Method for improving sterility test qualification rate of levofloxacin hydrochloride injection

Abstract

The invention belongs to the technical field of levofloxacin hydrochloride injection, and particularly relates to an off-grid vertical-axis wind driven generator with high wind resistance, which comprises the following specific steps: cleaning a beaker, a glass rod, a volumetric flask and a wide-mouth flask with purified water; the preparation method comprises the following steps: firstly, weighing 50.8 g of analytically pure magnesium chloride, adding the analytically pure magnesium chloride into a beaker, then adding a proper amount of purified water, and stirring with a glass rod until the magnesium chloride is completely dissolved; a glass rod is firstly used for drainage. The sterility test qualification rate of the levofloxacin hydrochloride injection is increased from 44.44% to 100%, the quality risk is greatly reduced, the market reputation and customer credibility of the product are enhanced, and powerful support is provided for long-term competitiveness of enterprises. And a detection optimization scheme formed in the attack process provides experience for prevention and solution of subsequent similar problems, and further improvement of a quality management system is promoted.

Description

Technical Field

[0001] This invention relates to the field of levofloxacin hydrochloride injection technology, specifically a method for improving the sterility test pass rate of levofloxacin hydrochloride injection. Background Technology

[0002] Levofloxacin hydrochloride injection is a quinolone antibiotic whose main function is to inhibit and kill bacteria. It exhibits good antibacterial activity against Gram-negative bacteria, Gram-positive bacteria, and some anaerobic bacteria. Clinically, it is commonly used to treat various infectious diseases caused by susceptible bacteria, such as respiratory infections (e.g., acute bronchitis, pneumonia), urinary tract infections (e.g., pyelonephritis, cystitis), reproductive system infections (e.g., pelvic inflammatory disease, epididymitis), skin and soft tissue infections, and intestinal infections.

[0003] According to the requirements of the 2020 edition of the Chinese Pharmacopoeia, Part IV, General Chapter 1101, Sterility Testing Method, sterility testing of levofloxacin hydrochloride injection is a crucial step in ensuring drug quality and safety. Furthermore, sterility testing must cover all production batches, with a pass rate of 100%. However, currently, the pass rate for sterility testing of levofloxacin hydrochloride injection is only 44.44%. The specific reasons are as follows:

[0004] 1. Low neutralizing agent dosage: Add 16 ml of 2.5 mol / L sterile magnesium chloride solution to every 400 ml of 0.9% sterile sodium chloride solution to eliminate antibacterial properties.

[0005] 2. Insufficient rinsing frequency: For both the test sample and positive control, the rinsing frequency should be: 800 ml of 0.9% sterile sodium chloride buffer (rinsing solution) per filter membrane, 100 ml each time, for a total of 8 rinses.

[0006] 3. Fewer shaking times: There is no requirement for the number of shaking times for "rinsing and shaking at the same time".

[0007] Based on the above, a method for improving the sterility test pass rate of levofloxacin hydrochloride injection is invented. Summary of the Invention

[0008] To address the aforementioned technical problems, according to one aspect of the present invention, the present invention provides the following technical solution:

[0009] A method for improving the sterility test pass rate of levofloxacin hydrochloride injection includes the following specific steps:

[0010] S100, Preparation: Prepare 2.5 mol / L sterile magnesium chloride solution:

[0011] S101: Rinse beakers, glass rods, volumetric flasks, and wide-mouth bottles with purified water;

[0012] S102: First, weigh 50.8g of analytical grade magnesium chloride and add it to a beaker. Then, add an appropriate amount of purified water and stir with a glass rod until completely dissolved.

[0013] S103: First, use a glass rod to guide the flow and transfer the S102 solution to a volumetric flask. Then, rinse the beaker and the glass rod used for guiding the flow with purified water. Transfer the rinsing solution into the volumetric flask and make up to the target volume with purified water. After mixing thoroughly, transfer the solution to a wide-mouth bottle.

[0014] S104: Slightly unscrew the cap of the wide-mouth bottle containing 2.5 mol / L magnesium chloride in S103, and sterilize it using an XG1.G type pulsating vacuum sterilizer;

[0015] S105: First, take out the wide-mouth bottle containing 2.5 mol / L magnesium chloride that was sterilized in S104, and tighten the cap. Then, observe whether the color change of the color strip on the sterilization indicator card matches the standard. Figure 1 To;

[0016] S106: First, seal the wide-mouth bottle containing 2.5mol / L sterile magnesium chloride that meets the requirements after sterilization in S105. Then, label the preparation date and expiration date and store it in a refrigerator for later use.

[0017] S200, Sample Testing Process:

[0018] S201: Consumables and samples used in the testing process are transferred to the clean area through a transfer window, and during the transfer, air shower and disinfection are performed based on the fan and ultraviolet lamp disinfection at the transfer window;

[0019] S202: Wipe the clean workbench with 75% ethanol;

[0020] S203: After spraying or wiping the petri dishes for disinfection, transfer them to the clean workbench disinfected in S202. Place three petri dishes in the order of left, center, and right to monitor the settling bacteria throughout the process and mark the petri dishes. Then, open the lids of the placed petri dishes one by one to expose the surface of the culture medium to the air. After all sampling is completed, invert the petri dishes in the incubator for incubation. Then, after sampling the petri dishes prepared with tryptic soy agar medium, incubate them in the incubator. After sampling the petri dishes prepared with Sabouraud dextrose agar medium, incubate them in the incubator.

[0021] S204: First, light an alcohol lamp in the clean workbench where S203 monitors the settling bacteria. Then, disinfect the tools that were passed through the transfer window in S201 by spraying or wiping them with disinfectant before moving them into the clean workbench.

[0022] S205: First, spray or wipe the drain tank with the outer packaging of the package that was passed through the transfer window to disinfect it, and then transfer it to the clean workbench. After removing the outer packaging of the drain tank, place it on the bacteria collection instrument. Then, place the drain outlet of the drain pipe in the water bucket on the clean workbench floor after each use for cleaning and disinfection. After that, take the sample to be tested, rinsing solution, and culture medium out of the trolley, spray or wipe them, and then place them on the clean workbench.

[0023] S206: First, spray or wipe the pH 7.0 sterile sodium chloride-peptone buffer solution or 0.9% sterile sodium chloride solution and the self-prepared 2.5 mol / L sterile magnesium chloride solution for disinfection, and then transfer them to the clean workbench. Next, remove the bottle cap under laminar flow, and use the outer flame of the alcohol lamp in the clean workbench to ignite the outer bottle mouth of the pH 7.0 sterile sodium chloride-peptone buffer solution or 0.9% sterile sodium chloride solution and the self-prepared 2.5 mol / L sterile magnesium chloride solution.

[0024] S207: Using a sterilized 30ml syringe, draw 16ml of 2.5mol / L sterile magnesium chloride solution from the mouth of the S206 flask and add it to 400ml of 0.9% sterile sodium chloride solution or pH 7.0 sterile sodium chloride-peptone buffer solution from the mouth of the S206 flask. Mix well and use it as a diluent for dissolving the test sample.

