A high-efficiency detection method for multiple microbial limits of tobramycin

Abstract

This invention discloses a highly efficient method for detecting multiple microbial limits of tobramycin, belonging to the field of microbial detection technology. This invention pioneers an enzyme-chemical synergistic neutralization system constructed from aminoglycoside-3-acetyltransferase, aminoglycoside-3'-phosphotransferase, magnesium chloride hexahydrate, and sodium chloride-peptone buffer. This system completely eliminates the antibacterial activity of tobramycin through a direct enzymatic inactivation pathway, overcoming the limitation of incomplete neutralization in traditional methods. Based on this system, and combined with process optimization involving post-inoculation of bacteria and the addition of inactivating enzymes and chemical neutralizing agents to the culture medium, a highly efficient detection method has been successfully developed. This method possesses four core advantages: high efficiency, specificity, environmental friendliness, and synergy. It can completely eliminate the antibacterial interference of tobramycin, significantly improving the accuracy and reliability of microbial limit test results.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, specifically to a highly efficient method for detecting multiple microbial limits of tobramycin. Background Technology

[0002] Tobramycin is an aminoglycoside antibiotic with potent antibacterial activity against Enterobacteriaceae, Pseudomonas aeruginosa, and Staphylococcus aureus. However, its antibacterial effect can lead to false negatives in microbial limit tests, affecting the accuracy of the assay. Therefore, appropriate methods must be employed to eliminate this interference.

[0003] Currently, microbial limit testing of tobramycin mainly relies on membrane filtration combined with extensive rinsing. For example, conventional methods require high-level dilution of the sample (1:1000 dilution level) and 500-900 mL of rinsing solution to partially eliminate antibacterial activity. However, this method is cumbersome and incomplete neutralization leads to low microbial recovery rates, affecting the accuracy of the results. In recent years, researchers have attempted to introduce chemical neutralizing agents into the diluent to optimize the antibacterial effect. For example, patent application CN116004759A, "A method for detecting the microbial limit of tobramycin," proposes adding polysorbate 80 as a neutralizing agent to the diluent; patent application CN116855573A, "A method for detecting the microbial limit of an intermediate of an aminoglycoside antibiotic," uses a composite neutralizing agent containing polysorbate 80, soybean lecithin, and metal salt ions. However, these chemical neutralization methods do not completely eliminate the antibacterial properties of tobramycin, especially its neutralization effect on Gram-positive bacteria such as Staphylococcus aureus and Bacillus subtilis is limited, leading to inaccurate final test results. In addition, these methods have problems such as poor effect on high-concentration samples and the need for extensive rinsing.

[0004] Therefore, developing a method that can completely eliminate the antibacterial properties of tobramycin and improve the accuracy of detection is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] Therefore, this invention provides a highly efficient method for detecting multiple microbial limits of tobramycin, which solves the technical problems of incomplete elimination of antibacterial activity and low detection sensitivity in the prior art through the synergistic effect of specific inactivating enzymes and chemical neutralizing agents.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention proposes a highly efficient method for detecting multiple microbial limits of tobramycin, comprising the following steps: (1) Take the target bacteria and prepare a bacterial suspension; (2) Mix the tobramycin test sample and diluent according to the mass-volume ratio to prepare the tobramycin test solution; (3) Take the tobramycin test solution obtained in step (2) and filter it through a filter membrane. Use the diluent as the rinsing solution to rinse the filter membrane. Repeat 5 times. Add the bacterial suspension prepared in step (1) in the last rinse and filter dry. Transfer the filter membrane to a culture medium containing a neutralizing agent and incubate at 30-35℃ for 18-24h to obtain the test sample. (4) Observe the test sample obtained in step (3), record the colony growth, and calculate the colony recovery ratio.

[0007] Further, the neutralizing agent mentioned in step (3) is at least one of an inactivating enzyme and a chemical neutralizing agent.

[0008] Further, the diluent mentioned in steps (2) and (3) is a synergistic neutralizing solution, which is composed of an inactivating enzyme, a chemical neutralizing agent and a solvent; the mass-volume ratio of the tobramycin test sample to the diluent is 1:5-1:10.

[0009] Furthermore, the inactivating enzyme is selected from at least one of aminoglycoside-3-acetyltransferase (AAC(3), such as AAC(3)-I, AAC(3)-II, AAC(3)-IV, etc.), aminoglycoside-3'-phosphotransferase (APH(3'), such as APH(3')-I and APH(3')-II) and aminoglycoside-4'-nucleotide transferase.

[0010] Further, the inactivating enzyme is aminoglycoside-3-acetyltransferase (AAC(3)) and aminoglycoside-3'-phosphotransferase (APH(3')), the ratio of the activity units of aminoglycoside-3-acetyltransferase and aminoglycoside-3'-phosphotransferase is 1:1 to 1:2, the amount of aminoglycoside-3-acetyltransferase (AAC(3)) added is 10-20U, and the amount of aminoglycoside-3'-phosphotransferase (APH(3')) added is 10-20U.

[0011] Furthermore, the chemical neutralizing agent is selected from at least one of divalent metal salts, polysorbate-80, and soybean lecithin.

[0012] Furthermore, the chemical neutralizing agent is a divalent metal salt, the divalent metal salt is magnesium chloride hexahydrate, and the concentration of magnesium chloride hexahydrate is 0.5 wt.%-7.5 wt.%.

[0013] Furthermore, the concentration of the magnesium chloride hexahydrate is 1 wt.%-7 wt.%.

[0014] Furthermore, the solvent is sodium chloride-peptone buffer, and the pH of the sodium chloride-peptone buffer is 7.0.

[0015] Further, the target bacteria in step (1) are selected from at least one of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger; the concentration condition of the bacterial suspension is: the number of colonies contained in each 0.1 mL bacterial suspension does not exceed 100 CFU.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention constructs an enzyme-chemical synergistic neutralization system based on aminoglycoside-3-acetyltransferase (AAC(3)), aminoglycoside-3'-phosphotransferase (APH(3')), magnesium chloride hexahydrate, and pH 7.0 sodium chloride-peptone buffer, and combines this with an optimized detection process, offering the following advantages: (1) Fundamental elimination mechanism By using AAC(3) and APH(3') to deactivate the enzyme, the activity of tobramycin is directly destroyed through irreversible covalent modification (acetylation / phosphorylation), rather than traditional competitive inhibition, resulting in a more thorough effect.

[0017] (2) Multi-dimensional performance advantages High efficiency: Enzyme catalysis is highly efficient, capable of processing high-concentration samples in a short time and reducing the dilution factor; Specificity: It only degrades the target antibiotic and has no adverse effects on microbial growth; Environmental friendliness: Reduces the amount of rinsing solution used; Synergy: When used in conjunction with a chemical neutralizing agent (magnesium chloride hexahydrate), Mg 2+ It acts as a cofactor to activate phosphotransferases and simultaneously degrade other antibacterial components (such as preservatives) in the formulation, thus achieving a comprehensive solution.

[0018] (3) Process optimization The post-inoculation method is used in the filtration stage to avoid premature inactivation of microorganisms; the neutralization system is used continuously in the culture stage, which together ensures a high bacterial recovery rate and reliable results.

[0019] Based on the above, a method that can completely eliminate the antibacterial properties of tobramycin and improve detection accuracy has been successfully developed. It has four core advantages: high efficiency, specificity, environmental friendliness, and synergy. Combined with process optimization of adding bacteria after the addition and adding inactivating enzymes and chemical neutralizing agents to the culture medium, the accuracy of detection and microbial recovery rate have been significantly improved. Detailed Implementation

[0020] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] This invention proposes a highly efficient method for detecting multiple microbial limits of tobramycin, comprising the following steps: (1) Take the target bacteria and prepare a bacterial suspension; (2) Mix the tobramycin test sample and diluent according to the mass-volume ratio to prepare the tobramycin test solution; (3) Take the tobramycin test solution obtained in step (2) and filter it through a filter membrane. Use the diluent as the rinsing solution to rinse the filter membrane. Repeat 5 times. Add the bacterial suspension prepared in step (1) in the last rinse and filter dry. Transfer the filter membrane to a culture medium containing a neutralizing agent and incubate at 30-35℃ for 18-24h to obtain the test sample. (4) Observe the test sample obtained in step (3), record the colony growth, and calculate the colony recovery ratio.

[0022] Furthermore, in step (3), the neutralizing agent is at least one of an inactivating enzyme and a chemical neutralizing agent.

[0023] Furthermore, in steps (2) and (3), the diluent is a synergistic neutralizing solution, which is composed of an inactivating enzyme, a chemical neutralizing agent and a solvent; the mass-volume ratio of tobramycin test sample to diluent is 1:5-1:10.

[0024] Furthermore, the inactivating enzyme is selected from at least one of aminoglycoside-3-acetyltransferase (AAC(3), such as AAC(3)-I, AAC(3)-II, AAC(3)-IV, etc.), aminoglycoside-3'-phosphotransferase (APH(3'), such as APH(3')-I and APH(3')-II) and aminoglycoside-4'-nucleotide transferase.

[0025] Furthermore, the inactivating enzymes are aminoglycoside-3-acetyltransferase (AAC(3)) and aminoglycoside-3'-phosphotransferase (APH(3')), with an activity unit ratio of 1:1 to 1:2. The amount of aminoglycoside-3-acetyltransferase (AAC(3)) added is 10-20U, and the amount of aminoglycoside-3'-phosphotransferase (APH(3')) added is 10-20U.

