Microbial limit inspection method for dalbavancin bulk drug

Abstract

The invention relates to the field of inspection and detection of dalbavancin, in particular to a microbial limit inspection method of dalbavancin. Comprising the following steps: S1, preparation of a test solution: dissolving a test sample with sterilized water, and respectively preparing a 1: 10 test solution and a 1: 50 test solution; s2, checking the total number of aerobic bacteria and the total number of mould and saccharomycetes; and S3, inspection of control bacteria Escherichia coli. The technical problem to be solved by the invention is to provide the microbial limit detection method for the dalbavancin, which is scientific and effective and is beneficial to detection, so that the microbial limit quality of the dalbavancin variety is controllable. According to the invention, sterile water is screened as a diluent, so that the problem of dissolution of the dalbavancin bulk drug is solved, and the recovery ratio of test bacteria is increased; a membrane filtration method is adopted, a proper neutralizer is added into a flushing fluid, and a proper neutralizer is added into a TSA / SDA culture medium of a film, so that the antibacterial activity of the dalbavancin is effectively removed, and the recovery ratio of five test bacteria in microbe counting meets the requirements of pharmacopeia.

Description

Technical Field

[0001] The present invention relates to the field of inspection and detection of dalbavancin, and in particular to a microbial limit inspection method for dalbavancin raw materials. Background Art

[0002] Dalbavancin is a new semisynthetic glycopeptide antibiotic that has antibacterial activity against vancomycin-resistant Staphylococcus aureus. It has a longer half-life than vancomycin and requires a lower dosage and frequency of use than vancomycin. It was approved by the FDA in May 2014 for the treatment of gram-positive bacteria (G + Acute bacterial skin and skin structure infections caused by bacteria (including methicillin-resistant Staphylococcus aureus (MRSA)). Existing technologies primarily focus on the chemical structure, mechanism of action, antibacterial activity, biosynthesis of the precursor A40926, high-yield strain selection, fermentation process optimization, isolation and purification, and genetic engineering modification of dalbavancin.

[0003] Article 1107 of the General Rules of Part IV of the 2020 edition of the "Chinese Pharmacopoeia" stipulates that pharmaceutical raw materials and excipients must undergo microbial limit testing. The inspection items include microbial counts (total aerobic bacteria, total mold and yeast counts) and control bacteria inspections.

[0004] However, dalbavancin API has strong antimicrobial activity, making it difficult to meet pharmacopoeial requirements using conventional microbial limit tests. Currently, there are no reports on how to effectively remove the antimicrobial activity of dalbavancin and rapidly establish a microbial limit test for it. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a scientific, effective and easy-to-detect microbial limit test method for dalbavancin.

[0006] The present invention is achieved in that:

[0007] A method for microbial limit test of dalbavancin comprises the following steps:

[0008] S1. Preparation of test solution: The test substance is dalbavancin API, which is dissolved in sterile water to prepare a 1:10 test solution and a 1:50 test solution, respectively;

[0009] S2. Total aerobic bacterial count, mold and yeast count: dilute the test solution at a ratio of 1:50 with 0.9% sterile sodium chloride solution, filter through a membrane, and rinse five times with 100 mL / membrane. Add bacterial suspension during the final rinse, ensuring the bacterial count is no greater than 100 cfu / mL. Transfer the membrane to a medium containing a neutralizer and observe the count results.

[0010] S3. Inspection of control bacteria Escherichia coli: Take 1:10 test solution, transfer it to tryptic soy liquid medium, and incubate it at 30-35℃ for 18-24 hours; take the above culture and inoculate it into MacConkey liquid medium, and incubate it at 42-44℃ for 24-48 hours; take the MacConkey liquid medium and streak it onto MacConkey agar medium plate, incubate it at 30-35℃ for 18-72 hours, and observe the growth.

[0011] Furthermore, the test bacteria for the total aerobic bacterial count include Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Aspergillus niger and Candida albicans; the test bacteria for the total mold and yeast count include Aspergillus niger and Candida albicans.