[0025] S208: Take a sterilized 30ml syringe, draw 20ml of 2.5mol / L sterile magnesium chloride solution from the mouth of the S206 flask, and add it to 400ml of 0.9% sterile sodium chloride solution or pH 7.0 sterile sodium chloride-peptone buffer solution from the mouth of the S206 flask. Mix well and use it as a rinsing solution for rinsing the filter membrane.

[0026] S209: Transfer the test sample, after being sprayed or wiped for disinfection, to a clean workbench;

[0027] S210: Take 30 test samples from S209 and transfer them to the clean workbench. Use a syringe to draw 10 samples at a time and add them to the 400ml test sample dissolving diluent prepared in S207. Shake thoroughly until the solution is homogeneous. Mix well and use it as the test solution for later use. Prepare 3 parallel solutions.

[0028] S211: Transfer the tryptic soy peptone liquid culture medium and thioglycolate fluid culture medium, which have been disinfected by spraying or wiping, into a clean workbench;

[0029] S212: Turn on the sterilizer, disinfect the well-packaged sterilizer by spraying or wiping it, and transfer it to the clean workbench. Check the integrity of the sterilizer packaging bag, then open the packaging bag and write the product name, batch number, and test date on the three tubes of the sterilizer.

[0030] S213: First, insert the base of the bacterial collector from the clean bench into the drain hole of the bacterial collector. Then, install the bacterial collector hose onto the peristaltic pump of the bacterial collector. After that, adjust the pump speed of the bacterial collector to 180 rpm / min to draw 50 ml of the rinsing solution prepared in S208 into each culture vessel to wet the filter membrane.

[0031] S214: Take 3 portions of the test solution prepared in S210 and filter them in 1 portion per tube of each bacterial collector. The amount of sample filtered by each filter membrane is equivalent to the total amount of 10 test samples. After filtering all the test solution, rinse the filter membrane with the rinsing solution prepared in S208. The amount of rinsing solution used for each filter membrane is 1000ml, 100ml each time, and rinse 10 times. Also, hold the bacterial collector horizontally with both hands and rotate and shake it 12 times to achieve rinsing and shaking at the same time.

[0032] S215: After the liquid in the collector is completely drained, stop the peristaltic pump of the collector with the collector tubing installed, and use the two clamps passed through the transfer window to clamp the upper tubing at the liquid inlet of the two filter cups respectively, and plug the bottom of the collector with the stopper. Then turn on the peristaltic pump that was just turned off, hold the culture medium bottle in your left hand and ignite it over a flame, and pull out the needle of the collector that has been drained of liquid with your right hand and insert it into the culture medium bottle. Invert the culture medium bottle and add tryptic soy peptone liquid culture medium to the 100ml mark on the cup.

[0033] S216: Use a disposable syringe to draw 5 ml of the self-prepared 2.5 mol / L sterile magnesium chloride solution and add it to S215 tryptic soybean liquid medium;

[0034] S217: First, place the bottle of tryptic soy broth culture medium containing 2.5 mol / L sterile magnesium chloride solution in S216 upright. After emptying the culture medium in the collection tube, stop the peristaltic pump started by S215 using the foot switch. Next, take another clamp and clamp the upper end of the tubing at the inlet of the filter cup containing tryptic soy broth culture medium. Then, remove the clamps from S215 that clamp the upper end of the tubing at the inlet of the two filter cups. Finally, hold the culture medium bottle in your left hand and ignite it over a flame. With your right hand, remove the needles from the collection tubes that have been emptied by S215 and insert them into the thioglycolate fluid culture medium bottle. Invert the culture medium bottle and add 100 ml of thioglycolate fluid culture medium to each of the other two filter cups.

[0035] S218: Use a disposable syringe to draw 5 ml of the self-prepared 2.5 mol / L sterile magnesium chloride solution and add it to two portions of thioglycolate fluid culture medium in S217. One portion is used as the test sample and the other portion is used as the positive control.

[0036] S219: First, use the two clips removed from S217 to clamp the upper end of the tubing at the inlet of the filter cup containing thioglycolate fluid culture medium. Then, take a pair of sterile scissors passed through the transfer window and cut the tubing 3-4 cm above the clips clamping the tubing of the filter cup containing thioglycolate fluid culture medium. Finally, insert the tail end into the exhaust port at the top of the filter cup.

[0037] S220: Remove all tubing from the peristaltic pump of the bacterial collector used on the device;

[0038] S221, negative control: Take another new collection tube and replace the test solution with an equal volume of diluent. The other steps are the same as adding thioglycolate fluid medium. Add tryptic soy peptone liquid medium to another tube.

[0039] S222: Place the test sample and negative control collection container into the transfer window and incubate the thioglycolate fluid medium tubes and tryptic soy peptone liquid medium used for the test sample and negative control examination.

[0040] S223: Transfer the thioglycolate fluid culture medium tube used for the positive control to the positive test room, add 1 ml of Staphylococcus aureus (not more than 100 CFU), and then incubate in a positive incubator.

[0041] S224: Daily, remove the test sample, negative control group, and positive control group from the incubator to observe whether bacteria grow.

[0042] S225, Result Interpretation: Positive control tubes should show good growth within 5 days of incubation; negative control tubes should not show bacterial growth, otherwise the experiment is invalid; if all test tubes are clear, or although they appear turbid but are confirmed to have no bacterial growth, the test samples are judged to meet the requirements; if any test tube is turbid and is confirmed to have bacterial growth, the test samples are judged to not meet the requirements.

[0043] As a preferred embodiment of the method for improving the sterility test pass rate of levofloxacin hydrochloride injection according to the present invention, wherein: the operating temperature of the XG1.G type pulsating vacuum sterilizer in S104 is 121°C, the operating time is 20 minutes, and the operating program is liquid mode.

[0044] As a preferred embodiment of the method for improving the sterility test pass rate of levofloxacin hydrochloride injection according to the present invention, wherein the temperature of the refrigerator in S106 is 2-8℃.

[0045] As a preferred embodiment of the method for improving the sterility test pass rate of levofloxacin hydrochloride injection according to the present invention, wherein: the air shower time in S201 is not less than 2 minutes and the disinfection time is not less than 30 minutes.

[0046] As a preferred embodiment of the method for improving the sterility test pass rate of levofloxacin hydrochloride injection according to the present invention, wherein: the monitoring time for settling bacteria in S203 is 0.5-4 hours; the culture time of the culture dishes prepared with tryptic soy peptone agar medium in S203 is not less than 3 days, and the culture temperature of the incubator is 30-35℃; the culture time of the culture dishes prepared with Sabouraud dextrose agar medium in S203 is not less than 5 days, and the culture temperature of the incubator is 20-25℃.

[0047] As a preferred embodiment of the method for improving the sterility test pass rate of levofloxacin hydrochloride injection according to the present invention, in step S214, during rinsing, the drain tank is lifted by 3-5 cm, and during shaking, it rotates every 2 revolutions / second.

