Medicinal material capable of being used in injection as well as preparation method and application of medicinal material
Through specific process parameters and purely physical technical means, the problem of high impurity content in food-grade ε-polylysine was solved, and ε-polylysine that meets injection-grade standards was prepared, achieving a high recovery rate and good safety, meeting the requirements for use in injections.
Patent Information
- Application Number
- CN202510735548.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-04
- Publication Date
- 2025-10-10
AI Technical Summary
It is difficult to prepare food-grade ε-polylysine that meets injection-grade standards using existing technologies, as there are problems with high bacterial endotoxin and impurity content.
Specific process parameters and purely physical technical means, including micropore filtration, anion exchange, ultrafiltration, etc., combined with specific pH adjustment and buffer treatment, are used to prepare ε-polylysine that meets injection-grade requirements.
A high recovery rate and good safety of ε-polylysine were achieved, and the bacterial endotoxin removal rate reached more than 99.38%, meeting the quality requirements of injection-grade pharmaceutical materials.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of medicine, and particularly relates to a medicinal material for injection and a preparation method and application thereof. BACKGROUND
[0002] In 1977, Japanese scholars S. Shima and H. Sakai found that a strain of actinomycete No. 346 could produce a large amount of stable Dragendo Positive (abbreviated as DP) substance in the process of screening from microorganisms. Through analysis of acid hydrolysis products and structural analysis, it was confirmed that the DP substance was a homopolymer monomer containing 25-30 lysine residues, which was called ε-polylysine (ε-PL).
[0003] The production of ε-polylysine is generally as follows: - Pretreatment: after fermentation, the fermentation broth is centrifuged or filtered to remove bacterial cells and insoluble impurities, and a clear fermentation broth is obtained.
[0004] - Adsorption: polylysine is adsorbed on the resin by using ion exchange resin, and impurities flow out with the solution.
[0005] - Elution: polylysine adsorbed on the resin is eluted with dilute hydrochloric acid or sodium chloride solution, and the eluate is collected.
[0006] - Concentration: the eluate is concentrated under reduced pressure to increase the concentration of polylysine.
[0007] - Purification: polylysine is further purified by gel filtration, ultrafiltration and other methods to remove small molecular impurities in the fermentation broth.
[0008] - Drying: the purified polylysine solution is spray dried or freeze dried to obtain the finished product of polylysine.
[0009] Currently, the purification of food-grade ε-polylysine is mainly achieved by strong acid cation exchange resin purification process and ultrafiltration, such as sulfonic acid type cation exchange resin. Polylysine is positively charged under acidic conditions and will exchange with the anions on the resin to be adsorbed; 1000-3000 Da membrane package ultrafiltration is used to remove small molecular fermentation impurities. Food-grade ε-polylysine has a high content of bacterial endotoxins, large particles and other impurities, and cannot be directly used for injection. Therefore, it is an urgent problem to prepare ε-polylysine for injection. SUMMARY
[0010] In view of the deficiencies of the prior art, the present application aims to provide a pharmaceutical material for use in injection and a preparation method and application thereof. The technical scheme of the present application does not use the repeated purification method of the existing production process, but on the basis of comprehensively considering the food-grade production process, without introducing any other impurities, uses several pure physical technical means, designs the process sequence combination according to the product characteristics, and sets the process space through specific process parameters, so as to realize the quality requirements of food-grade ε-polylysine reaching the injection grade use with low cost, simple and ingenious process design without impurity introduction.
[0011] The technical scheme adopted by the application is as follows: The present application provides a pharmaceutical material for use in injection, wherein the pharmaceutical material for use in injection comprises ε-polylysine. Preferably, the content of the ε-polylysine in the pharmaceutical material for use in injection is 1 mg / mL-100 mg / mL. Preferably, the content of the ε-polylysine in the pharmaceutical material for use in injection is 1 mg / mL-100 mg / mL. Preferably, the pH value of the pharmaceutical material for use in injection is 6.0-8.0. Preferably, the pharmaceutical material for use in injection further comprises water or a pH buffer which is tolerable to human body. Preferably, the pH buffer tolerable to human body is a PBS buffer with a concentration of 0.01-0.1 mol / L.
