CN108774285B - Preparation method of somatostatin and pharmaceutical composition thereof - Google Patents
Preparation method of somatostatin and pharmaceutical composition thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicine, and particularly relates to a preparation method of somatostatin and a pharmaceutical composition thereof, wherein the method adopts a method of eluting by a cation exchange resin column and an anion exchange resin column to obtain the somatostatin; the method for purifying somatostatin provided by the invention is simple and easy to operate, the purity of the obtained somatostatin can reach 99.7%, and the yield can reach more than 60%. The freeze-dried powder injection prepared by the composition has good solubility, good stability and good redissolution property; the freeze-dried powder injection prepared by the composition has the advantages of convenient use, easy storage and transportation, simple preparation method, easy industrial production and low production cost.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method of somatostatin and a pharmaceutical composition containing the somatostatin.
Background
Somatostatin (GHRIH, growth hormone release-excretion-inhibition hormone, or somatotatin) is a tetradecapeptide cleaved from a macromolecular peptide of 116 amino acids, which has a cyclic structure, a disulfide bond between the 3 rd and 14 th cysteines, and has the following chemical structure, and the molecular formula is C76H104N18O19S2, relative molecular mass 1637.89, white powder.
somatostatin is a somatostatin which is widely distributed in the central and peripheral nervous systems and gastrointestinal tract, and also present in rat and human hearts, and is released by D cells in gastrointestinal mucosa in the highest content in antrum and corpus, it inhibits secretion of bombesin, α -deoxyglucose, etc., reduces renin activity, produces a hypotensive effect, has negative inotropic effect on isolated guinea pig heart, further reduces cardiac index and arterial blood pressure in the presence of furosemide, stabilizes cell membranes by regulating secretion of gut hormones, protects them, and furthermore, somatostatin inhibits all gastrointestinal secretion.
The purity and yield of the somatostatin prepared at present are low, the requirements of industrial production cannot be met, especially the somatostatin cannot be purified on a large scale, and the application of the somatostatin is restricted. Therefore, it is of great significance to provide a method for purifying somatostatin.
Disclosure of Invention
In order to solve the problems in the prior art, a great deal of experimental exploration is carried out, a novel somatostatin purification method is found, and the obtained somatostatin has higher purity. The invention also aims to provide a somatostatin pharmaceutical composition and a preparation method thereof.
Specifically, the present invention provides:
a method of purifying somatostatin comprising the steps of:
step 1, washing a cation exchange resin column by using a first buffer solution, dissolving a somatostatin crude product by using the first buffer solution, and circularly loading the somatostatin crude product/L resin/sample according to the proportion of less than or equal to 50 g; eluting with a second buffer solution to obtain a first eluate; the first buffer solution and the second buffer solution are sodium phosphate buffer solutions;
step 2: using a phosphate buffer solution to adjust the pH value of the eluent to 4.5-6.5 to obtain a solution;
and step 3: washing the anion exchange resin column with a third buffer solution for pre-balancing, dissolving the product obtained in the step 2 with the third buffer solution, and loading according to 30g of somatostatin crude product/L resin/cycle; eluting with a fourth buffer solution to obtain a second eluate, concentrating, and drying to obtain somatostatin; the third buffer solution and the fourth buffer solution are sodium acetate buffer solutions.
The cation exchange resin is a carboxymethyl agarose column; the anion exchange resin is an agarose gel column.
The pH value of the phosphate buffer solution in the step 2 is 5.0-7.0.
The pH values of the first buffer solution, the second buffer solution, the third buffer solution and the fourth buffer solution are 4.0-6.0.
The second buffer solution and the fourth buffer solution contain sodium chloride.
A pharmaceutical composition containing somatostatin comprises somatostatin and an excipient, wherein the excipient is a mixture consisting of trehalose and galactose.
The composition comprises the following components in parts by weight: 0.8-1 part of somatostatin and 30-50 parts of excipient.