[0048] As a preferred embodiment of the method for improving the sterility test pass rate of levofloxacin hydrochloride injection according to the present invention, wherein: the culture temperature of the thioglycolate fluid culture medium tube in S222 is 30-35℃ and the culture time is not less than 14 days, and the culture temperature of the tryptic soy peptone liquid culture medium is 20-25℃ and the culture time is not less than 14 days.

[0049] As a preferred embodiment of the method for improving the sterility test pass rate of levofloxacin hydrochloride injection according to the present invention, wherein the incubation temperature of the incubator in S223 is 30-35℃.

[0050] Compared with existing technologies:

[0051] This invention improved the sterility test pass rate of levofloxacin hydrochloride injection from 44.44% to 100%, significantly reducing quality risks, enhancing the product's market reputation and customer trust, and providing strong support for the company's long-term competitiveness. Furthermore, the optimized testing solutions developed during the research and development process provided experience for the prevention and resolution of similar problems in the future, promoting the further improvement of the quality management system. Attached Figure Description

[0052] Figure 1 The graph shows the effect of different volumes of neutralizing agent of the present invention on the one-time pass rate of sterility of levofloxacin hydrochloride injection.

[0053] Figure 2 The graph shows the effect of different volumes of rinsing solution on the one-time pass rate of sterility testing of levofloxacin hydrochloride injection.

[0054] Figure 3 The graph shows the effect of different shaking times on the one-time pass rate of aseptic levofloxacin hydrochloride injection.

[0055] Figure 4This is a comparison chart showing the one-time pass rate of sterility testing for levofloxacin hydrochloride injection before and after the breakthrough of this invention. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below.

[0057] Example 1:

[0058] This invention provides a method for improving the sterility test pass rate of levofloxacin hydrochloride injection, comprising the following specific steps:

[0059] S100, Preparation: Prepare 2.5 mol / L sterile magnesium chloride solution:

[0060] S101: Rinse beakers, glass rods, volumetric flasks, and wide-mouth bottles with purified water;

[0061] S102: First, weigh 50.8g of analytical grade magnesium chloride and add it to a beaker. Then, add an appropriate amount of purified water and stir with a glass rod until completely dissolved.

[0062] S103: First, use a glass rod to guide the flow and transfer the S102 solution to a volumetric flask. Then, rinse the beaker and the glass rod used for guiding the flow with purified water. Transfer the rinsing solution into the volumetric flask and make up to the target volume (e.g., 100 mL) with purified water. After mixing thoroughly, transfer the solution to a wide-mouth bottle.

[0063] S104: Slightly unscrew the cap of the wide-mouth bottle containing 2.5 mol / L magnesium chloride in S103, and sterilize it using an XG1.G type pulsating vacuum sterilizer; wherein, the XG1.G type pulsating vacuum sterilizer operates at a temperature of 121℃, operates for 20 minutes, and operates in liquid mode.

[0064] S105: First, take out the wide-mouth bottle containing 2.5 mol / L magnesium chloride that was sterilized in S104, and tighten the cap. Then, observe whether the color change of the color strip on the sterilization indicator card matches the standard. Figure 1 To;

[0065] S106: First, seal the wide-mouth bottle containing 2.5 mol / L sterile magnesium chloride that meets the requirements after sterilization in S105. Then, label the preparation date and expiration date and store it in a refrigerator for later use. The temperature of the refrigerator should be 2°C.

[0066] S200, Sample Testing Process:

[0067] S201: Transfer consumables and samples used in the testing process to the clean area through the transfer window. When items are transferred from the general production area to the clean area or from the lower-level clean area to the higher-level clean area, turn on the transfer window fan button and the UV lamp disinfection switch to perform air shower and disinfection respectively. The air shower time is not less than 2 minutes and the UV lamp disinfection time is not less than 30 minutes. After completion, press the door opening button from the material picking side to open the transfer window door, take out the items, and close the transfer window door.

[0068] S202: Wipe the clean workbench with 75% ethanol or other proven disinfectant (compound peroxide concentrate type II 1:50);

[0069] S203: After spraying or wiping the petri dishes for disinfection, transfer them to the clean workbench disinfected in S202. Place three petri dishes in a left-center-right arrangement and monitor the settling bacteria throughout the process (no bacteria should grow; if bacteria grow, bacterial species identification is required). Mark the petri dishes. Then, open the lids of each petri dish one by one to expose the culture medium surface to air. The monitoring time is 0.5 hours. After all sampling is completed, invert the petri dishes in an incubator for incubation. Then, after sampling using petri dishes prepared with tryptic soy agar (TSA) medium, incubate... The samples were incubated in an incubator for at least 3 days at a temperature of 30°C. (When the environmental microbial population information is uncertain, different incubation conditions can be selected according to the monitoring focus, such as incubating at 20°C for 3 days and then transferring to 30°C for 2 days, or incubating at 30°C for 2 days and then transferring to 20°C for 3 days). After sampling, the samples were incubated in a culture dish prepared with Sabouraud dextrose agar (SDA) for at least 5 days at a temperature of 20°C.

[0070] S204: First, light an alcohol lamp in the clean workbench where S203 monitors the settling bacteria. Then, disinfect the tools that were passed through the transfer window in S201 by spraying or wiping them with disinfectant before moving them into the clean workbench.

[0071] S205: First, spray or wipe the drain tank with the outer packaging of the package that was passed through the transfer window to disinfect it, and then transfer it to the clean workbench. After removing the outer packaging of the drain tank, place it on the bacteria collection instrument. Then, place the drain outlet of the drain pipe in the water bucket on the clean workbench floor after each use for cleaning and disinfection. After that, take the sample to be tested, rinsing solution, and culture medium out of the trolley, spray or wipe them, and then place them on the clean workbench.

[0072] S206: First, spray or wipe the pH 7.0 sterile sodium chloride-peptone buffer solution or 0.9% sterile sodium chloride solution and the self-prepared 2.5 mol / L sterile magnesium chloride solution for disinfection, and then transfer them to the clean workbench. Next, remove the bottle cap under laminar flow, and use the outer flame of the alcohol lamp in the clean workbench to ignite the outer bottle mouth of the pH 7.0 sterile sodium chloride-peptone buffer solution or 0.9% sterile sodium chloride solution and the self-prepared 2.5 mol / L sterile magnesium chloride solution.

[0073] S207: Using a sterilized 30ml syringe, draw 16ml of 2.5mol / L sterile magnesium chloride solution from the mouth of the S206 flask and add it to 400ml of 0.9% sterile sodium chloride solution or pH 7.0 sterile sodium chloride-peptone buffer solution from the mouth of the S206 flask. Mix well and use it as a diluent for dissolving the test sample.

[0074] S208: Take a sterilized 30ml syringe, draw 20ml of 2.5mol / L sterile magnesium chloride solution from the end of the S206 flask, and add it to 400ml of 0.9% sterile sodium chloride solution or pH 7.0 sterile sodium chloride-peptone buffer solution from the end of the S206 flask. Mix well and use it as a rinsing solution for rinsing the filter membrane.

