A method for sterility checking of a microsphere injection

Abstract

The present application relates to the technical field of quality control of pharmaceutical preparations, and particularly relates to a sterile examination method of microsphere injection. The present application relates to a sterile examination method of microsphere injection, wherein the method comprises the step of crushing the microsphere injection. The sterile examination method of the present application is aimed at microspheres with strong microorganism adsorption capacity and which cannot be dissolved, can effectively release the microorganisms wrapped in the microspheres to realize the sterile examination inside and outside the microspheres, and has no inhibitory effect on microorganisms and high detection accuracy.

Description

Technical Field

[0001] This application relates to the field of quality control technology for pharmaceutical preparations, specifically to a method for sterility testing of microsphere injections. Background Technology

[0002] According to Chapter 9014 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, microspheres are defined as "tiny spherical entities formed when a drug is dissolved or dispersed in a carrier excipient." Their particle size is typically 1-250 μm. The carrier materials used to prepare microspheres are diverse, mainly divided into natural polymers (such as gelatin, starch, and albumin) and synthetic polymers (such as polylactic acid and PLGA), and also include carbon, resins, etc.

[0003] Microsphere injections are a type of sterile suspension injection in which drugs are encapsulated or dispersed in a carrier material to form tiny spherical entities. Their core function is to form a "drug reservoir" after injection or intervention in the body, achieving long-acting, sustained-release, or targeted drug release through the slow degradation of the carrier material or drug diffusion.

[0004] Sterility testing is a mandatory test to ensure the safe use of sterile products and a crucial step in determining the production cycle of sterile products. In the pharmaceutical field, for example, pharmacopoeias of various countries have strict requirements for sterility testing of injectable preparations, and have largely established internationally consistent testing standards and operating procedures, effectively improving the sterility assurance level of formulations. According to General Chapter 1101 of the 2020 edition of the Chinese Pharmacopoeia, there are two main methods for sterility testing: membrane filtration and direct inoculation. However, the Chinese Pharmacopoeia does not include specific provisions regarding sterility testing methods for injectable microspheres.

[0005] When the sterility testing methods of the current pharmacopoeia are directly applied to microsphere injections, the following systemic defects mainly exist: (1) Inability to distinguish between "outside" and "inside" sterility The sterility of microspheres needs to be examined simultaneously on both the "outer surface" and the "inner surface." Conventional testing methods, such as direct inoculation, are mainly for soluble or dispersible samples and cannot effectively disrupt the microsphere structure to release microorganisms that may be encapsulated within. Therefore, even if microorganisms are present inside the microspheres, conventional methods are often ineffective in detecting them, leading to false negatives.

[0006] (2) Obstacles to microsphere dissolution and filtration Microspheres are insoluble particles. When using conventional membrane filtration methods to test the sterility of microspheres, the microspheres will clog the filter membrane used to collect microorganisms, making the filtration method impossible.

[0007] (3) The introduction of detection reagents leads to antibacterial effect. Existing methods for aseptic testing within microspheres often introduce reagents that inhibit microbial growth. For example, the dimethyl sulfoxide (DMSO) assay requires a high concentration of DMSO, which is toxic to microbial colonies and has a significant inhibitory effect; similarly, the alkaline buffer assay introduces an alkaline solution that also inhibits microbial growth. These substances inhibit the growth of microorganisms in the culture medium, potentially leading to false negative results.

[0008] (4) There are gaps in the guiding principles. The current pharmacopoeia guidelines do not specify clear technical requirements for the microbial quality control of microspheres. The Chinese Pharmacopoeia only describes the formulation and manufacturing process of microspheres in the general rules for preparations, without mentioning specific provisions for sterility testing of microspheres.

[0009] Therefore, there is an urgent need in this field for a microsphere sterility testing method that can effectively release the microorganisms encapsulated within the microspheres, without inhibiting their activity, and with high detection accuracy, targeting microorganisms with strong adsorption capacity and insoluble microorganisms. Summary of the Invention

[0010] The inventors unexpectedly discovered that by using ball milling under appropriate conditions to break down microspheres with strong microbial adsorption capacity and insoluble structure to a suitable particle size range, bacteria and fungi inside the microspheres can be fully exposed, thereby enabling aseptic inspection of both the inside and outside of the microspheres. Furthermore, appropriate ball milling does not adversely affect microbial growth or introduce microorganisms.

[0011] Therefore, a first aspect of the present invention provides a method for sterility testing of microsphere injection solution, the method comprising the step of breaking up the microsphere injection solution to be tested, wherein the particle size of the microspheres to be tested ranges from 15 to 80 μm, and the average particle size of the broken microspheres is 3 to 6 μm.

[0012] Preferably, the broken microspheres are subjected to aseptic testing.

[0013] Preferably, the method includes the following steps: a) Provide a microsphere injection solution to be inspected, wherein the microspheres have a particle size range of 15-80 μm; b) The positive control group containing positive control bacteria, the test sample group containing microsphere injection, and the negative control group without microsphere injection and positive control bacteria were crushed separately so that the average particle size of the crushed microspheres was 3-6 μm. c) The fragmented positive control group, test sample group, and negative control group were inoculated into sterile culture medium and cultured at 20-35℃ to obtain cultures; and d) Observe whether the bacteria grow on the sterile culture medium. If the positive control group has bacterial growth, while the test sample group and the negative control group have no bacterial growth, then the microsphere injection solution to be tested is determined to be sterile.

[0014] Preferably, the particle size range of the microspheres to be inspected is 20-45 μm.

[0015] Preferably, the microsphere injection solution to be inspected is selected from one or more of the following: carbon microsphere injection solution, resin microspheres, lactic acid polymer microspheres, hydroxyapatite microspheres, gelatin microspheres, silica microspheres, and calcium alginate microspheres.

[0016] Preferably, the crushing is performed by ball milling at 300-500 rpm for 15-30 minutes, and more preferably by ball milling at 300-500 rpm for 20-30 minutes.

[0017] Preferably, the particle size distribution of the broken microsphere injection solution has a Dv(10) value of 1-2 μm, a Dv(50) value of 3-6 μm, a Dv(90) value of 6-14 μm, and a D[4,3] value of 3-7 μm.

[0018] Preferably, the positive control bacterium is Staphylococcus aureus; the sterile culture medium is selected from thioglycolate fluid medium and / or tryptic soy liquid medium.

[0019] Preferably, the positive control group, the test sample group, and the negative control group are all inoculated into thioglycolate fluid medium and tryptic soy peptone liquid medium, respectively.

[0020] Preferably, the inoculation amount of the positive control bacteria is no more than 100 cfu.

[0021] Preferably, the aseptic testing method is performed under aseptic conditions.

[0022] Preferably, if the sterile culture medium becomes turbid or if the microspheres are carbon microspheres, the method further includes the step of: after step c), taking a portion of the culture daily and continuing to culture it on a sterile culture medium at 20-35°C.

[0023] Preferably, the positive control group, the test sample group, and the negative control group all contain a buffer solution. The buffer solution is preferably a 0.9% sterile sodium chloride solution, a pH 7.0 sterile sodium chloride-peptone buffer, or sterile physiological saline.

[0024] Preferably, the positive control group contains positive control bacteria and buffer solution; the test sample group contains microsphere injection solution and buffer solution; and the negative control group contains only buffer solution.

[0025] Preferably, the aseptic testing method is an intraglobulin and extraglobulin aseptic testing method.

[0026] A second aspect of the present invention provides a sterile microsphere injection solution, wherein the microsphere injection solution is confirmed to be sterile both outside and inside the microspheres by the sterility testing method of the first aspect.