β the β trehalose β in β the β excipient β is β α β one β or β more β of β alpha β, β alpha β - β dicarboxyl β trehalose β, β beta β - β dicarboxyl β iso β - β trehalose β or β alpha β, β beta β - β dicarboxyl β trehalose β. β
The excipient comprises trehalose: the weight ratio of galactose is (0.5-0.9): 1.
the pH value of the composition is 4.5-6.5.
A method for preparing a freeze-dried powder injection from a pharmaceutical composition containing somatostatin comprises the following steps:
1) weighing excipient according to the prescription amount, dissolving the excipient in 2/3 of the prescription amount of water for injection, and uniformly stirring;
2) and (3) after the temperature of the preparation liquid is reduced to room temperature, adding the somatostatin according to the prescription amount, measuring the pH value, adjusting the pH value to 4.5-6.0, adding the water for injection to the full amount, finely filtering by using a microporous filter membrane, and freeze-drying to obtain the freeze-dried powder injection.
Compared with the prior art, the invention has the following advantages and positive effects:
1. the method for purifying somatostatin provided by the invention is simple and easy to operate, the purity of the obtained somatostatin can reach 99.7%, and the yield can reach more than 60%.
2. The freeze-dried powder injection prepared by the composition has good stability and good redissolution property; the freeze-dried powder injection prepared by the composition has the advantages of convenient use, easy storage and transportation, simple preparation method, easy industrial production and low production cost.
Detailed Description
The present invention is further described in the following description of the specific embodiments, which is not intended to limit the invention, but various modifications and improvements can be made by those skilled in the art according to the basic idea of the invention, within the scope of the invention, as long as they do not depart from the basic idea of the invention.
The somatostatin crude product was purchased from Hangzhou peptide Biotechnology Ltd and its HPLC purity was found to be 93.02%.
Example 1
Step 1: washing the carboxymethyl agarose column with 25mM sodium phosphate buffer (first buffer) with pH6.0 for pre-equilibration, dissolving the somatostatin crude product with the first buffer, and loading the sample according to 40g somatostatin crude product/L resin/cycle; passing through a gradient of 10-60% second buffer (20mM sodium phosphate, 110mM sodium chloride, pH6.5) over 20 column volumes; the flow rate was 7 mL/min and the eluate was monitored by detection at 215nm, collecting the characteristic peak eluate;
step 2: adjusting the pH value of the eluent to 5.0 by using a phosphate buffer solution with the pH value of 7.0 to obtain a solution;
and step 3: washing phenyl sepharose HP column with 20mM sodium acetate buffer (third buffer) with pH4.0 for pre-balancing, dissolving the solution obtained in step 2 with the third buffer, and loading according to 30g somatostatin crude product/L resin/cycle; eluting with a gradient of 0-20% fourth buffer (20mM sodium acetate, 1M sodium chloride, pH7.5) over 25 column volumes; the flow rate was 5 mL/min and the eluate was monitored by UV detection at 215 nm; collecting the characteristic peak eluent, concentrating and drying to obtain 25.73g of somatostatin with the purity of 99.93 percent, and the total purification yield is 64.32 percent.
Example 2
Step 1: washing the carboxymethyl agarose column with 25mM sodium phosphate buffer (first buffer) with pH6.0 for pre-equilibration, dissolving the crude somatostatin product with the first buffer, and loading the sample according to 100g crude somatostatin product/L resin/cycle; eluting with 0-40% gradient second buffer solution by 20 times column volume, and eluting with 40-80% second buffer solution by 3 times column volume gradient; the flow rate was 7 mL/min and the eluate was monitored by detection at 215 nm; collecting characteristic peak eluent;
step 2: the pH value of the eluent is adjusted to 6.0 by phosphate buffer solution with the pH value of 7.0 to obtain solution;
and step 3: washing phenyl sepharose HP column with 20mM sodium acetate buffer (third buffer) with pH4.0 for pre-balancing, dissolving the product obtained in step 2 with the third buffer, and loading according to 20g somatostatin crude product/L resin/cycle; eluting with a gradient of 0-20% fourth buffer (20mM sodium acetate, 1M sodium chloride, pH4.5) over 25 column volumes; the flow rate was 5 mL/min and the eluate was monitored by UV detection at 215 nm; and collecting the pure eluent of the characteristic peak, concentrating and drying to obtain 63.21g of somatostatin with the purity of 99.81 percent and the total purification yield of 63.21 percent.
