Spleen amino acid peptide oral lyophilized powder

Abstract

The application belongs to the technical field of biochemical pharmacy, and particularly relates to a spleen amino peptide oral freeze-dried powder, which is prepared from a spleen amino peptide solution through a freeze-drying step, wherein the spleen amino peptide solution comprises dry materials and water in a weight ratio of 1:80-100, the dry materials comprise 550 parts of animal spleen protein peptide powder, 200 parts of mannitol, 9 parts of sucralose and 150-300 parts of medicinal excipients. Through optimization of a formula and a freeze-drying process, the spleen amino peptide oral freeze-dried powder with good stability can be prepared, and the activity of the spleen amino peptide oral freeze-dried powder can be maintained for a long time at low temperature.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biochemical pharmacy, and particularly relates to a spleen amino peptide oral lyophilized powder. BACKGROUND

[0002] Spleen amino peptide is a general term of nucleotide and polypeptide complex from fresh animal spleen, which has the effects of activating lymphocytes through regulating immune system signal transmission and enhancing cell effect, and improves the resistance to bacterial, mold and viral intracellular infection. The lyophilization process can prolong the shelf life of the product by removing water between protein molecules, limiting the fluidity of molecules and slowing down the physical / chemical reaction of drug components. Therefore, the spleen amino peptide is usually prepared into a lyophilized powder dosage form. However, the formula is not always stable under the lyophilized powder dosage form, and the physical / chemical reaction is still prone to occur in the long-term storage process because the peptide or protein molecules are exposed to many stresses during the drying process. SUMMARY

[0003] In view of the above technical problems, the present application provides a spleen amino peptide oral lyophilized powder prepared from a spleen amino peptide solution through a lyophilization step, wherein the spleen amino peptide solution comprises dry materials and water in a weight ratio of 1:80-100, the dry materials comprise 550 parts by weight of animal spleen protein peptide powder, 200 parts by weight of mannitol, 9 parts by weight of sucralose and 150-300 parts by weight of pharmaceutical excipients.

[0004] By optimizing the ratio of protein / peptide to sugar and the lyophilization step, a spleen amino peptide oral lyophilized powder with good stability is prepared. BRIEF DESCRIPTION OF DRAWINGS

[0005] Figure 1 Process flow chart of Example 1. DETAILED DESCRIPTION

[0006] The following examples further illustrate the present application but should not be construed as limiting the present application. Modifications or substitutions of the method, steps or conditions of the present application without departing from the spirit and essence of the present application shall all fall within the scope of the present application.

[0007] Example 1

[0008] Firstly, the pig spleen protein peptide in the raw material is subjected to quality inspection: 5 g of pig spleen protein peptide test sample is taken and placed in a petri dish, and its color, morphology, impurities, smell and taste are observed with naked eyes under natural light. The test sample should be white to light yellow, the morphology is powder particle powder, there should be no caking phenomenon, it has the inherent taste and aroma of the product, and the impurities should be no visible foreign impurities under normal vision. The water content is less than 8%, the peptide content is not less than 0.1%, and the average molecular weight is 3000 daltons.

[0009] When the raw material quality inspection is qualified, the spleen amino acid peptide oral freeze-dried powder of Example 1 is prepared according to the following steps.

[0010] Liquid preparation step: add 80% of the total amount of purified water to the liquid preparation tank and heat. Add the prescribed amount of porcine spleen protein peptide powder to the purified water at 35℃ and stir until completely dissolved to prepare a porcine spleen protein peptide solution. Add the prescribed amount of sucralose, mannitol, essence, and sodium citrate in Table 1 to the porcine spleen protein peptide solution, stir until all dry materials are dissolved, then add purified water to the total amount and stir until uniform.

[0011] Table 1. Spleen amino acid peptide solution prescription 2 mg / bottle (polypeptide) for 5000 bottles

[0012] Raw material name Dosage Pig spleen protein peptide 550g Sucralose 9g Essence (blueberry) 180g Mannitol 200g Sodium citrate 80g Purified water Added to 90 kg

[0013] Filtering step: filter the spleen amino acid peptide solution using a 200-mesh filter bag. The filtered spleen amino acid peptide solution is completed within 8 hours.

[0014] Canning step: the filtered spleen amino acid peptide solution is then filled into bottles, each containing 18 ml of spleen amino acid peptide solution. The specific process is as follows: use purified water to clean the filter bottles, filling devices, silicone hoses, filling needles, etc. in the instrument cleaning room, and reserve them after cleaning. Turn on the equipment and adjust the speed of the bottle conveying net belt tray and the main machine. The oral solution bottles enter the walking beam in an orderly manner. After the oral solution bottles enter below the filling needle, turn on the filling pump switch to start filling. The oral solution bottles are directly used for filling under the protection of laminar flow. Adjust the needle filling amount to the qualified range, turn on the filling pressure plug machine, and perform half-pressure plug filling at a speed of ≤300 bottles / min. The intermediate product should meet the quality standards for intermediate product filling liquid. The filled bottles are placed in a stainless steel tray and then transferred to a freeze-drying box for pre-cooling every 15 minutes.