[0012] In addition, to solve the above problems, the present application further provides a preparation method of the pharmaceutical material for use in injection as described above, comprising: S1, dissolving food-grade ε-polylysine powder with a content of 98.0% or more with water or a pH buffer to prepare a solution; Preferably, in the step S1, the pH buffer is a PBS buffer with a concentration of 0.01-0.1 mol / L and a pH of 6.0-10.0. S2, clarifying the solution prepared in the step S1 by micro-pore filtration; Preferably, in the step S2, the micro-pore diameter is 0.45 μm-0.1 μm.
[0013] S3, adjusting the pH of the solution prepared in the step S2 and then purifying by anion exchange method, and collecting the flow-through peak at 210 nm. Preferably, in the step S3, the pH of the solution prepared in the step S2 is adjusted to 9.0-10.0. S4, taking the solution prepared in step S3, performing ultrafiltration purification, collecting the permeate peak at 210nm; Preferably, in step S4, the ultrafiltration pore size is 10kDa; Preferably, in step S4, during the ultrafiltration process, water or pH buffer is used for washing and filtering; S5, taking the solution prepared in step S4, adjusting the pH to 6.0-8.0, then performing filtration sterilization to obtain the pharmaceutical material for injection.
[0014] Preferably, in step S5, the pharmaceutical material for injection prepared in step S4 or S5 is concentrated, and then spray drying or freeze drying method is used to prepare dry powder.
[0015] In addition, the application also provides a use of the pharmaceutical material for injection as described above in the preparation of medical products, including adjuvants, vaccine products, drugs, diagnostic reagents and detection kits.
[0016] The application has the advantages that: the pharmaceutical material for injection, the preparation method and the use thereof, the main component of the pharmaceutical material for injection includes epsilon-polylysine. The application provides a preparation process of the pharmaceutical material for injection, which has a good recovery rate; bacterial endotoxin and safety experimental researches are carried out, and it is proved that the self-prepared pharmaceutical material for injection has good safety, which fills the blank of preparing the pharmaceutical material for injection by using food-grade epsilon-polylysine. BRIEF DESCRIPTION OF DRAWINGS
[0017] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.
[0018] Figure 1 The figure is the recovery rate of epsilon-polylysine in embodiment 2 of the application; Figure 2 The figure is the bacterial endotoxin removal rate in embodiment 3 of the application.
[0019] The implementation of the application, functional characteristics and advantages will be further described with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION
[0020] The above objects, characteristics and advantages of the application can be more obvious and easy to understand, and the following will be combined with the accompanying drawings to further describe the application. Figure 1 ,Figure 2 and the detailed description further illustrate the present application.
[0021] The present application provides a pharmaceutical material for injection, wherein the pharmaceutical material for injection comprises epsilon-polylysine; The epsilon-polylysine is an epsilon-amino homomeric polymer containing 25-30 lysine residues. Further, the content of the epsilon-polylysine in the pharmaceutical material for injection is 1 mg / mL-100 mg / mL, for example, 10 mg / mL, 20 mg / mL, 40 mg / mL, 60 mg / mL, 80 mg / mL, or 100 mg / mL. Further, the pH value of the pharmaceutical material for injection is 6.0-8.0, for example, 6.0, 6.5, 7.0, 7.5, or 8.0. Further, the pharmaceutical material for injection further comprises a human-tolerable water or pH buffer, for example, water, PBS, CBS, or Tris-HCl buffer. Further, the human-tolerable pH buffer is a PBS buffer with a concentration of 0.01-0.1 mol / L, for example, 0.01, 0.02, 0.03, 0.06, 0.08, or 0.1 mol / L.