[0012] Furthermore, the flushing solution is a 0.9% sterile sodium chloride solution containing 1% polysorbate 80 + 1%-3% sodium thiosulfate solution. The percentage of the above sodium thiosulfate is volume percentage, and the mother liquor concentration is 1 mol / L. The rest are mass percentages.

[0013] Furthermore, the formula of the medium containing the neutralizer is 4% polysorbate 80+0.5% soy lecithin+0.1% L-histidine TSA or SDA, and the above percentages are all mass percentages.

[0014] Furthermore, the aerobic bacteria are inoculated in a TSA medium containing a neutralizer and cultured at 30-35°C for no more than 3 days; the molds and yeasts are inoculated in an SDA medium containing a neutralizer and cultured at 20-25°C for no more than 5 days.

[0015] Furthermore, the membrane filtration uses a hydrophilic mixed cellulose sterile filter membrane.

[0016] The present invention has the following advantages:

[0017] (1) Sterile water was selected as the diluent in the "test solution preparation" step to solve the solubility problem of dalbavancin API and improve the recovery rate of test bacteria;

[0018] (2) In the “microbial count inspection” step, the membrane filtration method was used, and the neutralizing agent polysorbate 80 and a certain proportion of sodium thiosulfate solution were added to the rinsing liquid 0.9% sterile sodium chloride solution. Polysorbate 80, soybean lecithin and L-histidine were added to the film-coated tryptic soy peptone agar medium TSA / Sabouraud dextrose agar medium SDA culture medium, which effectively removed the antibacterial effect of dalbavancin. As a result, the recovery ratios of the five test bacteria (Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Aspergillus niger and Candida albicans) for microbial counts all fell between 0.5 and 2, which met the requirements of the pharmacopoeia. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Figure 1 The dissolution of the sample in 6 diluents.

[0021] Figure 2 To investigate the amount of sodium thiosulfate added to the flushing fluid. DETAILED DESCRIPTION

[0022] The technical solutions of the present invention will be described clearly and completely below in conjunction with the accompanying drawings and specific embodiments. Where specific conditions are not specified in the examples, conventional conditions or those recommended by the manufacturer were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0023] A microbial limit test method for dalbavancin comprises the following steps:

[0024] 1. Microbial limit standard: The total number of aerobic bacteria shall not exceed 10 3 cfu / g, the total number of molds and yeasts shall not exceed 10 2 cfu / g, no Escherichia coli shall be detected.

[0025] 2. Microbial limit items: total aerobic bacteria count, total mold and yeast count, and Escherichia coli.

[0026] 3. Established inspection methods:

[0027] 3.1 Total aerobic bacteria count: Add 1 mL of the test solution at a 1:50 dilution to 100 mL of 0.9% sterile sodium chloride solution. Filter through a membrane. Rinse each membrane with 500 mL of a rinse solution containing a neutralizer (100 mL / time). Remove the membrane with sterile tweezers and transfer to tryptic soy agar (TSA*) containing a neutralizer. Incubate inverted at 30-35°C for 5 days and then count the cells.

[0028] 3.2 Total mold and yeast count: Add 1 mL of the test solution at a 1:50 ratio to 100 mL of 0.9% sterile sodium chloride solution. Filter through a membrane. Rinse each membrane with 500 mL of a rinse solution containing a neutralizer (100 mL / time). Remove the membrane with sterile tweezers and transfer to Sabouraud dextrose agar (SDA*) containing a neutralizer. Incubate inverted at 20-25°C for 7 days and then count the cells.

[0029] 3.3 Control Bacteria Escherichia coli: Add 10 mL of the test solution at a 1:10 ratio to 100 mL of tryptic soy broth (TSB) and incubate at 30-35°C for 18-24 hours. Inoculate 1 mL of the above culture into 100 mL of MacConkey broth and incubate at 42-44°C for 24-48 hours. Streak the MacConkey broth onto a MacConkey agar plate and incubate at 30-35°C for 18-72 hours.

