Sterility testing method for solid medicine preparation

Abstract

The invention relates to a sterility testing method of a solid medicine preparation. The sterility test method of the solid medicinal preparation comprises the following steps: crushing the solid medicinal preparation, and mixing the crushed solid medicinal preparation with a dissolving reagent to prepare a to-be-tested sample solution; filtering the to-be-detected sample solution by adopting a thin film, and collecting a to-be-detected filter membrane; placing the to-be-detected filter membrane in a culture medium for culturing to obtain the growth condition of microorganisms; wherein the dissolving reagent comprises propylene carbonate with the volume percent of 10%-20% and bovine albumin with the mass percent of 1%-3%. The sterility test method can more accurately separate and detect microorganisms.

Description

Technical Field

[0001] The present application relates to the detection of pharmaceutical preparations, and in particular to a method for sterility testing of solid pharmaceutical preparations. Background Art

[0002] Sterility testing of solid pharmaceutical preparations is an important step in ensuring drug quality and safety. Its purpose is to detect whether there is microbial contamination in the drug, including bacteria, fungi, etc., to ensure that drugs used in humans do not cause adverse consequences such as infection.

[0003] Traditional sterility testing methods for solid pharmaceutical preparations include direct inoculation and membrane filtration. Direct inoculation involves inoculating a sample solution directly into a culture medium. However, the presence of antibacterial components in the sample can interfere with microbial culture results, leading to inaccurate test results. Membrane filtration involves filtering the sample solution to trap the microorganisms within a thin membrane before inoculation. However, in practical applications, this method also presents difficulties in completely separating the antibacterial components from the microorganisms. Furthermore, poorly soluble solid preparations often contain polymeric materials such as polylactic acid, poly(lactic-co-glycolic acid) (PLGA), and polycaprolactone. These polymers can encapsulate microorganisms, requiring a long period of time for the polymer to degrade before release. Both direct inoculation and membrane filtration methods struggle to effectively degrade the polymer and release the microorganisms quickly, potentially leading to missed detections. Furthermore, ensuring that microorganisms are not damaged during sample preparation is a prerequisite for accurate sterility testing. Summary of the Invention

[0004] Based on this, the present application provides a method for sterility inspection of solid pharmaceutical preparations that can effectively remove antibacterial components and polymer materials in solid pharmaceutical preparations, while causing little damage to microorganisms and enabling more accurate separation and detection of microorganisms.

[0005] The specific technical solutions are as follows:

[0006] A method for sterility testing of a solid pharmaceutical preparation, wherein the solid pharmaceutical preparation comprises at least one of a polymer material and an antibacterial component, wherein the polymer material comprises one or more of polylactic acid, polylactic-co-glycolic acid, and polycaprolactone, and the antibacterial component comprises one or more of dichloromethane, chloroform, and ethyl acetate;

[0007] The sterility inspection method comprises the following steps:

[0008] The solid pharmaceutical preparation is crushed and mixed with a dissolving reagent to prepare a sample solution to be tested;

[0009] Filtering the sample solution to be tested through a membrane to collect the filter membrane to be tested;

[0010] Placing the filter membrane to be tested in a culture medium for cultivation to obtain the growth status of the microorganisms;

[0011] The dissolving agent comprises 10% to 18% by volume of propylene carbonate and 1% to 3% by mass of bovine albumin.

[0012] In one embodiment, the dissolution reagent includes 13% to 17% by volume of propylene carbonate and 1.5% to 2.5% by mass of bovine albumin.

[0013] In one embodiment, the solvent of the dissolving reagent is water or a sodium chloride peptone buffer with a pH of 6.8 to 7.2.

[0014] In one embodiment, the mass volume ratio of the solid pharmaceutical preparation to the dissolving reagent is 1 g: (200-500) mL.

[0015] In one embodiment, the mass volume ratio of the solid pharmaceutical preparation to the dissolving reagent is 1 g: (200-400) mL.

[0016] In one embodiment, the particle size of the solid pharmaceutical preparation after pulverization is ≤850 μm.

[0017] In one embodiment, the film is a hydrophilic filter membrane with a pore size of 0.4 μm to 0.5 μm.

[0018] In one embodiment, the microorganisms include one or more of Staphylococcus aureus, Escherichia coli, Clostridium sporogenes, Bacillus subtilis, Candida albicans and Aspergillus niger.

[0019] In one embodiment, the culture medium comprises one or both of glycolate fluid medium and trypticase soy broth.

