High-purity subtilucent 37 peptide reference substance as well as production method and application thereof based on biosynthesis industrialization

Abstract

The invention relates to a high-purity subtilucent 37 peptide reference substance and a production method and application thereof based on biosynthesis industrialization, and belongs to the field of biomedicine.The production method comprises the steps that bacillus subtilis serves as a production strain, and fermentation liquor is obtained through primary seed tank fermentation and seed tank fermentation; carrying out ceramic membrane separation, double-effect concentration, salting-out and tubular centrifugation on the fermentation liquid to obtain a precipitate which is a crude product of the tri17 peptide; the preparation method comprises the following steps: redissolving a crude product of the withered grass tri17 peptide with a 20% ethanol aqueous solution, centrifuging, filtering, dewatering, carrying out ion exchange chromatography, carrying out ultrafiltration concentration, freeze-drying, and subpackaging to obtain a reference substance of the withered grass tri17 peptide; wherein the elution step of hydrophobic chromatography is water-5% ethanol water-50% ethanol water elution. Reversed phase chromatography, ion exchange chromatography and size exclusion chromatography detection results show that the purity of the reference substance is greater than 99.9%, the endotoxin is less than 10 EU / mL, the sterility test and the abnormal toxicity meet the high-purity bulk drug standard, and the reference substance can be used as a crude drug of the triticin.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a high-purity sublancin-37 peptide reference substance and a production method and application thereof based on biosynthesis. BACKGROUND

[0002] The English name of sublancin-37 peptide is Sublancin, which is composed of 37 amino acid residues and has a molecular weight of 3879.8 D a (according to the international relative atomic mass in 2018). The structure has two disulfide bonds formed by five cysteines, is stable, and has good resistance to high temperature, acid and alkali, and pepsin. Experiments show that sublancin-37 peptide can enhance the phagocytic function of macrophage RAW264.7. Effectiveness tests show that sublancin-37 peptide can promote growth, enhance immune function, and improve the morphological structure and bacterial flora composition of intestinal mucosa.

[0003] Sublancin-37 peptide is prepared by microbial fermentation based on biosynthesis. Sublancin-37 peptide has been approved as a new feed additive by the Ministry of Agriculture and Rural Affairs of the People's Republic of China (No. 614). The content of sublancin-37 peptide in feed additive sublancin-37 peptide is determined by high performance liquid chromatography with a reverse phase chromatography system, an ultraviolet detector or a diode array detector, and an external standard method. The quality and production cost of sublancin-37 peptide reference substance directly affect the detection results and detection cost of feed additive sublancin-37 peptide. High-purity sublancin-37 peptide reference substance is helpful for the development of sublancin-37 peptide reference substance research, the progress of sublancin-37 peptide drug research, and the more accurate monitoring of the production process of sublancin-37 peptide by enterprises, the optimization of production process, and the reduction of production cost. Therefore, the industrialized production of high-purity sublancin-37 peptide reference substance is particularly important.

[0004] Patent CN 115197304A discloses a method for separating and purifying sublancin-37 peptide. The method is to separate the sublancin-37 peptide sample with a concentration of 10-20 mg / mL by reverse phase C18 semi-preparative column, use trifluoroacetic acid and acetonitrile aqueous solution as mobile phase for gradient elution, collect the eluate for freeze-drying, and separate and prepare sublancin-37 peptide with a chromatographic purity of more than 99.7%, which can be used for mass spectrometry analysis. The method can obtain sublancin-37 peptide with a chromatographic purity of more than 99.7%, but the preparation amount is small, and it cannot realize industrialized production, can only meet the needs of scientific research experiments, and cannot meet the needs of feed enterprises and production enterprises for sublancin-37 peptide reference substance. Moreover, the method uses trifluoroacetic acid and acetonitrile organic reagents, which not only has high cost, but also brings great potential risk to post-processing and use.

[0005] In addition, the safety problem is also the biggest limit of the application of the peptide to the medical product, and the safety problem mainly includes three aspects: one is that the absorption through the gastrointestinal tract is slow, the degradation is more, the bioavailability is low, the second is that the residual allergen (including heat source, endotoxin) causes allergy to the body, and the third is the potential risk of some unknown impurities. Therefore, a new purification process is urgently needed to achieve high purity, less impurities, no heat source, and can be used as a reference for injection.

[0006] At present, the high-purity polypeptide reference is mostly prepared by chemical synthesis method, which has high cost and different spatial structures, causing great hidden troubles for later use. The conventional method for separating and purifying the peptide reference has low purity and cannot realize industrialized production. SUMMARY

[0007] The existing separation and purification technology of the peptide has low purity and cannot realize industrialized production. In order to solve the above technical problems, the application provides a high-purity peptide reference and an industrialized production method and application thereof.

[0008] The application is realized in the following manner: A high-purity peptide reference is prepared by the following steps: (1) Hydrophobic chromatography: the crude product of the peptide is dissolved with 15%-25% ethanol aqueous solution, stirred, centrifuged, and the supernatant is collected, filtered, and the obtained filtrate is the chromatography sample solution; the chromatography sample solution is adjusted to pH 4.5-5.5 without adjusting the conductivity, and hydrophobic chromatography purification is carried out, the filler is Butyl Hp, the loading is 70 g / L-80 g / L, the equilibration buffer is 50 mM NaAc-HAc with pH 5.0, 1.5 M NaCl, elution, and the elution program is divided into three steps, the first step: water elution, the second step: 3.5%-6.5% ethanol aqueous elution, the third step: 40%-60% ethanol aqueous elution, the eluate of the third step is collected, and the hydrophobic purified intermediate product of the peptide is obtained; (2) Ion exchange chromatography: the obtained peptide purification intermediate product is directly subjected to ion exchange chromatography purification without adjusting the conductivity, the filler is Capto S, the sample amount is 1-8 CV, the equilibration buffer is 50 mM NaAc-HAc with pH 5.0, the eluent is 50 mM NaAc-HAc with pH 5.0, 1 M NaCl, the flow-through liquid is collected, and the ion exchange intermediate product of the peptide is obtained; (3) Ultrafiltration purification: The obtained ion exchange purification intermediate of 37-peptide was purified by ultrafiltration. The membrane element was S-NF360, the transmembrane pressure was 3.5 to 4.0 bar, and the ultrafiltration buffer was ultrapure water. After ultrafiltration to a conductivity of 1.5 μm / cm to 3.5 μm / cm, the 37-peptide was concentrated to 1 g / L to 20 g / L to obtain the 37-peptide ultrafiltration intermediate. (4) Freeze-drying and packaging: Add 600 mL of the ultrafiltration intermediate of 37-peptide of kiwifruit to a clean freeze-drying tray, place it in a freeze dryer, and freeze-dry it by pre-freezing, freezing and slowly heating. After 36 h, turn off the heating, slowly introduce air through the air inlet valve, turn on the freeze dryer, collect the freeze-dried powder, and package it to obtain high-purity 37-peptide of kiwifruit reference standard.

[0009] Furthermore, the high-purity pyrimethamine reference standard is prepared by the following steps: (1) Hydrophobic chromatography: Dissolve crude 37-peptide in 20 times the volume of 20% ethanol aqueous solution, stir, centrifuge, collect the supernatant, filter, and the obtained filtrate is the chromatographic loading solution; take the chromatographic loading solution and adjust the pH to 5.0, without adjusting the conductivity, and perform hydrophobic chromatography purification. The packing material is ButylHp, and the loading is 75 g / L. The equilibration buffer is 50 mM NaAc-HAc and 1.5 M NaCl at pH 5.0. The elution program is carried out in three steps: the first step is to elute with 20 column volumes of water, the second step is to elute with 20 column volumes of 5% ethanol water, and the third step is to elute with 50% ethanol water for 5 column volumes. Collect the eluent of the UV 280 nm peak during the third step elution process to obtain the purified intermediate of 37-peptide. (2) Ion exchange chromatography: The obtained intermediate of 37-peptide of calcitriol was purified directly by ion exchange chromatography without adjusting the conductivity. The packing material was CaptoS, the loading volume was 5 CV, the equilibration buffer was 50 mM NaAc-HAc at pH 5.0, the eluent was 50 mM NaAc-Hac at pH 5.0 and 1 M NaCl, and the flow-through was collected to obtain the intermediate of 37-peptide of calcitriol ... (3) Ultrafiltration purification: The obtained ion exchange purification intermediate of 37-peptide was purified by ultrafiltration. The membrane element was S-NF360, the transmembrane pressure was 3.5-4.0 bar, and the ultrafiltration buffer was ultrapure water. After ultrafiltration to a conductivity of 2.5 μm / cm, the 37-peptide was concentrated to 10 g / L to obtain the 37-peptide ultrafiltration intermediate.

[0010] (4) Freeze-drying and packaging: Add the ultrafiltration intermediate of 37-peptide of calcinus thiol to a clean freeze-drying tray at a rate of 600 mL / tray, put it into a freeze dryer, and freeze-dry it by pre-freezing, freezing and slowly heating. After 36 h, turn off the heating, slowly introduce air through the air inlet valve, turn on the freeze dryer, collect the freeze-dried powder, and dispense it to obtain high-purity 37-peptide of calcinus thiol reference standard.