[0027] A third aspect of the invention provides a suitability test for a sterility testing method performed prior to the sterility testing method, to confirm whether the sterility testing method is applicable to the sterility testing of microsphere injections, i.e., to confirm whether the processing method and the sample itself will affect the growth of microorganisms. The suitability test includes setting one or more of the following groups, preferably multiple groups: Positive control of test sample: including the microsphere injection solution to be tested and the test bacteria (without ball milling or with ball milling). Positive control group: includes buffer solution and test bacteria (without ball milling or with ball milling treatment). Sample set: includes the microsphere injection solution to be tested and buffer (e.g., 0.9% sterile sodium chloride solution or pH 7.0 sterile sodium chloride-peptone buffer) (after ball milling); and Negative control group: includes buffer solution (unprocessed or ball-milled). Each group was inoculated into thioglycolate fluid medium or tryptic soy liquid medium and the growth of microorganisms in the medium was observed.

[0028] When the culture results show that the positive control and positive control group strains of the test sample grow well, while the sample group and negative control group show no sterile growth, it confirms that the method used can effectively eliminate interference (such as the infection of the test results by the sample itself and the processing method) and ensure the reliability of the detection results.

[0029] The purpose of setting up a positive control group containing test bacteria and buffer solution is to ensure that the strains are not affected by the processing during aseptic testing. If the strains in the positive control group grow well, it indicates that the aseptic testing process will not affect the growth of the strains (including test bacteria and any microorganisms that may be present in the sample).

[0030] The purpose of setting up a positive control group consisting of the sample and the test bacteria is to confirm whether the sample itself will affect the growth of the test bacteria, that is, to confirm whether there are substances in the sample that affect the growth of microorganisms. If the strains in the positive control group grow well, it indicates that there are no substances in the sample that affect the growth of microorganisms.

[0031] The technical solution of the present invention achieves the following technical effects: 1) This invention uses ball milling to break down microspheres with strong microbial adsorption capacity that are insoluble. After breaking down, 99.8% of the particles are smaller than 20 μm, and there are basically no particles larger than 20 μm. The average particle size is 4-5 μm, which allows bacteria or fungi to be fully exposed after the microspheres are broken down.

[0032] 2) The present invention demonstrates through the suitability test of the aseptic test method that microspheres and broken microspheres have no antibacterial effect, and the ball milling process under appropriate conditions has no significant effect on the growth of the strain. Both the positive control group and the positive control group of the test sample showed bacterial growth, proving that ball milling does not affect the growth of microorganisms.

[0033] 3) By using the method of the present invention to perform sterility testing on the microspheres, sterility testing can be performed on both the outside and inside of the microspheres, and sterile growth is observed in both cases, ensuring that the product meets sterility requirements.

[0034] 4) The sterility testing method of this invention can simultaneously perform sterility testing inside and outside microspheres that have strong microbial adsorption capacity and are insoluble, without introducing microorganisms, without adversely affecting microbial growth, and with high accuracy of test results. This sterility testing method for microspheres fills the gap in the pharmacopoeia regarding sterility testing of microspheres. Attached Figure Description

[0035] Figure 1 Microscopic images of the carbon microsphere injection solution before ball milling and under different ball milling conditions are shown.

[0036] Figure 2 The figure shows a particle size analysis report of carbon microspheres before ball milling of the carbon microsphere injection solution.

[0037] Figure 3 The figure shows a particle size analysis report of carbon microspheres after the carbon microsphere injection solution was ball-milled at 500 rpm for 20 min. Detailed Implementation

[0038] It should be understood that different applications of the disclosed products and methods may be adapted to specific needs in the art. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments of the invention only and is not intended to be limiting.

[0039] All publications, patents, and patent applications cited in this article—whether above or below—are incorporated herein by reference in their entirety.

[0040] definition To more clearly explain the embodiments of the present invention, some scientific terms and proper nouns are used herein. Unless explicitly defined herein, all such terms and nouns should be understood to have the meanings commonly understood by those skilled in the art. For clarity, some terms used herein are defined as follows.

[0041] Microsphere injection As described herein, the term "microsphere injection" refers to an injectable formulation in which solid microspheres are used as drug / radionoid carriers, and the microspheres are dispersed in an injectable solvent (such as physiological saline, injectable oil, suspension, etc.). In some embodiments, the particle size of the solid microspheres is typically 1 μm–250 μm, preferably 10 μm–200 μm, more preferably 15–80 μm, and most preferably 20–45 μm. The core functions of microsphere injections are targeted delivery, local embolization, sustained / controlled release of drugs, or internal radiation therapy. They can be administered via intravenous, arterial, intramuscular, subcutaneous, or intralesional injection, and are widely used in tumor treatment, pain management, long-acting drug delivery, and vascular embolization.

[0042] The carrier materials for preparing microspheres are mainly divided into natural polymer materials (such as gelatin, alginate, starch, albumin, chitosan, etc.), synthetic polymer materials (such as polylactic acid, polylactic acid-glycolic acid copolymer, etc.), and inorganic materials (such as hydroxyapatite, carbon materials, silicon microparticles, etc.). The microsphere injection solution of the present invention is listed below by way of example.

[0043] In some embodiments of the present invention, the microsphere injection solution includes microspheres that have a strong adsorption capacity for microorganisms and are insoluble. Such microsphere injection solutions are mainly selected from any one of carbon microsphere injection solutions, resin microspheres, lactic acid polymer microspheres, hydroxyapatite microspheres, gelatin microspheres, silica microspheres, and calcium alginate microspheres.

[0044] Carbon microsphere injection Carbon microspheres are particles with a spherical or near-spherical structure, primarily composed of carbon. They possess characteristics such as high packing density, good flowability, high mechanical strength, and large specific surface area, making them promising candidates for applications in drug delivery, adsorption, and catalyst carriers. Mesoporous carbon microspheres also exhibit exceptional chemical and thermal stability, excellent biocompatibility, unique pore structure, high specific surface area, and good thermal and electrical conductivity.

[0045] Currently known carbon microspheres mainly include: yttrium [ 90 Y] Carbon microsphere injection (disclosed in patent CN107715124A), such as NRT6003 injection (Chengdu Newrite Medical Technology Co., Ltd.), this drug has been approved by the NMPA for clinical trials for interventional internal irradiation therapy of primary liver cancer, liver metastases of colorectal cancer, and locally advanced pancreatic cancer. It is a domestically developed radioactive carbon microsphere drug with a particle size range of 20-45μm; Yttrium[ 89 Y] Carbon microsphere injection (Chengdu Newrite Medical Technology Co., Ltd.), particle size range 20-45μm; Technetium [ 99m[Tc] Carbon microsphere injection (disclosed in patent CN111603576A); Medical iodine 131 carbon microspheres (disclosed in patent CN106178006A); zirconium [ 89 Zr] Carbon microspheres (disclosed in patent CN111870707A); Medical phosphorus 32 carbon microspheres (disclosed in patent CN107715123A); medical-grade yttrium phosphate [ 90 Y 32 [PO4] Carbon microspheres (disclosed in patent CN107715122A), etc. The particle size of carbon microspheres is usually determined according to the application, and carbon microspheres of different sizes can be used for different applications. In some embodiments, the particle size range of carbon microspheres is 15-80 μm, preferably 20-45 μm.

[0046] Resin microsphere injection Resin microspheres are microspheres prepared using synthetic resins (such as acrylic polymers, polyvinyl alcohol, cross-linked polyvinyl alcohol, ethyl cellulose, etc.) as matrix materials. Resin microspheres possess excellent mechanical strength, chemical stability, and the ability to be functionalized, and are widely used in vascular embolization therapy and sustained drug release.

[0047] Several resin microsphere products and research reports exist in the current technology. Commercially available products include Embosphere® embolization microspheres, which are embolization microspheres made of acrylic polymers and porcine gel, stored in physiological saline, steam-sterilized, and packaged as an injection solution for the embolization treatment of arteriovenous malformations, highly vascularized tumors, and symptomatic uterine fibroids; DC Bead®, a drug-loaded embolization microsphere capable of loading and releasing chemotherapy drugs in a controlled manner for transarterial chemoembolization (TACE) therapy; and so on.