[0020] In one embodiment, in the solid pharmaceutical preparation, the mass percentage of the polymer material is 40% to 95%; and / or,

[0021] In the solid pharmaceutical preparation, the mass percentage of the antibacterial component is 0% to 0.5%.

[0022] The above-mentioned sterility inspection method for solid pharmaceutical preparations can fully separate microorganisms from preparation excipients, especially polymer materials and antibacterial components, by adopting a suitable dissolving reagent, and then combine it with membrane filtration to perform sterility inspection. At the same time, the dissolving reagent has little damage to microorganisms. Therefore, this sterility detection method can more accurately detect microorganisms in solid pharmaceutical preparations, especially poorly soluble solid pharmaceutical preparations (containing polymer materials), thereby effectively monitoring the quality and safety of drugs. DETAILED DESCRIPTION

[0023] Below in conjunction with specific embodiment, the sterility test method of solid pharmaceutical preparation of the present application is described in further detail.The application can be realized in many different forms, is not limited to embodiment described herein.On the contrary, the purpose of providing these embodiments is to make the present application disclosure be understood more thoroughly and comprehensively.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0025] As used herein, "one or more" refers to any one, any two, or any two or more of the listed items.

[0026] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0027] In this application, when referring to numerical ranges, unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0028] Unless otherwise specified, the percentage contents mentioned in this application refer to mass percentage for solid-liquid mixture and solid-solid mixture, and refer to volume percentage for liquid-liquid mixture.

[0029] Unless otherwise specified, percentage concentrations used in this application refer to final concentrations. Final concentrations refer to the percentage of an added component in the system after the addition of that component. Unless otherwise specified, all solvents used are water.

[0030] The temperature parameters in this application, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows for temperature fluctuations within the precision range of instrument control.

[0031] The room temperature in this application generally refers to 4°C to 30°C, preferably 20±5°C.

[0032] In this application, "little damage to microorganisms" means that after treatment with a dissolving reagent, the recovery rate of microorganisms must reach between 50% and 200% as required by regulations.

[0033] Some embodiments of the present application provide a method for sterility testing of a solid pharmaceutical preparation, wherein the solid pharmaceutical preparation comprises at least one of a polymer material and an antibacterial component, wherein the polymer material comprises one or more of polylactic acid, polylactic-co-glycolic acid, and polycaprolactone, and the antibacterial component comprises one or more of dichloromethane, chloroform, and ethyl acetate; the sterility testing method comprises the following steps:

[0034] The solid pharmaceutical preparation is crushed and mixed with a dissolving reagent to prepare a sample solution to be tested;

[0035] Filtering the sample solution to be tested through a membrane to collect the filter membrane to be tested;

[0036] Placing the filter membrane to be tested in a culture medium for cultivation to obtain the growth status of the microorganisms;

[0037] The dissolving agent comprises 10% to 18% by volume of propylene carbonate and 1% to 3% by mass of bovine albumin.

[0038] In some embodiments, the composition of the solid pharmaceutical formulation includes a polymer material and optionally includes an antibacterial component.

[0039] Specifically, in the dissolving agent, the volume percentage of the propylene carbonate includes but is not limited to: 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% or a range between any two of the foregoing.

[0040] Specifically, the mass percentage of the bovine albumin in the dissolution reagent includes but is not limited to: 1%, 1.5%, 2%, 2.5%, 3% or a range between any two of the foregoing.

[0041] In some embodiments, the dissolution reagent includes 13% to 17% by volume of propylene carbonate and 1.5% to 2.5% by mass of bovine albumin.

[0042] In some embodiments, the solvent of the dissolving reagent is water or a sodium chloride peptone buffer having a pH of 6.8 to 7.2. Specifically, the pH of the sodium chloride peptone buffer includes but is not limited to: 6.8, 6.9, 7, 7.1, 7.2, or a range between any two of the foregoing.

[0043] In some embodiments, the mass-to-volume ratio of the solid pharmaceutical preparation to the dissolving agent is ≤5 mg / mL.

[0044] In some embodiments, the mass volume ratio of the solid pharmaceutical preparation to the dissolving agent is 1g:(200-500)mL. Specifically, the mass volume ratio of the solid pharmaceutical preparation to the dissolving agent includes but is not limited to: 1g:200mL, 1g:250mL, 1g:267mL, 1g:300mL, 1g:350mL, 1g:400mL, 1g:450mL, 1g:500mL or a range between any two of the foregoing. Further, the mass volume ratio of the solid pharmaceutical preparation to the dissolving agent is 1g:(200-400)mL.