Example 3
Step 1: washing the carboxymethyl agarose column with 25mM sodium phosphate buffer (first buffer) of pH6.0 for pre-equilibration, dissolving somatostatin in the first buffer, and loading at 50g somatostatin/L resin/cycle; a gradient of 10-60% second buffer (20mM sodium phosphate, 110mM sodium chloride, pH6.5) was run through 20 column volumes; the flow rate was 7 mL/min and the eluate was monitored by detection at 215 nm; collecting characteristic peak eluent;
step 2: the pH value of the eluent is adjusted to 4.5 by phosphate buffer solution with the pH value of 6.0 to obtain solution;
and step 3: washing phenyl sepharose HP column with 20mM sodium acetate buffer (third buffer) with pH4.0 for pre-equilibration, dissolving the product obtained in step 2 with the third buffer, and loading according to 30g somatostatin/L resin/cycle; eluting with a gradient of 0-20% fourth buffer (20mM sodium acetate, 1M sodium chloride, pH4.5) over 25 column volumes; the flow rate was 5 mL/min and the eluate was monitored by UV detection at 215 nm; and collecting the pure eluent of the characteristic peak, concentrating and drying to obtain 31.89g of somatostatin with the purity of 99.81 percent and the total purification yield of 63.78 percent.
Test example 1:
dissolving the related substances in water to obtain solution containing 0.5mg per 1ml as test solution; precisely measuring 2ml, placing in a 100ml measuring flask, diluting with water to scale, shaking up, measuring by high performance liquid chromatography (2015 edition pharmacopoeia) with octadecylsilane chemically bonded silica as a control solution, using phosphoric acid solution (11 ml of phosphoric acid, 800ml of water, adjusting pH to 2.3 with triethylamine, and diluting to 1000ml of water) as a mobile phase A, and acetonitrile as a mobile phase B; flow rate was 1.5ml per minute; the detection wavelength was 215 nm. Gradient elution was performed as in table 1; taking about 10mg of somatostatin reference substance, placing the reference substance in a 20ml measuring flask, adding 1ml of 30% hydrogen peroxide solution, placing the reference substance at room temperature for 1 hour, adding water to dilute the reference substance to a scale, shaking up, filtering, taking 50ml of subsequent filtrate, injecting the subsequent filtrate into a liquid chromatograph, wherein the number of theoretical plates is not less than 2000 calculated according to a somatostatin peak, and the separation degree of the somatostatin main peak and an oxidative degradation product peak with the relative retention time of about 1.1 is not less than 2.0. Injecting 50ml of the control solution into a liquid chromatograph, and adjusting the detection sensitivity to make the main component chromatographic peak height about 20% of the full range. Precisely measuring the sample solution and the control solution by 50ml respectively, injecting into a liquid chromatograph, and recording the chromatogram.
TABLE 1 gradient elution
| Time (minutes) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 79 | 21 |
| 18 | 60 | 40 |
| 20 | 60 | 40 |
| 21 | 79 | 21 |
| 26 | 79 | 21 |
The content measurement is performed by high performance liquid chromatography (2015 pharmacopoeia).
Octadecylsilane chemically bonded silica is used as a filler for chromatographic conditions and system applicability tests; using phosphoric acid solution (taking 11ml of phosphoric acid, adding 800ml of water, adjusting the pH value to 2.3 by triethylamine, diluting to 1000ml by water) -acetonitrile (75: 25) as a mobile phase; flow rate was 1.5ml per minute; the detection wavelength was 215 nm. The number of theoretical plates is not less than 1500 calculated according to somatostatin peaks.