[0015] Freeze-drying step: including pre-freezing step, vacuum drying step, and nitrogen filling step.

[0016] The pre-freezing step includes: pre-cool the partition in the freeze-drying machine to 5℃, then load the spleen amino acid peptide solution canning bottles; each cycle is composed of cooling, holding, heating, and holding, and is repeated for 3 cycles. Cool the partition and spleen amino acid peptide solution canning bottles to -40℃ in 1 hour, then hold for 1 hour. During the heating period of each cycle, heat the partition and spleen amino acid peptide solution canning bottles to -3℃ for 1 hour, then hold for 1 hour.

[0017] Vacuum drying step: first perform sublimation drying on the pre-frozen product, then perform desorption drying.

[0018] Sublimation drying: the temperature of the refrigeration condenser is reduced to -55°C, the vacuum pump is started, and pre-evacuation is performed. When the vacuum degree in the freeze-drying box reaches 0.2 mbar, the temperature of the plate layer is increased. The temperature of the plate layer is increased to -20°C in 5 h, and the temperature is maintained for 10 h. The temperature of the plate layer is increased to -10°C in 2 h, and the temperature is maintained for 3 h.

[0019] Desorption drying: the limit vacuum is set, and the temperature of the plate layer is continuously increased. The temperature of the plate layer is increased to 0°C in 1 h, and the temperature is maintained for 3 h. The temperature of the plate layer is increased to 20°C in 1 h, and the temperature is maintained for 6 h. The drying stage is ended. The machine is stopped, nitrogen is filled into the freeze-drying box, and when the pressure in the freeze-drying box reaches 100 kPa, the vacuum is extracted to 10 Pa. Nitrogen is filled into the freeze-drying box again, and when the pressure in the freeze-drying box reaches 100 kPa, the nitrogen is pressurized, and the box is taken out and capped.

[0020] Nitrogen filling step: after the vacuum pump is stopped, nitrogen is filled into the freeze-drying box. When the pressure in the freeze-drying box reaches 100 kPa, the vacuum is extracted to 10 Pa. Nitrogen is filled into the freeze-drying box again, and when the pressure in the freeze-drying box reaches 100 kPa, the nitrogen is pressurized, and the box is taken out and capped.

[0021] Visual inspection step: the product transferred to the packaging room after sterilization is visually inspected, and the product with unqualified quality is picked out.

[0022] The above preparation steps mainly use the instruments and equipment in the following table:

[0023] Table 2. Main equipment model

[0024] Equipment Model Vertical ultrasonic cleaner YQCL20 / 4 Air flow type sterilization dryer KSZ620 / 43-EA Liquid preparation tank, scale PYG-800 Oral liquid bottle filling and rolling machine YGZ16 / 25 Sterilization cabinet YXQ.EAK-225-II Vacuum freeze dryer LYO-15 Horizontal labeling machine SML-880

[0025] The process flow is as follows: Figure 1 The process flow is as follows: Table 3. Quality inspection results

[0026]

[0027] Example 2

[0028]

[0029] The difference between Example 1 is that the porcine spleen protein peptide in the prescription is 600 g, and the sucralose is 50 g. The finished product is detected according to the national drug standard WS1-(X-022)-2002Z for the sample of Example 1. The results meet the standard.

[0030] Example 3

[0031] The difference between Example 1 is that the temperature is lowered-kept warm-raised-kept warm for 4 cycles. During the raising period of each cycle, the partition is raised to -5℃ for 1 h and then kept warm for 1 h. The finished product is detected according to the national drug standard WS1-(X-022)-2002Z for the sample of Example 1. The results meet the standard.

[0032] Control Example 1

[0033] The difference between Example 1 is that during the raising period of each cycle, the partition is raised to -1℃ for 1 h and then kept warm for 1 h. The appearance of the finished product is consistent with that of Example 1.

[0034] Control Example 2

[0035] The difference between Example 1 is that the sucralose is replaced by 10 g sorbitol. After the pre-freezing step, phase separation is observed. No spleen amino acid peptide freeze-dried powder is prepared.

[0036] Control Example 3

[0037] The difference between Example 1 is that the pre-freezing step: during the cooling period of each cycle, the nucleation temperature is -30℃, and the cooling rate is 2 h. After the pre-freezing step, the sample shows phase separation. No spleen amino acid peptide freeze-dried powder is prepared.

[0038] Comparing Control Examples 1-3 with Examples 1-3, it is proved that phase separation mainly occurs in the pre-freezing step of freeze-drying, which is dependent on the freeze-drying process and the formulation components to some extent.