[0045] In some embodiments, the particle size of the solid pharmaceutical preparation after pulverization is ≤850 μm.

[0046] In some embodiments, the membrane is a hydrophilic filter membrane with a pore size of 0.4 μm to 0.5 μm, and more specifically, a 0.45 μm hydrophilic filter membrane.

[0047] Without limitation, the microorganisms include one or more of Staphylococcus aureus, Escherichia coli, Clostridium sporogenes, Bacillus subtilis, Candida albicans, and Aspergillus niger. The type of culture medium can be selected according to the type of microorganism. In some embodiments, the culture medium includes one or both of glycolate fluid medium and trypticase soy liquid medium.

[0048] Without limitation, in the solid pharmaceutical preparation, the mass percentage of the polymer material is 40% to 95%. Specifically, the mass percentage of the polymer material includes, but is not limited to, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or a range between any two of the foregoing.

[0049] Without limitation, the mass percentage of the antibacterial component in the solid pharmaceutical preparation is 0% to 0.5%. Specifically, the mass percentage of the antibacterial component includes, but is not limited to, 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, or any range therebetween. It is understood that the antibacterial component is primarily derived from residual organic solvents during the preparation process.

[0050] For experimental parameters not specified in the following specific examples, reference is made to the guidance given in this application document, and reference may also be made to experimental manuals in the art or other experimental methods known in the art, or to the experimental conditions recommended by the manufacturer.

[0051] The raw materials and reagents involved in the following specific examples can be obtained commercially, or can be prepared by those skilled in the art according to known methods.

[0052] Investigation Example 1

[0053] This investigation is about the solubility study of the dissolving agent (propylene carbonate):

[0054] The test sample (naltrexone and risperidone combination solid implant, self-developed, containing 49.75wt% polylactic acid, 0.5wt% ethyl acetate, and 49.75wt% API) was placed in a sterile mortar and pestle and ground for 15 minutes to uniformly crush the test sample into a fine powder. The powder was then passed through a No. 2 sieve to accelerate the dissolution process. 2250 mg of the powder was weighed using a sterile stainless steel spoon and added to 600 mL of dissolution reagent (analytical grade propylene carbonate, 95% propylene carbonate aqueous solution, 20% propylene carbonate aqueous solution, and 10% propylene carbonate aqueous solution), respectively. The mixture was mixed and the dissolution was observed.

[0055] The results were: analytically pure propylene carbonate could completely dissolve the implant, 95% propylene carbonate aqueous solution could completely dissolve the implant, 20% propylene carbonate aqueous solution could completely dissolve the implant, and some batches of samples dissolved in 10% propylene carbonate aqueous solution, while some batches of samples showed swelling, indicating that when the concentration was less than 10%, the sample would be difficult to dissolve.

[0056] Investigation Example 2

[0057] This investigation is about the effect of a dissolving agent (propylene carbonate) on microorganisms.

[0058] Test group 1: Take 6 bottles of analytically pure propylene carbonate, add Escherichia coli, Staphylococcus aureus, Clostridium sporogenes, Bacillus subtilis, Candida albicans and Aspergillus niger prepared according to the requirements of the Chinese Pharmacopoeia with a bacterial count of 10-100 cfu / ml, and mix them evenly as test group 1.

[0059] Test group 2: Take 6 bottles of 95% propylene carbonate aqueous solution, add Escherichia coli, Staphylococcus aureus, Clostridium sporogenes, Bacillus subtilis, Candida albicans and Aspergillus niger prepared according to the requirements of the Chinese Pharmacopoeia with a bacterial count of 10-100 cfu / ml, and mix them evenly as test group 2.

[0060] Test group 3: Take 6 bottles of 20% propylene carbonate aqueous solution, add Escherichia coli, Staphylococcus aureus, Clostridium sporogenes, Bacillus subtilis, Candida albicans and Aspergillus niger prepared according to the requirements of the Chinese Pharmacopoeia with a bacterial count of 10-100 cfu / ml, and mix them evenly as test group 3.

[0061] Bacterial solution control group: Prepare six bottles of pH 7.0 sodium chloride-peptone buffer and add Escherichia coli, Staphylococcus aureus, Clostridium sporogenes, Bacillus subtilis, Candida albicans, and Aspergillus niger, prepared according to the Chinese Pharmacopoeia to a bacterial count of 10-100 cfu / ml. Mix thoroughly and set aside. Rinse the filter membrane with the flushing solution and directly fill with the corresponding culture medium. This will serve as the bacterial solution control group.