[0026] Furthermore, the chemical neutralizing agent is selected from at least one of divalent metal salts, polysorbate-80, and soybean lecithin.

[0027] Furthermore, the chemical neutralizing agent is a divalent metal salt, which is magnesium chloride hexahydrate, and the concentration of magnesium chloride hexahydrate is 0.5 wt.%-7.5 wt.%.

[0028] Furthermore, the concentration of magnesium chloride hexahydrate is 1 wt.%-7 wt.%.

[0029] Furthermore, the solvent is sodium chloride-peptone buffer, and the pH of the sodium chloride-peptone buffer is 7.0.

[0030] Further, in step (1), the target bacteria are selected from at least one of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger; the concentration conditions of the bacterial suspension are: the number of colonies contained in each 0.1 mL bacterial suspension does not exceed 100 CFU.

[0031] The experimental materials used in this experiment are as follows: reagents pH 7.0 Sodium Chloride-Peptone Buffer: Catalog No.: B682019-0250, Shanghai Sangon Biotech Co., Ltd. Magnesium chloride hexahydrate: Product No.: A601336-0500, Shanghai Sangon Biotech Co., Ltd.; Polysorbate-80: Product No.: Z100237-0001, Shanghai Sangon Biotech Co., Ltd.; Restriction endonucleases NcoI and XhoI, pMD 18-T vector, and T4 ligase were all purchased from Shanghai Sangon Biotech Co., Ltd.

[0032] strains The clinical isolate of E. coli was provided by our laboratory; Staphylococcus aureus ATCC6538; Bacillus subtilis A330692; The above strains were stored in glycerol tubes at -80℃ and activated into third generation (P3) strains by slant culture before use.

[0033] Preparation Example 1 Preparation of target bacterial suspension Take an appropriate amount of Staphylococcus aureus / Bacillus subtilis and add it to 1.2 mL of 0.05% polysorbate-80 in pH 7.0 sodium chloride-peptone buffer (prepared by mixing 0.5 mL of polysorbate-80 with 1000 mL of pH 7.0 sodium chloride-peptone buffer). Mix thoroughly to obtain Staphylococcus aureus / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria). Store at 2-8℃ and use within 2 hours.

[0034] Preparation Example 2 Preparation of AAC(3)-Ⅰ / APH(3')-Ⅱa protein (1) Construction and identification of pET28-AAC(3)-Ⅰ / pET28-APH(3')-Ⅱa recombinant plasmid Using genomic DNA from clinical isolate E. coli as a template, the target gene (AAC(3)-Ⅰ or APH(3')-Ⅱa gene) was amplified to obtain the target PCR product. The target PCR product was ligated into the pMD18-T vector to construct the pMD18-T-AAC(3)-Ⅰ / pMD18-T-APH(3')-Ⅱa recombinant plasmid, and its sequence correctness was verified by sequencing. The recombinant plasmid and pET28a expression vector that had been identified were digested with NcoⅠ and XhoⅠ, and the digestion products of the recombinant plasmid and pET28a expression vector were recovered and purified. They were ligated overnight at 16℃ using T4 ligase to obtain the ligation product. The ligation product was transformed into competent E. coli BL21(DE3) cells, plated on LB solid medium (containing 30 μg / mL kanamycin), and screened for positive clones after incubation at 37℃. The plasmid was extracted and verified by enzyme digestion, electrophoresis and sequencing. The antibiotic resistance function of the recombinant bacteria was confirmed by the minimum inhibitory concentration (MIC) experiment.

[0035] (2) Fermentation expression The identified recombinant bacteria were inoculated into 100 mL LB liquid medium (containing 30 μg / mL kanamycin), and cultured overnight at 37°C with shaking. The culture was then transferred to 1 L LB liquid medium at a 2% inoculation rate. When OD... 600 When the concentration is 0.6, add IPTG as an inducer to a final concentration of 1.0 mM, induce at 25°C for 6 h, and collect the fermentation broth.

[0036] (3) Affinity purification of recombinant proteins The fermentation broth was centrifuged at 4℃ and 8000 r / min for 20 min to collect the bacterial cells. The cells were resuspended in sterile PBS and then sonicated on ice to obtain the sonicated fragment. This sonicated fragment was centrifuged at 4℃ and 20000 r / min for 30 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtered supernatant was then filtered through a Ni... 2+- Slowly pass the NTA column (pre-equilibrated with buffer) and elute sequentially with the following buffers: 100 mL binding buffer, 60 mL wash buffer (composed of 0.5 M NaCl, 60 mM indole and 20 mM Tris-HCl, pH=7.9), and 100 mL elution buffer (composed of 1 M indole, 0.5 M NaCl and 20 mM Tris-HCl, pH=7.9). Collect the eluent to obtain the target protein (AAC(3)-Ⅰ or APH(3')-Ⅱa protein) and store at -80℃ for later use.

[0037] (4) Western imprint verification Mix the AAC(3)-Ⅰ or APH(3')-Ⅱa protein elution buffer with an equal volume of 2×SDS loading buffer, denature in a boiling water bath for 5 min, and perform 15% SDS-PAGE electrophoresis. Transfer the protein from the SDS-PAGE gel to a nitrocellulose membrane; incubate with anti-His-Tag monoclonal antibody, develop with ECL substrate, and expose to X-ray film for 10 min (room temperature). If the size of the developed band is consistent with the expected molecular weight (AAC(3)-Ⅰ or APH(3')-Ⅱa protein) and there are no nonspecific bands, it indicates that the high-purity target protein (AAC(3)-Ⅰ or APH(3')-Ⅱa protein) has been successfully obtained.

[0038] (5) Preparation of inactivating enzyme stock solution AAC(3)-Ⅰ protein / APH(3')-Ⅱa protein was prepared into AAC(3)-Ⅰ protein solution (10U / mL) / APH(3')-Ⅱa protein solution (10U / mL) using sterile water.

[0039] Preparation Example 3 Preparation of culture medium Tryptic soy broth (TSB, 1000 mL): Weigh an appropriate amount of tryptic soy broth (according to the label instructions on each batch of broth), add 1000 mL of water to dissolve it at a gentle temperature, dispense it into Erlenmeyer flasks (100 mL / flask), sterilize at 121 °C for 15 min, and then add 10 mL of 10 U / mL AAC(3)-Ⅰ protein solution (obtained in Preparation Example 2), 10 mL of 10 U / mL APH(3')-Ⅱa protein solution (obtained in Preparation Example 2) and 10 g of magnesium chloride hexahydrate to each 1000 mL of broth. Mix thoroughly and store at 4 °C for later use.

[0040] Mannitol sodium chloride agar medium (MSA): Commercially available pre-prepared medium was used. Before use, 10 mL of 10 U / mL AAC(3)-Ⅰ protein solution (obtained in Preparation Example 2), 10 mL of 10 U / mL APH(3')-Ⅱa protein solution (obtained in Preparation Example 2), and 10 g of magnesium chloride hexahydrate were added to every 1000 mL of medium. After thorough mixing, the mixture was poured into plates.

[0041] Preparation Example 4 Preparation of diluent (1) Chemical neutralizer Take 1000 mL of pH 7.0 sodium chloride-peptone buffer, add 10 g of magnesium chloride hexahydrate and 1 mL of polysorbate-80 in sequence, vortex at room temperature until completely dissolved, dispense into Erlenmeyer flasks, autoclave, cool to room temperature, and store at room temperature away from light for later use.

[0042] (2) Synergistic neutralizing liquid Add 10g of magnesium chloride hexahydrate to 1000mL of autoclaved sodium chloride-peptone buffer (pH 7.0) and vortex at room temperature until completely dissolved. Add 10mL of 10U / mL AAC(3)-Ⅰ protein solution (obtained in Preparation Example 2) and 10mL of 10U / mL LAPH(3')-Ⅱa protein solution (obtained in Preparation Example 2), and gently stir until completely dissolved. Store at 4°C for later use. The concentration of magnesium chloride hexahydrate is 1wt.%, designated as synergistic neutralization solution 1.

[0043] Add 40g of magnesium chloride hexahydrate to 1000mL of autoclaved sodium chloride-peptone buffer (pH 7.0) and vortex at room temperature until completely dissolved. Add 10mL of 10U / mL AAC(3)-Ⅰ protein solution (obtained in Preparation Example 2) and 10mL of 10U / mL LAPH(3')-Ⅱa protein solution (obtained in Preparation Example 2), and gently stir until completely dissolved. Store at 4°C for later use. The concentration of magnesium chloride hexahydrate is 4wt.%, designated as synergistic neutralization solution 2.

[0044] Add 70g of magnesium chloride hexahydrate to 1000mL of autoclaved sodium chloride-peptone buffer (pH 7.0) and vortex at room temperature until completely dissolved. Add 10mL of 10U / mL AAC(3)-Ⅰ protein solution (obtained in Preparation Example 2) and 10mL of 10U / mL LAPH(3')-Ⅱa protein solution (obtained in Preparation Example 2), and gently stir until completely dissolved. Store at 4°C for later use. The concentration of magnesium chloride hexahydrate is 7wt.%, designated as synergistic neutralization solution 3.

[0045] Add 5g of magnesium chloride hexahydrate to 1000mL of autoclaved sodium chloride-peptone buffer (pH 7.0) and vortex at room temperature until completely dissolved. Add 10mL of 10U / mL AAC(3)-Ⅰ protein solution (obtained in Preparation Example 2) and 10mL of 10U / mL LAPH(3')-Ⅱa protein solution (obtained in Preparation Example 2), and gently stir until completely dissolved. Store at 4°C for later use. The concentration of magnesium chloride hexahydrate is 0.5wt.%, denoted as synergistic neutralization solution 4.