[0030] 3.4 Testing basis

[0031] Pharmacopoeia of the People's Republic of China 2020 Edition, Part 4.

[0032] Recovery ratio formula

[0033] Recovery ratio of neutralizer group = average colony count of neutralizer group / average colony count of bacterial solution group

[0034] Recovery ratio of the test group = (average colony count of the test group - average colony count of the test sample group) / average colony count of the bacterial solution group

[0035] The recovery ratio should be between 0.5-2.

[0036] In this method, the average colony count of the test sample group was 0.

[0037] 4. Main problems encountered during method development

[0038] During the microbial limit test of dalbavancin (raw material), the sample inspection amount is 10g, the antibiotic content is high, and the antibacterial activity against Staphylococcus aureus and Bacillus subtilis is strong.

[0039] We selected the sensitive bacterium Staphylococcus aureus for preliminary experiments and encountered the following problems:

[0040] 4.1 The test solution prepared using conventional diluents, pH 7.0 sodium chloride-peptone buffer or tryptic soy broth (TSB), is a suspension.

[0041] 4.2 Neutralizer was added to the culture medium and recovery test was performed using the plate method. Sensitive bacteria did not grow.

[0042] 4.3 If the test solution is a suspension, sensitive bacteria will not grow during membrane filtration.

[0043] According to the preliminary experimental results, it is planned to conduct a microbial counting test using the membrane filtration method after inspecting the diluent, flushing fluid, filter membrane and culture medium.

[0044] 5. Solutions to the problems

[0045] Regarding question 4.1, regarding the appearance of an insoluble suspension during test solution preparation, we propose using sterile water as the diluent and conducting comparative tests with conventional diluents and rinsing agents. Regarding questions 4.2 and 4.3, we propose using membrane filtration and examining the membrane, rinsing solution, and membrane-attached culture medium.

[0046] 5.1 Diluent Investigation

[0047] When investigating the diluent, due to the high cost of sample synthesis, in order to save costs, 1 g of sample was weighed into 6 sterile test tubes, numbered 1, 2, 3, 4, 5, and 6. No. 1 was added with pH 7.0 sodium chloride-peptone buffer to 10 mL, No. 2 was added with tryptic soy broth (TSB) to 10 mL, No. 3 was added with pH 7.2 phosphate buffer to 10 mL, No. 4 was added with 0.1% peptone aqueous solution to 10 mL, No. 5 was added with 0.9% sodium chloride solution to 10 mL, and No. 6 was added with sterile water to 10 mL. The mixture was shaken for 1-5 minutes and the dissolution was observed, as shown in Table 1. Test tubes 1 to 6 all contained a 1:10 test solution. Conventional diluents include No. 1 pH 7.0 sodium chloride-peptone buffer, No. 2 TSB, and No. 3 pH 7.2 phosphate buffer. For the samples, these three diluents remained turbid after 3-5 minutes of oscillation, and there was sediment at the bottom of the test tube after standing. The recovery ratios of Staphylococcus aureus counted using 1:10 test solution and 1:50 test solution were all 0, which did not meet the requirements.

[0048] After shaking the sample for 3 minutes in the commonly used washing liquid No. 4 0.1% peptone aqueous solution and No. 5 0.9% sodium chloride solution, a nearly clear liquid can be obtained, which is better than the first three diluents. Figure 1 As can be seen, using sterile water with diluent No. 6 to prepare a 1:10 test solution yielded a clear liquid. Using diluents No. 4, 5, or 6 to prepare a 1:10 test solution or a 1:50 test solution resulted in recovery ratios for Staphylococcus aureus ranging from 0.5 to 2, meeting the requirements. Considering factors such as oscillation time, test solution state, and recovery ratio (preferably a recovery ratio greater than 0.7 for better stability), sterile water No. 6 was selected as the diluent for test solution preparation.