[0062] Negative control group: Take aqueous solution as the negative control group.

[0063] The samples from each group were tested for microorganisms using a membrane filtration method. The filter membrane was rinsed three times with 100 mL of pH 7.0 sodium chloride peptone buffer each time. The filter membrane was then placed on a culture medium plate with the bacterial surface facing upward for culture. Fluid thioglycollate medium (FTM) was used for Staphylococcus aureus, Escherichia coli, and Clostridium sporogenes, while tryptic soy broth (TSB) was used for Bacillus subtilis, Candida albicans, and Aspergillus niger. The microbial growth of experimental groups 1, 2, and 3, as well as the bacterial solution control group, was calculated. The results are shown in Table 1 below:

[0064] Table 1

[0065]

[0066] Conclusion: Using membrane filtration and qualitative sterility testing directly after filtration, the results showed that Staphylococcus aureus, Bacillus subtilis, and Aspergillus niger grew in the pure propylene carbonate group. In the 95% propylene carbonate aqueous solution group, all microorganisms grew except Clostridium sporogenes and Escherichia coli. In the 20% propylene carbonate group, all added microorganisms grew. This indicates that the toxic effect of propylene carbonate on microorganisms can be reduced by lowering its concentration. In combination with Example 1, it can be seen that 20% propylene carbonate aqueous solution has a greater solubility for implants. Further experiments can be used to confirm whether propylene carbonate is suitable as a solvent for sterility testing of implants.

[0067] Investigation Example 3

[0068] This investigation is a study on the recovery rate of microorganisms using a dissolving agent (propylene carbonate).

[0069] Test Group 1: Sterilize 6 bottles of analytically pure propylene carbonate (30 ml / bottle). After cooling, add Escherichia coli, Staphylococcus aureus, Clostridium sporogenes, Bacillus subtilis, Candida albicans, and Aspergillus niger, prepared according to the Chinese Pharmacopoeia with a bacterial count of 10-100 cfu / ml, and mix well to prepare this solution.

[0070] Test group 2: Take 95% propylene carbonate aqueous solution, divide it into 30mL / bottle, sterilize, cool, add Escherichia coli, Staphylococcus aureus, Clostridium sporogenes, Bacillus subtilis, Candida albicans and Aspergillus niger prepared according to the requirements of the Chinese Pharmacopoeia with a bacterial count of 10-100 cfu / ml, and mix them as test group 2.

[0071] Test group 3: Take a 30% propylene carbonate aqueous solution and divide it into 30 mL / bottle. After cooling, add Escherichia coli, Staphylococcus aureus, Clostridium sporogenes, Bacillus subtilis, Candida albicans and Aspergillus niger prepared according to the requirements of the Chinese Pharmacopoeia with a bacterial count of 10-100 cfu / ml respectively and mix them evenly as test group 3.

[0072] Test group 4: Take a 20% propylene carbonate aqueous solution, divide it into 30 mL / bottle, sterilize it, and after cooling, add Escherichia coli, Staphylococcus aureus, Clostridium sporogenes, Bacillus subtilis, Candida albicans and Aspergillus niger prepared according to the requirements of the Chinese Pharmacopoeia with a bacterial count of 10-100 cfu / ml respectively and mix them evenly as test group 4.

[0073] Test group 5: Take a 10% propylene carbonate aqueous solution, divide it into 30 mL / bottle, sterilize it, and after cooling, add Escherichia coli, Staphylococcus aureus, Clostridium sporogenes, Bacillus subtilis, Candida albicans and Aspergillus niger prepared according to the requirements of the Chinese Pharmacopoeia with a bacterial count of 10-100 cfu / ml respectively and mix them evenly as test group 5.

[0074] Bacterial solution control group: Prepare 6 bottles of 30 mL / bottle pH 7.0 sodium chloride-peptone buffer and add Escherichia coli, Staphylococcus aureus, Clostridium sporogenes, Bacillus subtilis, Candida albicans, and Aspergillus niger, prepared according to the Chinese Pharmacopoeia to a bacterial count of 10-100 cfu / ml. Mix thoroughly and set aside. Rinse the filter membrane with the flushing solution and directly fill with the corresponding culture medium. This will serve as the bacterial solution control group.

[0075] Negative control group: Take aqueous solution as the negative control group.