[0075] S209: Transfer the test sample, after being sprayed or wiped for disinfection, to a clean workbench;

[0076] S210: Take 30 test samples from S209 and transfer them to the clean bench. Use a syringe to draw 10 samples at a time and add them to the 400ml test sample dissolving diluent prepared in S207. Shake thoroughly until the solution is homogeneous. Mix well and use it as the test solution for later use. Prepare 3 parallel solutions.

[0077] S211: Transfer the tryptic soy peptone liquid culture medium and thioglycolate fluid culture medium, which have been disinfected by spraying or wiping, into a clean workbench;

[0078] S212: Turn on the microbial collector, and after spraying or wiping the well-packaged microbial collector (i.e., KSF330 triple disposable fully enclosed microbial culture device with 0.45μm microporous filter membrane) for disinfection, transfer it to the clean workbench and check the integrity of the microbial collector packaging bag. Then open the packaging bag and write the product name, batch number and test date on the three tubes of the microbial collector.

[0079] S213: First, insert the base of the bacterial collector from the clean bench into the drain hole of the bacterial collector. Then, install the bacterial collector hose onto the peristaltic pump of the bacterial collector. After that, adjust the pump speed of the bacterial collector to 180 rpm / min to draw 50 ml of the rinsing solution prepared in S208 into each culture vessel to wet the filter membrane.

[0080] S214: Take 3 portions of the test solution prepared in S210 and filter them in 1 portion per tube of each bacterial collector. The amount of sample filtered by each filter membrane is equivalent to the total amount of 10 test samples. After filtering all the test solution, rinse the filter membrane with the rinsing solution prepared in S208. The amount of rinsing solution used for each filter membrane is 1000ml, 100ml each time, and rinse 10 times. At the same time, hold the bacterial collector horizontally with both hands and rotate and shake it 12 times to achieve rinsing and shaking at the same time. During rinsing, lift the drain tank 3cm, and shake at 2 revolutions / second.

[0081] S215: After the liquid in the collector is completely drained, stop the peristaltic pump of the collector with the collector tubing installed, and use the two clamps passed through the transfer window to clamp the upper tubing at the inlet of the two filter cups respectively, and plug the bottom of the collector with the stopper. Then turn on the peristaltic pump that was just turned off, hold the culture medium bottle in your left hand and ignite it over a flame, and pull out the needle of the collector that has been drained of liquid with your right hand and insert it into the culture medium bottle. Invert the culture medium bottle and add tryptic soy peptone liquid culture medium that conforms to the 2020 edition of the Chinese Pharmacopoeia to the 100ml mark on the cup (three ring-shaped scalar graduation lines can be seen on the cup, from bottom to top, they are 50, 75 and 100ml respectively).

[0082] S216: Use a disposable syringe to draw 5 ml of the self-prepared 2.5 mol / L sterile magnesium chloride solution and add it to S215 tryptic soybean liquid medium;

[0083] S217: First, place the bottle containing tryptic soy broth culture medium (S216) with 2.5 mol / L sterile magnesium chloride solution upright. After emptying the culture medium from the collection tube, stop the peristaltic pump started by S215 using the foot switch. Next, take another clamp and clamp the upper end of the tubing at the inlet of the filter cup containing tryptic soy broth culture medium. Then, remove the clamps from S215 that clamp the upper end of the tubing at the inlet of the two filter cups. Finally, hold the culture medium bottle in your left hand and ignite it over a flame. With your right hand, remove the collection tube needles from the other two tubes (S215) and insert them into the thioglycolate fluid culture medium bottle. Invert the culture medium bottle and add 100 ml of thioglycolate fluid culture medium conforming to the 2020 edition of the Chinese Pharmacopoeia to each of the other two filter cups.

[0084] S218: Use a disposable syringe to draw 5 ml of the self-prepared 2.5 mol / L sterile magnesium chloride solution and add it to two portions of thioglycolate fluid culture medium in S217. One portion is used as the test sample and the other portion is used as the positive control.

[0085] S219: First, use the two clips removed from S217 to clamp the upper end of the tubing at the inlet of the filter cup containing thioglycolate fluid culture medium. Then, take a pair of sterile scissors passed through the transfer window and cut the tubing 3cm from the upper end of the clips that clamp the tubing of the filter cup containing thioglycolate fluid culture medium. Finally, insert the tail end into the exhaust port at the top of the filter cup.

[0086] S220: Remove all tubing from the peristaltic pump of the bacterial collector used on the device;

[0087] S221, Negative Control: Take another new collection tube and replace the test solution with an equal volume of diluent. Follow the same steps as adding thioglycolate fluid medium. Add tryptic soy peptone liquid medium (add 5 ml of 2.5 mol / L sterile magnesium chloride solution to every 100 ml of medium).

[0088] S222: Place the test sample and negative control collection container into the transfer window, and incubate the thioglycolate fluid medium tubes and tryptic soy liquid medium used for the test sample and negative control; wherein, the incubation temperature of the thioglycolate fluid medium tubes is 30℃ and the incubation time is not less than 14 days, and the incubation temperature of the tryptic soy liquid medium is 20℃ and the incubation time is not less than 14 days.

[0089] S223: According to the 2020 edition of the Chinese Pharmacopoeia 1101 sterility test method, the thioglycolate fluid culture medium tube used as a positive control was transferred to the positive test room, and 1 ml of Staphylococcus aureus not exceeding 100 CFU was added before incubation in a positive incubator; wherein, the incubation temperature of the incubator was 30℃.

[0090] S224: Daily, remove the test sample, negative control group, and positive control group from the incubator to observe whether bacteria grow.

[0091] S225, Result Interpretation: Positive control tubes should show good growth within 5 days of incubation; negative control tubes should not show bacterial growth, otherwise the experiment is invalid; if all test tubes are clear, or although they appear turbid but are confirmed to have no bacterial growth, the test samples are deemed to meet the requirements; if any test tube is turbid and is confirmed to have bacterial growth, the test samples are deemed to not meet the requirements, unless it can be sufficiently proven that the test results are invalid.

[0092] Example 2:

[0093] This invention provides a method for improving the sterility test pass rate of levofloxacin hydrochloride injection, comprising the following specific steps:

[0094] S100, Preparation: Prepare 2.5 mol / L sterile magnesium chloride solution:

[0095] S101: Rinse beakers, glass rods, volumetric flasks, and wide-mouth bottles with purified water;

[0096] S102: First, weigh 50.8g of analytical grade magnesium chloride and add it to a beaker. Then, add an appropriate amount of purified water and stir with a glass rod until completely dissolved.

[0097] S103: First, use a glass rod to guide the flow and transfer the S102 solution to a volumetric flask. Then, rinse the beaker and the glass rod used for guiding the flow with purified water. Transfer the rinsing solution into the volumetric flask and make up to the target volume (e.g., 100 mL) with purified water. After mixing thoroughly, transfer the solution to a wide-mouth bottle.

[0098] S104: Slightly unscrew the cap of the wide-mouth bottle containing 2.5 mol / L magnesium chloride in S103, and sterilize it using an XG1.G type pulsating vacuum sterilizer; wherein, the XG1.G type pulsating vacuum sterilizer operates at a temperature of 121℃, operates for 20 minutes, and operates in liquid mode.