[0011] A method for the industrial-scale production of high-purity kiwifruit peptide reference standard based on biosynthesis, the method comprising the following steps: (1) Hydrophobic chromatography: Dissolve crude 37-peptide in 15%~25% ethanol aqueous solution, stir, centrifuge, collect supernatant, filter, and the obtained filtrate is the chromatographic loading solution; take the chromatographic loading solution and adjust the pH to 4.5~5.5, without adjusting the conductivity, and perform hydrophobic chromatography purification. The packing material is Butyl Hp, and the loading is 70 g / L~80 g / L. The equilibration buffer is 50 mM NaAc-HAc and 1.5 M NaCl at pH 5.0. Elution is performed in three steps: first step: water elution, second step: 3.5%~6.5% ethanol water elution, third step: 40%~60% ethanol water elution. Collect the eluent from the third step to obtain the hydrophobic purified intermediate of 37-peptide. (2) Ion exchange chromatography: The obtained intermediate of 37-peptide of calcinus trichoseptum was purified directly by ion exchange chromatography without adjusting the conductivity. The packing material was CaptoS, the loading volume was 1~8CV, the equilibration buffer was 50 mM NaAc-HAc at pH 5.0, the eluent was 50 mM NaAc-Hac at pH 5.0 and 1 M NaCl, and the flow-through was collected to obtain the intermediate of 37-peptide of calcinus trichoseptum ion exchange. (3) Ultrafiltration purification: The obtained ion exchange purification intermediate of 37-peptide was purified by ultrafiltration. The membrane element was S-NF360, the transmembrane pressure was 3.5 to 4.0 bar, and the ultrafiltration buffer was ultrapure water. After ultrafiltration to a conductivity of 1.5 μm / cm to 3.5 μm / cm, the 37-peptide was concentrated to 1 g / L to 20 g / L to obtain a high-purity 37-peptide ultrafiltration intermediate.

[0012] (4) Freeze-drying and packaging: Add 600 mL of the ultrafiltration intermediate of 37-peptide of kiwifruit to a clean freeze-drying tray, place it in a Pudong GLZY80 freeze dryer, freeze-dry it by pre-freezing, freezing and slowly heating up, turn off the heating after 36 hours, slowly introduce air through the air inlet valve, turn on the freeze dryer, collect the freeze-dried powder, and package it to obtain high-purity 37-peptide of kiwifruit reference standard.

[0013] Furthermore, the production method of the high-purity pyrimethamine reference standard includes the following steps: (1) Hydrophobic chromatography: Dissolve crude 37-peptide in 20 times the volume of 20% ethanol aqueous solution, stir, centrifuge, collect the supernatant, filter, and the obtained filtrate is the chromatographic loading solution; take the chromatographic loading solution and adjust the pH to 5.0, without adjusting the conductivity, and perform hydrophobic chromatography purification. The packing material is ButylHp, and the loading is 75 g / L. The equilibration buffer is 50 mM NaAc-HAc and 1.5 M NaCl at pH 5.0. The elution program is carried out in three steps: the first step is to elute with 20 column volumes of water, the second step is to elute with 20 column volumes of 5% ethanol water, and the third step is to elute with 50% ethanol water for 5 column volumes. Collect the eluent of the UV 280 nm peak during the third step elution process to obtain the purified intermediate of 37-peptide. (2) Ion exchange chromatography: The obtained intermediate of 37-peptide of calcitriol was purified directly by ion exchange chromatography without adjusting the conductivity. The packing material was CaptoS, the loading volume was 5 CV, the equilibration buffer was 50 mM NaAc-HAc at pH 5.0, the eluent was 50 mM NaAc-Hac at pH 5.0 and 1 M NaCl, and the flow-through was collected to obtain the intermediate of 37-peptide of calcitriol ... (3) Ultrafiltration purification: The obtained ion exchange purification intermediate of 37-peptide was purified by ultrafiltration. The membrane element was S-NF360, the transmembrane pressure was 3.5-4.0 bar, and the ultrafiltration buffer was ultrapure water. After ultrafiltration to a conductivity of 2.5 μm / cm, the 37-peptide was concentrated to 10 g / L to obtain a high-purity 37-peptide ultrafiltration intermediate.

[0014] (4) Freeze-drying and packaging: Add 600 mL of the ultrafiltration intermediate of 37-peptide of kiwifruit to a clean freeze-drying tray, place it in a Pudong GLZY80 freeze dryer, freeze-dry it by pre-freezing, freezing and slowly heating up, turn off the heating after 36 hours, slowly introduce air through the air inlet valve, turn on the freeze dryer, collect the freeze-dried powder, and package it to obtain high-purity 37-peptide of kiwifruit reference standard.

[0015] Furthermore, the preparation method of the crude kiwifruit peptide includes the following steps: (1) Ceramic membrane filtration: The fermentation broth of 37-peptide of calcined ... Operating parameters: inlet pressure 0.5 MPa, outlet pressure 0.3 MPa, inlet temperature 40 ℃, feed temperature 35 ℃; ceramic membrane pore size 50 nm; (2) Double-effect concentration: The filtrate A was concentrated in three stages to a concentration of 40 g / L of tahinitropeptide to obtain a double-effect concentrate; The temperature of the first effect is 80 ℃, the temperature of the second effect is 70 ℃, the temperature of the third effect is 60 ℃, and the pressure is -0.1 MPa; (3) Ammonium sulfate salting out: Add ammonium sulfate to the obtained double-effect concentrate while stirring. The amount of ammonium sulfate added per liter of double-effect concentrate is 100~300g. Continue stirring for 2~6 h and salt out at 4 ℃ for 4~20 h. Then centrifuge with a tubular centrifuge to collect the precipitate and the clear liquid. The precipitate is crude 37-peptide of kiwifruit.

[0016] The reference standard was tested by reversed-phase chromatography, ion-exchange chromatography, and size exclusion chromatography. The purity was >99.9%, the endotoxin was <10 EU / mL, and the sterility and abnormal toxicity tests met the standards for injectable active pharmaceutical ingredients. Therefore, it can be used as an active pharmaceutical ingredient.

[0017] An injectable preparation comprising a reference standard of high-purity tahinitrofurantoin prepared by the above method, wherein the injectable preparation is selected from injection solutions, infusion solutions, concentrated solutions for injection, sterile powders for injection, and lyophilized powders for injection, and the route of administration of the injectable preparation is selected from subcutaneous injection, intramuscular injection, intravenous injection, or intravenous infusion.

[0018] The injection solution contains the following components per 100 mL of tahinitroglycerin injection: The following ingredients were used: 0.01–500 mg of tahinitroglycerin reference standard, 0.85–0.92 g of sodium chloride, 0.5–1 g of sodium formaldehyde sulfoxylate, 3–5 mL of propylene glycol, 0.3–0.8 g of sodium dihydrogen phosphate, and the remainder was water for injection.

[0019] The lyophilized powder for injection is prepared using the following steps: Take 90% water for injection, add the ultrafiltration intermediate of 37-peptide subtilis, stir and mix well, then add mannitol and tert-butanol successively, stirring until completely dissolved and clear. Adjust the pH value to between 6.5 and 7.5 with sodium dihydrogen phosphate, add water for injection quantitatively and mix well. After the semi-finished product is qualified, filter it through 0.45 µm and 0.22 µm water films respectively, and fill it into half-volume vials. Half-stop the vials and place them in a freeze dryer for freeze drying. After capping, the freeze-dried powder of 37-peptide subtilis is obtained. The method of use for the lyophilized powder injection formulation is as follows: mix the lyophilized powder with water for injection to form an injection solution; each 100 mL of the lyophilized powder injection solution contains the following components: The following ingredients were used: 0.01–500 mg of trichol 37-peptide reference standard, 10–20 g of mannitol, 5–10 g of tert-butanol, 0.3–0.8 g of sodium dihydrogen phosphate, and the remainder was water for injection.

[0020] Beneficial effects: This invention involves filtering the fermentation broth of 37-peptide (BPT) through a ceramic membrane, followed by double-effect concentration, salting out, and tubular centrifugation. The resulting precipitate is the crude BPT, with a content of 328.6 mg / g as determined by liquid chromatography. The crude BPT is then reconstituted with 20% ethanol aqueous solution, centrifuged, and filtered. Following hydrophobic and ion-exchange chromatography, the product is concentrated, desalted, and deethanolinated by ultrafiltration, and then freeze-dried and packaged to obtain a high-purity BPT reference standard. The high-purity BPT reference standard prepared using this method exhibits a purity >99.9% as determined by reversed-phase chromatography, ion-exchange chromatography, and size exclusion chromatography. Endotoxin levels are <10 EU / mL, related substances content is <0.1%, and sterility and abnormal toxicity tests meet the standards for injectable raw materials, laying the foundation for the production of BPT raw materials.

[0021] This invention uses 20 times its volume of 20% ethanol aqueous solution to reconstitute crude 37-peptide, which improves the dissolution rate, solubility, and hydrophobicity of 37-peptide and increases its binding ability with hydrophobic fillers. In addition, using ethanol aqueous solution for reconstitution allows the 37-peptide solution to be stored for a long time without the need to add other preservatives.

[0022] This invention employs a three-step elution method in the hydrophobic chromatography process of purifying 37-peptide of kiwifruit. The first step involves eluting with water for 20 column volumes to wash away most of the substances bound to the hydrophobic packing material. The second step involves eluting with 5% ethanol and water for 20 column volumes to wash away most of the bound pigments. The third step involves eluting with 50% ethanol and water for 5 column volumes to elute the bound 37-peptide of kiwifruit. The eluent from the UV 280 nm peak during the third elution process is collected to obtain the hydrophobic purified intermediate of 37-peptide of kiwifruit.

[0023] This invention directly performs ion exchange chromatography on the hydrophobic purification intermediate of 37-peptide. The ethanol content in the purified intermediate of 37-peptide is 35%. The ethanol binds to 37-peptide, so that 37-peptide does not bind to the ion exchange packing material and flows through directly. Trace impurities bind to the packing material and are attached to it, without flowing through. Attached Figure Description

[0024] Figure 1 This is a production flow chart for the reference standard of calcined 37-peptide.

[0025] Figure 2 The image shows the high-performance liquid chromatogram of the fermentation broth containing 37-peptide of calcinus thiamphetamine.

[0026] Figure 3 The image shows the high-performance liquid chromatogram of crude chlorophyll 37-peptide.