[0076] Membrane filtration detection:

[0077] The samples of each group were tested for microorganisms using the membrane filtration method. The filter membrane was rinsed three times with pH 7.0 sodium chloride peptone buffer, 100 mL each time. The filter membrane was placed on a culture medium plate with the bacterial side facing up. The culture medium was the same as that in Investigation Example 2. The microbial growth of test groups 1 to 5 and the bacterial solution control group was calculated. The microbial recovery rate of the test group was calculated according to the following formula:

[0078] Recovery rate of the experimental group = average colony count of the experimental group / average colony count of the bacterial solution control group * 100%

[0079] The results are shown in Table 2 below:

[0080] Table 2

[0081]

[0082] Conclusion: The higher the concentration of propylene carbonate, the greater the toxicity to microorganisms. The microbial recovery of the 10% propylene carbonate aqueous solution group met the requirements, and the toxic and side effects were negligible, so it can be used for sterility testing. However, combined with Example 1, it is known that its solubility is relatively poor. Therefore, it is necessary to continue to find a suitable concentration or investigate whether a suitable neutralizer can be added to make the microbial toxic and side effects within an acceptable range and meet the solubility requirements.

[0083] Investigation Example 4

[0084] This investigation example is a study on the recovery rate of microorganisms by adding the neutralizing agent Tween 80 to the dissolving reagent.

[0085] Test group 1: Take 1% Tween 80 in 20% propylene carbonate aqueous solution, divide it into 6 bottles, add 10-100 cfu / ml Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger respectively and mix well as test group 1.

[0086] Test group 2: Take 5% Tween 80 in 20% propylene carbonate aqueous solution, divide it into 6 bottles, add 10-100 cfu / ml of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger respectively and mix well as test group 2.

[0087] Test group 3: Take 20% propylene carbonate aqueous solution with 10% Tween 80, divide it into 6 bottles, add 10-100 cfu / ml of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger respectively and mix well as test group 3.

[0088] Bacterial solution control group: 6 bottles of aqueous solution were taken and 10-100 cfu / ml of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger were added and mixed to serve as the bacterial solution control group.

[0089] Negative control group: Take aqueous solution as the negative control group.

[0090] Membrane filtration detection:

[0091] The samples of each group were tested for microorganisms using the membrane filtration method. The filter membrane was rinsed three times with pH 7.0 sodium chloride peptone buffer, 100 mL each time. The filter membrane was placed on a culture medium plate with the bacterial side facing up. The culture medium was the same as that in Investigation Example 2. The microbial growth of test groups 1 to 3 and the bacterial solution control group was calculated. The microbial recovery rate of the test group was calculated according to the following formula:

[0092] Recovery rate of the experimental group = average colony count of the experimental group / average colony count of the bacterial solution control group * 100%

[0093] The results are shown in Table 3 below:

[0094] Table 3

[0095]

[0096] Conclusion: Solutions containing 1%, 5% and 10% Tween 80 could not neutralize the toxic effect of 20% propylene carbonate solution on microorganisms. Bacillus subtilis, Candida albicans and Aspergillus niger had a certain neutralizing effect in 5% and 10% Tween 80 solutions.

[0097] Investigation Example 5

[0098] This investigation example is a study on the recovery rate of microorganisms by adding the neutralizing agent bovine albumin to the dissolving reagent.

[0099] Test group 1: A 1% bovine albumin (mass percentage) aqueous solution containing 15% propylene carbonate (volume percentage) was divided into 6 bottles, and 10-100 cfu / ml of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger were added to each bottle and mixed evenly as test group 1.

[0100] Test group 2: 2% bovine albumin solution containing 15% propylene carbonate was divided into 6 bottles, and 10-100 cfu / ml of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger were added and mixed as test group 2.

[0101] Experimental Group 3: 2% bovine albumin solution containing 20% propylene carbonate was divided into 6 bottles, and 10-100 cfu / ml of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger were added and mixed as Experimental Group 2.

[0102] Bacterial solution control group: 6 bottles of aqueous solution were taken and 10-100 cfu / ml of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger were added and mixed to serve as the bacterial solution control group.

[0103] Negative control group: Take aqueous solution as the negative control group.