[0048] Lactic acid polymer microsphere injection Lactic acid polymer microspheres are microspheres prepared using polylactic acid (PLA) or lactic acid-glycolic acid copolymer (PLGA) as carrier materials. PLA and PLGA are biodegradable and biocompatible polymers widely used in the preparation of long-acting sustained-release microsphere formulations for injection. These microspheres can release drugs at a certain rate over several weeks or months, maintaining effective blood drug concentrations and reducing the frequency of dosing.

[0049] Commercially available products include Lizhenran® (injectable poly-L-lactic acid microsphere filler), which has been approved for marketing in China. It is used to inject into the deep dermis and superficial subcutaneous layer of the nasolabial folds to correct moderate to severe nasolabial fold wrinkles. Its main component, poly-L-lactic acid (PLLA), is a highly biocompatible and completely degradable polymer regenerative material.

[0050] Hydroxyapatite microsphere injection Hydroxyapatite (Ca 10 (PO4)6(OH)2,HA) microspheres refer to inorganic microspheres with hydroxyapatite as the main component. Hydroxyapatite is a major component of human bone and has good biocompatibility and bioactivity. Hydroxyapatite microspheres have advantages such as unique spherical structure, regular morphology, good fluidity, and large specific surface area, and are widely used in bone repair, drug delivery, and facial fillers.

[0051] Commercially available products include Moyang Bio's injectable hydroxyapatite microsphere facial filler, which is the first hydroxyapatite product in China for facial filling. Its main components are hydroxyapatite, sodium carboxymethyl cellulose, glycerin and water for injection. It is used for subcutaneous injection in the nasolabial fold area to correct moderate to severe nasolabial fold wrinkles.

[0052] Gelatin microsphere injection Gelatin microspheres are microspheres prepared using gelatin, a natural polymer, as a matrix. Gelatin has good biocompatibility and biodegradability, and is widely used in drug sustained release, vascular embolization, and targeted drug delivery. Gelatin microspheres can be used as embolic agents; after being injected via arterial cannulation, they remain at the tumor site, slowly releasing drugs and blocking tumor blood supply.

[0053] Silica microsphere injection Silica microspheres are microspheres prepared primarily from silicon materials (such as silica and calcium silicate). Silica-based microspheres possess excellent biocompatibility, chemical stability, and tunable pore structures, making them promising candidates for applications in drug delivery, bone tissue engineering, and bioimaging.

[0054] Calcium alginate microspheres injection Calcium alginate microspheres are calcium alginate gel microspheres formed by cross-linking sodium alginate with calcium ions. Calcium alginate microspheres possess excellent biocompatibility, hydrophilicity, and biodegradability. Their gel structure enables slow drug release, making them widely applicable in vascular embolization therapy and controlled drug release.

[0055] Aseptic examination The 2020 edition of the Chinese Pharmacopoeia, Part IV, General Chapter 1101, Sterility Test Method, defines the sterility test method as a method for checking whether drugs, biological products, medical devices, raw materials, excipients, and other products that are required to be sterile by the pharmacopoeia are sterile. If the test sample meets the requirements, it only indicates that no microbial contamination was found under the test conditions.

[0056] Sterility testing should be performed under aseptic conditions. The test environment must meet the requirements for sterility testing. Aseptic operation should be strictly followed throughout the entire testing process to prevent microbial contamination. Measures to prevent contamination should not affect the detection of microorganisms in the test sample.

[0057] The culture media used in aseptic testing include: thioglycolate liquid medium, which is mainly used for the culture of anaerobic bacteria, but can also be used for the culture of aerobic bacteria; and tryptic soy broth, which is used for the culture of fungi and aerobic bacteria.

[0058] The pharmacopoeia specifies two sterility testing methods: membrane filtration and direct inoculation. Membrane filtration should be used whenever the properties of the test sample permit. Microspheres are insoluble particles; if membrane filtration is used to test their sterility, the microspheres will clog the filter membrane used to collect microorganisms, rendering the filtration impossible. Therefore, membrane filtration cannot be used for microsphere sterility testing. If direct inoculation is used, the insoluble particle structure of the microspheres cannot be effectively disrupted to release any microorganisms that may be encapsulated within. Therefore, even if microorganisms are present inside the microspheres, they are difficult to detect using direct inoculation, easily leading to false negatives. Due to these limitations, none of the pharmacopoeia-specified sterility testing methods can be directly applied to microsphere sterility testing.

[0059] ball milling Ball milling is a processing method that utilizes the impact and grinding action of grinding media within a rotating container to pulverize, mix, and finely grind materials. It is widely used in fields such as new materials, refractory materials, and battery materials. The working principle of a ball mill is as follows: after the ball mill starts, the centrifugal force generated by its rotation causes the grinding media and materials to move along a specific trajectory, rapidly reducing the particle size of the material through collisions and friction between the media.

[0060] In this invention, the inventors unexpectedly discovered that by using ball milling under appropriate conditions to break down microbeads with strong microbial adsorption capacity and insoluble structure to a suitable particle size range, the adsorption of microorganisms on the carbon microbeads can be disrupted, fully exposing the bacteria and fungi inside the microbeads, thereby enabling aseptic inspection of both the inside and outside of the microbeads. Furthermore, appropriate ball milling does not adversely affect microbial growth or introduce microorganisms.

[0061] In some embodiments, the suitable particle size range is an average particle size of 3-6 μm, preferably 4-5 μm; particles with a diameter greater than 20 μm are substantially absent. In some embodiments, the suitable particle size range includes a particle size distribution Dv(10) value of 1-2 μm, preferably 1.2-1.8 μm, such as 1.29 μm, 1.42 μm, 1.49 μm, 1.5 μm, 1.52 μm, 1.6 μm, 1.7 μm, 1.79 μm; and a Dv(50) value of 3-6 μm, preferably 3.5-5 μm, such as 3.54 μm, 3.74 μm, 4.05 μm, 4.1 μm, 4.13 μm, 4.2 μm, 4.4 μm, 4.6μm, 4.74μm, 4.95μm; Dv(90) value is 6-14μm, preferably 6-11μm, for example 6.92μm, 7.92μm, 8μm, 8.68μm, 8.9μm, 9μm, 10μm, 11μm; D[4,3] value is 3-7μm, preferably 3.5-6μm, for example 3.61μm, 4.12μm, 4.74μm, 4.8μm, 4.9μm, 5.0μm, 5.2μm, 5.4μm, 5.79μm.

[0062] In some embodiments, the appropriate ball milling condition is ball milling at 300 rpm or higher and less than 800 rpm for 15-30 minutes. In some embodiments, the appropriate ball milling condition is ball milling at 300-500 rpm for 15-30 minutes, preferably ball milling at 300-500 rpm for 20-30 minutes, for example, ball milling at 300 rpm for 20 minutes, ball milling at 300 rpm for 25 minutes, ball milling at 300 rpm for 30 minutes, ball milling at 400 rpm for 20 minutes, ball milling at 400 rpm for 25 minutes, ball milling at 450 rpm for 20 minutes, ball milling at 450 rpm for 25 minutes, ball milling at 500 rpm for 20 minutes, ball milling at 500 rpm for 25 minutes.

[0063] In some embodiments, the aseptic testing method of the present invention includes a step of breaking up the microsphere injection solution to be tested. The microspheres to be tested have a particle size range of 15-80 μm, and the average particle size of the broken microspheres is 3-6 μm. The breaking is achieved by ball milling under appropriate conditions, so that the broken microspheres can reach a suitable particle size range, thereby fully exposing the bacteria and fungi inside the microspheres. Then, the aseptic testing method is used to perform aseptic testing on the broken microsphere injection solution, thereby achieving aseptic testing of both the inside and outside of the microspheres.