[0027] Figure 4 This is the high-performance liquid chromatogram of the reference standard, calcitriol 37-peptide.

[0028] Figure 5 Hydrophobic chromatography diagram of the purification process of 37-peptide of calcinolone.

[0029] Figure 6 This is an ion chromatography diagram of the purification process of 37-peptide of calcined kiwi. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only one embodiment of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] I. Preparation process of fermentation broth containing 37-peptide of calcined kiwi The fermentation broth of 37-peptide of sphagnum moss was prepared according to the method in patent application number: 202211725251.7.

[0032] II. Selection of Conditions in the Preparation Process of Crude Substantial Trichopeptide (1) Ceramic membrane filtration: The prepared chlorophyll 37-peptide fermentation broth was introduced from the fermenter into a 50 m³ membrane. 3 The solution is introduced into a storage tank under pressure from an air compressor into a ceramic membrane filter F01 (Shanghai Langji Chemical Technology Co., Ltd.) for membrane filtration, and the filtrate (named filtrate A) is collected. Operating parameters: inlet membrane pressure 0.5 MPa, outlet membrane pressure 0.3 MPa, inlet membrane temperature 40 ℃, feed temperature 35 ℃; ceramic membrane pore size 50 nm. (2) Double-effect concentration: The obtained filtrate A is concentrated in three stages to a concentration of 40 g / L of tahinitropeptide, wherein the temperature of the first effect is 80 ℃, the temperature of the second effect is 70 ℃, the temperature of the third effect is 60 ℃, and the pressure is -0.1 MPa; (3) Ammonium sulfate addition: Ammonium sulfate was added to the obtained double-effect concentrate while stirring. The amount of ammonium sulfate added per liter of double-effect concentrate was 100~300 g. Stirring was continued for 2 h, and the mixture was allowed to precipitate in a cold storage room (temperature 4 ℃) for 16 h. The precipitate was then collected by centrifugation using a tubular centrifuge. The content of thiamphetamine in the precipitate was determined by high performance liquid chromatography, and the recovery rate of thiamphetamine was calculated. The results showed that the optimal amount of ammonium sulfate added per liter of double-effect concentrate was 167 g, the content of thiamphetamine in the precipitate was 328.6 mg / g, and the recovery rate was 96.35%.

[0033] (4) Test method for ammonium sulfate salting-out conditions: Ammonium sulfate was added to the obtained double-effect concentrate while stirring. The amount of ammonium sulfate added per liter of double-effect concentrate was 176 g. Stirring was continued for 2, 4, and 6 h. The precipitates were then collected in a cold storage room (temperature 4 ℃) and at room temperature (temperature 4 ℃) for 4, 10, 16, and 20 h, respectively. The precipitates were then collected by centrifugation using a tubular centrifuge. The content of 37-peptide in the precipitates was determined by high performance liquid chromatography, and its recovery rate was calculated. The content and recovery rate of 37-peptide in the precipitates obtained under the above different conditions were not significantly different.

[0034] III. Preparation of chromatographic loading solution, optimization of hydrophobic chromatography, ion exchange chromatography and ultrafiltration concentration conditions during the purification of 37-peptide of calcinolone acetonide with a 20% ethanol aqueous solution as the reconstitution solution.

[0035] The entire purification process was carried out in a Grade C area of ​​the GMP workshop. The 37-peptide reconstituted solution was a 20% aqueous ethanol solution. All buffer solutions and water used were prepared with water for injection and filtered through a 0.22 μm membrane.

[0036] (I) Preparation of chromatographic loading solution and optimization of hydrophobic chromatography conditions in the purification process of 37-peptide of calcinolone (1) The crude product of 37-peptide of calcinolone was dissolved in 20 times the volume of 20% ethanol aqueous solution. After stirring at high speed for 3 hours, the supernatant was collected by centrifugation at 16000 rpm. The supernatant was then obtained by plate and frame filtration using ordinary filter paper, multilayer filter plate, and 0.22 μm mixed fiber membrane.

[0037] (2) Selection of hydrophobic chromatography packing Four hydrophobic packing materials, Butyl HP, Phenyl HP, Capto butyl, and Capto phenyl, were compared. The chromatographic loading solution was pumped into a pre-equilibrated chromatographic column packed with the corresponding packing material, and equilibration was continued for 5 CV. The column was then eluted directly with 100% water. The content of thiamphetamine in the flow-through, equilibration, and eluent was detected by HPLC. The binding rate and recovery rate were examined. The hydrophobic packing material Butyl HP showed the best binding rate, but the recovery rate was 0, which could be improved through subsequent elution.

[0038] (3) Determination of hydrophobic chromatography elution conditions After adjusting the hydrophobic chromatography loading solution to different pH and conductivity values, samples were loaded and equilibrated with buffer until no protein penetration occurred. Elution was then performed with 100% water and ethanol of different concentrations. Each elution peak was collected, and the distribution of thiamphetamine was determined by HPLC. The final determination was that the optimal pH for the hydrophobic chromatography loading solution was 5.0. Conductivity had no effect on the elution of thiamphetamine; higher conductivity resulted in less protein flow and more protein elution with water, while lower conductivity resulted in more protein flow and less protein elution with water. For ease of operation, increasing the packing material loading and not adjusting conductivity was recommended. A three-step elution method yielded the best results. Specifically, the three-step elution was: first, elution with 20 column volumes of water; second, elution with 5% ethanol and water for 20 column volumes; and third, elution with 50% ethanol and water for 5 column volumes.

[0039] (4) Selection of hydrophobic chromatography buffer Based on the characteristics of Butyl HP packing material and thiamphetamine, a buffer assay was designed. The buffer was equilibrated with the sample loading solution according to the table below. A three-step elution method was used. The buffer system with 50 mM NaAc-HAc, 1.5 M NaCl, and pH 5.0 showed high column loading and recovery, and was the optimal buffer system.

[0040] (5) Determination of hydrophobic chromatography loading Adjust the pH of the chromatographic loading solution to 5.0 with glacial acetic acid. No conductivity adjustment is required. Continuously load the solution, collect the flow-through, and use HPLC to detect breakthrough. Stop loading when 10% of the target thiamphetapeptide has flowed through; the calculated loading capacity is 75 g / L.

[0041] (6) Collection criteria for hydrophobic chromatography solution Adjust the pH of the chromatographic loading buffer to 5.0, continuously load the sample to the column capacity, then stop loading and continue equilibrating with buffer until no protein penetration occurs. Perform a three-step elution, collecting the eluent at the UV 280 nm peak during the third elution step. Collect when the UV absorption at 280 nm increases significantly and stop collecting when it decreases. HPLC analysis shows that the content of kilotonil in the eluent is ≥3000 ug / mL, with a purity ≥99.9%. This eluent is used as the hydrophobic purification intermediate A for the next purification step. A single chromatography step yields 70-75 g of eluent with a content ≥3000 ug / mL and a purity ≥99.9%, with a recovery rate of over 93%.

[0042] (II) Optimization of ion exchange chromatography conditions during the purification of 37-peptide of calcinus thiamphetamine.

[0043] The entire purification process was carried out in the Class C area of ​​the GMP workshop. The ion exchange loading solution of the 37-peptide of calcinus was the hydrophobic purification intermediate A (ethanol content of 35%) obtained in step (I). All buffers and water used were prepared with water for injection and filtered through a 0.22 μm membrane.

[0044] (1) Selection of packing material for ion exchange chromatography Comparing four ion exchange packing materials—Capto S, Capto Q, Capto MMC, and Capto DEAE—the hydrophobic purification intermediate A obtained in step (I) was directly pumped into a pre-equilibrated chromatography column packed with the corresponding packing material. After equilibration, elution was performed, and HPLC analysis revealed that the content of thiamphetamine was mainly concentrated in the flow-through, indicating varying degrees of purity improvement. Therefore, the purity and recovery rate of thiamphetamine in the flow-through, as well as the binding rate of trace impurities, were used as evaluation indicators. The Capto S ion exchange packing material showed the best performance.

[0045] (2) Selection of ion exchange chromatography buffer Based on the characteristics of Capto S packing material and thiamphetamine, a buffer assay was designed. The buffer solution was equilibrated and combined with the sample loading solution according to the table below, and eluted in one step with 1M NaCl. The buffer system with 50mM NaAc-HAc and pH 5.0 showed high binding and recovery rates of impurities and was the optimal buffer system.

[0046] (3) Determination of ion exchange chromatography loading The hydrophobic purification intermediate A obtained in step (I) above was directly loaded onto the sample, and the flow-through was collected. The purity of the 37-peptide in the flow-through was detected by HPLC to determine the loading amount. The results showed that the loading volume was within the range of 8 CV. Based on production practice and theoretical practice, the loading amount for ion exchange chromatography was determined to be 5 CV.

[0047] (4) Determination of elution conditions for ion exchange chromatography Ion exchange chromatography elution conditions were performed according to the recommendations of Capto S packing material. Equilibration was performed using 50 mM NaAc-HAc buffer at pH 5.0. The sample loading solution was the hydrophobic chromatography intermediate A obtained in step (I). Elution was performed using 50 mM NaAc-Hac and 1M NaCl buffer at pH 5.0. The flow-through was collected.

[0048] (5) Collection criteria for ion exchange chromatography solvent After loading the hydrophobic purification intermediate A obtained in step (I) above, the flow-through was collected, and the UV absorption elution peak at 280 nm was detected. The content, purity, and endotoxin of the subtilisin in the flow-through were determined. The subtilisin content was ≥5000 ug / mL, the purity was ≥99.99%, and the endotoxin was <10 EU / mL. This flow-through was used as the ion exchange purification intermediate B for the next purification step. A single chromatography step yielded 150 g of flow-through with a subtilisin content ≥5000 ug / mL and a purity ≥99.99%, with a recovery rate of 90%.