[0104] Membrane filtration detection:

[0105] The samples of each group were tested for microorganisms using the membrane filtration method. The filter membrane was rinsed three times with pH 7.0 sodium chloride peptone buffer, 100 mL each time. The filter membrane was placed on a culture medium plate with the bacterial side facing up. The culture medium was the same as that in Investigation Example 2. The microbial growth of test groups 1 to 3 and the bacterial solution control group was calculated. The microbial recovery rate of the test group was calculated according to the following formula:

[0106] Recovery rate of the experimental group = average colony count of the experimental group / average colony count of the bacterial solution control group * 100%

[0107] The results are shown in Table 4 below:

[0108] Table 4

[0109]

[0110] Conclusion: After 15% propylene carbonate solution was neutralized with 2% bovine albumin solution, the microbial recovery rate could reach 50%, and the toxic effect on microorganisms was negligible. The toxic effect of 20% propylene carbonate solution on Escherichia coli was still relatively large, and 2% bovine albumin could not neutralize the toxicity of 20% propylene carbonate solution.

[0111] The above experiments have shown that dissolving the sample with 15% propylene carbonate solution and adding 2% bovine albumin as a neutralizer has little toxicity to microorganisms and can be used as a solvent for sterility testing.

[0112] Investigation Example 6

[0113] This investigation example is the sterility test method of the test product.

[0114] Preparation of test solution: Take the test sample and place it in a sterile mortar and pestle. Grind it for 15 minutes to grind it into a fine powder. Then weigh 2250 mg of the powder with a sterile stainless steel spoon and add 600 mL of pH 7.0 sodium chloride peptone buffer containing 15% propylene carbonate (volume percentage) and 2% bovine albumin (mass percentage) to dissolve it. Mix well to obtain 600 mL of test solution. Prepare three samples in parallel.

[0115] Membrane filtration detection:

[0116] Test group and test sample control group: Take a triple sterile incubator, soak the filter membrane with a small amount of pH7.0 sodium chloride-peptone buffer, and filter. Then, evenly add the prepared 600mL test solution to the three filter cartridges of the triple sterile incubator (the inoculation amount of each filter membrane is about 750mg of the test sample, that is, 200mL of the test solution). After filtration, rinse the filter membrane with pH7.0 sodium chloride-peptone buffer, rinse each filter cartridge 3 times, each rinse volume 100mL, and filter dry; add 100mL of thioglycollate fluid culture medium to 4 of the filter cartridges, and add Staphylococcus aureus and Escherichia coli with a bacterial count of no more than 100cfu to three of the filter cartridges. 1 mL each of Escherichia coli and Clostridium sporogenes was added as the test group, and another filter cartridge was not added with challenge microorganisms and was used as the test control group. The cells were cultured at 30-35°C for no more than 5 days. 100 mL of tryptic soytone liquid culture medium was added to the other four filter cartridges, and 1 mL each of Bacillus subtilis, Candida albicans and Aspergillus niger with a bacterial count of no more than 100 cfu was added to three of the filter cartridges as the test group. The other filter cartridge was not added with challenge microorganisms and was used as the test control group. The cells were cultured at 20-25°C for no more than 5 days.

[0117] Neutralizer control group and negative control group: Take a triple sterile incubator, soak the filter membrane with a small amount of pH7.0 sodium chloride-peptone buffer, and filter. Then, evenly add the prepared 600mL of pH7.0 sodium chloride-peptone buffer containing 15% propylene carbonate and 2% bovine albumin to the three filter cartridges of the triple sterile incubator. After filtration, rinse the filter membrane with pH7.0 sodium chloride-peptone buffer. Rinse each filter cartridge 3 times with a rinse volume of 100mL each time, and filter dry; add 100mL of thioglycollate fluid culture medium to 4 of the filter cartridges, and add Staphylococcus aureus and Escherichia coli with a bacterial count of no more than 100cfu to three of the filter cartridges. and Clostridium sporogenes, as the neutralizer control group; another filter cartridge was not added with challenge microorganisms, as the negative control group, and cultured at 30-35°C for no more than 5 days. 100 mL of tryptic soytone liquid culture medium was added to the other four filter cartridges, and 1 mL each of Bacillus subtilis, Candida albicans and Aspergillus niger with a bacterial count of no more than 100 cfu were added to three of the filter cartridges as the neutralizer control group; the other filter cartridge was not added with challenge microorganisms, as the negative control group, and cultured at 20-25°C for no more than 5 days.

[0118] Positive control group: Prepare six bottles of pH 7.0 sodium chloride-peptone buffer and add Escherichia coli, Staphylococcus aureus, Clostridium sporogenes, Bacillus subtilis, Candida albicans, and Aspergillus niger, prepared according to the Chinese Pharmacopoeia with a bacterial count of 10-100 cfu / ml. Mix thoroughly and set aside. Rinse the filter membrane with the flushing solution and directly fill with the corresponding culture medium to serve as the bacterial solution control group.

