Method for improving product quality by optimizing posterior pituitary injection purification process

Abstract

The invention provides a method for improving product quality by optimizing a posterior pituitary injection purification process, and belongs to the technical field of medicines. According to the method, glacial acetic acid is adopted as a dissolving medium, and the influence of the concentration of glacial acetic acid, the extraction temperature and the filtering condition on the posterior pituitary injection is investigated, so that visible foreign matters and macromolecular substances are effectively removed, and the potency, the specific activity and the potency ratio of the product are improved. Besides, the method is simple to operate, ensures the stability and curative effect of the product, is suitable for industrial production of the posterior pituitary injection, and has important significance for improving the drug quality and meeting clinical requirements.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a method for optimizing the purification process of posterior pituitary injection to improve product quality. Background Art

[0002] Posterior pituitary injection is an injection preparation of a mixed polypeptide hormone extracted from the posterior pituitary of the brain, mainly containing two main active ingredients, vasopressin and oxytocin, and has important physiological functions.

[0003] When extracting the active ingredients in the posterior pituitary, filtration is difficult, and some related substances with larger molecular weights will be extracted simultaneously. Such high-molecular substances exceed the standard and are colored, have no therapeutic effect in the drug, may affect the stability and efficacy of the drug, and even be harmful to human health. Therefore, it is particularly crucial to remove such high-molecular impurities.

[0004] In the prior art, during the separation and purification of biological macromolecule active substances, in order to remove related substances with larger molecular weights, dialysis, gel chromatography or ultrafiltration technology is mainly used at present. However, in the crude extract of posterior pituitary hormone, a part of the related substances will aggregate with the posterior pituitary hormone, and another part of the related substances will remain separated from the posterior pituitary hormone. At the same time, the posterior pituitary hormone itself will also form polymers. If dialysis technology is used to purify the posterior pituitary hormone, the stock solution will be diluted, the recovery rate is low, and the production cycle is long. Using gel chromatography technology has a small loading capacity, high production cost, high operation technical requirements, and cannot completely separate the posterior pituitary hormone combined with the related substances, affecting the recovery rate. When using ultrafiltration technology, most of the posterior pituitary hormone cannot pass through the ultrafiltration membrane, but exists in the retentate together with the related substances, reducing the recovery rate of posterior pituitary hormone. Summary of the Invention

[0005] In order to solve the above problems of the prior art, the purpose of the present invention is to provide a method for optimizing the purification process of posterior pituitary injection to improve product quality.

[0006] The present invention provides a method for preparing posterior pituitary injection, and the method comprises the following steps: under the protection of inert gas, add water for injection into a container, mix posterior pituitary with an ice acetic acid solution with a concentration of 0.25% - 2%, heat and stir at 60 - 70 °C, add an antibacterial agent to form a suspension, centrifuge, separate the supernatant, add diatomaceous earth for filtration, freeze, thaw, and filter to remove impurities, thus obtaining the posterior pituitary injection.

[0007] Further, the mass ratio of the posterior pituitary to the antibacterial agent is 100:0.1 - 1; The mass ratio of the diatomaceous earth to the posterior pituitary is 0.1 - 0.3:100; The ratio of the posterior pituitary to the ice acetic acid solution is 4 - 8 potency units / ml.

[0008] Furthermore, the mass ratio of the posterior pituitary lobe to the bacteriostatic agent is 100:0.5; The mass ratio of the diatomaceous earth to the posterior pituitary lobe is 0.1:100; The ratio of the posterior pituitary lobe to the glacial acetic acid solution is 6 potency units / ml.

[0009] Furthermore, the concentration of the glacial acetic acid is 2%.

[0010] Furthermore, the heating temperature is 60°C.

[0011] Furthermore, the heating time is 0.5 - 2 hours.

[0012] Furthermore, the heating time is 1 hour.

[0013] Furthermore, the bacteriostatic agent is chlorobutanol.

[0014] Furthermore, the freezing conditions are: freezing at -50°C to -30°C for 0.5 - 4 hours.

[0015] Furthermore, the freezing conditions are: freezing at -40°C for 2 hours.

[0016] Furthermore, the conditions for filtration and impurity removal are: filtering under a filter membrane with a pore size of 0.1 μm to 0.45 μm.

[0017] Furthermore, the conditions for filtration and impurity removal are: filtering under a filter membrane with a pore size of 0.2 μm.