[0046] Add 75g of magnesium chloride hexahydrate to 1000mL of autoclaved sodium chloride-peptone buffer (pH 7.0) and vortex at room temperature until completely dissolved. Add 10mL of 10U / mL AAC(3)-Ⅰ protein solution (obtained in Preparation Example 2) and 10mL of 10U / mL LAPH(3')-Ⅱa protein solution (obtained in Preparation Example 2), and gently stir until completely dissolved. Store at 4°C for later use. The concentration of magnesium chloride hexahydrate is 7.5wt.%, designated as synergistic neutralization solution 5.

[0047] Add 10 mL of 10 U / mL AAC(3)-Ⅰ protein solution (obtained in Preparation Example 2) and 10 mL of 10 U / mL APH(3')-Ⅱa protein solution (obtained in Preparation Example 2) to 1000 mL of autoclaved pH 7.0 sodium chloride-peptone buffer, stir gently until completely dissolved, store at 4°C for later use, and designate it as Co-neutralization Solution 6.

[0048] The parameters for the above-mentioned autoclaving are: temperature 121℃, time 20min.

[0049] Example 1 1. Culture medium preparation The TSB (obtained in Preparation Example 3) / MSA (obtained in Preparation Example 3) from Preparation Example 3 were used as the test culture medium, and the control culture medium for both was kept on standby.

[0050] 2. Suitability test of culture medium for counting Referring to the 2020 edition of the Chinese Pharmacopoeia, section 1105 "Microbial Limit Tests for Non-Sterile Products: Microbial Counting Method," the suitability test of the culture medium for counting was conducted, and the following groups were established: Test group of culture medium: ≤100 CFU of Staphylococcus aureus / Bacillus subtilis were inoculated into TSB (obtained in Preparation Example 3) (two independent experiments) and incubated in an incubator at 30-35℃ for 18-24 h. Three parallel experiments were set up for each group. ≤100 CFU of Staphylococcus aureus / Bacillus subtilis were inoculated into MSA (obtained in Preparation Example 3) (two independent experiments) and incubated in an incubator at 30-35℃ for 18-72 h. Three parallel experiments were set up for each group. Control culture medium test group: The above test method was repeated by replacing the test culture medium with the control culture medium of each culture medium.

[0051] Record the average number of colonies on the culture medium of the above test group (expressed as mean ± standard deviation), and calculate the ratio of the average number of colonies on the tested solid culture medium to the average number of colonies on the control culture medium. If it is within the standard range of 0.5-2, and the colony morphology and size should be consistent with the colonies on the control culture medium, then the suitability of the culture medium for counting is confirmed to be in compliance with the regulations.

[0052] Table 1 below shows the results of the suitability test of the test culture medium for counting in the test group and the control culture medium of TSB (preparation example 3):

[0053] Table 2 below shows the results of the suitability test of the test culture medium for counting in the MSA (preparation example 3) test group and the control culture medium test group:

[0054] As shown in Tables 1 and 2 above, the ratio of the average colony count on the tested solid culture medium of TSB (obtained in Preparation Example 3) / MSA (obtained in Preparation Example 3) to the average colony count on the control culture medium is 0.97-1.00, which fully meets the range specified in the standard (0.5-2.0). Furthermore, the colony morphology and size should be consistent with those on the control culture medium. Therefore, the suitability of this counting method using TSB (obtained in Preparation Example 3) / MSA (obtained in Preparation Example 3) supplemented with inactivating enzymes (AAC(3)-Ⅰ protein and 10U APH(3')-Ⅱa protein) and 10g magnesium chloride hexahydrate meets the requirements.

[0055] Example 2 1. Preparation of tobramycin test solution Take 20g of tobramycin, add 100mL of diluent (the synergistic neutralizing solution 1 prepared in Preparation Example 4), mix well, and obtain a 1:5 tobramycin test solution.

[0056] 2. Suitability test of microbial limit test method 2.1 Experimental group setup 2.1.1 Test Sample Inspection Team Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (co-neutralizing solution 1); add 10 mL of 1:5 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (co-neutralizing solution 1) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (co-neutralizing solution 1) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (co-neutralizing solution 1) of the filter cartridge and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the test sample.

[0057] 2.1.2 Control group of test sample Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralization solution 1); add 10 mL of 1:5 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (synergistic neutralization solution 1) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (synergistic neutralization solution 1) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the test sample control sample.

[0058] 2.1.3 Positive control group Take one filter cartridge containing a filter membrane and wet the filter membrane with diluent (co-neutralizing solution 1); add 100 mL of diluent (co-neutralizing solution 1) to wash the filter cartridge and filter dry; use a pipette to draw 10 mL of diluent (co-neutralizing solution 1) to evenly rinse the inner wall of the filter cartridge and filter dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (co-neutralizing solution 1) of the filter cartridge and filter dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the positive control sample.

[0059] 2.1.4 Negative control group Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralization solution 1); add 100 mL of diluent (synergistic neutralization solution 1) to wash the filter cartridge and filter dry; use a pipette to draw 10 mL of diluent (synergistic neutralization solution 1) to rinse the inner wall of the filter cartridge evenly and filter dry; repeat the above rinsing steps 5 times and filter dry; transfer the filter membrane to 100 mL of TSB (obtained in preparation example 3) to obtain a negative control sample.

[0060] 2.1.5 Chemical neutralizing agent control group Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (chemical neutralizer); add 10 mL of 1:5 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (chemical neutralizer) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (chemical neutralizer) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (chemical neutralizer) of the filter cartridge and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the neutralizer control sample.

[0061] 2.2 Suitability Test of Aerobic Bacteria Counting Method The suitability test of the culture medium for counting was conducted in accordance with the 2020 edition of the Pharmacopoeia, "1105 Microbial Limit Tests for Non-sterile Products: Microbial Counting Method".

[0062] The samples from each of the above experimental groups (as shown in 2.1.1-2.1.5) were placed in an incubator at 30-35℃ and cultured for 18-24 hours to obtain cultures. After the culture was completed, each culture was streaked onto an MSA (preparation example 3) plate using an inoculation loop and cultured at 30-35℃ for 18-72 hours. Three parallel experiments were set up for each group.

[0063] Record the average colony count for each of the above test groups (expressed as mean ± standard deviation), and calculate the colony recovery ratio between the test sample group and the chemical neutralizing agent control group. The calculation formula is as follows:

[0064] The judgment criteria are as follows: a) The colony recovery ratio is in the range of 0.5-2.0; b) The size and shape of the colonies should be consistent with those on the control culture medium; c) The test sample control group and the negative control group should be free of bacterial growth.

[0065] Table 3 below shows the results of the total aerobic bacteria (Staphylococcus aureus / Bacillus subtilis) count test in the test sample group and the chemical neutralizer control group:

[0066] As shown in Table 3 above, when using 1:5 tobramycin test solution (10 mL) and synergistic neutralization solution 1 (magnesium chloride hexahydrate concentration of 1 wt.%) for microbial limit testing, the colony recovery ratios of all test sample groups (Staphylococcus aureus / Bacillus subtilis) were within the range specified in the standard (0.5-2.0), and the colony morphology and size should be consistent with the colonies on the control culture medium (in Example 1), indicating that the aerobic bacteria counting method complies with the regulations.

[0067] 2.3 Suitability test of control bacteria testing methods The suitability test of the control bacteria test method was conducted with reference to the 2020 edition of the Pharmacopoeia, "1106 Microbial Limit Tests for Non-sterile Products: Control Bacteria Test Method".

[0068] The samples from the above-mentioned test sample group, test sample control group, positive control group and negative control group were placed in an incubator at 30-35℃ and cultured for 18-24h to obtain cultures. After the culture was completed, the cultures of each test group were streaked onto MSA (obtained in Preparation Example 3) plates using an inoculation loop and cultured in an incubator at 30-35℃ for 18-72h. Three parallel experiments were set up for each group.

[0069] The judgment criteria are as follows: Test sample group and positive control group: Target control bacteria (Staphylococcus aureus / Bacillus subtilis) should be detected. The test sample control group and the negative control group should show no bacterial growth.

[0070] Table 4 below shows the results of the suitability test for the control bacteria testing method:

[0071] Note: "+" indicates that the target control bacteria were detected; "-" indicates that the target control bacteria were not detected.

[0072] As shown in Table 4 above, the target control bacteria (Staphylococcus aureus or Bacillus subtilis) were detected in both the positive control group and the test sample group, while no bacteria grew in both the negative control group and the test sample group, indicating that the test method for the control bacteria complies with the regulations.

[0073] Example 3 1. Preparation of tobramycin test solution Take 10g of tobramycin, add 100mL of diluent (synergistic neutralizing solution 1 prepared in Preparation Example 4), mix well, and obtain a 1:10 tobramycin test solution.

[0074] 2. Suitability test of microbial limit test method 2.1 Experimental group setup 2.1.1 Test Sample Inspection Team Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (co-neutralizing solution 1); add 10 mL of 1:10 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (co-neutralizing solution 1) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (co-neutralizing solution 1) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (co-neutralizing solution 1) of the filter cartridge and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the test sample.

[0075] 2.1.2 Control group of test sample Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralization solution 1); add 10 mL of 1:10 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (synergistic neutralization solution 1) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (synergistic neutralization solution 1) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the test sample control sample.