[0049] (III) Ultrafiltration Concentration Method in the Refining Process of Trichoseptapeptide The ion exchange purification intermediate B of subtilisin obtained in step (II) above was purified by ultrafiltration. The membrane element was S-NF360, the transmembrane pressure (TMP) was 3.5-4.0 bar, and the ultrafiltration buffer was ultrapure water. After ultrafiltration to a conductivity of 2.5 μm / cm, it was concentrated to the required concentration of subtilisin to obtain the ultrafiltration concentrated subtilisin intermediate.

[0050] The specific steps of ultrafiltration concentration are as follows: Mix an appropriate amount of qualified thiamphetapeptide ion exchange purification intermediate together (the conductivity after mixing is approximately 10 μs / cm), so that the total amount of thiamphetapeptide purified in a single ultrafiltration is 600 g. Slowly add this to the ultrafiltration purifier. After concentration to a water level of 60 cm, connect the inlet to ultrapure water (conductivity 1.8 μm / cm), maintaining the flow rate consistent with the filtrate flow rate. Continue ultrafiltration until the conductivity of the filtrate matches that of the ultrapure water. Continue ultrafiltration until the conductivity of the ultrafiltration concentrate in the ultrafiltration purifier reaches 2.5 μm / cm and is maintained at this level for at least 30 minutes. Concentrate to the required concentration of thiamphetapeptide and discharge from the outlet to obtain the thiamphetapeptide ultrafiltration concentrated intermediate. Then add 40 L of 20% ethanol aqueous solution to the ultrafiltration purifier to flush out any remaining trace amounts of thiamphetapeptide.

[0051] (iv) Freeze-drying and packaging methods in the refining process of 37-peptide of calcined tsampa Add 600 mL of the ultrafiltration intermediate of 37-peptide of calcinolone acetonide to a clean freeze-drying tray, place it in a Pudong GLZY80 freeze dryer, and freeze-dry it by pre-freezing, freezing and slow heating. After 36 h, turn off the heating, slowly introduce air through the air inlet valve, turn on the freeze dryer, collect the freeze-dried powder, and dispense it to obtain high-purity 37-peptide of calcinolone acetonide reference standard.

[0052] IV. Preparation method of chromatographic loading solution, optimization of ion exchange chromatography and ultrafiltration concentration conditions in the purification process of 37-peptide of calcitriol with water for injection as reconstitution solution.

[0053] The entire purification process was carried out in a Grade C area of ​​the GMP workshop. The 37-peptide reconstituted solution was water for injection. All buffer solutions and water used were prepared with water for injection and then filtered through a 0.22 μm membrane.

[0054] (I) Purification process of 37-peptide: preparation of chromatographic loading solution and optimization of hydrophobic chromatography conditions (1) The crude product of 37-peptide was dissolved in 20 times water for injection, stirred at high speed for 3 h, and centrifuged at 16000 rpm to collect the clear liquid. The chromatographic loading solution was prepared by plate and frame filtration using ordinary filter paper, multilayer filter plate, and 0.22 μm mixed fiber membrane.

[0055] (2) Selection of hydrophobic chromatography packing Four hydrophobic packing materials, Butyl HP, Phenyl HP, Capto butyl, and Capto phenyl, were compared. The chromatographic loading solution was pumped into a pre-equilibrated chromatographic column packed with the corresponding packing material, and equilibration was continued for 5 CV. The column was then eluted directly with 100% water. The content of thiamphetamine in the flow-through, equilibration, and eluent was detected by HPLC. The binding rate and recovery rate were investigated. The hydrophobic chromatographic packing material Butyl HP showed the best performance.

[0056] (3) Selection of hydrophobic chromatography buffer Based on the characteristics of Butyl HP packing material and thiamphetamine, a buffer assay was designed. The buffer was equilibrated with the sample loading solution according to the table below, and eluted with pure water in one step. The buffer system with 50mM NaAc-HAc, 1.5M NaCl, and pH 5.0 showed high column loading and recovery, and was the optimal buffer system.

[0057] (4) Determination of hydrophobic chromatography loading Adjust the pH and conductivity of the chromatographic loading solution to match the chromatography buffer. Adjust the pH to 5.0 with glacial acetic acid and the conductivity to 100-120 ms / cm with 3M NaCl. Load continuously, collect the flow-through, and detect breakthrough using HPLC. Stop loading when 10% of the target trefeptin has flowed through; the calculated loading capacity is 46 g / L.

[0058] (5) Determination of hydrophobic chromatography elution conditions Adjust the pH and conductivity of the chromatography loading solution to match the chromatography buffer. Load the sample continuously until the column capacity is reached, then stop loading and continue equilibrating with buffer until no protein penetration occurs.

[0059] Linear gradient elution was performed with water, controlled at 0-100%, 20 CV, and each elution peak was collected. The distribution of thiamphetamine was detected and determined by HPLC, and the linear elution mode and the target peak distribution of thiamphetamine were finally determined.

[0060] (6) Collection criteria for hydrophobic chromatography solution Adjust the pH and conductivity of the loading buffer to match the chromatography buffer. Load continuously to the column capacity, then stop loading and continue equilibrating with buffer until no protein penetration occurs. Perform linear elution with water for injection (0–100% B, 20 CV). Collect the eluent when the UV absorbance at 280 nm increases significantly, and stop collecting when it decreases. HPLC analysis shows that the collected eluent contains ≥1000 ug / mL of thiamphetamine with a purity ≥99.9%. This eluent is used as the hydrophobic purification intermediate C for the next purification step. Collect the eluent with a thiamphetamine content ≥500 ug / mL and a purity of 98.5%–99.9%, adjust the pH and conductivity, and perform a second hydrophobic chromatography. A single chromatography step yields 10–15 g of eluent with a content ≥1000 ug / mL and a purity ≥99.9%, and 8–12 g of eluent with a content ≥500 ug / mL and a purity of 98.5%–99.9%, with an overall recovery rate between 37% and 56%.

[0061] (ii) Optimization of ion exchange chromatography conditions during the purification process of 37-peptide of calcitriol with water for injection as the reconstitution solution.

[0062] The entire purification process was carried out in a Grade C area of ​​the GMP workshop. The 37-peptide reconstituted solution was water for injection. All buffer solutions and water used were prepared with water for injection and then filtered through a 0.22 μm membrane.

[0063] (1) Selection of packing material for ion exchange chromatography Comparing four ion exchange packing materials—Capto S, Capto Q, Capto MMC, and Capto DEAE—the hydrophobic purification intermediate obtained in step (I) was adjusted to a conductivity of 1.0 ms / cm using ultrapure water (C), and then pumped into a pre-equilibrated chromatography column packed with the corresponding packing material. After equilibration, elution was performed, and the content of thiamphetamine in the flow-through, equilibration, and eluent was detected by HPLC. The binding rate and recovery rate were examined. The ion exchange packing material Capto Q showed the best performance.

[0064] (2) Selection of ion exchange chromatography buffer Based on the characteristics of Capto Q packing material and thiamphetamine, a buffer assay was designed. The buffer was equilibrated with the sample loading solution according to the table below, and eluted with 1M NaCl in one step. The buffer system with 50mM NaAc-HAc and pH 5.0 showed high column loading and recovery, and was the optimal buffer system.

[0065] (3) Determination of ion exchange chromatography loading Adjust the pH and conductivity of the hydrophobic purification intermediate C obtained in step (I) above to be consistent with the chromatography buffer. Adjust the pH with glacial acetic acid and dilute the conductivity with water for injection. Load the sample continuously, collect the flow-through, and use HPLC to detect whether there is breakthrough. Stop loading when 10% of the target thiampheptacapeptide is flowed through, and calculate the loading capacity as 26 g / L.

[0066] (4) Determination of elution conditions for ion exchange chromatography Adjust the pH and conductivity of the hydrophobic purification intermediate C obtained in step (I) above to be consistent with the chromatography buffer. After continuously loading the sample to the column capacity, stop loading the sample and continue to equilibrate with buffer until no protein penetration occurs.

[0067] Linear gradient elution was performed using 1M NaCl, controlled at 0-100%, with 5 CV. Each elution peak was collected, and the distribution of thiamphetamine was detected and determined by HPLC. Finally, the linear elution mode and the target peak distribution of thiamphetamine were determined.

[0068] (5) Collection criteria for ion exchange chromatography solvent Adjust the pH and conductivity of the hydrophobic purification intermediate C obtained in step (I) above to match the chromatography buffer. Load the sample continuously to the column capacity, then stop loading and continue equilibrating with buffer until no protein penetration occurs. Perform linear elution with 1M NaCl using parameters 0–100% B and 5 CV. Collect the elution peak at 280 nm UV absorption. Detect the content and purity of thiamphetamine in the eluent. Use the eluent with a thiamphetamine content ≥800 μg / mL and a purity ≥99.9% as the ion exchange purification intermediate D for the next purification step. Perform a second ion exchange chromatography on the eluent with a content ≥500 μg / mL and a purity below 99.9%. A single chromatography step yields 8–10 g of eluent with a thiamphetamine content ≥800 μg / mL and a purity ≥99.99%, with a recovery rate between 30% and 40%.

[0069] (iii) Ultrafiltration concentration method in the purification process of 37-peptide of kiwifruit with water for injection as the reconstitution solution.

[0070] The ion-exchange purification intermediate D of subtilisin obtained in step (II) above was purified by ultrafiltration. The membrane element was S-NF360, the transmembrane pressure (TMP) was 3.5-4.0 bar, and the ultrafiltration buffer was ultrapure water. After ultrafiltration to a conductivity of 2.5 μm / cm, it was concentrated to the required concentration of subtilisin to obtain the ultrafiltration concentrated subtilisin intermediate.