[0049] Table 1 Diluent Investigation

[0050]

[0051]

[0052] 5.2 Flushing fluid inspection

[0053] Based on the diluent review, 0.9% sodium chloride solution No. 5 was selected as the base rinse solution for membrane filtration. The neutralizing agent polysorbate 80 (Tween 80) and sodium thiosulfate solution (Na2S2O3, using a stock solution concentration of 1 mol / L) were proposed for addition to the rinse solution. To prepare the rinse solution, Solution 1 and Solution 2 were prepared separately. After autoclaving, Solution 2 was added according to the volume percentage. Solution 1: 9.0g sodium chloride, 10.0g Tween 80, 1000mL water. Combine these ingredients, dissolve under lukewarm conditions, aliquot, and sterilize at 121°C for 15 minutes. Shake to mix thoroughly after autoclaving. Solution 2: 248.2g Na2S2O3·5H2O, 1000mL water. Combine these ingredients, dissolve under lukewarm conditions, aliquot, and sterilize at 121°C for 15 minutes. The filter membrane was attached to a TSA plate containing 4% Tween 80 + 0.5% lecithin. The recovery ratio of Staphylococcus aureus was used as an indicator to investigate the amount of neutralizer added. The results are shown in Table 2.

[0054] Table 2 Flushing fluid investigation

[0055]

[0056]

[0057] As can be seen from Table 2, when the flushing volume was 500 mL / membrane, the flushing solution was only 0.9% NaCl, the test bacteria did not grow, and the recovery ratio was 0; when 1% Tween 80 was added to 0.9% NaCl, the recovery ratio increased to 0.53; when 1% Tween 80 + 1% Na2S2O3 was added to 0.9% NaCl, the recovery ratio reached 0.75; with the increase of the amount of Na2S2O3 added, the recovery ratio decreased. Figure 2 When the addition amount of Na2S2O3 is 1%, 2% and 3%, the corresponding recovery ratios are 0.75, 0.73 and 0.72, which all meet the requirement of 0.5-2. When the addition amount is 10%, the recovery ratio drops to 0.48, and this addition amount is not recommended. Figure 2 According to the experimental situation, 0.9% NaCl containing 1% Tween 80 + 1% 1 mol / L Na2S2O3 was selected as the flushing solution.

[0058] 5.3 Culture medium investigation

[0059] Take 10g of sample and add diluent to 100mL to prepare a 1:10 test solution. This was further diluted to 1:20 and 1:50 test solutions. Dispense 1mL of each test solution into 100mL of 0.9% sterile sodium chloride solution (two membranes in parallel). Rinse the membranes five times with the rinse solution (100mL / membrane / time). Use sterile tweezers to transfer the membranes to different culture media. The recovery ratio of Bacillus subtilis was used as an indicator to evaluate the culture media. The results are shown in Table 3.

[0060] Table 3 Culture medium investigation

[0061]

[0062]

[0063] As shown in Table 3, both reducing the test solution concentration and adding a neutralizer effectively increased the recovery ratio of sensitive bacteria. When using TSA containing 4% Tween 80, 0.5% lecithin, and 0.1% L-histidine, the recovery ratio was 0.61 at a test solution concentration of 1:20, and increased to 0.75 at a test solution concentration of 1:50, both meeting the requirements. Therefore, TSA containing 4% Tween 80, 0.5% lecithin, and 0.1% L-histidine was selected as the medium for aerobic bacterial counts.

[0064] 5.4 Filter membrane inspection

[0065] Take 10g of sample and add sterile water to 100mL to prepare a 1:10 test solution, which is further diluted to a 1:50 test solution. Three different sterile filter membranes, hydrophilic mixed cellulose (MCE), hydrophilic nylon (N66), and hydrophilic polyethersulfone (PES), were attached to the pump head of the sterile filtration system. A filter cup was placed on each membrane and 100mL of 0.9% sterile sodium chloride solution was poured into each membrane. One mL of the 1:50 test solution was added to each of the 100mL solutions (two membranes in parallel). The membranes were then rinsed five times with the rinse solution (100mL / membrane / rinse). Using sterile tweezers, the membranes were transferred to TSA medium containing 4% Tween 80, 0.5% lecithin, and 0.1% L-histidine. The recovery ratios of Staphylococcus aureus and Bacillus subtilis were used as indicators to evaluate the performance of the different filter membranes. The results are shown in Table 4.