[0064] In some embodiments, the present invention provides a method for sterility testing of microsphere injection solutions, the method comprising the following steps: a) Provide a microsphere injection solution to be inspected, wherein the microspheres have a particle size range of 15-80 μm; b) The positive control group containing positive control bacteria, the test sample group containing microsphere injection, and the negative control group without microsphere injection and positive control bacteria were crushed separately so that the average particle size of the crushed microspheres was 4-6 μm. c) The fragmented positive control group, test sample group, and negative control group were inoculated into sterile culture medium and cultured at 20-35℃ to obtain cultures; and d) Observe whether the bacteria grow on the sterile culture medium. If the positive control group has bacterial growth, while the test sample group and the negative control group have no bacterial growth, then the microsphere injection solution to be tested is determined to be sterile.

[0065] In some implementations, the microsphere injection solution to be examined is a microsphere with strong adsorption capacity for microorganisms and which is insoluble, selected from any one of carbon microsphere injection solution, resin microspheres, lactic acid polymer microspheres, hydroxyapatite microspheres, gelatin microspheres, silica microspheres and calcium alginate microspheres.

[0066] In some implementations, microspheres with strong microbial adsorption capacity and insoluble structure cannot be dissolved, rendering existing sterility testing methods unusable for detecting their sterility. Even existing sterility testing methods for insoluble substances are ineffective for testing the sterility of these microspheres. Furthermore, the microspheres have a very strong adsorption capacity for microorganisms, making it difficult for microorganisms to be released from within, thus preventing the detection of sterility within the microspheres. For example, the microsphere injection solution to be tested is made of carbonized microspheres. The method of this invention solves this problem.

[0067] In some implementations, the particle size of the microspheres to be inspected ranges from 20 to 45 μm.

[0068] In some implementations, the positive control bacterium is Staphylococcus aureus; the sterile culture medium is selected from thioglycolate fluid medium and / or tryptic soy liquid medium. Thioglycolate fluid medium is mainly used for the culture of anaerobic bacteria, but can also be used for the culture of aerobic bacteria; tryptic soy liquid medium is used for the culture of fungi and aerobic bacteria.

[0069] In some implementation schemes, the positive control group, the test sample group, and the negative control group were all inoculated into thioglycolate fluid medium and tryptic soy peptone liquid medium, respectively.

[0070] In some implementation schemes, the inoculation amount of positive control bacteria is no more than 100 CFU.

[0071] The culture medium can be prepared according to the aseptic test method specified in Section 1101 of the General Chapter IV of the 2020 edition of the Chinese Pharmacopoeia, or a qualified dehydrated culture medium produced according to this formula or a commercially available pre-prepared culture medium can be used. After preparation, it should be sterilized using a validated sterilization procedure. If the prepared culture medium is not used immediately, it should be stored in a sterile, sealed container at 2-25°C in a light-protected environment and used within the validated shelf life.

[0072] In some implementations, thioglycolate fluid medium is incubated at 30-35°C, and tryptic soy peptone liquid medium is incubated at 20-25°C.

[0073] In some implementations, the culture time is no more than 5 days, for example, 5 days. In other implementations, the culture time is no less than 14 days, for example, 14 days.

[0074] In some implementations, the positive control group, the test sample group, and the negative control group all contain a buffer solution. The buffer solution is preferably a 0.9% sterile sodium chloride solution, a pH 7.0 sterile sodium chloride-peptone buffer, or sterile physiological saline.

[0075] In some implementation schemes, positive results The control group contained positive control bacteria and buffer solution; the test sample group contained microsphere injection solution and buffer solution; the negative control group contained only buffer solution.

[0076] In some implementation schemes, if the culture medium becomes turbid after the test sample is added or during the culture process, and it is not possible to determine whether microbial growth exists by appearance after 14 days of culture, at least 1 ml of the culture solution can be transferred to the same fresh culture medium. The original culture and the newly inoculated culture medium can be cultured for at least 4 days, and the turbidity of the inoculated fresh culture medium can be observed again. Alternatively, a smear of the culture solution can be taken, stained, and examined under a microscope to determine whether bacteria are present.

[0077] In some implementations, when the microspheres are carbon microspheres, the culture medium is black, and it is impossible to determine whether there is microbial growth from the appearance. At least 1 ml of the culture solution can be transferred to the same fresh culture medium, and the original culture and the newly inoculated culture medium can be cultured for at least 4 days. Observe whether the inoculated fresh culture medium becomes turbid again; or take a smear of the culture solution, stain it, and examine it under a microscope to determine whether there are bacteria.

[0078] In some implementations, the sterility testing method includes setting up a positive control group: comprising a buffer solution (e.g., 0.9% sterile sodium chloride solution, pH 7.0 sterile sodium chloride-peptone buffer, or sterile saline) and positive control bacteria (with or without ball milling); a test sample group: comprising the microsphere injection solution to be tested and a buffer solution (ball milled); and a negative control group: comprising a buffer solution (with or without ball milling); and inoculating each group into thioglycolate fluid medium or tryptic soy broth. When the culture results show that the positive control group strains grow well, while the test sample group and negative control group show no sterile growth, it indicates that the microsphere injection solution is sterile.

[0079] In some implementations, a suitability test for the sterility testing method is performed before the sterility testing method is applied to confirm whether the sterility testing method is applicable to the sterility testing of the microsphere injection solution, i.e., to confirm whether the processing method and the sample itself will affect the growth of microorganisms. The suitability test includes setting one or more of the following groups, preferably multiple: Positive control of test sample: including the microsphere injection solution to be tested and the test bacteria (without ball milling or with ball milling). Positive control group: includes buffer solution and test bacteria (without ball milling or with ball milling treatment). Sample set: includes the microsphere injection solution to be tested and buffer (e.g., 0.9% sterile sodium chloride solution or pH 7.0 sterile sodium chloride-peptone buffer) (after ball milling); and Negative control group: includes buffer solution (unprocessed or ball-milled). Each group was inoculated into thioglycolate fluid medium or tryptic soy liquid medium and the growth of microorganisms in the medium was observed.

[0080] When the culture results show that the positive control and positive control group strains of the test sample grow well, while the sample group and negative control group show no sterile growth, it confirms that the method used can effectively eliminate interference (such as the infection of the test results by the sample itself and the processing method) and ensure the reliability of the detection results.

[0081] The purpose of setting up a positive control group containing test bacteria and buffer solution is to ensure that the strains are not affected by the processing during aseptic testing. If the strains in the positive control group grow well, it indicates that the aseptic testing process will not affect the growth of the strains (including test bacteria and any microorganisms that may be present in the sample).

[0082] The purpose of setting up a positive control group consisting of the sample and the test bacteria is to confirm whether the sample itself will affect the growth of the test bacteria, that is, to confirm whether there are substances in the sample that affect the growth of microorganisms. If the strains in the positive control group grow well, it indicates that there are no substances in the sample that affect the growth of microorganisms.

[0083] For any procedures not specified in the suitability test for sterility testing methods, please refer to the above limitations regarding sterility testing methods.

[0084] Example For experimental procedures not specifically described in the following examples, please refer to the references cited herein. All experimental reagents and instruments used are commercially available. The experimental procedures in the following examples comply with the requirements of the Chinese Pharmacopoeia.