[0039] Test Example 1 Accelerated Test

[0040] Take Examples 1-3, Control Example 1 as 4 groups of test samples, 3 batches in each group, 1 bottle in each batch, a total of 12 samples. According to the "Guidelines for Stability Testing of Raw Materials and Preparations" (Chinese Pharmacopoeia 2015 Edition Volume IV General Rules 9001), the samples are subjected to accelerated testing. The samples are stored at -20℃±2℃, and the observation time is 24 months. The appearance and activity units of the first batch, the second batch, and the third batch of freeze-dried powder samples in each group are observed at the 6th, 12th, and 24th months, respectively. The results are as follows:

[0041] Table 4. Accelerated test results

[0042]

[0043] The test examples demonstrate that in addition to the lyophilization process having an impact on the stability of the sample, it was not anticipated by those skilled in the art that the spleen peptide solution diluted from the sample was also affected by the ratio of peptide content to sucralose content. The sucralose added can work in conjunction with the stabilizer (mannitol) to protect the protein from stress. It is possible that the sucralose assists in dispersing the protein molecules in the stabilizer such that both the protein and the stabilizer are in the same single amorphous phase and are less likely to phase separate.

[0044] While the present application has been described in some detail with specific reference to certain embodiments thereof, it will be understood that various modifications and changes can be made to the embodiments without departing from the broader spirit and scope of the application. Accordingly, the specification and drawings are to be regarded as illustrative in nature and that they are to be considered an exemplification of the application and are not to be regarded as being restrictive.

Claims

1. A spleen amino acid peptide oral lyophilized powder, characterized in that, Prepared by a freeze-drying step from a spleen amino acid peptide solution comprising dry material and water in a weight ratio of 1:80-100, the dry material comprising 550 parts by weight of porcine spleen protein peptide powder, 200 parts by weight of mannitol, 9 parts by weight of sucralose, 80 parts by weight of sodium citrate and 180 parts by weight of essence; The preparation method of the spleen amino acid peptide oral freeze-dried powder comprises the following steps: First, a spleen amino acid peptide solution is prepared by a liquid preparation step and a filtration step; Then, the filtered spleen amino acid peptide solution is subjected to a filling step to obtain a spleen amino acid peptide solution filling bottle; The spleen amino acid peptide solution filling bottle is subjected to a freeze-drying step, which comprises a pre-freezing step, a vacuum drying step and a nitrogen filling step; the pre-freezing step comprises several cycles of first temperature reduction and then temperature increase, and the vacuum drying step comprises a sublimation drying step and a desorption drying step; The water content of the porcine spleen protein peptide powder is less than 8%, the peptide content is not less than 0.1%, and the average molecular weight is 3000 Dalton; In the pre-freezing step, the partition plate in the freeze-drying machine is pre-cooled to 5℃, and then the spleen amino acid peptide solution filling bottle is loaded; During the temperature reduction period of each cycle, the partition plate and the spleen amino acid peptide solution filling bottle are cooled to -40℃ for 1 hour and then kept for 1 hour; During the temperature increase period of each cycle, the partition plate and the spleen amino acid peptide solution filling bottle are heated to -3℃ for 1 hour and then kept for 1 hour; The cycle is repeated for 3-5 times; In the sublimation drying step, the condenser temperature is reduced to -55℃, the vacuum pump is started, and when the vacuum degree in the freeze-drying box reaches 0.2 mbar, the plate layer is heated, the temperature of the plate layer is increased to -20℃ for 5 hours and then kept for 10 hours, and the temperature of the plate layer is increased to -10℃ for 2 hours and then kept for 3 hours; 2. The spleen peptide oral lyophilized powder according to claim 1, wherein, In the desorption drying step, the freeze-drying box is set to the limit vacuum degree, and the plate layer is continuously heated, the temperature of the plate layer is increased to 0℃ for 1 hour and then kept for 3 hours, and the temperature of the plate layer is increased to 20℃ for 1 hour and then kept for 6 hours.

3. The spleen peptide oral lyophilized powder according to claim 2, wherein, The nitrogen filling step comprises the following steps: after the vacuum pump is stopped, nitrogen is filled into the freeze-drying box, when the pressure in the freeze-drying box reaches 100 kPa, the vacuum pump is started to vacuum to 10 Pa, then nitrogen is filled into the freeze-drying box, when the pressure in the freeze-drying box reaches 100 kPa, the plug is closed, the nitrogen is kept at a constant pressure, and the box is taken out for capping. The liquid preparation step comprises the following steps: a prescribed amount of porcine spleen protein peptide powder is added to purified water with a temperature of 30-40℃ and stirred until all the dry material is dissolved to prepare a porcine spleen protein peptide solution; 4. The spleen peptide oral lyophilized powder according to claim 3, wherein the spleen peptide is spleen peptide of claim 1. A prescribed amount of sucralose, mannitol, essence and sodium citrate is added to the porcine spleen protein peptide solution, all the dry material is dissolved by stirring, purified water is added to the total amount and stirred uniformly. In the filtration step, a 200-mesh filter bag is used to filter the spleen amino acid peptide solution.

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