[0099] S105: First, take out the wide-mouth bottle containing 2.5 mol / L magnesium chloride that was sterilized in S104, and tighten the cap. Then, observe whether the color change of the color strip on the sterilization indicator card matches the standard. Figure 1 To;

[0100] S106: First, seal the wide-mouth bottle containing 2.5 mol / L sterile magnesium chloride that meets the requirements after sterilization in S105. Then, label it with the preparation date and expiration date and store it in a refrigerator for later use. The temperature of the refrigerator should be 4℃.

[0101] S200, Sample Testing Process:

[0102] S201: Transfer consumables and samples used in the testing process to the clean area through the transfer window. When items are transferred from the general production area to the clean area or from the lower-level clean area to the higher-level clean area, turn on the transfer window fan button and the UV lamp disinfection switch to perform air shower and disinfection respectively. The air shower time is not less than 2 minutes and the UV lamp disinfection time is not less than 30 minutes. After completion, press the door opening button from the material picking side to open the transfer window door, take out the items, and close the transfer window door.

[0103] S202: Wipe the clean workbench with 75% ethanol or other proven disinfectant (compound peroxide concentrate type II 1:50);

[0104] S203: After spraying or wiping the petri dishes for disinfection, transfer them to the clean workbench disinfected in S202. Place three petri dishes in a left-center-right arrangement and monitor the settling bacteria throughout the process (no bacteria should grow; if bacteria grow, bacterial identification is required). Mark the petri dishes. Then, open the lids of each petri dish one by one to expose the culture medium surface to air. The monitoring time is 2.5 hours. After all sampling is completed, invert the petri dishes and incubate them in an incubator. Then, after sampling using petri dishes prepared with tryptic soy agar (TSA) medium, incubate them in an incubator. The incubation period is no less than 3 days, and the incubation temperature is 32.5℃. (When the environmental microbial population information is uncertain, different incubation conditions can be selected according to different monitoring points of interest, such as first incubating at 27.5℃ for 4 days and then transferring to 32.5℃ for 2.5 days, or first incubating at 32.5℃ for 2.5 days and then transferring to 22.5℃ for 4 days). After sampling, the culture dishes prepared with Sabouraud dextrose agar (SDA) are incubated in an incubator for no less than 5 days, and the incubation temperature is 22.5℃.

[0105] S204: First, light an alcohol lamp in the clean workbench where S203 monitors the settling bacteria. Then, disinfect the tools that were passed through the transfer window in S201 by spraying or wiping them with disinfectant before moving them into the clean workbench.

[0106] S205: First, spray or wipe the drain tank with the outer packaging of the package that was passed through the transfer window to disinfect it, and then transfer it to the clean workbench. After removing the outer packaging of the drain tank, place it on the bacteria collection instrument. Then, place the drain outlet of the drain pipe in the water bucket on the clean workbench floor after each use for cleaning and disinfection. After that, take the sample to be tested, rinsing solution, and culture medium out of the trolley, spray or wipe them, and then place them on the clean workbench.

[0107] S206: First, spray or wipe the pH 7.0 sterile sodium chloride-peptone buffer solution or 0.9% sterile sodium chloride solution and the self-prepared 2.5 mol / L sterile magnesium chloride solution for disinfection, and then transfer them to the clean workbench. Next, remove the bottle cap under laminar flow, and use the outer flame of the alcohol lamp in the clean workbench to ignite the outer bottle mouth of the pH 7.0 sterile sodium chloride-peptone buffer solution or 0.9% sterile sodium chloride solution and the self-prepared 2.5 mol / L sterile magnesium chloride solution.

[0108] S207: Using a sterilized 30ml syringe, draw 16ml of 2.5mol / L sterile magnesium chloride solution from the mouth of the S206 flask and add it to 400ml of 0.9% sterile sodium chloride solution or pH 7.0 sterile sodium chloride-peptone buffer solution from the mouth of the S206 flask. Mix well and use it as a diluent for dissolving the test sample.

[0109] S208: Take a sterilized 30ml syringe, draw 20ml of 2.5mol / L sterile magnesium chloride solution from the mouth of the S206 flask, and add it to 400ml of 0.9% sterile sodium chloride solution or pH 7.0 sterile sodium chloride-peptone buffer solution from the mouth of the S206 flask. Mix well and use it as a rinsing solution for rinsing the filter membrane.

[0110] S209: Transfer the test sample, after being sprayed or wiped for disinfection, to a clean workbench;

[0111] S210: Take 30 test samples from S209 and transfer them to the clean workbench. Use a syringe to draw 10 samples at a time and add them to the 400ml test sample dissolving diluent prepared in S207. Shake thoroughly until the solution is homogeneous. Mix well and use it as the test solution for later use. Prepare 3 parallel solutions.

[0112] S211: Transfer the tryptic soy peptone liquid culture medium and thioglycolate fluid culture medium, which have been disinfected by spraying or wiping, into a clean workbench;

[0113] S212: Turn on the microbial collector, and after spraying or wiping the well-packaged microbial collector (i.e., KSF330 triple disposable fully enclosed microbial culture device with 0.45μm microporous filter membrane) for disinfection, transfer it to the clean workbench and check the integrity of the microbial collector packaging bag. Then open the packaging bag and write the product name, batch number and test date on the three tubes of the microbial collector.

[0114] S213: First, insert the base of the bacterial collector from the clean bench into the drain hole of the bacterial collector. Then, install the bacterial collector hose onto the peristaltic pump of the bacterial collector. After that, adjust the pump speed of the bacterial collector to 180 rpm / min to draw 50 ml of the rinsing solution prepared in S208 into each culture vessel to wet the filter membrane.

[0115] S214: Take 3 portions of the test solution prepared in S210 and filter them in 1 portion per tube of each bacterial collector. The amount of sample filtered by each filter membrane is equivalent to the total amount of 10 test samples. After filtering all the test solution, rinse the filter membrane with the rinsing solution prepared in S208. The amount of rinsing solution used for each filter membrane is 1000ml, 100ml each time, and rinse 10 times. At the same time, hold the bacterial collector horizontally with both hands and rotate and shake it 12 times to achieve rinsing and shaking at the same time. During rinsing, lift the drain tank 4cm, and shake it at 2 revolutions / second.

[0116] S215: After the liquid in the collector is completely drained, stop the peristaltic pump of the collector with the collector tubing installed, and use the two clamps passed through the transfer window to clamp the upper tubing at the inlet of the two filter cups respectively, and plug the bottom of the collector with the stopper. Then turn on the peristaltic pump that was just turned off, hold the culture medium bottle in your left hand and ignite it over a flame, and pull out the needle of the collector that has been drained of liquid with your right hand and insert it into the culture medium bottle. Invert the culture medium bottle and add tryptic soy peptone liquid culture medium that conforms to the 2020 edition of the Chinese Pharmacopoeia to the 100ml mark on the cup (three ring-shaped scalar graduation lines can be seen on the cup, from bottom to top, they are 50, 75 and 100ml respectively).

