference substance is each appropriate, adds 0.02mol/L
Squama phthalate buffer (pH 7.0), which is dissolved and diluted, to be made in every lml containing [D-Ser1]-thymalfasin and [D-Asn28]-thymus gland method
The mixed solution of new each 50 μ g, the 0.5mg containing thymalfasin, take 20 μ l to inject liquid chromatograph, adjust gradient table 45 minutes
The ratio of the Mobile phase B at place makes the retention time of thymalfasin be about 30 minutes, records chromatogram.Number of theoretical plate presses thymalfasin
Peak, which calculates, is not less than 2000, and the separating degree between the peak impurity I and thymalfasin peak should be greater than 1.2.
1 gradient mobile phase of table
| Time (minute) | Mobile phase A | Mobile phase B (%) |
| 0 | 88 | 12 |
| 45 | 82 | 18 |
| 50 | 50 | 50 |
| 51 | 88 | 12 |
| 60 | 88 | 12 |
Measuring method takes this product appropriate, accurately weighed, and 0.02mol/L phosphate buffer (pH 7.0) is added to dissolve and quantify dilute
The solution that the 0.5mg containing thymalfasin in every lm l is made is released, as test solution；Precision measures 20 μ l of test solution injection
Liquid chromatograph records chromatogram；It separately takes thymalfasin reference substance appropriate, is measured in the same method.By external standard method with calculated by peak area, i.e.,
?.As a result table 2.
The thymalfasin impurity content detection that the different preparation methods of table 2 obtain
Comparative example 1:CN201210052504 embodiment 1
Conclusion: the above results show that total related substance of thymalfasin prepared by the present invention is respectively less than 0.2%, [D-
Asn28]-thymalfasin is less than 0.07%, [D-Ser1Less than 0.05%, other single maximum contaminants are less than]-thymalfasin
0.05%, it is superior to raw material and comparative example 1.
Embodiment 4
Under conditions of cleaning, by 37.5g dextrosan and 12.5g glycine investment liquid dispenser tool, injection is added
Stirring and dissolving in water 700ml is added 1.6g thymalfasin (being made by 3 the method for embodiment), pH value is surveyed, with pH=7.0's
Disodium hydrogen phosphate-citrate buffer solution adjusts pH to 7.0, and water for injection is added and obtains to full dose through 0.22 μm of filtering with microporous membrane
To thymalfasin filtrate.3ml is dispensed in ampere bottle, aseptic freeze-dried powder-injection is made in 72 hours in freeze-drying.
Embodiment 5
Under conditions of cleaning, by 46.4g dextrosan and 11.6g glycine investment liquid dispenser tool, injection is added
Stirring and dissolving in water 700ml is added 1.9g thymalfasin (being made by 2 the method for embodiment), pH value is surveyed, with pH=7.0's
Disodium hydrogen phosphate-citrate buffer solution adjusts pH to 7.0, and water for injection is added and obtains to full dose through 0.22 μm of filtering with microporous membrane
To thymalfasin filtrate.3ml is dispensed in ampere bottle, aseptic freeze-dried powder-injection is made in 72 hours in freeze-drying.
Embodiment 6
Under conditions of cleaning, by 50g dextrosan and 10g glycine investment liquid dispenser tool, water for injection is added
Stirring and dissolving in 700ml is added 2.0g thymalfasin (being made by 1 the method for embodiment), pH value is surveyed, with the phosphorus of pH=7.0
Sour disodium hydrogen-citrate buffer solution adjusts pH to 6.9, and water for injection is added and obtains to full dose through 0.22 μm of filtering with microporous membrane
Thymalfasin filtrate.3ml is dispensed in ampere bottle, aseptic freeze-dried powder-injection is made in 72 hours in freeze-drying.
Embodiment 7
Under conditions of cleaning, by 35g dextrosan and 10g glycine investment liquid dispenser tool, water for injection is added
1.9g thymalfasin is added in stirring and dissolving in 700ml, pH value is surveyed, with disodium hydrogen phosphate-citrate buffer solution tune of pH=7.0
PH to 6.9 is saved, water for injection is added to full dose and obtains thymalfasin filtrate through 0.22 μm of filtering with microporous membrane.Dispense 3ml in
In ampere bottle, aseptic freeze-dried powder-injection is made in 72 hours in freeze-drying.