[0071] The specific steps of ultrafiltration concentration are as follows: Mix an appropriate amount of qualified thiamphetapeptide ion exchange purification intermediate (with a conductivity between 30 and 45 ms / cm after mixing) to obtain a total of 600g of thiamphetapeptide for a single ultrafiltration purification. Slowly add this mixture to the ultrafiltration purifier. After concentration to a water level of 60 cm, connect the inlet to ultrapure water (conductivity 1.8 μm / cm). The flow rate should be consistent with the filtrate flow rate. Continue ultrafiltration until the conductivity of the filtrate matches that of the ultrapure water after multiple consecutive tests. Then continue ultrafiltration until the conductivity of the ultrafiltration concentrate in the ultrafiltration purifier reaches 2.5 μm / cm and is maintained for at least 30 minutes. Concentrate to the required concentration of thiamphetapeptide and discharge from the outlet to obtain the thiamphetapeptide ultrafiltration concentration intermediate. Add 40 L of ultrapure water to the ultrafiltration purifier to flush out any remaining trace amounts of thiamphetapeptide.

[0072] (iv) Freeze-drying and packaging methods in the refining process of 37-peptide of calcined tsampa Add 600 mL of the ultrafiltration intermediate of 37-peptide of calcinolone acetonide to a clean freeze-drying tray, place it in a Pudong GLZY80 freeze dryer, and freeze-dry it by pre-freezing, freezing and slow heating. After 36 h, turn off the heating, slowly introduce air through the air inlet valve, turn on the freeze dryer, collect the freeze-dried powder, and dispense it to obtain high-purity 37-peptide of calcinolone acetonide reference standard.

[0073] Example 1 This embodiment illustrates the preparation process of the preferred sphagnum molybdate peptide.

[0074] (I) Preparation process of crude 37-peptide of calcined ... (1) Ceramic membrane filtration: The fermentation broth of 37-peptide prepared according to Example 1 in the prior art (202211725251.7) was introduced from the fermenter into a 50 m³ ceramic membrane filtration system. 3 The solution is introduced into a storage tank under pressure from an air compressor into a ceramic membrane filter F01 (Shanghai Langji Chemical Technology Co., Ltd.) for membrane filtration, and the filtrate (named filtrate A) is collected. Operating parameters: inlet membrane pressure 0.5 MPa, outlet membrane pressure 0.3 MPa, inlet membrane temperature 40 ℃, feed temperature 35 ℃; ceramic membrane pore size 50 nm. (2) Double-effect concentration: The obtained filtrate A is concentrated in three stages to a concentration of 40 g / L of tahinitropeptide, wherein the temperature of the first effect is 80 ℃, the temperature of the second effect is 70 ℃, the temperature of the third effect is 60 ℃, and the pressure is -0.1 MPa; (3) Ammonium sulfate salting-out: Ammonium sulfate was added to the obtained double-effect concentrate at a ratio of 176 g / L, and the mixture was stirred for another 4 h. Salting-out was carried out at 4℃ for 10 h. The precipitate and supernatant were then collected by centrifugation using a tubular centrifuge. The precipitate was crude tahinitrofurantoin, and the content of tahinitrofurantoin was determined to be 328.6 mg / g by high performance liquid chromatography, with a recovery rate of 96.35%.

[0075] (II) Refining process of 37-peptide of calcined oxaliplatin The entire purification process was carried out in a Grade C area of ​​the GMP workshop. The 37-peptide reconstituted solution was a 20% aqueous ethanol solution. All buffer solutions and water used were prepared with water for injection and filtered through a 0.22 μm membrane.

[0076] (1) Hydrophobic chromatography: The crude product of tahinitrophenylephrine obtained in the above steps was dissolved in 20 times the volume of tahinitrophenylephrine reconstituted solution. After stirring at high speed for 3 h, the supernatant was collected by centrifugation at 16000 rpm. The supernatant was then obtained by plate and frame filtration using ordinary filter paper, multilayer filter plate, and 0.22 μm mixed fiber membrane. The pH of the chromatographic sample solution was adjusted to 5.0, and hydrophobic chromatography purification was performed using Butyl Hp as the packing material. The sample was loaded at a concentration of 75 g / L, and the equilibration buffer consisted of 50 mM NaAc-Hac and 1.5 M NaCl at pH 5.0. The elution program consisted of three steps: first, elution with water for 20 column volumes; second, elution with 5% ethanol-water for 20 column volumes; and third, elution with 50% ethanol-water for 5 column volumes. The eluent from the UV 280 nm peak during the third elution step was collected and analyzed by HPLC C8 column to obtain the hydrophobic purified intermediate of tahinitrophenylephrine, with a HPLC purity of 99.9%.

[0077] (2) Ion exchange chromatography: The obtained intermediate product of 35% ethanol was purified by ion exchange chromatography without adjusting the conductivity. The packing material was Capto S, the loading volume was 5 CV, the equilibration buffer was 50 mM NaAc-HAc at pH 5.0, and the eluent was 50 mM NaAc-Hac and 1 M NaCl at pH 5.0. The flow-through was collected to obtain the intermediate product of 35% ethanol of 37% ethanol.

[0078] (3) Ultrafiltration Concentration: The obtained ion exchange purification intermediate of 37-peptide subtilis is purified by ultrafiltration. The membrane element is S-NF360, the transmembrane pressure (TMP) is 3.5-4.0 bar, and the ultrafiltration buffer is ultrapure water. After ultrafiltration to a conductivity of 2.5 μm / cm, it is concentrated to the required concentration of 37-peptide subtilis, to 1 g / L-20 g / L, generally to 10 g / L, to obtain the 37-peptide subtilis ultrafiltration intermediate. The specific steps of ultrafiltration concentration are as follows: Step (III) in "III. Preparation of chromatographic loading solution, hydrophobic chromatography, ion exchange chromatography and optimization of ultrafiltration concentration conditions in the purification process of 37-peptide subtilis with 20% ethanol aqueous solution". (4) Freeze-drying and packaging: Add 600 mL of the ultrafiltration intermediate of 37-peptide of calcinus thiol to a clean freeze-drying tray, place it in a Pudong GLZY80 freeze dryer, freeze-dry it by pre-freezing, freezing and slowly heating up, turn off the heating after 36 hours, slowly introduce air through the air inlet valve, turn on the freeze dryer, collect the freeze-dried powder, and package it to obtain high-purity 37-peptide of calcinus thiol reference standard.

[0079] The test results of the pyrimethamine reference standard obtained by the above steps are shown in Table 1.

[0080] Comparative Example 1 This embodiment illustrates the purification process of 37-peptide of kiwifruit, in which the reconstitution solution is water for injection.

[0081] The entire purification process was carried out in a GMP Class C area. All buffer solutions and water used were prepared with water for injection and filtered through a 0.22 μm membrane.

[0082] (1) Hydrophobic chromatography: The crude subtilisin obtained in Example 1 was dissolved in 20 times the amount of water for injection. After stirring at high speed for 3 h, the supernatant was collected by centrifugation at 16000 rpm. The supernatant was then filtered through ordinary filter paper, multilayer filter plate, and 0.22 μm mixed fiber membrane to obtain the chromatographic loading solution. The pH of the chromatographic loading solution was adjusted to 5.0 and the conductivity was adjusted to 100~120 ms / cm. Hydrophobic chromatography purification was performed using Butyl Hp as the packing material and loading at a loading of 46 g / L. The equilibration buffer was 50 mM NaAc-HAc and 1.5 M NaCl at pH 5.0. The eluent was water for injection. The UV 280 nm peak was collected and detected by HPLC. The subtilisin eluents that met the standards were combined to obtain the hydrophobic purified intermediate of subtilisin.

[0083] (2) Ultrafiltration concentration: The obtained hydrophobic purification intermediate of 37-peptide is purified by ultrafiltration and concentrated to the required concentration of 37-peptide, generally concentrated to 10 g / L, to obtain the ultrafiltration intermediate of 37-peptide.

[0084] The specific steps for ultrafiltration concentration are as follows: Step (III) in "IV. Preparation of chromatographic loading solution, hydrophobic chromatography, ion exchange chromatography and optimization of ultrafiltration concentration conditions in the purification process of thiamphetamine subtilis with water for injection as the reconstitution solution".

[0085] (3) Freeze-drying and packaging: Add 600 mL of the ultrafiltration intermediate of 37-peptide of kiwifruit to a clean freeze-drying tray, place it in a Pudong GLZY80 freeze dryer, freeze-dry it by pre-freezing, freezing and slowly heating up, turn off the heating after 36 hours, slowly introduce air through the air inlet valve, turn on the freeze dryer, collect the freeze-dried powder, and package it to obtain high-purity 37-peptide of kiwifruit reference standard.

[0086] The test results of the 37-peptide reference standards obtained by the above steps are shown in Table 1.

[0087] Comparative Example 2 This embodiment illustrates the purification process of 37-peptide of kiwifruit, in which the reconstitution solution is water for injection.

[0088] The entire purification process was carried out in a GMP Class C area. All buffer solutions and water used were prepared with water for injection and filtered through a 0.22 μm membrane.

[0089] (1) Ion exchange chromatography: The crude subtilisin obtained in Example 1 was dissolved in 20 times the amount of water for injection. After stirring at high speed for 3 h, the supernatant was collected by centrifugation at 16000 rpm. The supernatant was then filtered through ordinary filter paper, multilayer filter plate, and 0.22 μm mixed fiber membrane to obtain the chromatographic loading solution. The pH of the chromatographic loading solution was adjusted to 5.0 and the conductivity to 1.0 ms / cm. Ion exchange chromatography purification was performed using Capto Q packing material at a loading of 26 g / L. The equilibration buffer was 50 mM NaAc-HAc at pH 5.0, and the eluent was 50 mM NaAc-Hac and 1 M NaCl at pH 5.0. The elution peak was collected and detected by HPLC. The subtilisin eluents that met the standards were combined to obtain the subtilisin ion exchange purified intermediate.