[0085] Example 1: Screening of carbon microsphere breakage conditions Yttrium was analyzed using a vertical planetary ball mill (model: DECO-PBM-V-2L, Changsha DECO Instrument Equipment Co., Ltd.) at different speeds and times. 89 [Y] Carbon microsphere injection solution (batch number: YCM20200928, Chengdu Newrite Medical Technology Co., Ltd.) was crushed, and the particle size distribution of the carbon microspheres was measured by wet method using a laser particle size analyzer. The ball milling conditions and particle size distribution results are listed in Table 1. The morphology of the carbon microspheres before and after ball milling was observed under a microscope, and the results are as follows. Figure 1 As shown. Particle size analysis was performed on the carbon microspheres before and after ball milling. Figure 2 and Figure 3 The particle size analysis report of carbon microspheres before ball milling and after ball milling at 500 rpm for 20 min is shown (particle size analysis report of other ball milling conditions is not shown).

[0086] Table 1. Ball milling conditions and particle size distribution of carbon microspheres From Table 1 above and Figures 1 to 3It can be seen that within a certain range of rotation speed and time, increasing the rotation speed and extending the time can make the carbon microspheres more thoroughly broken. After ball milling the carbon microspheres at 300 rpm for 5 min, the microscopic observation results showed that there were no intact spheres, but the particle size distribution data showed that 10% of them were larger than 20 μm, which may be unbroken spheres, posing a risk that it is impossible to fully check whether the carbon microspheres are sterile. After ball milling the carbon microspheres at 300 rpm for 20-30 min and at 400 rpm for 20-25 min, the particle size distribution Dv(90) values ​​of the carbon microspheres were all below 14 μm, and there were basically no particles with a particle size greater than 20 μm. The Dv(50) value was 3-5 μm, and the carbon microspheres were basically broken, which destroyed the adsorption of microorganisms on the carbon microspheres, allowing bacteria and fungi to be basically exposed. After ball milling at 500 rpm for 20 min, the carbon microspheres had a particle size distribution Dv(90) value of 8.68 μm, with 99.8% of the particles having a diameter less than 20 μm and virtually no particles larger than 20 μm, indicating good microsphere fragmentation. Furthermore, after fragmentation at 500 rpm for 20 min, the average particle size of the microspheres was 4-5 μm, disrupting the adsorption of microorganisms on the carbon microspheres and allowing bacteria and fungi to be fully exposed after microsphere fragmentation. While ball milling at 500 rpm for 25 min resulted in even finer microspheres with a better particle size distribution, disrupting microbial adsorption, the longer milling time was required.

[0087] Example 2: Suitability Test of Sterility Testing Method 1 sample NRT6003 Injection (This product is decayed to a non-radioactive state), batch number: YCM20220425, specification: 2ml / bottle, manufacturer: Chengdu Newrite Medical Technology Co., Ltd.

[0088] 2 strains Staphylococcus aureus [CMCC(B) 26003], Escherichia coli [CMCC(B) 44102], Clostridium sporogenes [CMCC(B) 64941], Bacillus subtilis [CMCC(B) 63501], Candida albicans [CMCC(F) 98001], and Aspergillus niger [CMCC(F) 98003] were sourced from the China National Institutes for Food and Drug Control.

[0089] 3. Culture medium Information on the culture media used in this study is shown in Table 2.

[0090] Table 2 Information on Culture Media Used in Research The results of the culture medium suitability test meet the requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0091] 4. Instruments The main equipment used in this study is shown in Table 3.

[0092] Table 3 Information on Research Instruments and Equipment 5 methods Perform the procedure according to the requirements of Section 1101, General Chapter, Aseptic Test Method, Radiopharmaceuticals, Part IV of the 2020 edition of the Chinese Pharmacopoeia (take one vial of the test sample and inoculate it in equal amounts into 7.5 ml thioglycolate fluid medium and tryptic soy peptone liquid medium. The inoculation volume per tube is 0.2 ml). All operations must be performed in a qualified clean environment, i.e., in a biosafety cabinet. All experimental instruments and reagents have been sterilized in an autoclave. Simultaneously, the suitability of the culture medium used must be checked to ensure its sterility.

[0093] 6 Consumables Anaerobic bag, model: AnaeroPack-Anaero, batch number: 2138LJ-2, manufacturer: MITSUBISHI GASCHEMICAL CO., INC., used for Clostridium sporogenes culture.

[0094] 7. Preparation of bacterial culture 7.1 Take 1 ml of tryptone liquid culture of Staphylococcus aureus, Escherichia coli and Bacillus subtilis after culturing at 33℃ for 24 hours, and dilute it with 0.9% sterile sodium chloride solution to prepare bacterial suspensions with bacterial counts of approximately 2500-5000 cfu / ml and 250-500 cfu / ml, respectively, for later use.

[0095] 7.2 Take 1 ml of Clostridium sporogenes thioglycolate fluid culture that has been cultured at 33℃ for 24 hours, and dilute it with 0.9% sterile sodium chloride solution to prepare bacterial suspensions with bacterial counts of approximately 2500-5000 cfu / ml and 250-500 cfu / ml, for later use.

[0096] 7.3 Take an appropriate amount of Sabouraud dextrose agar culture of Candida albicans that has been cultured at 23℃ for 48 hours, and dilute it with 0.9% sterile sodium chloride solution to prepare bacterial suspensions with bacterial counts of approximately 2500-5000 cfu / ml and 250-500 cfu / ml, for later use.

[0097] 7.4 Take a Sabouraud dextrose agar culture of Aspergillus niger that has been cultured at 23℃ for 7 days, add 3-5 ml of 0.9% sterile sodium chloride solution containing 0.05% polysorbate 80, and wash away the spores. Then aspirate 1 ml of the spore suspension and dilute it with 0.9% sterile sodium chloride solution containing 0.05% polysorbate 80 to prepare bacterial suspensions with a bacterial count of approximately 2500-5000 CFU / ml and 250-500 CFU / ml, for later use.

[0098] 8. Method suitability test 8.1 Preliminary Experiment 1 8.1.1 Experimental Group This product is NRT6003 injection, a carbon microsphere product. To simultaneously check the sterility of the microspheres inside and out, the product was first crushed using a ball mill.

[0099] Take one bottle (2ml) of this product and add it to a sterile ball mill jar. Then add 0.2ml of the prepared test bacterial suspension (about 2500-50000 cfu / ml). Ball mill at 800rpm for 30min. Take 0.2ml of the ball-milled sample and aseptically inoculate it into 7.5ml of thioglycolate fluid medium and tryptic soy liquid medium, respectively.

[0100] 8.1.2 Sample Group Use 0.9% sterile sodium chloride solution instead of bacterial solution, and follow the same procedure as the experimental group.

[0101] 8.1.3 Positive Control Use 0.9% sterile sodium chloride solution instead of the sample, and follow the same procedure as the experimental group.

[0102] 8.1.4 Negative Control Replace the sample and bacterial solution with 0.9% sterile sodium chloride solution, and follow the same procedure as the experimental group.

[0103] Thioglycolate fluid medium was incubated at 33℃ for 5 days, and tryptic soy broth was incubated at 23℃ for 5 days. Because the carbon microsphere samples were very turbid, the presence of microbial growth could not be determined visually. Therefore, 1 ml of culture was taken daily and transferred to 100 ml of thioglycolate fluid medium and tryptic soy broth, and incubated at the corresponding temperatures for another 4 days before observation. The results are shown in Table 4. All cultures were conducted in an incubator.

[0104] Table 4. Observation results of the first culture in the preliminary experiment. The experimental results showed that no Aspergillus niger grew in the test group when operated according to this method, therefore this method cannot be used for sterility testing of this product. Because the surface tension of the sample is relatively high after ball milling, in order to facilitate more accurate sampling, it is proposed to conduct a second preliminary experiment using an equal volume dilution of the sample.

[0105] 8.2 Preliminary Experiment Two 8.2.1 Experimental Group Take one bottle (2ml) of this product and add it to a sterile ball mill jar. Then add 2ml of the prepared test bacterial suspension (about 250-500cfu / ml). Ball mill at 800rpm for 30min. Take 0.4ml of the ball-milled sample (equivalent to 0.2ml test volume) and aseptically inoculate it into 15ml of thioglycolate fluid medium and tryptic soy liquid medium, respectively.