[0076] 2.1.3 Positive control group Take one filter cartridge containing a filter membrane and wet the filter membrane with diluent (co-neutralizing solution 1); add 100 mL of diluent (co-neutralizing solution 1) to wash the filter cartridge and filter dry; use a pipette to draw 10 mL of diluent (co-neutralizing solution 1) to evenly rinse the inner wall of the filter cartridge and filter dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (co-neutralizing solution 1) of the filter cartridge and filter dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the positive control sample.

[0077] 2.1.4 Negative control group Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralization solution 1); add 100 mL of diluent (synergistic neutralization solution 1) to wash the filter cartridge and filter dry; use a pipette to draw 10 mL of diluent (synergistic neutralization solution 1) to rinse the inner wall of the filter cartridge evenly and filter dry; repeat the above rinsing steps 5 times and filter dry; transfer the filter membrane to 100 mL of TSB (obtained in preparation example 3) to obtain a negative control sample.

[0078] 2.1.5 Chemical neutralizing agent control group Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (chemical neutralizer); add 10 mL of 1:10 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (chemical neutralizer) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (chemical neutralizer) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (chemical neutralizer) of the filter cartridge and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the neutralizer control sample.

[0079] 2.2 Suitability Test of Aerobic Bacteria Counting Method The suitability test of the culture medium for counting was conducted in accordance with the 2020 edition of the Pharmacopoeia, "1105 Microbial Limit Tests for Non-sterile Products: Microbial Counting Method".

[0080] The samples from each of the above experimental groups (as shown in 2.1.1-2.1.5) were placed in an incubator at 30-35℃ and cultured for 18-24 hours to obtain cultures. After the culture was completed, each culture was streaked onto an MSA (preparation example 3) plate using an inoculation loop and cultured at 30-35℃ for 18-72 hours. Three parallel experiments were set up for each group.

[0081] Suitability test of aerobic bacteria counting method Record the average colony count for each of the above test groups (expressed as mean ± standard deviation), and calculate the colony recovery ratio between the test sample group and the chemical neutralizing agent control group. The calculation formula is as follows:

[0082] The judgment criteria are as follows: a) The colony recovery ratio is in the range of 0.5-2.0; b) The size and shape of the colonies should be consistent with those on the control culture medium; c) The test sample control group and the negative control group should be free of bacterial growth.

[0083] Table 5 below shows the results of the total aerobic bacteria (Staphylococcus aureus / Bacillus subtilis) count test in the test sample group and the chemical neutralizer control group:

[0084] As shown in Table 5 above, when using 1:10 tobramycin test solution (10 mL) and synergistic neutralization solution 1 (with a magnesium chloride hexahydrate concentration of 1 wt.%) for microbial limit testing, the colony recovery ratios of all test sample groups (Staphylococcus aureus / Bacillus subtilis) were within the range specified in the standard (0.5-2.0), and the colony morphology and size should be consistent with the colonies on the control culture medium (in Example 1), indicating that the aerobic bacteria counting method complies with the regulations.

[0085] 2.3 Suitability test of control bacteria testing methods The suitability test of the control bacteria test method was conducted with reference to the 2020 edition of the Pharmacopoeia, "1106 Microbial Limit Tests for Non-sterile Products: Control Bacteria Test Method".

[0086] The samples from the above-mentioned test sample group, test sample control group, positive control group and negative control group were placed in an incubator at 30-35℃ and cultured for 18-24h to obtain cultures. After the culture was completed, the cultures of each test group were streaked onto MSA (obtained in Preparation Example 3) plates using an inoculation loop and cultured in an incubator at 30-35℃ for 18-72h. Three parallel experiments were set up for each group.

[0087] The judgment criteria are as follows: Test sample group and positive control group: Target control bacteria (Staphylococcus aureus / Bacillus subtilis) should be detected. The test sample control group and the negative control group should show no bacterial growth.

[0088] Table 6 below shows the results of the suitability test for the control bacteria:

[0089] Note: "+" indicates that the target control bacteria were detected; "-" indicates that the target control bacteria were not detected.

[0090] As shown in Table 6 above, the target control bacteria (Staphylococcus aureus or Bacillus subtilis) were detected in both the positive control group and the test sample group, while no bacterial growth was observed in both the negative control group and the test sample group, indicating that the test method for the control bacteria complies with the regulations.

[0091] Example 4 1. Preparation of tobramycin test solution Take 5g of tobramycin, add 100mL of diluent (synergistic neutralizing solution 1 prepared in Preparation Example 4), mix well, and obtain a 1:20 tobramycin test solution.

[0092] 2. Suitability test of microbial limit test method 2.1 Experimental group setup 2.1.1 Test Sample Inspection Team Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (co-neutralizing solution 1); add 10 mL of 1:20 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (co-neutralizing solution 1) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (co-neutralizing solution 1) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (co-neutralizing solution 1) of the filter cartridge and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the test sample.

[0093] 2.1.2 Control group of test sample Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralization solution 1); add 10 mL of 1:20 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (synergistic neutralization solution 1) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (synergistic neutralization solution 1) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the test sample control sample.

[0094] 2.1.3 Positive control group Take one filter cartridge containing a filter membrane and wet the filter membrane with diluent (co-neutralizing solution 1); add 100 mL of diluent (co-neutralizing solution 1) to wash the filter cartridge and filter dry; use a pipette to draw 10 mL of diluent (co-neutralizing solution 1) to evenly rinse the inner wall of the filter cartridge and filter dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (co-neutralizing solution 1) of the filter cartridge and filter dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the positive control sample.

[0095] 2.1.4 Negative control group Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralization solution 1); add 100 mL of diluent (synergistic neutralization solution 1) to wash the filter cartridge and filter dry; use a pipette to draw 10 mL of diluent (synergistic neutralization solution 1) to rinse the inner wall of the filter cartridge evenly and filter dry; repeat the above rinsing steps 5 times and filter dry; transfer the filter membrane to 100 mL of TSB (obtained in preparation example 3) to obtain a negative control sample.

[0096] 2.1.5 Chemical neutralizing agent control group Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (chemical neutralizer); add 10 mL of 1:20 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (chemical neutralizer) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (chemical neutralizer) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (chemical neutralizer) of the filter cartridge and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the neutralizer control sample.

[0097] 2.2 Suitability Test of Aerobic Bacteria Counting Method The suitability test of the culture medium for counting was conducted in accordance with the 2020 edition of the Pharmacopoeia, "1105 Microbial Limit Tests for Non-sterile Products: Microbial Counting Method".

[0098] The samples from each of the above experimental groups (as shown in 2.1.1-2.1.5) were placed in an incubator at 30-35℃ and cultured for 18-24 hours to obtain cultures. After the culture was completed, each culture was streaked onto an MSA (preparation example 3) plate using an inoculation loop and cultured at 30-35℃ for 18-72 hours. Three parallel experiments were set up for each group.

[0099] Suitability test of aerobic bacteria counting method Record the average colony count for each of the above test groups (expressed as mean ± standard deviation), and calculate the colony recovery ratio between the test sample group and the chemical neutralizing agent control group. The calculation formula is as follows:

[0100] The judgment criteria are as follows: a) The colony recovery ratio is in the range of 0.5-2.0; b) The size and shape of the colonies should be consistent with those on the control culture medium; c) The test sample control group and the negative control group should be free of bacterial growth.

[0101] Table 7 below shows the results of the total aerobic bacteria (Staphylococcus aureus / Bacillus subtilis) count test in the test sample group and the chemical neutralizer control group:

[0102] As shown in Table 7 above, when using 1:20 tobramycin test solution (10 mL) and synergistic neutralization solution 1 (with a magnesium chloride hexahydrate concentration of 1 wt.%) for microbial limit testing, the colony recovery ratios of all test sample groups (Staphylococcus aureus / Bacillus subtilis) were within the range specified in the standard (0.5-2.0), and the colony morphology and size should be consistent with the colonies on the control culture medium (in Example 1), indicating that the aerobic bacteria counting method complies with the regulations.

[0103] 2.3 Suitability test of control bacteria testing methods The suitability test of the control bacteria test method was conducted with reference to the 2020 edition of the Pharmacopoeia, "1106 Microbial Limit Tests for Non-sterile Products: Control Bacteria Test Method".

[0104] The samples from the above-mentioned test sample group, test sample control group, positive control group and negative control group were placed in an incubator at 30-35℃ and cultured for 18-24h to obtain cultures. After the culture was completed, the cultures of each test group were streaked onto MSA (obtained in Preparation Example 3) plates using an inoculation loop and cultured in an incubator at 30-35℃ for 18-72h. Three parallel experiments were set up for each group.

[0105] The judgment criteria are as follows: Test sample group and positive control group: Target control bacteria (Staphylococcus aureus / Bacillus subtilis) should be detected. The test sample control group and the negative control group should show no bacterial growth.

[0106] Table 8 below shows the results of the suitability test for the control bacteria:

[0107] Note: "+" indicates that the target control bacteria were detected; "-" indicates that the target control bacteria were not detected.

[0108] As shown in Table 8 above, the target control bacteria (Staphylococcus aureus or Bacillus subtilis) were detected in both the positive control group and the test sample group, while no bacteria grew in both the negative control group and the test sample group, indicating that the test method for the control bacteria complies with the regulations.

[0109] Example 5 1. Preparation of tobramycin test solution Take 10g of tobramycin, add 100mL of diluent (synergistic neutralizing solution 2 prepared in Preparation Example 4), mix well, and obtain a 1:10 tobramycin test solution.

[0110] 2. Suitability test of microbial limit test method 2.1 Experimental group setup 2.1.1 Test Sample Inspection Team Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (co-neutralizing solution 2); add 10 mL of 1:10 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (co-neutralizing solution 2) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (co-neutralizing solution 2) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (co-neutralizing solution 2) of the filter cartridge and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the test sample.