[0090] (2) Hydrophobic chromatography: The conductivity of the obtained thiamphetapeptide ion exchange intermediate was adjusted to 100~120 ms / cm and purified by hydrophobic chromatography. Butyl Hp was used as the packing material and the sample was loaded at a loading of 46 g / L. The equilibration buffer was 50 mM NaAc-HAc and 1.5 M NaCl at pH 5.0. The eluent was water for injection. The UV 280 nm peak was collected and detected by HPLC. The eluents of thiamphetapeptide that met the standard were combined to obtain the hydrophobic purified intermediate of thiamphetapeptide.

[0091] (3) Ultrafiltration concentration: The obtained hydrophobic purification intermediate of 37-peptide is concentrated by ultrafiltration to the required concentration of 37-peptide, generally to 10 g / L, to obtain the ultrafiltration intermediate of 37-peptide.

[0092] The specific steps for ultrafiltration concentration are as follows: Step (III) in "IV. Preparation of chromatographic loading solution, hydrophobic chromatography, ion exchange chromatography and optimization of ultrafiltration concentration conditions in the purification process of thiamphetamine subtilis with water for injection as the reconstitution solution".

[0093] (4) Freeze-drying and packaging: Add 600 mL of the ultrafiltration intermediate of 37-peptide of calcinus thiol to a clean freeze-drying tray, place it in a Pudong GLZY80 freeze dryer, freeze-dry it by pre-freezing, freezing and slowly heating up, turn off the heating after 36 hours, slowly introduce air through the air inlet valve, turn on the freeze dryer, collect the freeze-dried powder, and package it to obtain high-purity 37-peptide of calcinus thiol reference standard.

[0094] The test results of the pyrimethamine reference standard obtained by the above steps are shown in Table 1.

[0095] Comparative Example 3 This embodiment illustrates the purification process of 37-peptide of kiwifruit, in which the reconstitution solution is water for injection.

[0096] The entire purification process was carried out in a GMP Class C area. All buffer solutions and water used were prepared with water for injection and filtered through a 0.22 μm membrane.

[0097] (1) Hydrophobic chromatography: The crude subtilisin obtained in Example 1 was dissolved in 20 times the amount of water for injection. After stirring at high speed for 3 h, the supernatant was collected by centrifugation at 16000 rpm. The supernatant was then filtered through ordinary filter paper, multilayer filter plate, and 0.22 μm mixed fiber membrane to obtain the chromatographic loading solution. The pH of the chromatographic loading solution was adjusted to 5.0 and the conductivity to 100~120 ms / cm. Hydrophobic chromatography purification was performed using Butyl Hp as the packing material and loading at a capacity of 46 g / L. The equilibration buffer was 50 mM NaAc-HAc and 1.5 M NaCl at pH 5.0. The eluent was water for injection. The UV 280 nm peak was collected and detected by HPLC. The subtilisin eluents that met the standards were combined to obtain the hydrophobic purified intermediate of subtilisin.

[0098] (2) Ion exchange chromatography: The conductivity of the obtained 37-peptide purified intermediate was adjusted to 1.0 ms / cm and purified by ion exchange chromatography. The packing material was Capto Q, and the sample was loaded at a loading of 26 g / L. The equilibration buffer was 50 mM NaAc-HAc at pH 5.0, and the eluent was 50 mM NaAc-Hac and 1M NaCl at pH 5.0. The UV absorption peak at 280 nm was collected and detected by HPLC. The eluents of 37-peptide that met the standard were combined to obtain the 37-peptide purified intermediate by ion exchange chromatography.

[0099] (3) Ultrafiltration concentration: The obtained ion exchange purified intermediate of 37-peptide is concentrated by ultrafiltration to the required concentration of 37-peptide, generally to 10 g / L, to obtain the 37-peptide ultrafiltration intermediate.

[0100] The specific steps for ultrafiltration concentration are as follows: Step (III) in "IV. Preparation of chromatographic loading solution, hydrophobic chromatography, ion exchange chromatography and optimization of ultrafiltration concentration conditions in the purification process of thiamphetamine subtilis with water for injection as the reconstitution solution".

[0101] (4) Freeze-drying and packaging: Add 600 mL of the ultrafiltration intermediate of 37-peptide of calcinus thiol to a clean freeze-drying tray, place it in a Pudong GLZY80 freeze dryer, freeze-dry it by pre-freezing, freezing and slowly heating up, turn off the heating after 36 hours, slowly introduce air through the air inlet valve, turn on the freeze dryer, collect the freeze-dried powder, and package it to obtain high-purity 37-peptide of calcinus thiol reference standard.

[0102] The test results of the pyrimethamine reference standard obtained by the above steps are shown in Table 1.

[0103] Table 1. Detection results of refined pyrimethamine triseptapeptide reference standard V. Quality standards for chloroquine 37-peptide reference standard.

[0104] Based on Appendix 4, "Special Requirements for the Quality Management of Raw Materials," of the Good Manufacturing Practice for Veterinary Drugs (2020 Revision), and in conjunction with the test results of the tahinitroglycerin reference standard, the quality standard for the tahinitroglycerin reference standard is formulated, as detailed in Table 2 below.

[0105] Table 2 Quality Standards for Trichoseptapeptide VI. Preparation method of 37-peptide injection.

[0106] The 37-peptide subtilis injection contains the following per 100 mL: 0.01-500 mg 37-peptide subtilis reference standard, 0.85-0.92 g sodium chloride, 0.5-1 g sodium formaldehyde sulfoxylate, 3-5 mL propylene glycol, 0.3-0.8 g sodium dihydrogen phosphate, and the remainder being water for injection.

[0107] The preparation method of the above-mentioned tahinitrofurantoin injection includes the following steps: Take an appropriate amount of water for injection, add formaldehyde and sodium bisulfite and stir to dissolve, then add propylene glycol and stir to mix well. Adjust the pH to 5.0~7.0 with sodium dihydrogen phosphate, purge with nitrogen until saturated, add 0.001~50 mL of 10 mg / mL tahinitrofurantoin ultrafiltration intermediate, stir until completely dissolved and clear, and always purge with nitrogen to saturate the solution tank. Add water for injection and mix quantitatively. Adjust the pH value to 6.5~7.5 with sodium dihydrogen phosphate. The two pH adjustments can prevent the precipitation of tahinitrofurantoin by the first adjustment and improve the stability of the product by the second adjustment. After the semi-finished product is qualified, filter it with 0.45 µm and 0.22 µm water films respectively and fill it with nitrogen. When filling, fill the solution and vial with nitrogen, stopper and crimp the cap. After the clarity is qualified, package it.

[0108] VII. Preparation method of lyophilized powder for injection of 37-peptide of calcinolone.

[0109] The lyophilized powder of 37-peptide contains, per 100 mL injection: 0.01-500 mL of 37-peptide reference standard, 10-20 g of mannitol, 5-10 g of tert-butanol, 0.3-0.8 g of sodium dihydrogen phosphate, and the remainder is water for injection.

[0110] The preparation method of the above-mentioned lyophilized powder of 37-peptide subtilis includes the following steps: Take 90% water for injection, add 0.001~50 mL of 10 mg / mL 37-peptide subtilis ultrafiltration intermediate, stir and mix well, then add mannitol and tert-butanol successively, stir until completely dissolved and clear, then adjust the pH value to between 6.5 and 7.5 with sodium dihydrogen phosphate, add water for injection quantitatively and mix well, test the semi-finished product to be qualified, filter with 0.45 µm and 0.22 µm water films respectively, and fill into vials with half volume; half-stop the vials and place them in a freeze dryer for freeze drying, then cap them to obtain lyophilized powder of 37-peptide subtilis.

[0111] VIII. Purity Detection Method for Bacillus Trichopeptide Reference Standard.

[0112] (1) Size exclusion chromatography Column: TSK G2000SWXl Mobile phase A: 0.1% TFA, Mobile phase B: 80% ACN + 20% H2O + 0.085% TFA.

[0113] Flow rate: 1.0 mL / min, detection wavelength: 280 nm, column temperature: 25℃.

[0114] Chromatographic conditions: 50% A + 50% B, injection volume 10~50 μL. Analysis time: 23 min.

[0115] (2) Reversed-phase chromatography Column: ZOBAX 300SB-C8 Mobile phase A: 0.1% TFA, Mobile phase B: 80% ACN + 20% H2O + 0.085% TFA.

[0116] The flow rate was 1.0 mL / min, the detection wavelength was 280 nm, and the column temperature was 25℃.

[0117] mobile phase gradient conditions: (3) Ion exchange chromatography Column: BioSuite NP CXC Mobile phase A: 20 mM NaAc, pH 5.0; Mobile phase B: 20 ​​mM NaAc + 1 M NaCl, pH 5.0.

[0118] Flow rate: 1.0 mL / min, detection wavelength: 280 nm, column temperature: 25℃.

[0119] mobile phase gradient conditions: IX. Method for detecting endotoxin in 37-peptide reference standard.

[0120] Endotoxin detection of the 37-peptide reference standard was performed according to Appendix 1143 of the 2020 edition of the Chinese Veterinary Pharmacopoeia, using the method for bacterial endotoxin testing. (1) Interference test Prepare solutions A, B, C, and D according to Table 3. The test solutions used should be solutions that do not detect endotoxins and do not exceed the maximum effective dilution (MVD). Refer to the Limulus Amebocyte Lysate (LAL) reagent instructions for operation. The test is valid only if all parallel tubes of solution A and negative control solution D are negative, and the results of series solution C are within the sensitivity verification range of the LAL reagent.

[0121] Table 3 Preparation of interference test solutions by gelation method Note: A is the test solution; B is the interference test series; C is the control series of Limulus amebocyte lysate (LAL) reagent labeled sensitivity; D is the negative control.