[0066] Table 4 Filter membrane inspection

[0067]

[0068] Note: The filter membrane size is Φ47mm in diameter and 0.45μm in pore size.

[0069] Table 4 shows that when using hydrophilic mixed cellulose (MCE), hydrophilic nylon (N66), or hydrophilic polyethersulfone (PES) for membrane filtration, only when the membrane was MCE did the recovery ratios for sensitive bacteria Staphylococcus aureus and Bacillus subtilis exceed 0.5, meeting the requirements. MCE was selected as the membrane for subsequent filtration.

[0070] Based on the test results of sensitive bacteria on diluent, flushing solution, culture medium and filter membrane, the suitability test of the method for microbial enumeration of 5 test bacteria and control bacteria was carried out.

[0071] 6. Method applicability test

[0072] 6.1 Preparation of test solution

[0073] Test solution (1): Take 10 g of the test sample and add sterile water to 100 mL. Oscillate at 44°C to disperse it evenly. Prepare the 1:10 test solution (1).

[0074] Test solution (2): Take 20 mL of test solution (1) and add 80 mL of sterile water to dilute it into a 1:50 test solution (2) for later use.

[0075] Irrigation solution containing neutralizer: 1% polysorbate 80 + 1% (mL / mL) 1 mol / L sodium thiosulfate solution (sterilized separately and added according to volume percentage) in 0.9% sterile sodium chloride solution.

[0076] Neutralizer-containing culture medium (TSA* / SDA*): 4% polysorbate 80 + 0.5% soy lecithin + 0.1% L-histidine in TSA / SDA.

[0077] 6.2 Bacterial liquid preparation

[0078] 1. Take the liquid culture of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, and Escherichia coli cultured at 30-35°C for 18-24 hours and dilute them with 0.9% sterile sodium chloride solution to make a bacterial suspension containing no more than 100 cfu per 1 mL, and set aside.

[0079] 2. Take a liquid culture of Candida albicans cultured at 20-25°C for 2-3 days and dilute it with 0.9% sterile sodium chloride solution to a bacterial suspension containing no more than 100 cfu per 1 mL, and set aside.

[0080] 3. Take the Aspergillus niger slant culture after culturing at 20-25°C for 7 days, add 3-5 mL of 0.9% sterile sodium chloride solution containing 0.05% (mL / mL) polysorbate 80 to elute the spores, aspirate the spore suspension and dilute it with 0.9% sterile sodium chloride solution containing 0.05% (mL / mL) polysorbate 80 to a bacterial suspension containing no more than 100 cfu per 1 mL, and set aside.

[0081] 6.3 Applicability test of the method for counting total aerobic bacteria, molds and yeasts

[0082] Recovery ratio determination

[0083] experimental group

[0084] Experimental group (1)

[0085] Take 1 mL of the test solution (2) and add 0.9% sterile sodium chloride solution to 100 mL. Add 1 mL of Staphylococcus aureus suspension (the bacterial count is not greater than 100 cfu), filter through a membrane, rinse 10 times with a rinse solution containing a neutralizer, 100 mL / membrane / time, remove the membrane with sterile tweezers and transfer it to a culture medium containing a neutralizer (prepare two in parallel), incubate at the specified temperature and for the specified time, and observe the counting results. Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger are all operated according to the same method (TSA* cultures are incubated at 30-35°C for no more than 3 days, and SDA* cultures are incubated at 20-25°C for no more than 5 days).

[0086] Experimental group (2)

[0087] Rinse 5 times with a rinse solution containing a neutralizer, 100 mL / membrane / time. Add 1 mL of the test bacterial suspension (the bacterial count is not more than 100 cfu) during the last rinse. The rest of the operation is the same as that of the test group (1).