[0111] 2.1.2 Control group of test sample Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralization solution 2); add 10 mL of 1:10 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (synergistic neutralization solution 2) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (synergistic neutralization solution 2) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in preparation example 3) to obtain the test sample control sample.

[0112] 2.1.3 Positive control group Take one filter cartridge containing a filter membrane and wet the filter membrane with diluent (co-neutralizing solution 2); add 100 mL of diluent (co-neutralizing solution 2) to wash the filter cartridge and filter dry; use a pipette to draw 10 mL of diluent (co-neutralizing solution 2) to evenly rinse the inner wall of the filter cartridge and filter dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (co-neutralizing solution 2) of the filter cartridge and filter dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the positive control sample.

[0113] 2.1.4 Negative control group Take one filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralization solution 2); add 100 mL of diluent (synergistic neutralization solution 2) to wash the filter cartridge and filter dry; use a pipette to draw 10 mL of diluent (synergistic neutralization solution 2) to rinse the inner wall of the filter cartridge evenly and filter dry; repeat the above rinsing steps 5 times and filter dry; transfer the filter membrane to 100 mL of TSB (obtained in preparation example 3) to obtain the negative control sample.

[0114] 2.1.5 Chemical neutralizing agent control group Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (chemical neutralizer); add 10 mL of 1:10 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (chemical neutralizer) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (chemical neutralizer) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (chemical neutralizer) of the filter cartridge and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the neutralizer control sample.

[0115] 2.2 Suitability Test of Aerobic Bacteria Counting Method The suitability test of the culture medium for counting was conducted in accordance with the 2020 edition of the Pharmacopoeia, "1105 Microbial Limit Tests for Non-sterile Products: Microbial Counting Method".

[0116] The samples from each of the above experimental groups (as shown in 2.1.1-2.1.5) were placed in an incubator at 30-35℃ and cultured for 18-24 hours to obtain cultures. After the culture was completed, each culture was streaked onto an MSA (preparation example 3) plate using an inoculation loop and cultured at 30-35℃ for 18-72 hours. Three parallel experiments were set up for each group.

[0117] Suitability test of aerobic bacteria counting method Record the average colony count for each of the above test groups (expressed as mean ± standard deviation), and calculate the colony recovery ratio between the test sample group and the chemical neutralizing agent control group. The calculation formula is as follows:

[0118] The judgment criteria are as follows: a) The colony recovery ratio is in the range of 0.5-2.0; b) The size and shape of the colonies should be consistent with those on the control culture medium; c) The test sample control group and the negative control group should be free of bacterial growth.

[0119] Table 9 below shows the results of the total aerobic bacteria (Staphylococcus aureus / Bacillus subtilis) count test in the test sample group and the chemical neutralizer control group:

[0120] As shown in Table 9 above, when using 1:10 tobramycin test solution (10 mL) and synergistic neutralization solution 2 (magnesium chloride hexahydrate concentration of 4 wt.%) for microbial limit testing, the colony recovery ratios of all test sample groups (Staphylococcus aureus / Bacillus subtilis) were within the range specified in the standard (0.5-2.0), and the colony morphology and size should be consistent with the colonies on the control culture medium (in Example 1), indicating that the aerobic bacteria counting method complies with the regulations.

[0121] 2.3 Suitability test of control bacteria testing methods The suitability test of the control bacteria test method was conducted with reference to the 2020 edition of the Pharmacopoeia, "1106 Microbial Limit Tests for Non-sterile Products: Control Bacteria Test Method".

[0122] The samples from the above-mentioned test sample group, test sample control group, positive control group and negative control group were placed in an incubator at 30-35℃ and cultured for 18-24h to obtain cultures. After the culture was completed, the cultures of each test group were streaked onto MSA (obtained in Preparation Example 3) plates using an inoculation loop and cultured in an incubator at 30-35℃ for 18-72h. Three parallel experiments were set up for each group.

[0123] The judgment criteria are as follows: Test sample group and positive control group: Target control bacteria (Staphylococcus aureus / Bacillus subtilis) should be detected. The test sample control group and the negative control group should show no bacterial growth.

[0124] Table 10 below shows the results of the suitability test for the control bacteria:

[0125] Note: "+" indicates that the target control bacteria were detected; "-" indicates that the target control bacteria were not detected.

[0126] As shown in Table 10 above, the target control bacteria (Staphylococcus aureus or Bacillus subtilis) were detected in both the positive control group and the test sample group, while no bacteria grew in both the negative control group and the test sample group, indicating that the test method for the control bacteria complies with the regulations.

[0127] Example 6 1. Preparation of tobramycin test solution Take 10g of tobramycin, add 100mL of diluent (the synergistic neutralizing solution 3 prepared in Preparation Example 4), mix well, and obtain a 1:10 tobramycin test solution.

[0128] 2. Suitability test of microbial limit test method 2.1 Experimental group setup 2.1.1 Test Sample Inspection Team Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralization solution 3); add 10 mL of 1:10 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (synergistic neutralization solution 3) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (synergistic neutralization solution 3) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (synergistic neutralization solution 3) of the filter cartridge and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the test sample.

[0129] 2.1.2 Control group of test sample Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralizing solution 3); add 10 mL of 1:10 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (synergistic neutralizing solution 3) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (synergistic neutralizing solution 3) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in preparation example 3) to obtain the test sample control sample.

[0130] 2.1.3 Positive control group Take one filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralizing solution 3); add 100 mL of diluent (synergistic neutralizing solution 3) to wash the filter cartridge and filter dry; use a pipette to draw 10 mL of diluent (synergistic neutralizing solution 3) to evenly rinse the inner wall of the filter cartridge and filter dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (synergistic neutralizing solution 3) of the filter cartridge and filter dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the positive control sample.

[0131] 2.1.4 Negative control group Take one filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralizing solution 3); add 100 mL of diluent (synergistic neutralizing solution 3) to wash the filter cartridge and filter dry; use a pipette to draw 10 mL of diluent (synergistic neutralizing solution 3) to rinse the inner wall of the filter cartridge evenly and filter dry; repeat the above rinsing steps 5 times and filter dry; transfer the filter membrane to 100 mL of TSB (obtained in preparation example 3) to obtain the negative control sample.

[0132] 2.1.5 Chemical neutralizing agent control group Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (chemical neutralizer); add 10 mL of 1:10 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (chemical neutralizer) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (chemical neutralizer) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (chemical neutralizer) of the filter cartridge and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the neutralizer control sample.

[0133] 2.2 Suitability Test of Aerobic Bacteria Counting Method The suitability test of the culture medium for counting was conducted in accordance with the 2020 edition of the Pharmacopoeia, "1105 Microbial Limit Tests for Non-sterile Products: Microbial Counting Method".

[0134] The samples from each of the above experimental groups (as shown in 2.1.1-2.1.5) were placed in an incubator at 30-35℃ and cultured for 18-24 hours to obtain cultures. After the culture was completed, each culture was streaked onto an MSA (preparation example 3) plate using an inoculation loop and cultured at 30-35℃ for 18-72 hours. Three parallel experiments were set up for each group.

[0135] Suitability test of aerobic bacteria counting method Record the average colony count for each of the above test groups (expressed as mean ± standard deviation), and calculate the colony recovery ratio between the test sample group and the chemical neutralizing agent control group. The calculation formula is as follows:

[0136] The judgment criteria are as follows: a) The colony recovery ratio is in the range of 0.5-2.0; b) The size and shape of the colonies should be consistent with those on the control culture medium; c) The test sample control group and the negative control group should be free of bacterial growth.

[0137] Table 11 below shows the results of the total aerobic bacteria (Staphylococcus aureus / Bacillus subtilis) count test in the test sample group and the chemical neutralizer control group:

[0138] As shown in Table 11 above, when using 1:10 tobramycin test solution (10 mL) and synergistic neutralization solution 3 (with a magnesium chloride hexahydrate concentration of 7 wt.%) for microbial limit testing, the colony recovery ratios of all test sample groups (Staphylococcus aureus / Bacillus subtilis) were within the range specified in the standard (0.5-2.0), and the colony morphology and size should be consistent with the colonies on the control culture medium (in Example 1), indicating that the aerobic bacteria counting method complies with the regulations.

[0139] 2.3 Suitability test of control bacteria testing methods The suitability test of the control bacteria test method was conducted with reference to the 2020 edition of the Pharmacopoeia, "1106 Microbial Limit Tests for Non-sterile Products: Control Bacteria Test Method".

[0140] The samples from the above-mentioned test sample group, test sample control group, positive control group and negative control group were placed in an incubator at 30-35℃ and cultured for 18-24h to obtain cultures. After the culture was completed, the cultures of each test group were streaked onto MSA (obtained in Preparation Example 3) plates using an inoculation loop and cultured in an incubator at 30-35℃ for 18-72h. Three parallel experiments were set up for each group.

[0141] The judgment criteria are as follows: Test sample group and positive control group: Target control bacteria (Staphylococcus aureus / Bacillus subtilis) should be detected. The test sample control group and the negative control group should show no bacterial growth.

[0142] Table 12 below shows the results of the suitability test for the control bacteria:

[0143] Note: "+" indicates that the target control bacteria were detected; "-" indicates that the target control bacteria were not detected.

[0144] As shown in Table 12 above, the target control bacteria (Staphylococcus aureus or Bacillus subtilis) were detected in both the positive control group and the test sample group, while no bacteria grew in both the negative control group and the test sample group, indicating that the test method for the control bacteria complies with the regulations.