[0018] The present invention has achieved the following beneficial effects: The present invention provides a method for optimizing the purification process of posterior pituitary injection to improve product quality. This method uses glacial acetic acid as a dissolution medium, and investigates the effects of glacial acetic acid concentration, extraction temperature, and filtration conditions on posterior pituitary injection, effectively removing visible foreign matters and high-molecular substances, and improving the vasopressin titer, specific activity, and titer ratio of the product. In addition, this method is simple to operate, ensures the stability and efficacy of the product, is suitable for the industrial production of posterior pituitary injection, and is of great significance for improving drug quality and meeting clinical needs.

[0019] Obviously, based on the above content of the present invention, according to the common general knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modification, substitution, or variation can be made.

[0020] The following is a further detailed description of the above content of the present invention in the form of specific embodiments. However, it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments. Any technology implemented based on the above content of the present invention belongs to the scope of the present invention. Specific Embodiments

[0021] The raw materials and equipment used in the present invention are all known products, obtained by purchasing commercially available products.

[0022] In the embodiments of the present invention, the "potency unit" refers to a measure of the biological activity intensity of the active ingredients (such as vasopressin and oxytocin) in the posterior pituitary. According to the Chinese Pharmacopoeia, for posterior pituitary injection, calculated based on vasopressin, the potency should be 87% - 115% of the labeled amount, and the vasopressin potency per 1 mg of protein should not be less than 4.0 units; the ratio of the potency of oxytocin to vasopressin should be 0.9 - 1.7. Those skilled in the art can determine the potency unit of the posterior pituitary according to the biological assay methods in the prior art (such as the general rules for vasopressin biological assay 1205 and the general rules for oxytocin biological assay 1210).

[0023] Example 1. Preparation Process of the Posterior Pituitary Injection of the Present Invention Prepare the posterior pituitary injection in the prescription amount (100 ml): In the dilution tank, introduce nitrogen. Place the posterior pituitary powder in the dilution tank at the weighed amount (6 potency units / ml of glacial acetic acid solution), add the weighed glacial acetic acid solution (concentration 2%, 50 ml), continuously heat and stir at 60 °C for 1 h, add the weighed chlorobutanol (the added amount of chlorobutanol is 0.5% of the mass of the posterior pituitary powder), then centrifuge the suspension, take the supernatant, add the weighed diatomaceous earth (the added amount of diatomaceous earth is 0.1% of the mass of the posterior pituitary powder), filter, take the filtrate, freeze it at -40 °C for 2 h. After taking it out, quickly thaw (thaw by natural temperature rise), filter and remove impurities (0.2 μm) from the thawed solution, and make up the volume to 100 ml with injection water to obtain the posterior pituitary injection.

[0024] The beneficial effects of the present invention are demonstrated by the following experimental examples.

[0025] Experimental Example 1. Screening of the Purification Process of the Posterior Pituitary Injection 1. Test Indexes The specific test methods and standards are as shown in the Pharmacopoeia (Part 1 of the Second Section of the Text of the 2020 Edition, "Posterior Pituitary Injection"): High molecular weight substances: Determined by size exclusion chromatography (General Rule 0514). Limit: Calculated by area normalization method, high molecular weight substances with a molecular weight greater than 10,000 shall not exceed 10.0%; Visible foreign matters: Unless otherwise specified, it shall be examined according to the method for the examination of visible foreign matters (General Principles 0904) and shall comply with the provisions; Calculated as vasopressin, the potency shall be 87% to 115% of the labeled amount, and the vasopressin potency per 1 mg of protein shall not be less than 4.0 units; the ratio of oxytocin to vasopressin potency shall be 0.9 to 1.7.

[0026] 2. Screening of purification process (1)Screening the concentration of glacial acetic acid solution In the present invention, glacial acetic acid solution is used as a medium to dissolve and extract posterior pituitary. Different concentrations of glacial acetic acid solution (2%, 1%, 0.5%, 0.25%) were investigated. The specific operation is as follows: referring to the process of Example 1, the difference is only that different concentrations of glacial acetic acid solution are controlled according to Table 1 to prepare posterior pituitary injection under different conditions.

[0027] Table 1 Influence of different concentrations of glacial acetic acid solution on posterior pituitary injection The data in Table 1 show that the higher the concentration of the added glacial acetic acid solution, the better the vasopressin potency, specific activity and potency ratio of the posterior pituitary injection. Therefore, glacial acetic acid solution with a concentration of 2% was selected as the medium to study the purification process of posterior pituitary injection.