[0022] In addition, the present application further provides a preparation method of the pharmaceutical material for injection, comprising: S1, dissolving food-grade epsilon-polylysine powder with a content of 98.0% or above in water or a pH buffer to prepare a solution; Further, in the step S1, the pH buffer is a PBS buffer with a concentration of 0.01-0.1 mol / L and a pH of 6.0-10.0, for example, 0.01, 0.02, 0.03, 0.06, 0.08, or 0.1 mol / L; for example, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0. S2, clarifying the solution prepared in the step S1 by micro-pore filtration; Further, in the step S2, the micro-pore diameter for filtration is 0.45 μm-0.1 μm, for example, 0.45 μm, 0.22 μm, or 0.1 μm.
[0023] S3, adjusting the pH of the solution prepared in the step S2 and then purifying by anion exchange method, for example, Cap Q ion exchange gel or DEAE, and collecting the flow-through peak at 210 nm. Furthermore, in step S3, the solution prepared in step S2 is taken and the pH is adjusted to 9.0-10.0, for example, 9.0, 9.5, or 10.0; S4. The solution prepared in step S3 was purified by ultrafiltration, and the permeate peak was collected at 210 nm; Furthermore, in step S4, the ultrafiltration pore size is 10KDa; Furthermore, in step S4, during the ultrafiltration process, diafiltration is performed using water or pH buffer; Furthermore, in step S4, the pH buffer is a PBS buffer of 0.01-0.1 mol / L and a pH value of 6.0-8.0, for example, the concentration may be 0.01, 0.02, 0.03, 0.06, 0.08, 0.1 mol / L; the pH value may be 6.0, 6.5, 7.0, 7.5, 8.0; S5. Take the solution prepared in step S4, adjust the pH to 6.0-8.0, and filter and sterilize to obtain a medicinal material that can be used for injection.
[0024] Furthermore, in step S5, in order to facilitate storage and use of the medicinal material that can be used in the injection, the medicinal material that can be used in the injection prepared in step S4 or S5 can be concentrated and then prepared into a dry powder by spray drying or freeze drying.
[0025] In addition, the present invention also provides a use of the above-mentioned medicinal material for use in injections in the preparation of pharmaceutical products, including adjuvants, vaccine products, drugs, diagnostic reagents and detection kits.
[0026] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.
[0027] Example 1: Preparation of a medicinal material that can be used in an injection.
[0028] 1. Dissolve food-grade ε-polylysine powder with a content of more than 98.0% in 0.06 mol / L PBS buffer at a pH of 9.0 to prepare a solution; 2. Take the solution prepared in step S1 and filter it through a 0.45 μm pore size filter to clarify; 3. Take the solution prepared in step S2, adjust the pH to 9.0 with 1 mol / L sodium hydroxide solution, and purify it by anion exchange method using Cap Q ion exchange gel, and collect the flow-through peak at 210 nm; 4. Take the solution prepared in step S3, and purify by ultrafiltration using an ultrafiltration membrane with a pore size of 10 kDa. During the ultrafiltration process, repeatedly wash and filter using 0.06 mol / L PBS buffer with a pH of 6.0, and collect the permeate peak at 210 nm. 5. Take the solution prepared in step S4, filter and sterilize using a filter with a pore size of 0.22 μm, and prepare a pharmaceutical material that can be used for injection.
[0029] Example 2: Preparation of a pharmaceutical material that can be used for injection.