[0145] Example 7 1. Preparation of tobramycin test solution Take 10g of tobramycin, add 100mL of diluent (synergistic neutralizing solution 4 prepared in preparation example 4), mix well, and obtain a 1:10 tobramycin test solution.

[0146] 2. Suitability test of microbial limit test method 2.1 Experimental group setup 2.1.1 Test Sample Inspection Team Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralization solution 4); add 10 mL of 1:10 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (synergistic neutralization solution 4) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (synergistic neutralization solution 4) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (synergistic neutralization solution 4) of the filter cartridge and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the test sample.

[0147] 2.1.2 Control group of test sample Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralizing solution 4); add 10 mL of 1:10 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (synergistic neutralizing solution 4) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (synergistic neutralizing solution 4) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the test sample control sample.

[0148] 2.1.3 Positive control group Take one filter cartridge containing a filter membrane and wet the filter membrane with diluent (co-neutralizing solution 4); add 100 mL of diluent (co-neutralizing solution 4) to wash the filter cartridge and filter dry; use a pipette to draw 10 mL of diluent (co-neutralizing solution 4) to evenly rinse the inner wall of the filter cartridge and filter dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (co-neutralizing solution 4) of the filter cartridge and filter dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the positive control sample.

[0149] 2.1.4 Negative control group Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralizing solution 4); add 100 mL of diluent (synergistic neutralizing solution 4) to wash the filter cartridge and filter dry; use a pipette to draw 10 mL of diluent (synergistic neutralizing solution 4) to rinse the inner wall of the filter cartridge evenly and filter dry; repeat the above rinsing steps 5 times and filter dry; transfer the filter membrane to 100 mL of TSB (obtained in preparation example 3) to obtain the negative control sample.

[0150] 2.1.5 Chemical neutralizing agent control group Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (chemical neutralizer); add 10 mL of 1:10 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (chemical neutralizer) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (chemical neutralizer) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (chemical neutralizer) of the filter cartridge and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the neutralizer control sample.

[0151] 2.2 Suitability Test of Aerobic Bacteria Counting Method The suitability test of the culture medium for counting was conducted in accordance with the 2020 edition of the Pharmacopoeia, "1105 Microbial Limit Tests for Non-sterile Products: Microbial Counting Method".

[0152] The samples from each of the above experimental groups (as shown in 2.1.1-2.1.5) were placed in an incubator at 30-35℃ and cultured for 18-24 hours to obtain cultures. After the culture was completed, each culture was streaked onto an MSA (preparation example 3) plate using an inoculation loop and cultured at 30-35℃ for 18-72 hours. Three parallel experiments were set up for each group.

[0153] Suitability test of aerobic bacteria counting method Record the average colony count for each of the above test groups (expressed as mean ± standard deviation), and calculate the colony recovery ratio between the test sample group and the chemical neutralizing agent control group. The calculation formula is as follows:

[0154] The judgment criteria are as follows: a) The colony recovery ratio is in the range of 0.5-2.0; b) The size and shape of the colonies should be consistent with those on the control culture medium; c) The test sample control group and the negative control group should be free of bacterial growth.

[0155] Table 13 below shows the results of the total aerobic bacteria (Staphylococcus aureus / Bacillus subtilis) count test in the test sample group and the chemical neutralizer control group:

[0156] As shown in Table 13 above, when using 1:10 tobramycin test solution (10 mL) and synergistic neutralization solution 4 (with a magnesium chloride hexahydrate concentration of 0.5 wt.%) for microbial limit testing, the colony recovery ratios of all test sample groups (Staphylococcus aureus / Bacillus subtilis) were within the range specified in the standard (0.5-2.0), and the colony morphology and size should be consistent with the colonies on the control culture medium (in Example 1), indicating that the aerobic bacteria counting method complies with the regulations.

[0157] 2.3 Suitability test of control bacteria testing methods The suitability test of the control bacteria test method was conducted with reference to the 2020 edition of the Pharmacopoeia, "1106 Microbial Limit Tests for Non-sterile Products: Control Bacteria Test Method".

[0158] The samples from the above-mentioned test sample group, test sample control group, positive control group and negative control group were placed in an incubator at 30-35℃ and cultured for 18-24h to obtain cultures. After the culture was completed, the cultures of each test group were streaked onto MSA (obtained in Preparation Example 3) plates using an inoculation loop and cultured in an incubator at 30-35℃ for 18-72h. Three parallel experiments were set up for each group.

[0159] The judgment criteria are as follows: Test sample group and positive control group: Target control bacteria (Staphylococcus aureus / Bacillus subtilis) should be detected. The test sample control group and the negative control group should show no bacterial growth.

[0160] Table 14 below shows the results of the suitability test for the control bacteria:

[0161] Note: "+" indicates that the target control bacteria were detected; "-" indicates that the target control bacteria were not detected.

[0162] As shown in Table 14 above, the target control bacteria (Staphylococcus aureus or Bacillus subtilis) were detected in both the positive control group and the test sample group, while no bacteria grew in both the negative control group and the test sample group, indicating that the test method for the control bacteria complies with the regulations.

[0163] Example 8 1. Preparation of tobramycin test solution Take 10g of tobramycin, add 100mL of diluent (synergistic neutralizing solution 5 prepared in Preparation Example 4), mix well, and obtain a 1:10 tobramycin test solution.

[0164] 2. Suitability test of microbial limit test method 2.1 Experimental group setup 2.1.1 Test Sample Inspection Team Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralizing solution 5); add 10 mL of 1:10 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (synergistic neutralizing solution 5) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (synergistic neutralizing solution 5) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (synergistic neutralizing solution 5) of the filter cartridge and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the test sample.

[0165] 2.1.2 Control group of test sample Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralizing solution 5); add 10 mL of 1:10 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (synergistic neutralizing solution 5) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (synergistic neutralizing solution 5) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the test sample control sample.

[0166] 2.1.3 Positive control group Take one filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralizing solution 5); add 100 mL of diluent (synergistic neutralizing solution 5) to wash the filter cartridge and filter dry; use a pipette to draw 10 mL of diluent (synergistic neutralizing solution 5) to evenly rinse the inner wall of the filter cartridge and filter dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (synergistic neutralizing solution 5) of the filter cartridge and filter dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the positive control sample.

[0167] 2.1.4 Negative control group Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralizing solution 5); add 100 mL of diluent (synergistic neutralizing solution 5) to wash the filter cartridge and filter dry; use a pipette to draw 10 mL of diluent (synergistic neutralizing solution 5) to rinse the inner wall of the filter cartridge evenly and filter dry; repeat the above rinsing steps 5 times and filter dry; transfer the filter membrane to 100 mL of TSB (obtained in preparation example 3) to obtain a negative control sample.

[0168] 2.1.5 Chemical neutralizing agent control group Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (chemical neutralizer); add 10 mL of 1:10 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (chemical neutralizer) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (chemical neutralizer) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (chemical neutralizer) of the filter cartridge and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the neutralizer control sample.

[0169] 2.2 Suitability Test of Aerobic Bacteria Counting Method The suitability test of the culture medium for counting was conducted in accordance with the 2020 edition of the Pharmacopoeia, "1105 Microbial Limit Tests for Non-sterile Products: Microbial Counting Method".

[0170] The samples from each of the above experimental groups (as shown in 2.1.1-2.1.5) were placed in an incubator at 30-35℃ and cultured for 18-24 hours to obtain cultures. After the culture was completed, each culture was streaked onto an MSA (preparation example 3) plate using an inoculation loop and cultured at 30-35℃ for 18-72 hours. Three parallel experiments were set up for each group.

[0171] Suitability test of aerobic bacteria counting method Record the average colony count for each of the above test groups (expressed as mean ± standard deviation), and calculate the colony recovery ratio between the test sample group and the chemical neutralizing agent control group. The calculation formula is as follows:

[0172] The judgment criteria are as follows: a) The colony recovery ratio is in the range of 0.5-2.0; b) The size and shape of the colonies should be consistent with those on the control culture medium; c) The test sample control group and the negative control group should be free of bacterial growth.

[0173] Table 15 below shows the results of the total aerobic bacteria (Staphylococcus aureus / Bacillus subtilis) count test in the test sample group and the chemical neutralizer control group:

[0174] As shown in Table 15 above, when using 1:10 tobramycin test solution (10 mL) and synergistic neutralization solution 5 (with a magnesium chloride hexahydrate concentration of 7.5 wt.%) for microbial limit testing, the colony recovery ratios of all test sample groups (Staphylococcus aureus / Bacillus subtilis) were within the range specified in the standard (0.5-2.0), and the colony morphology and size should be consistent with the colonies on the control culture medium (in Example 1), indicating that the aerobic bacteria counting method complies with the regulations.

[0175] 2.3 Suitability test of control bacteria testing methods The suitability test of the control bacteria test method was conducted with reference to the 2020 edition of the Pharmacopoeia, "1106 Microbial Limit Tests for Non-sterile Products: Control Bacteria Test Method".

[0176] The samples from the above-mentioned test sample group, test sample control group, positive control group and negative control group were placed in an incubator at 30-35℃ and cultured for 18-24h to obtain cultures. After the culture was completed, the cultures of each test group were streaked onto MSA (obtained in Preparation Example 3) plates using an inoculation loop and cultured in an incubator at 30-35℃ for 18-72h. Three parallel experiments were set up for each group.