[0119] The results are shown in Table 5 below:

[0120] Table 5

[0121]

[0122] It can be seen that the experimental results meet the requirements after the sample was dissolved in pH 7.0 sodium chloride peptone buffer containing 2% bovine albumin and 15% propylene carbonate.

[0123] Investigation Example 7

[0124] This investigation example is a screening study of different types of dissolution reagents.

[0125] (1) Solubility investigation:

[0126] Take the test sample, place it in a sterile mortar, and grind it for 15 minutes to grind it into a fine powder. Then pass it through a No. 2 sieve to accelerate the dissolution process. Then use a sterile stainless steel spoon to weigh 2250 mg of powder, add it to 600 mL of dissolving reagent, mix well, and observe the dissolution. The results are as follows:

[0127] Ethanol: Analytical grade 95% or more ethanol can dissolve the test sample. When the concentration is reduced to 20% in aqueous solution, it is only partially soluble.

[0128] Dimethyl sulfoxide (DMSO): Analytical grade can completely dissolve the test sample, and partially dissolve when the concentration is reduced to 50% in aqueous solution.

[0129] Sulfolane: Analytically pure solvent can partially dissolve the test sample, but after dissolution, the analytically pure solvent will recrystallize. When the concentration is reduced, the dissolution effect becomes worse, so this solvent is not used.

[0130] (2) Investigation of the effect of dissolving reagent ethanol on microorganisms:

[0131] Test group 1: 20% ethanol containing 0.1% polysorbate 80 was dispensed into 30 ml bottles, 6 bottles each, and 10-100 cfu / ml of Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Clostridium sporogenes, Candida albicans, and Aspergillus niger solutions were added to the bottles as test group 1.

[0132] Test group 2: 30% ethanol containing 0.1% polysorbate 80 was dispensed into 30 ml bottles, 6 bottles each, and 10-100 cfu / ml of Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Clostridium sporogenes, Candida albicans, and Aspergillus niger solutions were added to the bottles as test group 2.

[0133] Bacterial solution control group: 6 bottles of pH 7.0 sodium chloride peptone buffer were taken and 10-100 cfu / ml of Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Clostridium sporogenes, Candida albicans and Aspergillus niger were added respectively as bacterial solution control groups.

[0134] Membrane filtration detection:

[0135] The samples of each group were tested for microorganisms using the membrane filtration method. The filter membrane was rinsed three times with pH 7.0 sodium chloride peptone buffer, 100 mL each time. The filter membrane was placed on a culture medium plate with the bacterial side facing up. The culture medium was the same as that in Investigation Example 2. The microbial growth of test group 1, test group 2, and the bacterial solution control group was calculated. The microbial recovery rate of the test group was calculated according to the following formula:

[0136] Recovery rate of the experimental group = average colony count of the experimental group / average colony count of the bacterial solution control group * 100%

[0137] The results are shown in Table 6 below:

[0138] Table 6

[0139]

[0140] Conclusion: Although 20% ethanol containing 0.1% polysorbate 80 and 30% ethanol containing 0.1% polysorbate 80 have certain solubility for samples, they have certain effects on microorganisms. The recovery rates of Escherichia coli and Clostridium sporogenes in 20% ethanol cannot reach the requirement of 50% recovery rate. When the concentration of 30% ethanol containing 0.1% polysorbate 80 is high, the recovery rate of Aspergillus niger reaches 50%, while the others cannot meet the requirements. Therefore, ethanol with a concentration of more than 20% cannot be used as a solvent for sterility testing.

[0141] (3) Investigation of the effect of dissolving agent dimethyl sulfoxide on microorganisms:

[0142] Test group 1: Take 6 bottles of analytical grade dimethyl sulfoxide solution, add Escherichia coli, Staphylococcus aureus, Clostridium sporogenes, Bacillus subtilis, Candida albicans and Aspergillus niger prepared as required and mix well as test group 1.

[0143] Test Group 2: Prepare six bottles of 95% dimethyl sulfoxide aqueous solution. Add Escherichia coli, Staphylococcus aureus, Clostridium sporogenes, Bacillus subtilis, Candida albicans, and Aspergillus niger, prepared as required, to each solution and mix thoroughly to form Test Group 2.

[0144] Test Group 3: Prepare six bottles of 50% dimethyl sulfoxide aqueous solution. Add Escherichia coli, Staphylococcus aureus, Clostridium sporogenes, Bacillus subtilis, Candida albicans, and Aspergillus niger, prepared as required, to each solution and mix thoroughly to form Test Group 3.