[0088] Test sample group

[0089] Take 1 mL of the test solution (2) and replace the test bacterial suspension with sterile water. Perform the same operation as the test group (2), incubate at the specified temperature and for the specified time, and observe the counting results.

[0090] Neutralizer group

[0091] Take 1 mL of sterile water instead of test solution (2) and perform the same operation as test group (2).

[0092] Bacterial liquid group

[0093] Take 1 mL of sterile water instead of test solution (2) and perform the same operation as test group (2).

[0094] A negative and blank control was also performed.

[0095] Test results

[0096] The results of the neutralizer applicability test are shown in Table 5.

[0097] Table 5 Applicability results of the neutralizer control group

[0098]

[0099] Note: Both negative and blank controls showed no bacterial growth; * indicates culture medium containing neutralizer.

[0100] The results in Table 5 show that when the rinsing solution containing the neutralizer and the culture medium containing the neutralizer were used, the recovery ratios of the five test bacteria were all between 0.5 and 2, which met the requirements. It can be seen that the added neutralizer can be used for the applicability test of the counting method.

[0101] Considering that dalbavancin is a new semi-synthetic glycopeptide antibiotic with strong bactericidal activity against Gram-positive bacteria, Staphylococcus aureus and Bacillus subtilis were selected to conduct the recovery ratio test of the test group (1). The results are shown in Table 6.

[0102] Table 6 Results of recovery ratios of some test bacteria in test group (1)

[0103]

[0104]

[0105] Note: No colonies grew in the negative and blank controls; * indicates culture medium containing neutralizer.

[0106] The results in Table 6 show that, according to the test group (1), 1 mL of the test solution (2) was added to 100 mL of 0.9% sterile sodium chloride solution, 1 mL of a Staphylococcus aureus (or Bacillus subtilis) bacterial suspension (with a bacterial count of no more than 100 cfu) was added, and the membrane was filtered. The recovery ratios of the two test bacteria were both less than 0.5, which did not meet the requirements. Therefore, a recovery ratio test was conducted on the test group (2), i.e., the membrane was rinsed 5 times with a rinse solution containing a neutralizer, 100 mL / membrane / time, and the bacterial suspension was added during the last rinse. The results are shown in Table 7.

[0107] Table 7 Recovery ratio results of test group (2)

[0108]

[0109] Note: No colonies grew in the negative and blank controls; * indicates culture medium containing neutralizer.

[0110] The results in Table 7 show that when the total aerobic bacteria count, mold and yeast count test was conducted using a 1:50 test solution with the addition of a neutralizer combined with membrane filtration, the recovery ratio of each test bacteria was between 0.5 and 2, which met the requirements of the pharmacopoeia.

[0111] 6.4 Applicability test of Escherichia coli test method

[0112] Test solution

[0113] Same as the counting method suitability test 1:10 test solution (1).

[0114] Experimental process

[0115] experimental group

[0116] Take 10 mL of the test solution (1) and add it to 100 mL of tryptic soy broth, add 1 mL of Escherichia coli suspension (the bacterial count is not more than 100 cfu), and incubate at 30-35°C for 18-24 hours. Take 1 mL of the above culture and inoculate it into 100 mL of MacConkey broth and incubate it at 42-44°C for 24-48 hours. Take the MacConkey broth and streak it onto a MacConkey agar plate and incubate it at 30-35°C for 18-72 hours.

[0117] Test sample control group

[0118] Take 10 mL of the test solution (1) and replace the test bacterial suspension with sterile water, and perform the same operation as the test group.

[0119] Negative control group

[0120] Take 10 mL of sterile water instead of the test solution (1), and replace the test bacterial suspension with sterile water, and operate in the same way as the test group.

[0121] Test results

[0122] The results of three tests on Escherichia coli are shown in Table 8.