The determination method comprises precisely weighing appropriate amount of the product, dissolving in water, quantitatively diluting to obtain solution containing 0.1mg per 1ml, precisely measuring 20ml, injecting into liquid chromatograph, and recording chromatogram; and taking another appropriate amount of somatostatin control, and determining by the same method. Calculating according to the peak area by an external standard method to obtain the product. Results are shown in Table 2.
TABLE 2 different samples
| Sample (I) | Total related substances (%) | Single maximum impurity (%) | Number of impurities |
| Example 1 | 0.11 | 0.02 | 3 |
| Example 2 | 0.10 | 0.03 | 3 |
| Example 3 | 0.09 | 0.03 | 4 |
| Raw materials | 4.67 | 1.32 | 24 |
And (4) conclusion: the results show that the somatostatin prepared by the invention has less than 0.1 percent of total related substances and less than 0.05 percent of single maximum impurity, and is superior to the raw materials.
Example 4
under clean conditions, 18.5g α alpha, alpha-dicarbonyl trehalose and 26.5g α galactose are put into a liquid preparation device, 700ml α water for injection is added and stirred for dissolution, the solution is cooled to room temperature, 1g α somatostatin (prepared by the method described in example 1) is added after the solution is cooled to room temperature, the pH value is measured, the pH value is adjusted to 5.2 by acetic acid-sodium acetate buffer (0.1M, pH4.5), the water for injection is added to full dose, and the solution is filtered by a 0.22 mu M microporous filter membrane to obtain somatostatin filtrate, and the filtrate is subpackaged into 3ml α ampoules, and is frozen and dried for 72 hours to prepare the sterile freeze-dried powder injection.
Example 5
under clean conditions, 18g of α, beta-dicarbonyl trehalose and 22g of galactose are put into a liquid preparation device, 700ml of water for injection are added and stirred for dissolution, after the preparation liquid is cooled to room temperature, 1.0g of somatostatin (prepared by the method described in example 3) is added, the pH value is measured, the pH value is adjusted to 5.1 by using acetic acid-sodium acetate buffer (0.1M, pH4.5), the water for injection is added to the full dose, the obtained product is filtered by a 0.22 mu M microporous membrane to obtain a somatostatin filtrate, 3ml of the filtrate is subpackaged in an ampere bottle, and the aseptic freeze-dried powder injection is prepared after freeze drying for 72 hours.
Example 6
under clean condition, putting 23.7g β, β -dicarboxyitrehalose and 26.3g galactose into a liquid preparation device, adding 700ml of water for injection, stirring and dissolving, cooling the prepared liquid to room temperature, adding 1.0g somatostatin (prepared by the method described in example 2), measuring pH value, adding acetic acid-sodium acetate buffer (0.1M, pH4.5) to 5.0, adding water for injection to full volume, filtering through a 0.22 mu M microporous membrane to obtain somatostatin filtrate, subpackaging 3ml in an ampere bottle, and freeze-drying for 72 hours to prepare the sterile freeze-dried powder injection.
Example 7
under clean conditions, 5g of α, α -dicarbonyl trehalose, 5g of β, β -dicarbonyl isoceralose and 20g of galactose are put into a liquid preparation device, 700ml of water for injection is added, stirring and dissolving are carried out, 1.0g of somatostatin (prepared according to the method described in example 1) is added after the prepared liquid is cooled to room temperature, the pH value is measured, the pH value is adjusted to 4.9 by acetic acid-sodium acetate buffer solution (0.1M, the pH value is 4.5), the water for injection is added to the full volume, the obtained product is filtered by a 0.22 mu M microporous membrane to obtain somatostatin filtrate, the obtained product is subpackaged into 3ml of ampoules, and freeze-dried for 72 hours to prepare the sterile freeze-dried powder injection.