[0145] Bacterial solution control group: Prepare six bottles of pH 7.0 sodium chloride-peptone buffer and add 10-100 cfu of Escherichia coli, Staphylococcus aureus, Clostridium sporogenes, Bacillus subtilis, Candida albicans, and Aspergillus niger, prepared according to the Chinese Pharmacopoeia requirements. Mix thoroughly and set aside. Rinse the filter membrane with the flushing solution and directly fill with the corresponding culture medium. This will serve as the bacterial solution control group.

[0146] Negative control group: Take aqueous solution as the negative control group.

[0147] Membrane filtration detection:

[0148] The samples of each group were tested for microorganisms using the membrane filtration method. The filter membrane was rinsed three times with pH 7.0 sodium chloride peptone buffer, 100 mL each time. The filter membrane was placed with the bacterial side facing up on a culture medium plate for culture. The culture medium was the same as that in Investigation Example 2. After filtration, the sample was directly tested using the qualitative sterility test method.

[0149] The results are shown in Table 7 below:

[0150] Table 7

[0151]

[0152] Conclusion: Only Bacillus subtilis can grow in 95% dimethyl sulfoxide solution, and only Bacillus subtilis and Aspergillus niger can grow in 50% dimethyl sulfoxide solution. The rest of the microorganisms cannot grow. Therefore, dimethyl sulfoxide cannot be used as a solvent.

[0153] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0154] The embodiments described above only express several implementation methods of the present application, which are convenient for understanding the technical solutions of the present application in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of this application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A method for sterility testing of solid pharmaceutical preparations, characterized in that: The solid pharmaceutical preparation comprises at least one of a polymer material and an antibacterial component, wherein the polymer material comprises one or more of polylactic acid, polylactic-co-glycolic acid, and polycaprolactone, and the antibacterial component comprises one or more of dichloromethane, chloroform, and ethyl acetate; The sterility inspection method comprises the following steps: The solid pharmaceutical preparation is crushed and mixed with a dissolving reagent to prepare a sample solution to be tested; Filtering the sample solution to be tested through a membrane to collect the filter membrane to be tested; Placing the filter membrane to be tested in a culture medium for cultivation to obtain the growth status of the microorganisms; The dissolving agent comprises 10% to 18% by volume of propylene carbonate and 1% to 3% by mass of bovine albumin.

2. The method for sterility inspection of solid pharmaceutical preparations according to claim 1, characterized in that: The dissolving agent includes 13% to 17% by volume of propylene carbonate and 1.5% to 2.5% by mass of bovine albumin.

3. The method for sterility inspection of solid pharmaceutical preparations according to claim 1 or 2, characterized in that: The solvent of the dissolving reagent is water or a sodium chloride peptone buffer solution with a pH of 6.8 to 7.

2.

4. The method for sterility inspection of solid pharmaceutical preparations according to claim 1 or 2, characterized in that: The mass volume ratio of the solid pharmaceutical preparation to the dissolving reagent is 1 g: (200-500) mL.

5. The method for sterility inspection of solid pharmaceutical preparations according to claim 4, characterized in that: The mass volume ratio of the solid pharmaceutical preparation to the dissolving reagent is 1 g: (200-400) mL.

6. The method for sterility inspection of solid pharmaceutical preparations according to claim 1 or 2, characterized in that: The particle size of the solid pharmaceutical preparation after pulverization is ≤850 μm.

7. The method for sterility inspection of solid pharmaceutical preparations according to claim 1 or 2, characterized in that: The film is a hydrophilic filter membrane with a pore size of 0.4 μm to 0.5 μm.

8. The method for sterility inspection of solid pharmaceutical preparations according to claim 1 or 2, characterized in that: The microorganisms include one or more of Staphylococcus aureus, Escherichia coli, Clostridium sporogenes, Bacillus subtilis, Candida albicans and Aspergillus niger.

9. The method for sterility inspection of solid pharmaceutical preparations according to claim 7, characterized in that: The culture medium includes one or both of glycolate fluid medium and trypticase soy liquid medium.

10. The method for sterility inspection of solid pharmaceutical preparations according to claim 1 or 2, characterized in that: In the solid pharmaceutical preparation, the mass percentage of the polymer material is 40% to 95%; and / or, In the solid pharmaceutical preparation, the mass percentage of the antibacterial component is 0% to 0.5%.

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