[0177] The judgment criteria are as follows: Test sample group and positive control group: Target control bacteria (Staphylococcus aureus / Bacillus subtilis) should be detected. The test sample control group and the negative control group should show no bacterial growth.

[0178] Table 16 below shows the results of the suitability test for the control bacteria examination method:

[0179] Note: "+" indicates that the target control bacteria were detected; "-" indicates that the target control bacteria were not detected.

[0180] As shown in Table 16 above, the target control bacteria (Staphylococcus aureus or Bacillus subtilis) were detected in both the positive control group and the test sample group, while no bacterial growth was observed in both the negative control group and the test sample group, indicating that the test method for the control bacteria complies with the regulations.

[0181] Comparative Example 1 1. Preparation of tobramycin test solution Take 10g of tobramycin, add 100mL of diluent (synergistic neutralizing solution 6 prepared in Preparation Example 4), mix well, and obtain a 1:10 tobramycin test solution.

[0182] 2. Suitability test of microbial limit test method 2.1 Experimental group setup 2.1.1 Test Sample Inspection Team Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralizing solution 6); add 10 mL of 1:10 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (synergistic neutralizing solution 6) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (synergistic neutralizing solution 6) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (synergistic neutralizing solution 6) of the filter cartridge and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the test sample.

[0183] 2.1.2 Control group of test sample Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralizing solution 6); add 10 mL of 1:10 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (synergistic neutralizing solution 6) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (synergistic neutralizing solution 6) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the test sample control sample.

[0184] 2.1.3 Positive control group Take one filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralizing solution 6); add 100 mL of diluent (synergistic neutralizing solution 6) to wash the filter cartridge and filter dry; use a pipette to draw 10 mL of diluent (synergistic neutralizing solution 6) to evenly rinse the inner wall of the filter cartridge and filter dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (synergistic neutralizing solution 6) of the filter cartridge and filter dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the positive control sample.

[0185] 2.1.4 Negative control group Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (synergistic neutralizing solution 6); add 100 mL of diluent (synergistic neutralizing solution 6) to wash the filter cartridge and filter dry; use a pipette to draw 10 mL of diluent (synergistic neutralizing solution 6) to rinse the inner wall of the filter cartridge evenly and filter dry; repeat the above rinsing steps 5 times and filter dry; transfer the filter membrane to 100 mL of TSB (obtained in preparation example 3) to obtain the negative control sample.

[0186] 2.1.5 Chemical neutralizing agent control group Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (chemical neutralizer); add 10 mL of 1:10 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (chemical neutralizer) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (chemical neutralizer) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (chemical neutralizer) of the filter cartridge and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the neutralizer control sample.

[0187] 2.2 Suitability Test of Aerobic Bacteria Counting Method The suitability test of the culture medium for counting was conducted in accordance with the 2020 edition of the Pharmacopoeia, "1105 Microbial Limit Tests for Non-sterile Products: Microbial Counting Method".

[0188] The samples from each of the above experimental groups (as shown in 2.1.1-2.1.5) were placed in an incubator at 30-35℃ and cultured for 18-24 hours to obtain cultures. After the culture was completed, each culture was streaked onto an MSA (preparation example 3) plate using an inoculation loop and cultured at 30-35℃ for 18-72 hours. Three parallel experiments were set up for each group.

[0189] Record the average colony count for each of the above test groups (expressed as mean ± standard deviation), and calculate the colony recovery ratio between the test sample group and the chemical neutralizing agent control group. The calculation formula is as follows:

[0190] The judgment criteria are as follows: a) The colony recovery ratio is in the range of 0.5-2.0; b) The size and shape of the colonies should be consistent with those on the control culture medium; c) The test sample control group and the negative control group should be free of bacterial growth.

[0191] Table 17 below shows the results of the total aerobic bacteria (Staphylococcus aureus / Bacillus subtilis) count test in the test sample group and the chemical neutralizer control group:

[0192] As shown in Table 17 above, when using 1:10 tobramycin test solution (10 mL) and synergistic neutralization solution 6 (without magnesium chloride hexahydrate) for microbial limit testing, the colony recovery ratios of all test sample groups (Staphylococcus aureus / Bacillus subtilis) were within the range specified in the standard (0.5-2.0), and the colony morphology and size should be consistent with the colonies on the control culture medium (in Example 1), indicating that the aerobic bacteria counting method complies with the regulations.

[0193] 2.3 Suitability test of control bacteria testing methods The suitability test of the control bacteria test method was conducted with reference to the 2020 edition of the Pharmacopoeia, "1106 Microbial Limit Tests for Non-sterile Products: Control Bacteria Test Method".

[0194] The samples from the above-mentioned test sample group, test sample control group, positive control group and negative control group were placed in an incubator at 30-35℃ and cultured for 18-24h to obtain cultures. After the culture was completed, the cultures of each test group were streaked onto MSA (obtained in Preparation Example 3) plates using an inoculation loop and cultured in an incubator at 30-35℃ for 18-72h. Three parallel experiments were set up for each group.

[0195] The judgment criteria are as follows: Test sample group and positive control group: Target control bacteria (Staphylococcus aureus / Bacillus subtilis) should be detected. The test sample control group and the negative control group should show no bacterial growth.

[0196] Table 18 below shows the results of the suitability test for the control bacteria:

[0197] Note: "+" indicates that the target control bacteria were detected; "-" indicates that the target control bacteria were not detected.

[0198] As shown in Table 18 above, the target control bacteria (Staphylococcus aureus or Bacillus subtilis) were detected in both the positive control group and the test sample group, while no bacteria grew in both the negative control group and the test sample group, indicating that the test method for the control bacteria complies with the regulations.

[0199] Comparative Example 2 1. Preparation of tobramycin test solution Take 10g of tobramycin, add 100mL of pH 7.0 sodium chloride-peptone buffer (sterilized), mix well, and obtain a 1:10 tobramycin test solution.

[0200] 2. Suitability test of microbial limit test method 2.1 Experimental group setup 2.1.1 Test Sample Inspection Team Take one filter cartridge containing a filter membrane and wet the filter membrane with pH 7.0 sodium chloride-peptone buffer (sterilized); add 100 mL of pH 7.0 sodium chloride-peptone buffer (sterilized) to wash the filter cartridge and filter dry; use a pipette to draw 10 mL of pH 7.0 sodium chloride-peptone buffer (sterilized) to evenly rinse the inner wall of the filter cartridge and filter dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus bacterial suspension / Bacillus subtilis suspension (bacterial count not exceeding 100 CFU / 0.1 mL) to the pH 7.0 sodium chloride-peptone buffer (sterilized) in the filter cartridge and filter dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the test sample.

[0201] 2.1.2 Control group of test sample Take a filter cartridge containing a filter membrane and wet the filter membrane with pH 7.0 sodium chloride-peptone buffer (sterilized). Add 10 mL of 1:10 tobramycin test solution to the filter cartridge and filter until dry. Add 100 mL of pH 7.0 sodium chloride-peptone buffer (sterilized) to wash the filter cartridge and filter until dry. Use a pipette to draw 10 mL of pH 7.0 sodium chloride-peptone buffer (sterilized) to rinse the inner wall of the filter cartridge evenly and filter until dry. Repeat the above rinsing steps 5 times and filter until dry. Transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the test sample control sample.

[0202] 2.1.3 Positive control group Take one filter cartridge containing a filter membrane and wet the filter membrane with pH 7.0 sodium chloride-peptone buffer (sterilized); add 100 mL of pH 7.0 sodium chloride-peptone buffer (sterilized) to wash the filter cartridge and filter dry; use a pipette to draw 10 mL of pH 7.0 sodium chloride-peptone buffer (sterilized) to evenly rinse the inner wall of the filter cartridge and filter dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus bacterial suspension / Bacillus subtilis suspension (bacterial count not exceeding 100 CFU / 0.1 mL) to the pH 7.0 sodium chloride-peptone buffer (sterilized) in the filter cartridge and filter dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the positive control sample.

[0203] 2.1.4 Negative control group Take one filter cartridge containing a filter membrane and wet the filter membrane with pH 7.0 sodium chloride-peptone buffer (sterilized); add 100 mL of pH 7.0 sodium chloride-peptone buffer (sterilized) to wash the filter cartridge and filter dry; use a pipette to draw 10 mL of pH 7.0 sodium chloride-peptone buffer (sterilized) to rinse the inner wall of the filter cartridge evenly and filter dry; repeat the above rinsing steps 5 times and filter dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the negative control sample.

[0204] 2.1.5 Chemical neutralizing agent control group Take a filter cartridge containing a filter membrane and wet the filter membrane with diluent (chemical neutralizer); add 10 mL of 1:10 tobramycin test solution to the filter cartridge and filter until dry; add 100 mL of diluent (chemical neutralizer) to wash the filter cartridge and filter until dry; use a pipette to draw 10 mL of diluent (chemical neutralizer) to evenly rinse the inner wall of the filter cartridge and filter until dry; repeat the above rinsing steps 5 times. In the last rinse, add Staphylococcus aureus suspension / Bacillus subtilis suspension (containing no more than 100 CFU / 0.1 mL of bacteria) to the diluent (chemical neutralizer) of the filter cartridge and filter until dry; transfer the filter membrane to 100 mL of TSB (obtained in Preparation Example 3) to obtain the neutralizer control sample.

[0205] 2.2 Suitability Test of Aerobic Bacteria Counting Method The suitability test of the culture medium for counting was conducted in accordance with the 2020 edition of the Pharmacopoeia, "1105 Microbial Limit Tests for Non-sterile Products: Microbial Counting Method".