Example 8
under clean condition, 3g of α, α -dicarboxyl trehalose, 7g of β, β -dicarboxyl isotrehalose, 8g of α, β -dicarboxyl trehalose and 20g of galactose are put into a liquid preparation device, 700ml of water for injection is added to be stirred and dissolved, after the prepared liquid is cooled to room temperature, 1.0g of somatostatin is added, the pH value is measured, acetic acid-sodium acetate buffer solution (0.1M, pH is equal to 4.5) is used for regulating the pH value to 4.9, water for injection is added to the full dose, the solution is filtered by a 0.22 mu M microporous filter membrane to obtain somatostatin filtrate, 3ml of the somatostatin filtrate is subpackaged in an ampere bottle, and freeze drying is carried out for 72 hours to prepare the sterile freeze-dried powder injection.
Prescription screening test
1. Screening of excipients in formulation solutions
Contains somatostatin 1 mg/ml. And respectively contains 4% (W/V) of mannitol, trehalose and galactose (weight ratio of 3:1), trehalose and galactose as excipient. Placing the solution in an incubator at 37 ℃ for incubation, and detecting the stability of the somatostatin in the solution at each time point. And freeze-drying (freeze-drying procedure: pre-freezing at-40 ℃ for 4 hours, vacuumizing to 300mT, setting temperature-raising procedure of 1 ℃/minute to-20 ℃, freeze-drying for 12 hours, setting temperature-raising procedure of 1 ℃/minute to 30 ℃, drying for 4 hours, and corking and capping) was carried out to observe the appearance of the freeze-dried form preparation, and the test results are shown in Table 3. The formulation solution is preferably a combination of mannitol or trehalose with galactose as an excipient, as judged from the viewpoint of stability and lyophilized appearance.
TABLE 3 purity (%) and lyophilized appearance of somatostatin in the excipient-containing formulation solution
2. Screening of excipients in formulation solutions
Contains 0, 0.1, 0.25, 0.5, 1.0, 2.0, 3.2 or 6.0mg/ml of somatostatin. And respectively contains 4% (W/V) of mannitol, trehalose and galactose composition, and trehalose and galactose as excipient. At the same time, a preparation solution without excipients is prepared. And (4) taking a glass bottle, subpackaging the preparation solution according to 1 ml/bottle, and freeze-drying. Reconstitution of the lyophilized form of the pharmaceutical preparation was examined. The test results are shown in Table 4.
TABLE 4 clarity of lyophilized formulations when different excipients are included in the formulation solution, after reconstitution
Note: <1 indicates that the turbidity is less than that of the No. 1 standard solution; standard solution with turbidity greater than No. 1
The test result shows that solute molecules such as mannitol, a composition of trehalose and galactose (in a weight ratio of 3:1), excipients such as trehalose and galactose are introduced, the concentration range of somatostatin in a pharmaceutical preparation solution for preparing a soluble and unfrozen dry preparation is only increased from 0-0.1 mg/ml to 0-0.25 mg/ml, and the concentration of somatostatin in a lyophilized re-soluble pharmaceutical preparation is slightly increased; the introduction of some excipients (e.g., galactose) has little effect on increasing the concentration of somatostatin in the reconstituted pharmaceutical formulation after lyophilization.