Prescription screening test
1, the screening of formulation buffer system
Prepare acetic acid-sodium acetate buffer solution (0.1M, pH=4.5), citric acid-sodium citrate buffer solution (0.1M, pH=
4.8), disodium hydrogen phosphate-citrate buffer solution (0.1M, pH=7.0), disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution (0.1M,
PH=7.4).Referring to dissolution of the method measurement thymalfasin in each buffer under 2010 editions two note on the use items of Chinese Pharmacopoeia
Degree, specifically: appropriate amount of sample is weighed, in 25 DEG C of ± 1 DEG C of a certain amount of solution, every strength oscillation in 5 minutes 30 seconds, observation 30
Dissolution situation in minute is considered as dissolution when without visual visible particles of solute.Test result is shown in Table 3, sentences from solubility angle
Disconnected, the preferred disodium hydrogen phosphate-citrate buffer solution of preparation solution, disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution are as buffer.
Solubility of 3 thymalfasin of table in buffer
2, the stability analysis in disodium hydrogen phosphate-citrate buffer solution, disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution
Prepare disodium hydrogen phosphate-citrate buffer solution (0.1M, pH=7.0), disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution
(0.1M, pH=7.4) weighs a certain amount of thymalfasin and dissolves in wherein, and forming concentration is 2.1mg/ml solution, is placed in 37 DEG C and incubates
Case incubates bath, carries out HPLC detection to wherein thymalfasin purity in 0,1,2,4,8,12 hour, test result is shown in Table 4.From stabilization
Property angle judgement, the preferred disodium hydrogen phosphate-citrate buffer solution of preparation solution is as buffer.
Purity (%) of 4 thymalfasin of table in buffer
3, in preparation solution excipients screening (one)
It prepares disodium hydrogen phosphate-citrate buffer solution (0.1M, pH=7.0), contains thymalfasin 1.6mg/ml.And respectively
Contain the mannitol of 4% (W/V), the composition of dextrosan and glycine (weight ratio 4:1), sorbierite, lactose as excipient
Agent.It is placed in 37 DEG C of incubators and incubates bath, detect the stability of thymalfasin in each time point solution.And (freeze-drying program is lyophilized
Are as follows: -40 DEG C pre-freeze 4 hours；It is evacuated to 300mT；1 DEG C/min of temperature program of setting, rises to -20 DEG C；Freeze-drying 12 hours；
1 DEG C/min of temperature program of setting, rises to 30 DEG C；It is 4 hours dry；Tamponade simultaneously rolls lid.), observe the outer of its lyophilized form preparation
It sees, test result such as table 5.From stability and freeze-drying appearance angle judgement, the preferred mannitol of preparation solution or dextrosan with it is sweet
The composition of propylhomoserin is as excipients.
5 thymalfasin of table is containing the purity (%) and freeze-drying appearance in excipient formulation solution buffer
4, in preparation solution excipients screening (two)
Prepare disodium hydrogen phosphate-citrate buffer solution (0.1M, pH=7.0), containing thymalfasin 0,0.1,0.25,0.5,
1.0,1.6,3.2 or 6.0mg/ml.And contain the composition of the sweet mannitol of 4% (W/V), dextrosan and glycine respectively
(weight ratio 4:1), sorbierite, lactose are as excipients.The preparation solution for not containing excipients is prepared simultaneously.Vial is taken, is pressed
The 1ml/ bottles of above-mentioned preparation solutions of packing, freeze-drying.The pharmaceutical preparation for investigating lyophilized form redissolves situation.Test result such as table 6.
Lyophilized preparation when containing different excipients in 6 preparation solution of table, the clarity of preparation after redissolution
Note: < 1 refers to less than No. 1 titer of turbidity；> 1 refers to that turbidity is greater than No. 1 titer
The display of this test result, introduces the solute molecules such as mannitol or the excipients such as dextrosan or sorbierite, only will
Prepare the concentration range of thymalfasin in the pharmaceutical preparation solution that can dissolve lyophilized preparation be increased to 0 by 0~0.1mg/ml~
0.25mg/ml can redissolve thymalfasin concentration in pharmaceutical preparation after slightly increasing freeze-drying；Some excipients introducing (such as
Lactose) it is little to the influence that can redissolve thymalfasin concentration in pharmaceutical preparation after raising freeze-drying.