[0206] The samples from each of the above experimental groups (as shown in 2.1.1-2.1.5) were placed in an incubator at 30-35℃ and cultured for 18-24 hours to obtain cultures. After the culture was completed, each culture was streaked onto an MSA (preparation example 3) plate using an inoculation loop and cultured at 30-35℃ for 18-72 hours. Three parallel experiments were set up for each group.

[0207] Record the average colony count for each of the above test groups (expressed as mean ± standard deviation), and calculate the colony recovery ratio between the test sample group and the chemical neutralizing agent control group. The calculation formula is as follows:

[0208] The judgment criteria are as follows: a) The colony recovery ratio is in the range of 0.5-2.0; b) The size and shape of the colonies should be consistent with those on the control culture medium; c) The test sample control group and the negative control group should be free of bacterial growth.

[0209] Table 19 below shows the results of the total aerobic bacteria (Staphylococcus aureus / Bacillus subtilis) count test in the test sample group and the chemical neutralizer control group:

[0210] As shown in Table 19 above, when using 1:10 tobramycin test solution (10 mL) and pH 7.0 sodium chloride-peptone buffer (sterilized) for microbial limit testing, the colony recovery ratios of all test groups (Staphylococcus aureus / Bacillus subtilis) were not within the range specified in the standard (0.5-2.0), and the colony morphology and size were inconsistent with those on the control culture medium (in Example 1), indicating that the aerobic bacteria counting method did not meet the requirements.

[0211] 2.3 Suitability test of control bacteria testing methods The suitability test of the control bacteria test method was conducted with reference to the 2020 edition of the Pharmacopoeia, "1106 Microbial Limit Tests for Non-sterile Products: Control Bacteria Test Method".

[0212] The samples from the above-mentioned test sample group, test sample control group, positive control group and negative control group were placed in an incubator at 30-35℃ and cultured for 18-24h to obtain cultures. After the culture was completed, the cultures of each test group were streaked onto MSA (obtained in Preparation Example 3) plates using an inoculation loop and cultured in an incubator at 30-35℃ for 18-72h. Three parallel experiments were set up for each group.

[0213] The judgment criteria are as follows: Test sample group and positive control group: Target control bacteria (Staphylococcus aureus / Bacillus subtilis) should be detected. The test sample control group and the negative control group should show no bacterial growth.

[0214] Table 20 below shows the results of the suitability test for the control bacteria:

[0215] Note: "+" indicates that the target control bacteria were detected; "-" indicates that the target control bacteria were not detected.

[0216] As shown in Table 20 above, the target control bacteria (Staphylococcus aureus or Bacillus subtilis) were detected in all positive control groups, while no bacteria grew in the test sample test group, test sample control group, and negative control group, indicating that the test method for the control bacteria did not meet the requirements.

[0217] The experimental results of the above embodiments (1-8) and comparative examples (1 and 2) show that: 1. Validation of the effectiveness of the enzyme-chemical synergistic neutralization system (1) Microbial counting method (Pharmacopoeia 1105 standard) Suitability of counting media: In Example 1, TSB (obtained in Preparation Example 3) and MSA (obtained in Preparation Example 3) containing inactivating enzymes (AAC(3)-Ⅰ protein and APH(3')-Ⅱa protein) and magnesium chloride hexahydrate were added. The ratio of the average number of colonies to their respective control media was in the range of 0.5-2.0, and the colony morphology and size were consistent with the control media. This confirms that the suitability of TSB and MSA media with added neutralizing components meets the pharmacopoeia requirements. Colony recovery ratio: In Examples 2-8, the colony recovery ratios of all test groups were in the range of 0.5-2.0, indicating that the synergistic neutralization system can effectively neutralize the antibacterial activity of tobramycin, meeting the pharmacopoeia standards. It is noteworthy that, in contrast, the control group using only the chemical neutralizing agent (polysorbate 80) generally had lower colony recovery ratios and poorer stability (increased standard deviation). This phenomenon indicates that single chemical neutralization is insufficient, while in this invention, the inactivating enzyme and the chemical neutralizing agent play a crucial synergistic role. Stability: The recovery ratio of Example 2 (1:5 tobramycin test solution) was closest to 1.0 and had the smallest standard deviation, indicating that the enzyme-chemical synergistic neutralization system has better advantages under high concentration test solution conditions.

[0218] (2) Control bacteria test method (Pharmacopoeia 1106 standard) In Examples 2-8, the target bacteria were detected in all test sample groups and the positive control group, while no growth was observed in the negative control group, which met the pharmacopoeia standards and confirmed the reliability of the enzyme-chemical synergistic neutralization system for the detection of control bacteria.

[0219] 2. Magnesium chloride hexahydrate (Mg) 2+ The key role of synergistic neutralizing liquid (1) Effect of concentration Optimal range (1wt.%-7wt.%): Within this range, Mg 2+ It forms the functional substrate Mg-ATP with ATP, activates aminoglycoside-3'-phosphotransferase, and significantly improves neutralization efficiency (Examples 3-6). Insufficient or excessive concentration: In Examples 7-8 (Mg 2+ When the concentration exceeded 1 wt.%–7 wt.%, the colony recovery rate of all test samples decreased (0.81–0.86), and the stability decreased (standard deviation increased); while Comparative Example 1 (without Mg) showed a different trend. 2+ The colony recovery rates of all test sample groups in the study further decreased (0.77-0.85), confirming that Mg... 2+ It is an essential factor for synergistic effects.

[0220] This phenomenon is mainly due to Mg 2+By binding with ATP to form the functional complex Mg-ATP, which acts as an essential cofactor for APH(3'), it directly activates the enzyme's catalytic activity. When Mg is deficient... 2+ If the concentration exceeds the optimum concentration, Mg-ATP production is limited or enzyme conformational stability is reduced, leading to a decrease in the phosphorylation efficiency of APH(3'), which in turn leads to a decrease in the colony recovery ratio and an increase in the standard deviation.

[0221] 3. Advantages compared to traditional methods In Comparative Example 2 (using only pH 7.0 sodium chloride-peptone buffer), the colony recovery ratios of all test groups were significantly lower (0.04-0.10), and control bacteria were not detected, demonstrating that simple dilution cannot neutralize the strong antibacterial activity of tobramycin, further highlighting the significant advantages of the synergistic system.

[0222] In summary, this invention successfully developed a highly efficient method for the microbial limit detection of tobramycin based on an enzyme-chemical synergistic neutralization system constructed from aminoglycoside-3-acetyltransferase (AAC(3)), aminoglycoside-3'-phosphotransferase (APH(3')), magnesium chloride hexahydrate, and pH 7.0 sodium chloride-peptone buffer. This system fundamentally eliminates the antibacterial activity of tobramycin through the covalent modification of specific enzymes. Combined with a detection process optimized by post-inoculation and culture medium, it not only significantly improves the colony recovery rate but also ensures the accuracy and reliability of the detection results, providing an excellent solution for the quality control of tobramycin preparations (such as eye drops and injections).

[0223] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting multiple microbial limits of tobramycin, characterized in that, Includes the following steps: (1) Take the target bacteria and prepare a bacterial suspension; (2) Mix the tobramycin test sample and diluent according to the mass-volume ratio to prepare the tobramycin test solution; (3) Take the tobramycin test solution obtained in step (2) and filter it through a filter membrane. Use the diluent as the rinsing solution to rinse the filter membrane. Repeat 5 times. Add the bacterial suspension prepared in step (1) in the last rinse and filter dry. Transfer the filter membrane to a culture medium containing a neutralizing agent and incubate at 30-35℃ for 18-24h to obtain the test sample. (4) Observe the test sample obtained in step (3), record the colony growth, and calculate the colony recovery ratio. The target bacteria mentioned in step (1) are selected from Staphylococcus aureus and / or Bacillus subtilis; The diluent mentioned in steps (2) and (3) is a synergistic neutralizing solution, which is composed of an inactivating enzyme, a chemical neutralizing agent and a solvent; The neutralizing agent described in step (3) consists of an inactivating enzyme and a chemical neutralizing agent; The inactivating enzymes mentioned in steps (2) and (3) are aminoglycoside-3-acetyltransferase and aminoglycoside-3'-phosphotransferase, the ratio of the activity units of aminoglycoside-3-acetyltransferase and aminoglycoside-3'-phosphotransferase is 1:1 to 1:2, the amount of aminoglycoside-3-acetyltransferase added is 10-20U, and the amount of aminoglycoside-3'-phosphotransferase added is 10-20U; The chemical neutralizing agent mentioned in steps (2) and (3) is a divalent metal salt, wherein the divalent metal salt is magnesium chloride hexahydrate, and the concentration of magnesium chloride hexahydrate is 0.5 wt.%-7.5 wt.%; The solvent mentioned in steps (2) and (3) is sodium chloride-peptone buffer.

2. The method for detecting multiple microbial limits of tobramycin as described in claim 1, characterized in that, The mass-to-volume ratio of the tobramycin test sample to the diluent is 1:5 to 1:

10.

3. The method for detecting multiple microbial limits of tobramycin as described in claim 1, characterized in that, The concentration of the magnesium chloride hexahydrate is 1 wt.%-7 wt.%.

4. The method for detecting multiple microbial limits of tobramycin as described in claim 1, characterized in that, The pH of the sodium chloride-peptone buffer solution is 7.

0.

5. The method for detecting multiple microbial limits of tobramycin as described in claim 1, characterized in that, The concentration condition of the bacterial suspension is: the number of colonies contained in each 0.1 mL bacterial suspension does not exceed 100 CFU.

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