3. Screening of excipients in formulation solutions
Preparing preparation solution, wherein the trehalose and galactose composition and the somatostatin are mixed according to different weight ratios and are prepared by water. The pH value was measured by using a PB-10 type pH meter (Sartorius, USA) according to the requirement of pH value measurement in pharmacopoeia 2015 edition of the people's republic of China. And (3) detecting the solubility of the somatostatin in the preparation solution. The formulation solution was filled into 2ml glass freeze-drying bottles, 1 ml/bottle, and transferred into a cryo-freeze dryer (Supermodulyo type, 0.4 m)2E-CApparatus company) product cabinet. The lyophilized preparation was reconstituted with 1ml of water for injection, and the reconstitution of the lyophilized form of the pharmaceutical preparation was examined. And (3) putting the freeze-dried preparation into an incubator at 37 ℃, re-dissolving the freeze-dried preparation in 1ml of water for injection in each bottle after 8 weeks of incubation, and detecting the purity of the somatostatin in the preparation solution. The pH values of the preparation solutions of trehalose and galactose in different weight ratios, the appearance and reconstitution of the lyophilized preparations, and the purity of somatostatin after the lyophilized preparations were left at 37 ℃ for 8 weeks are shown in table 5, and the purity of somatostatin after lyophilization of each preparation solution was measured to be 99.86%. Comprehensively judging the pH value of a preparation solution, the appearance of a freeze-dried preparation and the stability of the freeze-dried preparation, wherein the weight ratio of trehalose to galactose is preferably (0.5-0.9): 1. the lyophilized pharmaceutical preparations can be reconstituted with 1ml to 5ml of water for injection and reconstituted into liquid pharmaceutical preparations.
TABLE 5 pH, lyophilized appearance, reconstitution time and 8 week stability with different excipients
Note: <1 indicates that the turbidity is less than that of the No. 1 standard solution; standard solution with turbidity greater than No. 1
And (4) test conclusion: the test shows that the weight ratio of (0.5-0.9): 1, trehalose: the galactose excipient is used as the excipient of the somatostatin preparation, and the product quality is more excellent.
Test example of somatostatin for injection
1. Apparatus and medicine
High performance liquid chromatograph (model HP1100, hewlett packard, usa), DAD detector, AgilentChemstation chromatography workstation: a clarity tester type YB-2 (precision instruments works of Tianjin university); DU640 type ultraviolet spectrophotometer (beckmann, usa); pHS-3C type digital acidimeters (Shanghai Lei magnetic Instrument works); WS/08-0l type temperature and humidity regulating box (Hangzhou blue sky instrument production Co., Ltd.); analytical balance model mayler.ae200 (switzerland); somatostatin (samples prepared as described in examples 4-8) was injected.
2. Method of producing a composite material
And (3) taking the product, adding water to dissolve the product, preparing a solution containing 0.5mg of the product in each 1ml, uniformly mixing, and measuring according to a law (appendix VI H), wherein the pH value is 4.5-6.5.
Measuring content by taking 10 bottles of the product, adding appropriate amount of water respectively, dissolving the content and quantitatively diluting to obtain a solution containing about 0.1mg per 1ml, measuring according to the method under somatostatin, calculating the content of each bottle, and calculating the average content of 10 bottles.
2.3 influencing factor experiments
Under the packaging conditions of the marketed drugs, three batches of the samples of example 5 (batch Nos. 160321, 160407, and 160610) were examined under high temperature (60 ℃), high light (4500lx), and high humidity conditions for 5 days and 10 days, and the acidity, content, and related substances were examined to determine the indexes. The results are shown in Table 6.
TABLE 6 influence factor test results
A. B, C represents three batches of samples from example 5, batches 160321, 160407, 160610.
2.6 accelerated test
Under the packaging conditions of the marketed drugs, three batches of the samples of example 5 (batch numbers 160321, 160407 and 160610) were stored at 40 +/-2 ℃ and 75% relative humidity at 5% soil, and sampled at the end of 0, 1, 2, 3 and 6 months, respectively, to determine the indexes. All samples were white loose blocks in character. The results of the acidity, related substances and content measurement are shown in Table 7.
2.7 Long term experiments
Under the packaging conditions of the marketed drugs, three batches of the samples of example 5 (batch numbers 160321, 160407 and 160610) were stored at 25. + -. 2 ℃ and 60. + -. 10% relative humidity, and samples were taken at the end of 0, 3, 6, 12, 18 and 24 months, respectively, to determine the indices. All the samples are white loose blocks, and the results of measuring acidity, related substances and content are shown in Table 7.