5, in preparation solution excipients screening (three)
Formulated solution, wherein disodium hydrogen phosphate-citrate buffer solution (0.1M, pH=7.0), different weight are righter than poly-
Rotation sugar and glycine compositions, thymalfasin 1.6mg/ml, are deployed with water.Using the PB-10 type pH meter (U.S.
Sartorius company), according to " Pharmacopoeia of People's Republic of China 2010 editions two " annex VI H related pH value detection requirement into
Row detection.Detect the solubility of thymalfasin in preparation solution.Preparation solution is packed into 2ml glass, bottle is lyophilized, 1ml/ bottles, moved into
The freeze-drying of low-temperature freeze-drying machine (Supermodulyo type, 0.4m2, E-C Apparatus company) product bin.With 1ml injection
Water redissolves the preparation of freeze-drying, and the pharmaceutical preparation for investigating lyophilized form redissolves situation.The preparation of freeze-drying is put into 37 DEG C of incubators, incubates bath 8
It is redissolved with 1ml water for injection for every bottle after week, detects the purity of thymalfasin in preparation solution.The dextrosan of different weight ratio with
The appearance of lyophilized form preparation and situation is redissolved and the preparation of lyophilized form exists after the preparation solution pH value of glycine, freeze-drying
37 DEG C place 8 weeks after thymalfasin purity it is as shown in table 7, the purity detecting of thymalfasin is after the freeze-drying of each preparation solution
99.86%.By the stability comprehensive descision of preparation solution pH, lyophilized form preparation shape and lyophilized preparation, preferably poly- dextrorotation
Sugar and glycine weight ratio are (3~5): 1.The available 1ml of the pharmaceutical preparation of lyophilized form is redissolved to 5ml water for injection, again
Liquid pharmaceutical formulation is made.
Contain pH, freeze-drying appearance, weight molten time and 8 peripheral stabilities under different excipient concentrations in 7 preparation solution of table
Note: < 1 refers to less than No. 1 titer of turbidity；> 1 refers to that turbidity is greater than No. 1 titer.
Conclusion (of pressure testing): experiments have shown that, it is figuration that select the weight ratio of dextrosan and glycine, which be 3~5:1, by above-mentioned
Agent has better product quality.
Injection thymalfasin test example
1, instrument and drug
High performance liquid chromatograph (HP1100 type, hewlette-packard), DAD detector, AgilentChemstation color
Compose work station: YB-2 type clarity test instrument (Precision Instrument Factory, Tianjin Univ.)；640 type UV detector (U.S. shellfish of DU
Gram Mann)；PHS-3C type digital ph (Shanghai Lei Ci instrument plant)；WS/08-0l type temperature and humidity regulator (Hangzhou blue sky instrument
Device production Co., Ltd)；200 type analysis balance (Switzerland) of METYLER.AE；Injection thymalfasin is (according to embodiment 6
The sample of preparation).
2, method
2.1 chromatographic condition octadecylsilane chemically bonded silicas are filler: being filling with octadecylsilane chemically bonded silica
Agent；With acetonitrile-water-phosphoric acid (140:860:1) for mobile phase A；With acetonitrile-water-phosphoric acid (250:750:1) for Mobile phase B, detection
Wavelength is 210nm.According to the form below carries out gradient elution；Number of theoretical plate is calculated by thymalfasin peak is not less than 2000.Thymalfasin peak
It should meet the requirements with the separating degree at other impurities peak.
2.2 investigate project and method
2.2.1 acidity: injection thymalfasin thymalfasin sample is taken, adds water that the solution of 0.5mg/ml is made, using acid
Degree meter directly measures.As a result pH value is 6.6~7.5.
2.2.2 the clarity and color of solution: taking the new thymus gland method of injection 5, and respectively plus the solution of 1mg/ml is made in water,
According to solution colour inspection technique, each batch of sample solution is clarified, is colourless.