[0123] Table 8 Results of the applicability test of the Escherichia coli inspection method

[0124]

[0125]

[0126] The results in Table 8 show that the control bacteria Escherichia coli of this product can be tested according to the method by adding 10 mL of the test solution at a ratio of 1:10 to 100 mL of tryptic soy peptone liquid culture medium.

[0127] 7. Conclusion of the method applicability test

[0128] The microbial limit test of dalbavancin can be performed using the following methods:

[0129] (1) Take 10 g of the test sample, add sterile water to 100 mL, and vortex to mix to make a 1:10 test solution. Take 2 mL of the 1:10 test solution and add 8 mL of sterile water to make a 1:50 test solution.

[0130] (2) Total aerobic bacteria count, mold and yeast count: Take 1 mL of the test solution at a ratio of 1:50 and add it to 100 mL of 0.9% sterile sodium chloride solution. Filter through the membrane and rinse five times with 0.9% sterile sodium chloride solution containing 1% polysorbate 80 + 1% (mL / mL) 1 mol / L sodium thiosulfate solution (100 mL / membrane / time). Move the membrane to TSA / SDA plate culture medium containing 4% polysorbate 80 + 0.5% soy lecithin + 0.1% L-histidine and inspect according to the regulations.

[0131] (3) For the control bacterium Escherichia coli, take 10 mL of the 1:10 test solution and add it to 100 mL of trypticase soy peptone liquid culture medium and inspect according to the method.

[0132] The total number of aerobic bacteria in 1g of test sample shall not exceed 10 3 The total number of cfu, mold and yeast should not exceed 10 2 cfu, no Escherichia coli shall be detected.

[0133] Although the specific embodiments of the present invention are described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for microbial limit test of dalbavancin raw material, characterized in that: The following steps are involved: S1. Preparation of test solution: The test substance is dalbavancin API, which is dissolved in sterile water to prepare a 1:10 test solution and a 1:50 test solution, respectively; S2. Total aerobic bacteria count, mold and yeast count: dilute the test solution at a ratio of 1:50 with 0.9% sterile sodium chloride solution, filter through a membrane, and rinse five times with 100 mL / membrane. Add bacterial suspension during the final rinse, ensuring the bacterial count is no greater than 100 cfu / mL. Transfer the membrane to a medium containing a neutralizer and observe the count results. S3. Inspection of control bacteria Escherichia coli: Take 1:10 of the test solution, transfer it to tryptic soy liquid medium, and incubate it at 30-35℃ for 18-24 hours; take the above culture and inoculate it into MacConkey liquid medium, and incubate it at 42-44℃ for 24-48 hours; take the MacConkey liquid medium and streak it onto MacConkey agar medium plate, and incubate it at 30-35℃ for 18-72 hours.

2. The microbial limit test method for dalbavancin according to claim 1, wherein: The test bacteria for the total aerobic bacterial count include Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Aspergillus niger and Candida albicans; the test bacteria for the total mold and yeast count include Aspergillus niger and Candida albicans.

3. The microbial limit test method for dalbavancin according to claim 1, wherein: The flushing solution is a 0.9% sterile sodium chloride solution containing 1% polysorbate 80 + 1%-3% sodium thiosulfate solution. The above sodium thiosulfate is prepared separately, the percentage is volume percentage, and the mother liquor concentration is 1 mol / L. The rest are mass percentages.

4. The method for microbial limit test of dalbavancin according to claim 1, wherein: The formula of the medium containing the neutralizer is TSA or SDA containing 4% polysorbate 80, 0.5% soy lecithin, and 0.1% L-histidine, and the above percentages are all weight percentages.

5. The microbial limit test method for dalbavancin according to claim 1, wherein: The aerobic bacteria are inoculated in a TSA medium containing a neutralizer and cultured at 30-35°C for no more than 3 days; the molds and yeasts are inoculated in an SDA medium containing a neutralizer and cultured at 20-25°C for no more than 5 days.

6. The method for microbial limit test of dalbavancin according to claim 1, wherein: The membrane filtration adopts a hydrophilic mixed cellulose sterile filter membrane.

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