TABLE 7 accelerated test and Long term test investigation results
A. B, C represents three batches of samples from example 5, batches 160321, 160407, 160610.
And (4) conclusion: the results of the influence factor test and the accelerated test show that the somatostatin for injection has no obvious change in each determination index and good stability; the somatostatin for injection is placed at room temperature for a long time for 24 months, the quality indexes of the somatostatin are not obviously changed, and the product stability is good.
Stability determination test after reconstitution of somatostatin for injection
The samples 160321, 160407, and 160610 were reconstituted with 250ml of water for injection, placed in a refrigerator at 4 ℃ and measured once a day for 14 days, and subjected to linear regression analysis for time (X) based on the measured value (Y). The results are shown in Table 8.
TABLE 8 stability of somatostatin for injection after reconstitution
P >0.05 indicates that the concentration of somatostatin is substantially stable over the time measured.
And (4) conclusion: as can be seen from Table 8, somatostatin remained stable after three batches of the product of the invention were reconstituted and continuously measured for 14 days. The method shows that the somatostatin for injection prepared by the method has stable property after redissolution.
Claims (3)
1. A preparation method of a somatostatin freeze-dried powder injection is characterized by comprising the following steps: the freeze-dried powder injection is prepared from 0.8-1 part by weight of somatostatin and 30-50 parts by weight of excipient, wherein the excipient is a mixture consisting of trehalose and galactose, and the ratio of trehalose: the weight ratio of galactose is 0.5-0.9: 1;
the preparation method of the freeze-dried powder injection comprises the following steps:
1) dissolving excipient in 2/3 of the prescribed amount of water for injection, and stirring to obtain a preparation solution;
2) after the temperature of the preparation liquid is reduced to room temperature, adding somatostatin, measuring the pH value, adjusting the pH value to 4.5-6.0, adding water for injection to full volume, finely filtering by using a microporous filter membrane, and freeze-drying to obtain a freeze-dried powder injection;
the preparation method of the somatostatin comprises the following steps:
step 1, washing a cation exchange resin column by using a first buffer solution, dissolving a somatostatin crude product by using the first buffer solution, and circularly loading the somatostatin crude product/L resin/sample according to the proportion of less than or equal to 50 g; eluting with a second buffer solution to obtain a first eluent; the first buffer solution and the second buffer solution are sodium phosphate buffer solutions;
step 2: using a phosphate buffer solution to adjust the pH value of the eluent to 4.5-6.5 to obtain a solution;
and step 3: washing the anion exchange resin column with a third buffer solution for pre-balancing, dissolving the solution in the step (2) with the third buffer solution, and loading according to 30g of somatostatin crude product/L resin/cycle; eluting with a fourth buffer solution to obtain a second eluate, concentrating, and drying to obtain somatostatin; the third buffer solution and the fourth buffer solution are sodium acetate buffer solutions;
the cation exchange resin is a carboxymethyl agarose column; the anion exchange resin is an agarose gel column; the pH values of the first buffer solution, the second buffer solution, the third buffer solution and the fourth buffer solution are 4.0-6.0; the second buffer solution and the fourth buffer solution contain sodium chloride.
2. The method for preparing a somatostatin freeze-dried powder injection according to claim 1, wherein the pH value of the phosphate buffer solution in the step 2 is 5.0-7.0.
3. the method for preparing a somatostatin freeze-dried powder injection according to claim 1, wherein trehalose in the excipient is one or more of α, α -dicarboxyl trehalose, beta-dicarboxyl trehalose, β, beta-dicarboxyl trehalose.
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| CN107698676A (en) * | 2017-11-07 | 2018-02-16 | 江西浩然生物医药有限公司 | A kind of extraction preparation method of high-purity menotropins |
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| CN107698676A (en) * | 2017-11-07 | 2018-02-16 | 江西浩然生物医药有限公司 | A kind of extraction preparation method of high-purity menotropins |
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| Purificationand Characterization of a 43-kDa Transcription Factor Required for Rat SomatostatinGene Expression;Zheng Zhu等;《THEJOURNAL OF BIOLOGICAL CHEMISTR》;19890415;第264卷(第11期);6550-6556 * |
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