2.2.3 clarity: taking the new thymus gland method of injection 5, and respectively plus the solution of 1mg/ml is made in water for injection, according to China
Pharmacopeia inspection meets regulation.
2.2.4 in relation to substance: take this product, add 0.02mol/L phosphate buffer (pH7.0) to dissolve and dilute be made it is every
The solution of the 0.5mg containing thymalfasin is as test solution in 1ml；Precision measures test solution 2ml, sets in 100ml measuring bottle,
Add 0.02mol/L phosphate buffer (pH7.0) to be diluted to scale, shakes up, as contrast solution.According to the color under content determination item
Spectral condition, takes 20 μ l of contrast solution, injects liquid chromatograph, adjusts detection sensitivity, makes the peak height of principal component chromatographic peak be about
The 10% of full scale.It is accurate again to measure test solution and each 20 μ l of contrast solution, it is injected separately into liquid chromatograph, records chromatography
Figure.If any impurity peaks in the chromatogram of test solution, in addition to solvent peak and acetic acid peak, single impurity peak area is not greater than pair
According to solution main peak area (2.0%), each impurity peak area summation is not greater than 2 times (4.0%) of contrast solution main peak area.
2.2.5 it assay: is measured according to high performance liquid chromatography (two V D of annex of Chinese Pharmacopoeia version in 2010).
The experiment of 2.3 influence factors
Under marketed products terms of packing, three batches of 6 samples of embodiment (lot number 170305,170307,170309) are existed
Investigated 5,10 days under high temperature (60 DEG C), Qiang Guang (4500lx), super-humid conditions, to the indexs such as its acidity, content and related substance into
Row is investigated, and indices meet regulation.Measurement result is shown in Table 9.
9 influence factor test result of table
A, B, C represent three batches of samples of embodiment 6, the sample of lot number 170305,170307,170309.
2.6 Acceleration study
Under marketed products terms of packing, three batches of 6 samples of embodiment (lot number 170305,170307,170309) are existed
It stores under the conditions of temperature (40 ± 2) DEG C, 75% soil 5% of relative humidity, is sampled respectively at 0,1,2,3,6 the end of month, measurement is every
Index.Each batch of sample property is white loose block.Meet regulation, clarity of solution and color, clarity meet rule
It is fixed.Acidity the results are shown in Table 10 in relation to substance and assay.
2.7 long-term experiment
Under marketed products terms of packing, three batches of 6 samples of embodiment (lot number 170305,170307,170309) are existed
It stores under the conditions of temperature (25 ± 2) DEG C, relative humidity 60% ± 10%, is sampled respectively at 0,3,6,12,18,24 the end of month, surveyed
Determine indices.Each batch of sample property is white loose block, meets regulation, and clarity of solution and color, clarity accord with
Regulation is closed, acidity the results are shown in Table 10 in relation to substance and assay.
10 accelerated test of table and long term test investigate result
A, B, C represent three batches of samples of embodiment 6, the sample of lot number 170305,170307,170309.
Conclusion: influence factor test, accelerated test are the results show that injection thymalfasin items testing index becomes without obvious
Change, has good stability；Injection thymus gland method 24 months every quality index are placed under the conditions of long-term room-temperature without significant change, product
It has good stability.
Stability Determination after injection thymalfasin redissolves is tested
By 170305,170307,170309, the sample water for injection 250ml of Chinese patent application 201210052708
It after redissolution, is placed in 4 DEG C of refrigerators, measurement is primary daily, METHOD FOR CONTINUOUS DETERMINATION 14 days, carries out straight line to time (X) with measured value (Y) and returns
Return analysis.As a result as shown in table 11.
Stability after the redissolution of 11 injection thymalfasin of table
Note: P > 0.05 illustrates that thymalfasin is basicly stable within the time measured.
Conclusion: as known from Table 11, after three batches of products redissolution of the present invention after METHOD FOR CONTINUOUS DETERMINATION 14 days, thymalfasin keeps content steady
It is fixed.Property is stablized after illustrating the injection thymalfasin redissolution of the preparation of method shown in the present invention.