[0117] S216: Use a disposable syringe to draw 5 ml of the self-prepared 2.5 mol / L sterile magnesium chloride solution and add it to S215 tryptic soybean liquid medium;

[0118] S217: First, place the bottle containing tryptic soy broth culture medium (S216) with 2.5 mol / L sterile magnesium chloride solution upright. After emptying the culture medium from the collection tube, stop the peristaltic pump started by S215 using the foot switch. Next, take another clamp and clamp the upper end of the tubing at the inlet of the filter cup containing tryptic soy broth culture medium. Then, remove the clamps from S215 that clamp the upper end of the tubing at the inlet of the two filter cups. Finally, hold the culture medium bottle in your left hand and ignite it over a flame. With your right hand, remove the collection tube needles from the other two tubes (S215) and insert them into the thioglycolate fluid culture medium bottle. Invert the culture medium bottle and add 100 ml of thioglycolate fluid culture medium conforming to the 2020 edition of the Chinese Pharmacopoeia to each of the other two filter cups.

[0119] S218: Use a disposable syringe to draw 5 ml of the self-prepared 2.5 mol / L sterile magnesium chloride solution and add it to two portions of thioglycolate fluid culture medium in S217. One portion is used as the test sample and the other portion is used as the positive control.

[0120] S219: First, use the two clips removed from S217 to clamp the upper end of the tubing at the inlet of the filter cup containing thioglycolate fluid culture medium. Then, take a pair of sterile scissors that have been passed through the transfer window and cut the tubing 3.5cm from the top of the clips that clamp the tubing of the filter cup containing thioglycolate fluid culture medium. Finally, insert the tail end into the exhaust port at the top of the filter cup.

[0121] S220: Remove all tubing from the peristaltic pump of the bacterial collector used on the device;

[0122] S221, Negative Control: Take another new collection tube and replace the test solution with an equal volume of diluent. Follow the same steps as adding thioglycolate fluid medium. Add tryptic soy peptone liquid medium (add 5 ml of 2.5 mol / L sterile magnesium chloride solution to every 100 ml of medium).

[0123] S222: Place the test sample and negative control collection container into the transfer window, and incubate the thioglycolate fluid medium tubes and tryptic soy liquid medium used for the test sample and negative control; wherein the incubation temperature of the thioglycolate fluid medium tubes is 32.5℃ and the incubation time is not less than 14 days, and the incubation temperature of the tryptic soy liquid medium is 22.5℃ and the incubation time is not less than 14 days;

[0124] S223: According to the 2020 edition of the Chinese Pharmacopoeia 1101 sterility test method, the thioglycolate fluid culture medium tube used as a positive control was transferred to the positive test room, and 1 ml of Staphylococcus aureus not exceeding 100 CFU was added before incubation in a positive incubator; wherein, the incubation temperature of the incubator was 32.5℃.

[0125] S224: Daily, remove the test sample, negative control group, and positive control group from the incubator to observe whether bacteria grow.

[0126] S225, Result Interpretation: Positive control tubes should show good growth within 5 days of incubation; negative control tubes should not show bacterial growth, otherwise the experiment is invalid; if all test tubes are clear, or although they appear turbid but are confirmed to have no bacterial growth, the test samples are deemed to meet the requirements; if any test tube is turbid and is confirmed to have bacterial growth, the test samples are deemed to not meet the requirements, unless it can be sufficiently proven that the test results are invalid.

[0127] Example 3:

[0128] This invention provides a method for improving the sterility test pass rate of levofloxacin hydrochloride injection, comprising the following specific steps:

[0129] S100, Preparation: Prepare 2.5 mol / L sterile magnesium chloride solution:

[0130] S101: Rinse beakers, glass rods, volumetric flasks, and wide-mouth bottles with purified water;

[0131] S102: First, weigh 50.8g of analytical grade magnesium chloride and add it to a beaker. Then, add an appropriate amount of purified water and stir with a glass rod until completely dissolved.

[0132] S103: First, use a glass rod to guide the flow and transfer the S102 solution to a volumetric flask. Then, rinse the beaker and the glass rod used for guiding the flow with purified water. Transfer the rinsing solution into the volumetric flask and make up to the target volume (e.g., 100 mL) with purified water. After mixing thoroughly, transfer the solution to a wide-mouth bottle.

[0133] S104: Slightly unscrew the cap of the wide-mouth bottle containing 2.5 mol / L magnesium chloride in S103, and sterilize it using an XG1.G type pulsating vacuum sterilizer; wherein, the XG1.G type pulsating vacuum sterilizer operates at a temperature of 121℃, operates for 20 minutes, and operates in liquid mode.

[0134] S105: First, take out the wide-mouth bottle containing 2.5 mol / L magnesium chloride that was sterilized in S104, and tighten the cap. Then, observe whether the color change of the color strip on the sterilization indicator card matches the standard. Figure 1 To;

[0135] S106: First, seal the wide-mouth bottle containing 2.5 mol / L sterile magnesium chloride that meets the requirements after sterilization in S105. Then, label it with the preparation date and expiration date and store it in a refrigerator for later use. The temperature of the refrigerator should be 8°C.

[0136] S200, Sample Testing Process:

[0137] S201: Transfer consumables and samples used in the testing process to the clean area through the transfer window. When items are transferred from the general production area to the clean area or from the lower-level clean area to the higher-level clean area, turn on the transfer window fan button and the UV lamp disinfection switch to perform air shower and disinfection respectively. The air shower time is not less than 2 minutes and the UV lamp disinfection time is not less than 30 minutes. After completion, press the door opening button from the material picking side to open the transfer window door, take out the items, and close the transfer window door.

[0138] S202: Wipe the clean workbench with 75% ethanol or other proven disinfectant (compound peroxide concentrate type II 1:50);

[0139] S203: After spraying or wiping the petri dishes for disinfection, transfer them to the clean workbench disinfected in S202. Place three petri dishes in a left-center-right arrangement and monitor the settling bacteria throughout the process (no bacteria should grow; if bacteria grow, bacterial identification is required). Mark the petri dishes. Then, open the lids of each petri dish one by one to expose the culture medium surface to air. The monitoring time is 4 hours. After all sampling is completed, invert the petri dishes and incubate them in an incubator. Then, after sampling using petri dishes prepared with tryptic soy agar (TSA) medium, incubate them in an incubator. The samples were cultured in a medium for at least 3 days at a temperature of 35°C. (When the environmental microbial population information is uncertain, different culture conditions can be selected according to the monitoring focus, such as culturing at 35°C for 5 days and then transferring to 35°C for 3 days, or culturing at 35°C for 3 days and then transferring to 25°C for 5 days). After sampling, the culture dishes prepared with Sabouraud dextrose agar (SDA) were cultured in an incubator for at least 5 days at a temperature of 25°C.

[0140] S204: First, light an alcohol lamp in the clean workbench where S203 monitors the settling bacteria. Then, disinfect the tools that were passed through the transfer window in S201 by spraying or wiping them with disinfectant before moving them into the clean workbench.