[0122] (2) Endotoxin detection of the test sample Prepare solutions A, B, C, and D according to Table 4. Use a dilution factor (MVD) of 1 and an interference-free solution to prepare solutions A and B. Follow the instructions for use of the Limulus Amebocyte Lysate (LAL) reagent. Incubate for 60 minutes and observe the results within 2 minutes. If all parallel tubes of negative control solution D are negative, all parallel tubes of positive control solution B are positive, and all parallel tubes of positive control solution C are positive, the test is valid. If both parallel tubes of solution A are negative, the test sample is considered compliant; if both parallel tubes of solution A are positive, the test sample is considered non-compliant. If one of the two parallel tubes of solution A is positive and the other is negative, a retest is required. For the retest, four parallel tubes of solution A should be prepared. If all parallel tubes are negative, the test sample is considered compliant; otherwise, the test sample is considered non-compliant.

[0123] Table 4. Preparation of test solutions for endotoxin content detection by gel electrophoresis Number Concentration of endotoxin / solution of endotoxin preparation Number of parallel tubes A None / test solution 2 B 2λ / test solution 2 C 2λ / inspection water 2 D None / inspection water 2 Note: A is the test solution; B is the positive control; C is the positive control; D is the negative control.

[0124] XI. Sterility test method for thiamphetamine reference standard.

[0125] (1) Inactivation of pyrimethamine reference standard Before using microbial methods to detect antibiotic residues, the antibacterial subtilisin reference standard should be inactivated. Because subtilisin has excellent thermal and pH stability, enzymatic digestion is used for inactivation. The enzymatic digestion reaction system is shown in Table 5 below.

[0126] Table 5. Enzymatic digestion reaction system of calcined 37-peptide reference standard Name Bacitracin Control Trypsin 10% NH4HCO3 Ultrapure water Amount added 0.40 mL 0.050 mL 0.10 mL 0.45 mL After enzymatic digestion in a 37 ℃ water bath overnight, the light absorption of the test sample was detected by HPLC. The characteristic absorption peak of the test sample completely disappeared after overnight digestion. At the same time, 100 µl was taken for activity detection using turbidimetric assay. The undigested test sample served as a positive control. After overnight digestion, the antibacterial activity of the subtilisin reference standard completely disappeared. This proves that the subtilisin reference standard was successfully inactivated.

[0127] (2) The sterility test shall be performed in accordance with the sterility test method in Appendix 1101 of Part I of the 2020 edition of the Chinese Veterinary Pharmacopoeia.

[0128] XII. Detection method for abnormal toxicity of 37-peptide reference standard.

[0129] The abnormal toxicity test of the 37-peptide reference standard was conducted according to Appendix 1141 of the 2020 edition of the Chinese Veterinary Pharmacopoeia. Five healthy mice were used as the experimental subjects. The injection dose was 5 mg / mL. All five mice survived within 48 hours after the tail vein injection of 0.5 mL, with no deaths, and the mice's final weight increased, which indicated that they met the requirements for abnormal toxicity.

[0130] The production method of the thiamphetamine reference standard obtained by this invention includes preparing thiamphetamine fermentation broth, preparing crude thiamphetamine, and refining thiamphetamine. The preparation process of the thiamphetamine fermentation broth includes fermentation with Bacillus subtilis (B. subtilis). Bacillus Subtilis CGMCC15404) has undergone strain rejuvenation and activation, and is now available as primary and secondary seed culture in a 30 L seed tank. 3 Seed container, 30 M 3 Fermentation in fermenters yielded a 6330 mg / L content of thiamphetamine, more than double the highest yield currently achieved. The crude thiamphetamine was prepared by filtering the fermentation broth through a ceramic membrane, followed by double-effect falling membrane evaporation and concentration, salting out, and tubular centrifugation. The resulting precipitate was the crude thiamphetamine, with a content of 328.6 mg / g as determined by liquid chromatography. The purification process was conducted in a GMP-certified Class C area. All buffer solutions and water used were prepared with water for injection. The crude thiamphetamine was reconstituted with 20% ethanol aqueous solution, centrifuged, filtered, and then subjected to hydrophobic and ion-exchange two-step chromatography. Following ultrafiltration concentration, desalting, de-alcoholization, and freeze-drying, the thiamphetamine reference standard was obtained.

[0131] The reference standard for 37-peptide subtilis can be used for the quality evaluation of 37-peptide subtilis and the quality evaluation of products containing 37-peptide subtilis.

[0132] The purity of the tahinitroglycerin reference standard prepared by the method of the present invention was >99.9% as determined by reversed-phase chromatography, ion-exchange chromatography, and size exclusion chromatography. The endotoxin content was <10 EU / mL, the related substances content was <0.1%, and the sterility test and abnormal toxicity met the standards for high-purity reference standards. Compared with the comparative example, the method used in the present invention has a higher recovery rate, all above 90%.

[0133] This invention uses 20 times its volume of 20% ethanol aqueous solution to reconstitute crude 37-peptide, which improves the dissolution rate, solubility, and hydrophobicity of 37-peptide and increases its binding ability with hydrophobic fillers. In addition, using ethanol aqueous solution for reconstitution allows the 37-peptide solution to be stored for a long time without the need to add other preservatives.

[0134] This invention employs a three-step elution method in the hydrophobic chromatography process for the purification of subtilisin: Step 1: elution with water for 20 column volumes to remove most of the substance bound to the hydrophobic packing material; Step 2: elution with 5% ethanol-water for 20 column volumes to remove most of the bound pigments; Step 3: elution with 50% ethanol-water for 5 column volumes to elute the bound subtilisin. The eluent from the 280 nm UV peak during the third elution step is collected to obtain the purified subtilisin intermediate. This invention selects packing material and elution method based on the characteristics of subtilisin to maximize the binding force between subtilisin and the packing material, resulting in successful elution.

[0135] This invention directly performs ion exchange chromatography on the purified intermediate of 37-peptide of calcinus trichoseptide. The ethanol content in the purified intermediate of 37-peptide of calcinus trichoseptide is 35%. Ethanol binds to 37-peptide of calcinus trichoseptide, so that 37-peptide of calcinus trichoseptide does not bind to the ion exchange packing material and flows through directly; while trace impurities and residual proteins bind to the packing material and hang on the packing material, and do not flow through.

[0136] This invention provides a high-purity thiamphetamine reference standard. The reference standard, as determined by reversed-phase chromatography, ion-exchange chromatography, and size exclusion chromatography, exhibits a purity >99.9%, endotoxin <10 EU / mL, and meets the standards for injectable active pharmaceutical ingredients (APIs) in terms of sterility and abnormal toxicity. Therefore, it can be used as an API. In other words, a high-purity thiamphetamine reference standard is equivalent to an injectable thiamphetamine API. Furthermore, a method for industrializing the high-purity thiamphetamine reference standard based on biosynthesis is a method for producing an injectable thiamphetamine API. The biosynthesis method refers to fermentation as described in this field.

[0137] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention. This includes any combination of specific technical features. To avoid unnecessary repetition, the present invention will not further describe the various possible combinations. However, these simple modifications and combinations should also be considered as part of the content disclosed in this invention and are all within the protection scope of this invention.

Claims

1. A high-purity pyrimethamine reference standard, characterized in that, The high-purity pyrimethamine reference standard is prepared by the following steps: (1) Hydrophobic chromatography: Dissolve crude tahinitrofurantoin in 15%~25% ethanol aqueous solution, stir, centrifuge, collect supernatant, filter, and the obtained filtrate is the chromatographic loading solution; take the chromatographic loading solution and adjust the pH to 4.5~5.5, without adjusting the conductivity, and perform hydrophobic chromatography purification. The packing material is Butyl Hp, and the loading is 70 g / L~80 g / L. The equilibration buffer is 50 mM NaAc-HAc and 1.5 M NaCl at pH 5.

0. Elution is performed in three steps: first step: water elution, second step: 3.5%~6.5% ethanol water elution, third step: 40%~60% ethanol water elution. Collect the eluent from the third step to obtain the hydrophobic purified intermediate of tahinitrofurantoin. (2) Ion exchange chromatography: The obtained intermediate of 37-peptide of calcinus trichoseptum was purified directly by ion exchange chromatography without adjusting the conductivity. The packing material was Capto S, the loading volume was 1~8 CV, the equilibration buffer was 50 mM NaAc-HAc at pH 5.0, the eluent was 50 mM NaAc-Hac at pH 5.0 and 1 M NaCl, and the flow-through was collected to obtain the intermediate of 37-peptide of calcinus trichoseptum ion exchange. (3) Ultrafiltration purification: The obtained ion exchange purification intermediate of 37-peptide was purified by ultrafiltration. The membrane element was S-NF360, the transmembrane pressure was 3.5 to 4.0 bar, and the ultrafiltration buffer was ultrapure water. After ultrafiltration to a conductivity of 1.5 μm / cm to 3.5 μm / cm, the 37-peptide was concentrated to 1 g / L to 20 g / L to obtain the 37-peptide ultrafiltration intermediate. (4) Freeze-drying and packaging: Add 600 mL of the ultrafiltration intermediate of 37-peptide of kiwifruit to a clean freeze-drying tray, place it in a freeze dryer, and freeze-dry it by pre-freezing, freezing and slowly heating. After 36 h, turn off the heating, slowly introduce air through the air inlet valve, turn on the freeze dryer, collect the freeze-dried powder, and package it to obtain high-purity 37-peptide of kiwifruit reference standard.

2. The reference standard as described in claim 1, characterized in that, The high-purity pyrimethamine reference standard was prepared by the following steps: (1) Hydrophobic chromatography: Dissolve crude 37-peptide in 20 times the volume of 20% ethanol aqueous solution, stir, centrifuge, collect the supernatant, filter, and the obtained filtrate is the chromatographic loading solution; take the chromatographic loading solution and adjust the pH to 5.0, without adjusting the conductivity, and perform hydrophobic chromatography purification. The packing material is Butyl Hp, and the loading is 75 g / L. The equilibration buffer is 50 mM NaAc-HAc and 1.5 M NaCl at pH 5.