[0030] 1. Dissolve food-grade ε-polylysine powder with a content of 98.0% or more using 0.06 mol / L PBS buffer with a pH of 10.0 to prepare a solution. 2. Filter and clarify the solution prepared in step S1 using a filter with a pore size of 0.45 μm. 3. Take the solution prepared in step S2, adjust the pH to 10.0 using 1 mol / L sodium hydroxide solution, purify by anion exchange using Cap Q ion exchange resin, and collect the flow-through peak at 210 nm. 4. Take the solution prepared in step S3, purify by ultrafiltration using an ultrafiltration membrane with a pore size of 10 kDa. During the ultrafiltration process, repeatedly wash and filter using 0.06 mol / L PBS buffer with a pH of 8.0, and collect the permeate peak at 210 nm. 5. Take the solution prepared in step S4, filter and sterilize using a filter with a pore size of 0.22 μm, and prepare a pharmaceutical material that can be used for injection.
[0031] Example 3: Detection of ε-polylysine recovery rate Method for detecting the content of ε-polylysine 1. Preparation of reference solution Take 0.4 g of ε-polylysine reference material, accurately weigh, and transfer to a 100 ml volumetric flask. Add an appropriate amount of 0.06 mol / L PBS buffer (pH 8.0) to dissolve, dilute to the calibration line, and shake well to obtain a reference stock solution. Take 1.0 ml, 2.0 ml, 3.0 ml, 4.0 ml, and 5.0 ml of the solution, respectively, and transfer to 100 ml volumetric flasks. Dilute to the calibration line with PBS to obtain 0.04 mg / ml, 0.08 mg / ml, 0.12 mg / ml, 0.16 mg / ml, and 0.20 mg / ml reference linear solutions.
[0032] 2. Preparation of test solution Take the test solution, add 0.06 mol / L PBS Buffer (pH 8.0) dilution, quantitative preparation of ε-polylysine containing about 0.1 mg / ml solution, shake well for standby.
[0033] 3. Test sample inspection According to the "Chinese Pharmacopoeia" UV-visible spectrophotometry (general 0401) is carried out. With solvent as blank, at 210 nm, the absorbance of the reference linear solution and the test sample is measured. With the reference linear solution concentration as the abscissa, the absorbance as the ordinate, linear regression is carried out, and the regression straight line equation is obtained (the correlation coefficient r is required to be greater than or equal to 0.99, that is, R2 is greater than or equal to 0.98). According to the regression straight line equation and the absorbance of the test sample solution, the dilution multiple, the content of ε-polylysine in the test sample solution is calculated.
[0034] Table 1, 12 group sample ε-polylysine content detection results
[0035] The detection results are shown in Table 1 and Figure 1 It can be seen from Table 1 that the recovery rate of the 12 groups of samples of the food-grade ε-polylysine powder after being treated by the method of the present application is more than 86%, the loss of ε-polylysine is small, and the recovery rate is good.
[0036] Example 4: Comparison of bacterial endotoxin detection results Bacterial endotoxin detection is carried out according to the "Chinese Pharmacopoeia" bacterial endotoxin detection method (general 1143).
[0037] Table 2, 12 group sample bacterial endotoxin detection results
[0038] The detection results are shown in Table 2 and Figure 2 It can be seen from Table 2 that the bacterial endotoxin removal rate of the 12 groups of samples of the food-grade ε-polylysine powder after being treated by the method of the present application is more than 99.38%, the bacterial endotoxin detection result is less than 0.25 EU / mg, which meets the use requirements of injection-grade pharmaceutical materials.
[0039] Example 5: Abnormal toxicity detection of pharmaceutical materials that can be used in injection Abnormal toxicity detection is carried out according to the "Chinese Pharmacopoeia" abnormal toxicity detection method (general 1141), and the mouse tail vein is injected with 0.05 mg per mouse.
[0040] Table 3, 3 group sample safety detection results
[0041] As can be seen from the above table, the abnormal toxicity detection results of the 3 batches of medicinal materials for use in injection meet the requirements of the Chinese Pharmacopoeia.