[0106] 8.2.2 Sample Group Use 0.9% sterile sodium chloride solution instead of bacterial solution, and follow the same procedure as the experimental group.

[0107] 8.2.3 Positive control Use 0.9% sterile sodium chloride solution instead of the sample, and follow the same procedure as the experimental group.

[0108] 8.2.4 Negative Control Replace the sample and bacterial solution with 0.9% sterile sodium chloride solution, and follow the same procedure as the experimental group.

[0109] Thioglycolate fluid medium was incubated at 33℃ for 5 days, and tryptic soy broth was incubated at 23℃ for 5 days. Because the carbon microsphere samples were very turbid, the presence of microbial growth could not be determined visually. Therefore, 1 ml of culture was taken daily and transferred to 100 ml of thioglycolate fluid medium and tryptic soy broth, and incubated at the corresponding temperatures for another 4 days before observation. The results are shown in Table 5. All cultures were conducted in an incubator.

[0110] Table 5. Observation results of culture in the second preliminary experiment The experimental results showed that, following this method, neither Aspergillus niger nor Candida albicans grew in the experimental group; therefore, this method cannot be used for sterility testing of this product. The experiment revealed that ball milling at 800 rpm for 30 minutes had a significant impact on molds and yeasts; therefore, molds and yeasts will be used in preliminary experiment three.

[0111] 3. Preliminary Experiment Three 8.3.1 Experimental Group Take one bottle (2ml) of this product and add it to a sterile ball mill jar. Then add 2ml of the prepared Candida albicans and Aspergillus niger suspension (about 250-500 cfu / ml). After ball milling at 500rpm for 20min, take 0.4ml of the ball-milled sample and aseptically inoculate it into 15ml of tryptic soy liquid culture medium.

[0112] 8.3.2 Sample Group Use 0.9% sterile sodium chloride solution instead of bacterial culture, and take 0.4 ml of the ball-milled sample and aseptically inoculate it into 15 ml of thioglycolate fluid culture medium. The rest of the procedure is the same as the experimental group.

[0113] 8.3.3 Positive Control Use 0.9% sterile sodium chloride solution instead of the sample, and follow the same procedure as the experimental group.

[0114] 8.3.4 Negative Control Replace the sample and bacterial culture with 0.9% sterile sodium chloride solution. Take another 0.4 ml of the ball-milled sample and aseptically inoculate it into 15 ml of thioglycolate fluid medium. The rest of the procedure is the same as the experimental group.

[0115] Thioglycolate fluid medium was incubated at 33℃ for 5 days, and tryptic soy broth was incubated at 23℃ for 5 days. Because the carbon microsphere samples were very turbid, the presence of microbial growth could not be determined visually. Therefore, 1 ml of culture was taken daily and transferred to 100 ml of thioglycolate fluid medium and tryptic soy broth, and incubated at the corresponding temperatures for another 4 days before observation. The results are shown in Table 6. All cultures were conducted in an incubator.

[0116] Table 6. Observation results of the three preliminary experiments The experimental results show that both test bacteria grew well when operated according to this method, and this method is intended to be used for the sterility test of this product.

[0117] 8.4 Suitability Test for Aseptic Methods 8.4.1 Experimental Group Take one bottle (2ml) of this product and add it to a sterile ball milling jar. Then add 2ml of the prepared test bacterial suspension (about 250-500cfu / ml). Ball mill at 500rpm for 20min. Take 0.4ml of the ball-milled sample and aseptically inoculate it into 15ml of thioglycolate fluid medium and tryptic soy liquid medium, respectively.

[0118] 8.4.2 Sample Group Use 0.9% sterile sodium chloride solution instead of bacterial solution, and follow the same procedure as the experimental group.

[0119] 8.4.3 Positive Control Use 0.9% sterile sodium chloride solution instead of the sample, and follow the same procedure as the experimental group.

[0120] 8.4.4 Negative Control Replace the sample and bacterial solution with 0.9% sterile sodium chloride solution, and follow the same procedure as the experimental group.

[0121] Thioglycolate fluid medium was incubated at 33℃ for 5 days, and tryptic soy broth was incubated at 23℃ for 5 days. Because the carbon microsphere samples were very turbid, the presence of microbial growth could not be determined visually. Therefore, 1 ml of culture was taken daily and transferred to 100 ml of thioglycolate fluid medium and tryptic soy broth, and incubated at the corresponding temperatures for another 4 days before observation. The results are shown in Table 7. All cultures were conducted in an incubator.

[0122] Table 7. Results of culture observation for aseptic suitability test The test results showed that, when operated according to this method, the product had no antibacterial properties, and all six test bacteria grew well. Therefore, it can be used... This method is used to test the sterility of this product.

[0123] The above suitability tests eliminated the risk of false negatives. If the product itself contains antimicrobial components (such as antibiotics or preservatives), it will inhibit the growth of any potentially present microorganisms. In this case, without validation, even if the product contains bacteria, it might not be detectable. Furthermore, the suitability tests demonstrated that the method used does not affect the growth of microorganisms (including test bacteria and microorganisms that may be present in the sample). Therefore, this embodiment confirms through suitability testing that the method effectively eliminates interference and ensures the reliability of the detection results.

[0124] Example 3: Suitability Test of Sterility Testing Method 1 Sample yttrium[ 89 Y] Carbon microsphere injection, batch number: YCM20200928 / YCM20220425, manufacturer: Chengdu Newrite Medical Technology Co., Ltd.

[0125] 2 strains Staphylococcus aureus [CMCC(B) 26003], Escherichia coli [CMCC(B) 44102], Clostridium sporogenes [CMCC(B) 64941], Bacillus subtilis [CMCC(B) 63501], Candida albicans [CMCC(F) 98001], and Aspergillus niger [CMCC(F) 98003] were sourced from the China National Institutes for Food and Drug Control.

[0126] 3. Culture media and reagents Information on the culture medium and reagents used in this embodiment is shown in Table 8.

[0127] Table 8 Information on culture media and reagents used in this embodiment 4. Instruments The main equipment in this embodiment is shown in Table 9.

[0128] Table 9. Information on instruments and equipment used in this embodiment. All operations must be performed in a qualified clean environment, i.e., within a biosafety cabinet. All laboratory equipment and reagents must be sterilized in an autoclave. Furthermore, the suitability of the culture media used must be checked to ensure their sterility.

[0129] 5. Recovery test and aseptic method 5.1 Preparation of bacterial culture Fresh cultures of Staphylococcus aureus, Bacillus subtilis, and Escherichia coli were inoculated into tryptic soy broth; fresh cultures of Clostridium sporogenes were inoculated into thioglycolate fluid medium and incubated at 33°C for 18-24 hours; fresh cultures of Candida albicans were inoculated into Sabouraud dextrose broth and incubated at 23°C for 18-24 hours; appropriate amounts of the above cultures were diluted with sterile sodium chloride-peptone buffer (pH 7.0) to a suitable gradient to prepare a culture with a bacterial count not exceeding 100 μL per ml. CFU bacterial suspension; Fresh culture of Aspergillus niger was inoculated onto Sabouraud dextrose agar slant and cultured at 23°C for 5 days. The spores were washed off with sterile sodium chloride-peptone buffer at pH 7.0 containing 0.05% (ml / ml) polysorbate 80. The spore suspension was then transferred to a sterile test tube and diluted with sterile sodium chloride-peptone buffer at pH 7.0 containing 0.05% (ml / ml) polysorbate 80 to prepare a spore suspension containing no more than 100 CFU per ml.