0. The elution program is carried out in three steps: the first step is to elute with 20 column volumes of water, the second step is to elute with 20 column volumes of 5% ethanol water, and the third step is to elute with 50% ethanol water for 5 column volumes. Collect the eluent of the UV 280 nm peak during the third step elution process to obtain the purified intermediate of 37-peptide. (2) Ion exchange chromatography: The obtained intermediate of 37-peptide of calcitriol was purified directly by ion exchange chromatography without adjusting the conductivity. The packing material was Capto S, the loading volume was 5 CV, the equilibration buffer was 50 mM NaAc-HAc at pH 5.0, the eluent was 50 mM NaAc-Hac at pH 5.0 and 1 M NaCl, and the flow-through was collected to obtain the intermediate of 37-peptide of calcitriol ... (3) Ultrafiltration purification: The obtained ion exchange purification intermediate of 37-peptide was purified by ultrafiltration. The membrane element was S-NF360, the transmembrane pressure was 3.5-4.0 bar, and the ultrafiltration buffer was ultrapure water. After ultrafiltration to a conductivity of 2.5 μm / cm, the 37-peptide was concentrated to 10 g / L to obtain the 37-peptide ultrafiltration intermediate. (4) Freeze-drying and packaging: Add the ultrafiltration intermediate of 37-peptide of calcinus thiol to a clean freeze-drying tray at a rate of 600 mL / tray, put it into a freeze dryer, and freeze-dry it by pre-freezing, freezing and slowly heating. After 36 h, turn off the heating, slowly introduce air through the air inlet valve, turn on the freeze dryer, collect the freeze-dried powder, and dispense it to obtain high-purity 37-peptide of calcinus thiol reference standard.

3. A production method for industrial-scale production of high-purity pyridostigmine reference standard based on biosynthesis, characterized in that, The production method of the high-purity pyrimethamine reference standard includes the following steps: (1) Hydrophobic chromatography: Dissolve crude tahinitrofurantoin in 15%~25% ethanol aqueous solution, stir, centrifuge, collect supernatant, filter, and the obtained filtrate is the chromatographic loading solution; take the chromatographic loading solution and adjust the pH to 4.5~5.5, without adjusting the conductivity, and perform hydrophobic chromatography purification. The packing material is Butyl Hp, and the loading is 70 g / L~80 g / L. The equilibration buffer is 50 mM NaAc-HAc and 1.5 M NaCl at pH 5.

0. Elution is performed in three steps: first step: water elution, second step: 3.5%~6.5% ethanol water elution, third step: 40%~60% ethanol water elution. Collect the eluent from the third step to obtain the hydrophobic purified intermediate of tahinitrofurantoin. (2) Ion exchange chromatography: The obtained intermediate of 37-peptide of calcinus trichoseptum was purified directly by ion exchange chromatography without adjusting the conductivity. The packing material was Capto S, the loading volume was 1~8 CV, the equilibration buffer was 50 mM NaAc-HAc at pH 5.0, the eluent was 50 mM NaAc-Hac at pH 5.0 and 1 M NaCl, and the flow-through was collected to obtain the intermediate of 37-peptide of calcinus trichoseptum ion exchange. (3) Ultrafiltration purification: The obtained ion exchange purification intermediate of 37-peptide was purified by ultrafiltration. The membrane element was S-NF360, the transmembrane pressure was 3.5 to 4.0 bar, and the ultrafiltration buffer was ultrapure water. After ultrafiltration to a conductivity of 1.5 μm / cm to 3.5 μm / cm, the 37-peptide was concentrated to 1 g / L to 20 g / L to obtain the 37-peptide ultrafiltration intermediate. (4) Freeze-drying and packaging: Add 600 mL of the ultrafiltration intermediate of 37-peptide of kiwifruit to a clean freeze-drying tray, place it in a freeze dryer, and freeze-dry it by pre-freezing, freezing and slowly heating. After 36 h, turn off the heating, slowly introduce air through the air inlet valve, turn on the freeze dryer, collect the freeze-dried powder, and package it to obtain high-purity 37-peptide of kiwifruit reference standard.

4. The production method as described in claim 3, characterized in that, The production method of the high-purity pyrimethamine reference standard includes the following steps: (1) Hydrophobic chromatography: Dissolve crude 37-peptide in 20 times the volume of 20% ethanol aqueous solution, stir, centrifuge, collect the supernatant, filter, and the obtained filtrate is the chromatographic loading solution; take the chromatographic loading solution and adjust the pH to 5.0, without adjusting the conductivity, and perform hydrophobic chromatography purification. The packing material is Butyl Hp, and the loading is 75 g / L. The equilibration buffer is 50 mM NaAc-HAc and 1.5 M NaCl at pH 5.

0. The elution program is carried out in three steps: the first step is to elute with 20 column volumes of water, the second step is to elute with 20 column volumes of 5% ethanol water, and the third step is to elute with 50% ethanol water for 5 column volumes. Collect the eluent of the UV 280 nm peak during the third step elution process to obtain the purified intermediate of 37-peptide. (2) Ion exchange chromatography: The obtained intermediate of 37-peptide of calcitriol was purified directly by ion exchange chromatography without adjusting the conductivity. The packing material was Capto S, the loading volume was 5 CV, the equilibration buffer was 50 mM NaAc-HAc at pH 5.0, the eluent was 50 mM NaAc-Hac at pH 5.0 and 1 M NaCl, and the flow-through was collected to obtain the intermediate of 37-peptide of calcitriol ... (3) Ultrafiltration purification: The obtained ion exchange purification intermediate of 37-peptide was purified by ultrafiltration. The membrane element was S-NF360, the transmembrane pressure was 3.5-4.0 bar, and the ultrafiltration buffer was ultrapure water. After ultrafiltration to a conductivity of 2.5 μm / cm, the 37-peptide was concentrated to 10 g / L to obtain the 37-peptide ultrafiltration intermediate. (4) Freeze-drying and packaging: Add the ultrafiltration intermediate of 37-peptide of calcinus thiol to a clean freeze-drying tray at a rate of 600 mL / tray, put it into a freeze dryer, and freeze-dry it by pre-freezing, freezing and slowly heating. After 36 h, turn off the heating, slowly introduce air through the air inlet valve, turn on the freeze dryer, collect the freeze-dried powder, and dispense it to obtain high-purity 37-peptide of calcinus thiol reference standard.

5. The reference standard or production method as described in any one of claims 1 to 4, characterized in that, The preparation method of the crude kiwifruit peptide includes the following steps: (1) Ceramic membrane filtration: The fermentation broth of 37-peptide of calcined ... Operating parameters: inlet pressure 0.5 MPa, outlet pressure 0.3 MPa, inlet temperature 40 ℃, feed temperature 35 ℃; ceramic membrane pore size 50 nm; (2) Double-effect concentration: The filtrate A was concentrated in three stages to a concentration of 40 g / L of tahinitropeptide to obtain a double-effect concentrate; The temperature of the first effect is 80 ℃, the temperature of the second effect is 70 ℃, the temperature of the third effect is 60 ℃, and the pressure is -0.1 MPa; (3) Ammonium sulfate salting out: Add ammonium sulfate to the obtained double-effect concentrate while stirring. The amount of ammonium sulfate added per liter of double-effect concentrate is 100~300 g. Continue stirring for 2~6 h and salt out at 4 ℃ for 4~20 h. Then centrifuge with a tubular centrifuge to collect the precipitate and the clear liquid. The precipitate is crude 37-peptide of kiwifruit.

6. The high-purity pyrimethamine reference standard as described in claim 1, characterized in that, The reference standard was tested by reversed-phase chromatography, ion-exchange chromatography, and size exclusion chromatography. The purity was >99.9%, the endotoxin was <10 EU / mL, and the sterility and abnormal toxicity tests met the standards for injectable active pharmaceutical ingredients. It was used as an active pharmaceutical ingredient.

7. An injectable preparation comprising the high-purity pyrimethamine reference standard as described in claim 6, characterized in that, The injectable is selected from injection solutions, infusion solutions, concentrated solutions for injection, sterile powders for injection, and lyophilized powders for injection. The route of administration of the injectable is selected from subcutaneous injection, intramuscular injection, intravenous injection, or intravenous drip.

8. An injectable preparation as described in claim 7, characterized in that, The injection contains the following components per 100 mL of tahinitroglycerin injection: The following ingredients were used: 0.01–500 mg of tahinitroglycerin reference standard, 0.85–0.92 g of sodium chloride, 0.5–1 g of sodium formaldehyde sulfoxylate, 3–5 mL of propylene glycol, 0.3–0.8 g of sodium dihydrogen phosphate, and the remainder was water for injection.

9. An injectable preparation as described in claim 7, characterized in that, The lyophilized powder for injection is prepared using the following steps: Take 90% water for injection, add the ultrafiltration intermediate of 37-peptide subtilis, stir and mix well, then add mannitol and tert-butanol successively, stirring until completely dissolved and clear. Adjust the pH value to between 6.5 and 7.5 with sodium dihydrogen phosphate, add water for injection quantitatively and mix well. After the semi-finished product is qualified, filter it through 0.45 µm and 0.22 µm water films respectively, and fill it into half-volume vials. Half-stop the vials and place them in a freeze dryer for freeze drying. After capping, the freeze-dried powder of 37-peptide subtilis is obtained. The method of using the lyophilized powder for injection is as follows: mix the lyophilized powder with water for injection to form an injection solution; each 100 mL of the lyophilized powder injection solution contains the following components: The following ingredients were used: 0.01–500 mg of trichol 37-peptide reference standard, 10–20 g of mannitol, 5–10 g of tert-butanol, 0.3–0.8 g of sodium dihydrogen phosphate, and the remainder was water for injection.

Images