Claims (3)
1. a kind of preparation method of thymalfasin, which is characterized in that the preparation method the following steps are included:
Step 1: thymalfasin crude product being dissolved in disodium hydrogen phosphate-citrate buffer solution, is to fix with glucan G type gel
Phase, disodium hydrogen phosphate-citrate buffer solution are mobile phase, collect purpose peak and obtain the first eluent, freeze-drying；
Step 2: the freeze-dried powder of the first eluent being dissolved in mobile phase A, using cyanoalkysilane bonded silica gel as stationary phase, with matter
Amount concentration is that 1% acetonitrile solution of 0.05~0.1% perchloric acid is A phase, and mass concentration is 0.05~0.1% perchloric acid
95% acetonitrile solution is B phase, and gradient is 40%B → 70%B, carries out second and purifies, and linear gradient elution collects elution
Liquid, by eluent membrane filtration, concentration；
Step 3: ether being added into concentrate, stirs, after standing 10-12 hours at 4-6 DEG C, thymalfasin is precipitated.
2. a kind of preparation method of thymalfasin according to claim 1, which is characterized in that the wherein phosphoric acid hydrogen two
Sodium-citrate buffer solution pH is 2.0~4.0.
3. a kind of pharmaceutical composition of the thymalfasin containing claim 1, which is characterized in that described pharmaceutical composition includes chest
Gland method is new, excipient, buffer, and the excipient is the mixture that dextrosan and glycine form；The composition packet
Ingredient containing following parts by weight: 1.5~2 parts by weight thymalfasins, 40-60 parts by weight excipient, buffer；In the excipient
The weight ratio of dextrosan and glycine is 3~5:1；Dextrosan is spray drying dextrosan in the excipient；It is described
Buffer is disodium hydrogen phosphate-citrate buffer solution；The pH value of the composition is 6.6~7.5；
The pharmaceutical composition be freeze-dried powder, the freeze drying powder injection the preparation method comprises the following steps:
It takes excipient to be dissolved in the water for injection of 2/3 recipe quantity, stirs evenly；Thymalfasin is added, pH value is surveyed, with phosphoric acid hydrogen two
Sodium-citrate buffer solution adjusts pH to 6.6~7.5, and addition water for injection to full dose is filling with 0.22 μm of miillpore filter refined filtration,
Freeze-drying, obtains freeze drying powder injection.
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| CN117849224A (en) * | 2024-01-09 | 2024-04-09 | 哈尔滨吉象隆生物技术有限公司 | Thymalfasin impurity detection method |
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| CN101318011A (en) * | 2007-04-17 | 2008-12-10 | 海南中和药业股份有限公司 | Pentapeptide nose spraying agent for thymus gland, preparation method and application thereof |
| CN101346135A (en) * | 2005-12-27 | 2009-01-14 | 莫诺索尔克斯有限公司 | PH modulated films for delivery of actives |
| CN101683519A (en) * | 2008-08-25 | 2010-03-31 | 海南中和多肽药物研发有限公司 | Composition of thymic peptide alpha 1 and thymopeptide-5 and preparation method thereof |
| CN102167738A (en) * | 2011-01-26 | 2011-08-31 | 浙江大学 | Method for separating and purifying thymosin beta4 |
| CN104587452A (en) * | 2015-01-21 | 2015-05-06 | 上海苏豪逸明制药有限公司 | Solution for preparing thymalfasin preparation and method for preparing thymalfasin preparation by virtue of solution |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101346135A (en) * | 2005-12-27 | 2009-01-14 | 莫诺索尔克斯有限公司 | PH modulated films for delivery of actives |
| CN101318011A (en) * | 2007-04-17 | 2008-12-10 | 海南中和药业股份有限公司 | Pentapeptide nose spraying agent for thymus gland, preparation method and application thereof |
| CN101683519A (en) * | 2008-08-25 | 2010-03-31 | 海南中和多肽药物研发有限公司 | Composition of thymic peptide alpha 1 and thymopeptide-5 and preparation method thereof |
| CN102167738A (en) * | 2011-01-26 | 2011-08-31 | 浙江大学 | Method for separating and purifying thymosin beta4 |
| CN104587452A (en) * | 2015-01-21 | 2015-05-06 | 上海苏豪逸明制药有限公司 | Solution for preparing thymalfasin preparation and method for preparing thymalfasin preparation by virtue of solution |
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