[0130] Dilution factor: Staphylococcus aureus (diluted to 10) -7 Bacillus subtilis (diluted to 10) -5 Escherichia coli (diluted to 10) -7 Clostridium sporogenes (diluted to 10) -6 Candida albicans (diluted to 10) -5 Aspergillus niger (diluted to 10) -4 When diluting, a vortex mixer should be used to mix thoroughly.

[0131] Prepare the bacterial solution using the above method and set aside for later use.

[0132] The above bacterial suspensions were inoculated onto tryptic soy agar, thioglycolate fluid medium, or Sabouraud dextrose agar, respectively, and cultured and counted. Staphylococcus aureus, Bacillus subtilis, and Escherichia coli were cultured on tryptic soy agar, Clostridium sporogenes on thioglycolate fluid medium, and Candida albicans and Aspergillus niger on Sabouraud dextrose agar.

[0133] 5.2 Recovery Rate Test To measure the bacterial culture recovery rate, the following groups were set up: Sample group: Yttrium added [ 89 One vial (2 ml) of carbon microsphere injection solution and 2 ml of pH 7.0 sterile sodium chloride-peptone buffer.

[0134] Positive control group: Add 2 ml of pH 7.0 sterile sodium chloride-peptone buffer and 2 ml of bacterial dilution solution from step 5.1.

[0135] Positive control group for test sample: Add one sample bottle (2ml) and 2ml of bacterial diluent from step 5.1.

[0136] For Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Aspergillus niger, and Candida albicans, take 0.5 ml of the diluted bacterial solution from step 5.1 and place it in the corresponding culture dish. Prepare two parallel dishes for each bacterial solution. The dishes containing Staphylococcus aureus, Escherichia coli, and Bacillus subtilis should be inoculated with tryptic soy agar, while the dishes containing Aspergillus niger and Candida albicans should be inoculated with Sabouraud dextrose agar. Stir thoroughly with a sterile glass rod and allow the dishes to cool and solidify. For Clostridium sporogenes, inoculate 0.5 ml of the well-mixed Clostridium sporogenes culture into 500 ml of thioglycolate fluid medium. Place the above media in a biochemical incubator or a water-jacketed constant temperature incubator and incubate at the specified temperature, then count the bacteria. Thioglycolate fluid medium and tryptic soy agar are incubated at 33°C, while Sabouraud dextrose agar is incubated at 23°C. The obtained bacterial solution is called bacterial solution (stock solution), which is the diluted bacterial solution without any treatment.

[0137] Add the positive control group material to the ball mill jar and shake well by hand. For Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Aspergillus niger, and Candida albicans, take 1 ml of the well-shaken bacterial solution and place it in the corresponding culture dish. Prepare two parallel dishes for each bacterial solution. The dishes containing Staphylococcus aureus, Escherichia coli, and Bacillus subtilis are inoculated with tryptic soy agar, while the dishes containing Aspergillus niger and Candida albicans are inoculated with Sabouraud dextrose agar. Stir well with a sterile glass rod and allow the dishes to cool and solidify. For Clostridium sporogenes, take 1 ml of the well-shaken Clostridium sporogenes bacterial solution and inoculate it into 500 ml of thioglycolate fluid medium. Place the above media in a biochemical incubator or a water-jacketed constant temperature incubator and incubate at the specified temperature, then count the bacteria. Thioglycolate fluid medium and tryptic soy agar are incubated at 33°C, and Sabouraud dextrose agar is incubated at 23°C. The obtained bacterial solution is called bacterial solution (after rinsing), which is the bacterial solution after dilution and mixing in the ball mill jar.

[0138] The ball mill jars containing the positive control group and the test sample positive control group materials were placed in a vertical planetary ball mill and milled at 500 rpm for 20 min. For Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Aspergillus niger, and Candida albicans, 0.1 ml of the milled material was placed in petri dishes, with two parallel dishes prepared for each species. The dishes containing Staphylococcus aureus, Escherichia coli, and Bacillus subtilis were inoculated with tryptophan-soybean agar, while the dishes containing Aspergillus niger and Candida albicans were inoculated with Sabouraud dextrose agar. The dishes were stirred thoroughly with a sterile glass rod and allowed to cool and solidify. For Clostridium sporogenes, 0.1 ml of the milled material was inoculated into 500 ml of thioglycolate fluid medium. The above media were placed in a biochemical incubator or a water-jacketed constant temperature incubator and cultured and counted at the specified temperature. Thioglycolate fluid medium and tryptophan-soybean agar were cultured at 33°C, and Sabouraud dextrose agar was cultured at 23°C. The bacterial solution obtained in the positive control group is called bacterial solution (after ball milling), which is the bacterial solution after dilution and ball milling. The bacterial solution obtained in the positive control group of the test sample is called bacterial solution + sample (after ball milling), which is the solution after grinding the diluted bacterial solution and the sample together in a ball mill.

[0139] The microbial count and bacterial recovery rate in the bacterial culture are shown in Table 10 below (the values ​​are the average of two parallel plates).

[0140] Table 10 Results of microbial count and bacterial culture recovery (500 rpm 20 min) Note: Clostridium sporogenes is an anaerobic bacterium, and counting it during culture is difficult, so it was not counted; Bacillus subtilis was over-diluted, and the bacterial concentration was too low, so counting it was meaningless; no growth was observed in the sample group, i.e., the microbial count was 0.

[0141] Recovery rate of bacterial solution after wetting (%) = (bacterial solution (after wetting) - sample group) / bacterial solution (original solution) × 100%.

[0142] Recovery rate of bacterial solution after ball milling (%) = (bacterial solution (after ball milling) - sample group) / bacterial solution (after wetting) × 100%.

[0143] Recovery rate of bacterial solution + sample (%) = (bacterial solution + sample (after ball milling) - sample group) / bacterial solution (after ball milling) × 100%.

[0144] According to General Chapter 1105 of the 2020 edition of the Chinese Pharmacopoeia, a counting method is acceptable when its recovery rate is not less than 50%.

[0145] A recovery rate of ≥50% indicates that the sample processing method is effective, and the sample can be processed using this method (ball milling).

[0146] A recovery rate of <50% indicates that the sample processing method has a significant damaging effect on microorganisms and should not be used to process samples (ball milling).

[0147] Under the condition of 300 rpm for 5 min, microscopic observation showed no intact spheres, but particle size distribution data showed that 10% of the particles were larger than 20 μm, which may contain unbroken spheres. This posed a risk of not being able to fully inspect whether the carbon microspheres were sterile, so this condition was excluded. Under the condition of ball milling at 500 rpm for 20 min, the carbon microsphere particle size distribution data showed that 99.8% of the particles were smaller than 20 μm, and there were virtually no particles larger than 20 μm. The carbon microspheres were well broken down, and the bacterial solution recovery rate was greater than 50%. Therefore, this sample treatment method was effective.

[0148] Observe whether there is bacterial growth in the bacterial solution (after ball milling) and the bacterial solution + sample (after ball milling) group. The results are shown in Table 11 below.

[0149] Table 11 shows the culture results of the bacterial suspension (after ball milling) and the bacterial suspension + sample (after ball milling) group (500 rpm for 20 min). The results above indicate that the carbon microsphere injection has no inhibitory effect on the tested strains. Furthermore, after ball milling at 500 rpm for 20 min, the positive control group of the test sample still showed positive results after co-treatment with the strain, indicating that ball milling itself does not affect the normal growth of microorganisms.

[0150] 5.3 Aseptic Methods The testing methods are based on the 2020 edition of the Chinese Pharmacopoeia, Part IV, General Chapter 1101, Sterility Test Method, and General Chapter 1401, Radiopharmaceutical Assay Method.

[0151] The following groups were set up for aseptic testing methods: Sample group: Add one sample vial (2ml) and 2ml of pH7.0 sterile sodium chloride-peptone buffer to the ball mill jar.