[0042] Example 6: Application of a medicinal material for use in injection to adjuvants and vaccine products 1. Preparation of EP adjuvant sample: The prepared ε-polylysine solution was used to prepare the EP adjuvant sample. First, 7.0 ml of ε-polylysine solution at 4.83 mg / ml was added to 50 ml of Poly IC solution at 2 mg / ml, and then 0.4 ml of 0.1 mol / L calcium chloride solution was added. The solution was supplemented with 0.01 mol / L PBS solution (pH = 7.6) to 100 ml, and the pH was adjusted to 7.6 with a pH adjusting solution. Finally, the solution was filtered through a filter membrane with a pore size of 0.45 μm to remove bacteria and sterilized to obtain a sterile EP adjuvant sample.
[0043] 2. Preparation of EP adjuvant rabies vaccine sample: The EP adjuvant and inactivated rabies virus antigen were mixed at a ratio of 7:3 to prepare an EP adjuvant rabies vaccine sample.
[0044] 3. Detection of immune effect of adjuvant rabies vaccine sample: The titer detection was performed according to the rabies vaccine titer determination method (NIH method). The Poly IC solution + vaccine was used as the control group, and the inactivated rabies virus antigen at 0.3 ml / dose was used as the simple vaccine control group.
[0045] Table 4: Results of immune effect detection of samples
[0046] As can be seen from the above table, the titer of the adjuvant rabies vaccine group is much higher than that of the simple vaccine control group, and is also significantly higher than that of the Poly IC solution + vaccine control group, which proves that the prepared ε-polylysine plays a good stabilizer role in resisting the degradation of Poly IC by nucleases.
[0047] 4. Abnormal toxicity detection of EP adjuvant and adjuvant rabies vaccine sample: The detection was performed according to the method of the Chinese Pharmacopoeia.
[0048] Table 5: Results of abnormal toxicity detection of samples
[0049] As can be seen from the above table, the abnormal toxicity detection results of the EP adjuvant and the adjuvant rabies vaccine sample meet the requirements of the Chinese Pharmacopoeia.
[0050] The above merely illustrates the specific embodiments of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of the changes or replacements within the technical range disclosed by the present application, which should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.
Claims
1. A medicinal material that can be used in an injection, characterized in that: The pharmaceutical materials that can be used in injections include ε-polylysine; Wherein, the ε-polylysine is an ε-amino homomonomer polymer containing 25-30 lysine residues; The content of the ε-polylysine in the pharmaceutical material that can be used in the injection is 1 mg / mL-100 mg / mL; The pH value of the medicinal material that can be used in injection is 6.0-8.0; The medicinal materials that can be used in injections also include: water or pH buffer solution that can be tolerated by the human body; The pH buffer solution that can be tolerated by the human body is 0.01-0.1 mol / L PBS buffer solution.
2. A method for preparing the medicinal material for injection according to claim 1, characterized in that: include: S1. Dissolve food-grade ε-polylysine powder with a content of more than 98.0% in water or a pH buffer to prepare a solution, wherein the pH buffer is a PBS buffer with a pH of 6.0-10.0 and a concentration of 0.01-0.1 mol / L. S2, taking the solution prepared in step S1 and performing microfiltration to clarify, wherein the filtration micropore size is 0.45 μm-0.1 μm; S3. Take the solution prepared in step S2, adjust the pH to 9.0-10.0, purify it by anion exchange method, and collect the flow-through peak at 210 nm; S4. The solution prepared in step S3 was purified by ultrafiltration, and the permeate peak was collected at 210 nm. The ultrafiltration pore size was 10 kDa. During ultrafiltration, water or pH buffer is used for filtration; S5. Take the solution prepared in step S4, adjust the pH to 6.0-8.0, and filter and sterilize to obtain a medicinal material that can be used in an injection; The medicinal material for injection prepared in step S4 or S5 is concentrated and then prepared into a dry powder by spray drying or freeze drying.
3. A use of the medicinal material for injection according to any one of claims 1 to 2 in the preparation of a pharmaceutical product, characterized in that: The products include adjuvants, vaccine products, pharmaceuticals, diagnostic reagents and test kits.