[0141] S205: First, spray or wipe the drain tank with the outer packaging of the package that was passed through the transfer window to disinfect it, and then transfer it to the clean workbench. After removing the outer packaging of the drain tank, place it on the bacteria collection instrument. Then, place the drain outlet of the drain pipe in the water bucket on the clean workbench floor after each use for cleaning and disinfection. After that, take the sample to be tested, rinsing solution, and culture medium out of the trolley, spray or wipe them, and then place them on the clean workbench.

[0142] S206: First, spray or wipe the pH 7.0 sterile sodium chloride-peptone buffer solution or 0.9% sterile sodium chloride solution and the self-prepared 2.5 mol / L sterile magnesium chloride solution for disinfection, and then transfer them to the clean workbench. Next, remove the bottle cap under laminar flow, and use the outer flame of the alcohol lamp in the clean workbench to ignite the outer bottle mouth of the pH 7.0 sterile sodium chloride-peptone buffer solution or 0.9% sterile sodium chloride solution and the self-prepared 2.5 mol / L sterile magnesium chloride solution.

[0143] S207: Using a sterilized 30ml syringe, draw 16ml of 2.5mol / L sterile magnesium chloride solution from the mouth of the S206 flask and add it to 400ml of 0.9% sterile sodium chloride solution or pH 7.0 sterile sodium chloride-peptone buffer solution from the mouth of the S206 flask. Mix well and use it as a diluent for dissolving the test sample.

[0144] S208: Take a sterilized 30ml syringe, draw 20ml of 2.5mol / L sterile magnesium chloride solution from the mouth of the S206 flask, and add it to 400ml of 0.9% sterile sodium chloride solution or pH 7.0 sterile sodium chloride-peptone buffer solution from the mouth of the S206 flask. Mix well and use it as a rinsing solution for rinsing the filter membrane.

[0145] S209: Transfer the test sample, after being sprayed or wiped for disinfection, to a clean workbench;

[0146] S210: Take 30 test samples from S209 and transfer them to the clean workbench. Use a syringe to draw 10 samples at a time and add them to the 400ml test sample dissolving diluent prepared in S207. Shake thoroughly until the solution is homogeneous. Mix well and use it as the test solution for later use. Prepare 3 parallel solutions.

[0147] S211: Transfer the tryptic soy peptone liquid culture medium and thioglycolate fluid culture medium, which have been disinfected by spraying or wiping, into a clean workbench;

[0148] S212: Turn on the microbial collector, and after spraying or wiping the well-packaged microbial collector (i.e., KSF330 triple disposable fully enclosed microbial culture device with 0.45μm microporous filter membrane) for disinfection, transfer it to the clean workbench and check the integrity of the microbial collector packaging bag. Then open the packaging bag and write the product name, batch number and test date on the three tubes of the microbial collector.

[0149] S213: First, insert the base of the bacterial collector from the clean bench into the drain hole of the bacterial collector. Then, install the bacterial collector hose onto the peristaltic pump of the bacterial collector. After that, adjust the pump speed of the bacterial collector to 180 rpm / min to draw 50 ml of the rinsing solution prepared in S208 into each culture vessel to wet the filter membrane.

[0150] S214: Take 3 portions of the test solution prepared in S210 and filter them in 1 portion per tube of each bacterial collector. The amount of sample filtered by each filter membrane is equivalent to the total amount of 10 test samples. After filtering all the test solution, rinse the filter membrane with the rinsing solution prepared in S208. The amount of rinsing solution used for each filter membrane is 1000ml, 100ml each time, and rinse 10 times. At the same time, hold the bacterial collector horizontally and rotate it 12 times to achieve rinsing and shaking at the same time. During rinsing, lift the drain tank 5cm, and shake at 2 revolutions / second.

[0151] S215: After the liquid in the collector is completely drained, stop the peristaltic pump of the collector with the collector tubing installed, and use the two clamps passed through the transfer window to clamp the upper tubing at the inlet of the two filter cups respectively, and plug the bottom of the collector with the stopper. Then turn on the peristaltic pump that was just turned off, hold the culture medium bottle in your left hand and ignite it over a flame, and pull out the needle of the collector that has been drained of liquid with your right hand and insert it into the culture medium bottle. Invert the culture medium bottle and add tryptic soy peptone liquid culture medium that conforms to the 2020 edition of the Chinese Pharmacopoeia to the 100ml mark on the cup (three ring-shaped scalar graduation lines can be seen on the cup, from bottom to top, they are 50, 75 and 100ml respectively).

[0152] S216: Use a disposable syringe to draw 5 ml of the self-prepared 2.5 mol / L sterile magnesium chloride solution and add it to S215 tryptic soybean liquid medium;

[0153] S217: First, place the bottle containing tryptic soy broth culture medium (S216) with 2.5 mol / L sterile magnesium chloride solution upright. After emptying the culture medium from the collection tube, stop the peristaltic pump started by S215 using the foot switch. Next, take another clamp and clamp the upper end of the tubing at the inlet of the filter cup containing tryptic soy broth culture medium. Then, remove the clamps from S215 that clamp the upper end of the tubing at the inlet of the two filter cups. Finally, hold the culture medium bottle in your left hand and ignite it over a flame. With your right hand, remove the collection tube needles from the other two tubes (S215) and insert them into the thioglycolate fluid culture medium bottle. Invert the culture medium bottle and add 100 ml of thioglycolate fluid culture medium conforming to the 2020 edition of the Chinese Pharmacopoeia to each of the other two filter cups.

[0154] S218: Use a disposable syringe to draw 5 ml of the self-prepared 2.5 mol / L sterile magnesium chloride solution and add it to two portions of thioglycolate fluid culture medium in S217. One portion is used as the test sample and the other portion is used as the positive control.

[0155] S219: First, use the two clips removed from S217 to clamp the upper end of the tubing at the inlet of the filter cup containing thioglycolate fluid culture medium. Then, take a pair of sterile scissors that have been passed through the transfer window and cut the tubing 4cm from the upper end of the clips that clamp the tubing of the filter cup containing thioglycolate fluid culture medium. Finally, insert the tail end into the exhaust port at the top of the filter cup.

[0156] S220: Remove all tubing from the peristaltic pump of the bacterial collector used on the device;

[0157] S221, Negative Control: Take another new collection tube and replace the test solution with an equal volume of diluent. Follow the same steps as adding thioglycolate fluid medium. Add tryptic soy peptone liquid medium (add 5 ml of 2.5 mol / L sterile magnesium chloride solution to every 100 ml of medium).

[0158] S222: Place the test sample and negative control collection container into the transfer window, and incubate the thioglycolate fluid medium tubes and tryptic soy liquid medium used for the test sample and negative control; wherein the incubation temperature of the thioglycolate fluid medium tubes is 35℃ and the incubation time is not less than 14 days, and the incubation temperature of the tryptic soy liquid medium is 25℃ and the incubation time is not less than 14 days.

[0159] S223: According to the 2020 edition of the Chinese Pharmacopoeia 1101 sterility test method, the thioglycolate fluid culture medium tube used as a positive control was transferred to the positive test room, and 1 ml of Staphylococcus aureus not exceeding 100 CFU was added before incubation in a positive incubator; wherein, the incubation temperature of the incubator was 35℃.