[0152] Positive control group: Add 2 ml of pH 7.0 sterile sodium chloride-peptone buffer and 2 ml of bacterial dilution solution from step 5.1 to the ball mill jar.

[0153] Positive control group for test sample: Add one sample bottle (2 ml) and 2 ml of bacterial diluent from step 5.1 to the ball mill jar.

[0154] Two sets of ball milling conditions were set: ball milling at 300 rpm for 5 minutes and ball milling at 500 rpm for 20 minutes.

[0155] Positive control group and test sample positive control group: Take 6 test tubes containing 7.5 ml of thioglycolate fluid medium. Inoculate 0.2 ml of the test sample positive control group containing Staphylococcus aureus, Escherichia coli, and Clostridium sporogenes in 3 test tubes respectively. Inoculate 0.2 ml of the test sample positive control group containing Staphylococcus aureus, Escherichia coli, and Clostridium sporogenes in 3 test tubes respectively. Take 6 test tubes containing 7.5 ml of tryptic soy broth. Inoculate 0.2 ml of the test sample positive control group containing Aspergillus niger, Candida albicans, and Bacillus subtilis in 3 test tubes respectively. Inoculate 0.2 ml of the test sample positive control group containing Aspergillus niger, Candida albicans, and Bacillus subtilis in 3 test tubes respectively.

[0156] Sample groups: 0.2 ml of the ball-milled sample group was inoculated into test tubes containing 7.5 ml of thioglycolate fluid medium and tryptic soy peptone liquid medium, respectively.

[0157] Thioglycolate fluid culture medium was placed in a water-jacketed constant temperature incubator and cultured at 33°C for observation. The culture time should not exceed 5 days.

[0158] Tryptic soybean peptone liquid culture medium was placed in a biochemical incubator and incubated at 23°C for observation. The incubation time should not exceed 5 days.

[0159] After the culture is completed, observe whether there is bacterial growth, and record the results in Tables 12 and 13 below.

[0160] Table 12 Observation results of aseptic method (300 rpm 5 min) Table 13 Observation results of aseptic method (500 rpm 20 min) The culture results showed that bacteria grew in both the positive control group and the positive control group of the test sample, while no bacteria grew in either of the two culture media of the sample group. This indicates that the microspheres and broken microspheres have no antibacterial effect or the antibacterial effect is negligible. The sterility test of the test sample can be carried out according to this test method and test conditions.

[0161] Example 4: Sterility test of NRT6003 injection solution Sample: NRT6003 injection (this product is decayed to a non-radioactive state), batch number: RCM20201101 / RCM20201018 / RCM20210805, manufacturer: Chengdu Newrite Medical Technology Co., Ltd.

[0162] Three batches of NRT6003 injection solution (batch numbers: RCM20201101 / RCM20201018 / RCM20210805) were subjected to sterility testing and cultured for 14 days to observe for bacterial growth. Unless otherwise specified in this example, other specific operating methods are described in Example 2.

[0163] The sterility testing method is as follows: Test sample group: Aseptically take 1 bottle (2ml) of this product and add it to a sterile ball mill jar, then add 2ml of sterile physiological saline, and ball mill at 500rpm for 20min. Take 0.4ml of the ball-milled sample and aseptically inoculate it into 15ml of thioglycolate fluid medium and tryptic soy liquid medium, respectively.

[0164] Positive control group: Staphylococcus aureus was used as the positive control bacterium. The preparation of the bacterial suspension was the same as in the applicability test 5.1 of Example 2. Take Staphylococcus aureus bacterial suspension (2 ml), add 2 ml of sterile physiological saline and mix well. Ball mill at 500 rpm for 20 min. Take 0.4 ml of the mixture and aseptically inoculate it into 15 ml of thioglycolate fluid medium and tryptic soy liquid medium, respectively.

[0165] Negative control group: Take 0.4 ml of sterile physiological saline, ball mill at 500 rpm for 20 min, and aseptically inoculate into 15 ml of thioglycolate fluid medium and tryptic soy broth.

[0166] Thioglycolate fluid medium was incubated at 33℃ for 14 days, and tryptic soy saturated liquid medium was incubated at 23℃ for 14 days. The presence of bacterial growth was observed in the culture media. The test results of three batches of NRT6003 injection solution according to the above method are shown in Table 14.

[0167] Table 14. Sterility test results of three batches of NRT6003 injection solution This method was used to perform sterility testing on microspheres of multiple batches of NRT6003 injection. All batches showed no bacterial growth. The positive control group showed good bacterial growth, while the negative control group showed no bacterial growth. Furthermore, ball milling disrupted the adsorption of microorganisms onto the carbon microspheres, allowing bacteria and fungi to be fully exposed after the microspheres were broken. Therefore, in addition to testing the sterility of the outside of the microspheres, the sterility of the inside of the microspheres was also tested. These results demonstrate that the NRT6003 injection microspheres are sterile both externally and internally using the sterility testing method defined in this invention, and the product sterility test results meet the requirements.

[0168] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A method for aseptic testing of a microsphere injection solution, characterized in that, The method includes a step of breaking up the microsphere injection solution to be examined. The microspheres to be examined have a particle size range of 15-80 μm, and the average particle size of the broken microspheres is 3-6 μm.

2. The method according to claim 1, characterized in that, The method includes the following steps: a) Provide a microsphere injection solution to be inspected, wherein the microspheres have a particle size range of 15-80 μm; b) The positive control group containing positive control bacteria, the test sample group containing microsphere injection, and the negative control group without microsphere injection and positive control bacteria were crushed separately so that the average particle size of the crushed microspheres was 3-6 μm. c) The fragmented positive control group, test sample group, and negative control group were inoculated into sterile culture medium and cultured at 20-35℃ to obtain cultures; and d) Observe whether the bacteria grow on the sterile culture medium. If the positive control group has bacterial growth, while the test sample group and the negative control group have no bacterial growth, then the microsphere injection solution to be tested is determined to be sterile.

3. The method according to claim 1 or 2, characterized in that, The microspheres to be inspected have a particle size range of 20-45 μm.

4. The method according to claim 1 or 2, characterized in that, The microsphere injection solution to be inspected is selected from one or more of the following: carbon microsphere injection solution, resin microspheres, lactic acid polymer microspheres, hydroxyapatite microspheres, gelatin microspheres, silica microspheres, and calcium alginate microspheres.

5. The method according to claim 1 or 2, characterized in that, The crushing was carried out by ball milling at 300-500 rpm for 15-30 minutes.

6. The method according to claim 1 or 2, characterized in that, The particle size distribution of the fragmented microsphere injection solution is as follows: Dv(10) value is 1-2 μm, Dv(50) value is 3-6 μm, Dv(90) value is 6-14 μm, and D[4,3] value is 3-7 μm.

7. The method according to claim 2, characterized in that, The positive control bacterium is Staphylococcus aureus; the sterile culture medium is selected from thioglycolate fluid medium and / or tryptic soy peptone liquid medium.

8. The method according to claim 7, characterized in that, The positive control group, the test sample group, and the negative control group were all inoculated into thioglycolate fluid medium and tryptic soy peptone liquid medium, respectively.

9. The method according to claim 2, characterized in that, The inoculation amount of the positive control bacteria shall not exceed 100 cfu.

10. The method according to claim 1 or 2, characterized in that, The aseptic testing method is performed under aseptic conditions.

11. The method according to claim 4, characterized in that, If the sterile culture medium becomes turbid or if the microspheres are carbon microspheres, the method further includes the step of: after step c), taking a portion of the culture daily and continuing to culture it on a sterile culture medium at 20-35°C.

12. The method according to claim 1 or 2, characterized in that, The sterility testing methods are intraglobal and extraglobal sterility testing methods.

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