[0028] (2)Screening temperature In the present invention, glacial acetic acid solution with a concentration of 2% was selected as the medium to continue to investigate the effects of different extraction temperatures (60 °C, 70 °C) on the potency, specific activity, potency ratio and high molecular substances of the medicinal liquid. The specific operation is as follows: referring to the process of Example 1, the difference is only that different temperatures are controlled according to Table 2 to prepare posterior pituitary injection under different conditions.

[0029] Table 2 Influence of different temperatures on posterior pituitary injection The data in Table 2 show that compared with the extraction temperature of 70 °C, when preparing posterior pituitary injection at the extraction temperature of 60 °C, a posterior pituitary injection with better vasopressin potency, specific activity and potency ratio and lower content of high molecular substances can be obtained. Therefore, the extraction temperature of 60 °C was selected to study the purification process of posterior pituitary injection.

[0030] (3)Screening filtration conditions The present invention further examines the effects of adding diatomaceous earth on the visible foreign matters, macromolecular substances, vasopressin titer, specific activity, and titer ratio of posterior pituitary injection. The specific operation is as follows: referring to the process of Example 1, the difference is only that different filtration conditions are controlled according to Table 3 to prepare posterior pituitary injections under different conditions. Among them, the posterior pituitary injection prepared without adding diatomaceous earth is consistent with the posterior pituitary injection described in Example 1 of the patent application document CN119792350A.

[0031] Table 3 Effects of Different Filtration Conditions on Posterior Pituitary Injection The data in Table 3 show that the posterior pituitary injection without adding diatomaceous earth contains 9.2% - 9.8% of macromolecular substances, the product is turbid and has white dots; while no macromolecular substances are detected in the posterior pituitary injection after adding diatomaceous earth, the product is clear and has no white dots. Moreover, the posterior pituitary injection after adding 0.1% of diatomaceous earth has better effects on vasopressin titer, specific activity, and titer ratio than the posterior pituitary injection after adding 0.3% of diatomaceous earth. Therefore, the present invention selects to add 0.1% of diatomaceous earth in the filtration process.

[0032] In summary, the present invention obtains an optimized purification process for posterior pituitary injection, that is: using a 2% glacial acetic acid solution as the dissolution medium, selecting 60°C as the extraction temperature, and selecting to add 0.1% of diatomaceous earth for the filtration condition.

[0033] In summary, the present invention provides a method for optimizing the purification process of posterior pituitary injection to improve product quality. This method uses glacial acetic acid as the dissolution medium, and examines the effects of glacial acetic acid concentration, extraction temperature, and filtration conditions on posterior pituitary injection, effectively removing visible foreign matters and macromolecular substances, and improving the vasopressin titer, specific activity, and titer ratio of the product. In addition, this method is simple to operate, ensures the stability and efficacy of the product, is applicable to the industrial production of posterior pituitary injection, and is of great significance for improving drug quality and meeting clinical needs.

Claims

1. A method for preparing posterior pituitary injection, characterized in that, The method comprises the following steps: Under the protection of inert gas, add water for injection into a container, mix posterior pituitary with an acetic acid solution having a concentration of 0.25% - 2%, heat and stir at 60 - 70°C, add a bacteriostatic agent to form a suspension, centrifuge, separate the supernatant, filter by adding diatomaceous earth, freeze, thaw, and filter to remove impurities, thus obtaining the posterior pituitary injection.

2. The method according to claim 1, characterized in that The mass ratio of the posterior pituitary to the bacteriostatic agent is 100:0.1 - 1; The mass ratio of the diatomaceous earth to the posterior pituitary is 0.1 - 0.3:100; The ratio of the posterior pituitary to the acetic acid solution is 4 - 8 potency units / ml.

3. The method according to claim 2, wherein The mass ratio of the posterior pituitary to the bacteriostatic agent is 100:0.5; The mass ratio of the diatomaceous earth to the posterior pituitary is 0.1:100; The ratio of the posterior pituitary to the acetic acid solution is 6 potency units / ml.

4. The method according to claim 1, wherein The concentration of the acetic acid is 2%; 5. The method according to claim 1, wherein The temperature of the heating is 60°C; 6. The method according to claim 1, wherein The time of the heating is 0.5 - 2 hours; 7. The method according to claim 6, characterized in that, The time of the heating is 1 hour; 8. The method according to claim 1, characterized in that, The bacteriostatic agent is chlorobutanol; 9. The method according to claim 1, characterized in that, The conditions for freezing are: freeze at -50°C to -30°C for 0.5 - 4 hours; 10. The method according to claim 1, characterized in that The conditions for filtering to remove impurities are: filter under a filter membrane of 0.1 μm - 0.45μm.

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