[0160] S224: Daily, remove the test sample, negative control group, and positive control group from the incubator to observe whether bacteria grow.

[0161] S225, Result Interpretation: Positive control tubes should show good growth within 5 days of incubation; negative control tubes should not show bacterial growth, otherwise the experiment is invalid; if all test tubes are clear, or although they appear turbid but are confirmed to have no bacterial growth, the test samples are deemed to meet the requirements; if any test tube is turbid and is confirmed to have bacterial growth, the test samples are deemed to not meet the requirements, unless it can be sufficiently proven that the test results are invalid.

[0162] Compared to traditional methods, this approach incorporates the following improvements:

[0163] 1. Single-factor experiment to verify the amount of neutralizing agent added.

[0164] Because levofloxacin hydrochloride injection has significant antibacterial properties, it strongly inhibits the growth of standard strains used in testing (such as Staphylococcus aureus and Escherichia coli). Even if the sample is actually contaminated with microorganisms, the antibacterial components will still inhibit the growth of the strains, leading to false negative results (inflated pass rates).

[0165] Traditional detection methods involve adding 16 ml of 2.5 mol / L sterile magnesium chloride solution to every 400 ml of 0.9% sterile sodium chloride solution to eliminate antibacterial activity.

[0166] To investigate the effect of different concentrations of neutralizing agent on the "one-time pass rate of sterility testing for levofloxacin hydrochloride injection," experiments were conducted on the sterility of levofloxacin hydrochloride injection by varying the volume of neutralizing agent added (14 ml, 16 ml, and 18 ml) (6 times per group). The specific procedure involved adding different volumes of neutralizing agent to the samples, followed by sterility testing, incubation for 14 days, and then calculating the pass rate.

[0167] Table 1: Statistical table of the single-pass pass rate of sterility test for different volumes of neutralizing agent and X injection solution.

[0168]

[0169] 2. Single-factor verification test of flushing frequency

[0170] In membrane filtration sterility testing, the rinsing solution (e.g., 0.9% sterile sodium chloride buffer, pH 7.0 peptone buffer, etc.) is the core method for eliminating the antibacterial properties of the sample (such as β-lactam drugs in levofloxacin hydrochloride injection). β-lactam antibiotics in levofloxacin hydrochloride injection adsorb onto the filter membrane surface. Even if a neutralizing agent can partially neutralize their activity, a sufficient amount of rinsing solution is still needed to thoroughly remove residual drugs. Residual drugs inhibit the growth of microorganisms in subsequent cultures, avoiding false negative results.

[0171] The traditional method involves rinsing each filter membrane with 800 ml of 0.9% sterile sodium chloride buffer (rinsing solution), 100 ml each time, for a total of 8 rinses.

[0172] To investigate the effect of the number of rinses on the "one-time pass rate of sterility testing for levofloxacin hydrochloride injection," the rinsing volume of 0.9% sterile sodium chloride buffer per filter membrane was varied (600ml, 800ml, and 1000ml), and sterility testing of levofloxacin hydrochloride injection was conducted (6 times per group). The specific procedure involved adding different volumes of rinsing solution to the samples, followed by sterility testing, incubation for 14 days, and then calculating the pass rate.

[0173] Table 2: Statistical table of the single-pass pass rate of sterility of different volumes of rinsing solution and levofloxacin hydrochloride injection.

[0174]

[0175] 3. Number of shaking cycles

[0176] Shaking physically disrupts the adsorption between microorganisms and the filter membrane, suspending the microorganisms in the rinsing solution. These microorganisms are then discharged with the rinsing solution or evenly distributed on the filter membrane surface, improving the detection rate and avoiding false negatives (microorganisms not detected due to adsorption). Traditional methods are described as "rinsing and shaking simultaneously," where each collection container is lifted and the liquid inside is shaken vigorously with the wrist. The number of shakes is not specified. For highly adsorbent samples (such as antibiotic injections), "small amounts, multiple times" of shaking are necessary to ensure sufficient release of microorganisms.

[0177] To investigate the effect of the number of shaking cycles on the "one-time pass rate of sterility testing for levofloxacin hydrochloride injection," a sterility test of levofloxacin hydrochloride injection was conducted with three different shaking cycles (6, 8, and 10 cycles per group). The specific procedure involved processing samples with three different shaking cycles, followed by sterility testing, incubation for 14 days, and then calculating the pass rate.

[0178] Table 3: Statistical table of the pass rate of sterility test for levofloxacin hydrochloride injection with different shaking times.

[0179]

[0180] Conclusion: Through the single-factor verification experiments on the amount of neutralizing agent, the volume of rinsing solution, and the number of shaking times, it was found that as the amount of neutralizing agent, the volume of rinsing solution, and the number of shaking times increased, the first-pass yield of levofloxacin hydrochloride injection gradually increased, indicating that this factor has a significant impact on the "low first-pass yield of levofloxacin hydrochloride injection".

[0181] 4. Select the neutralizing agent concentration, number of rinses, and number of shaking cycles through orthogonal experiments.

[0182] After preliminary screening: (1) The amount of neutralizing agent added was selected as 18 ml of 2.5 mol / L sterile magnesium chloride solution, 20 ml of 2.5 mol / L sterile magnesium chloride solution, and 22 ml of 2.5 mol / L sterile magnesium chloride solution in 400 ml; (2) The number of rinses in 1000 ml of rinsing solution were 6, 8, and 10 times, respectively; (3) The number of shaking times were 8, 10, and 12 times, respectively. Six test groups were prepared for each group, and the growth of six test bacteria (species: Staphylococcus aureus, Escherichia coli, Clostridium sporogenes, Bacillus subtilis, Candida albicans, Aspergillus niger) in the six test groups under the same conditions was statistically analyzed to analyze the influence of three parameters on the stability of the test results (grown bacteria count 1, no grown bacteria count 0, and the number of grown bacteria in the same group is accumulated).

[0183] Table 4: Factor Ranking Table for Orthogonal Experiments

[0184]

[0185] Table 5: Statistical Table of Experimental Results

[0186]

[0187]

[0188] Table 6: Calculation Table for Intuitive Analysis of Orthogonal Experiments

[0189]

[0190]

[0191] Table 7: Optimal Parameter Combinations

[0192] parameter Number of shaking times Number of flushes with the flushing solution Neutralizer addition amount Optimal combination 12 times 8 times 20ml

[0193] Through the orthogonal array, we can see the influencing factors: number of shaking times > number of rinsing times with rinsing solution > amount of neutralizing agent added. The optimal combination is A2B2C3, that is, the method parameters are set as follows: the amount of neutralizing agent added is 20ml of 2.5mol / L sterile magnesium chloride solution, the number of rinsing times with 100ml of rinsing solution is 8, and the number of shaking times is 12. All 6 bacterial species in the 6 experimental groups grew bacteria, that is, the antibacterial properties of levofloxacin hydrochloride injection were eliminated.

[0194] Conclusions: 1. The neutralizing agent dosage is 20 ml of 2.5 mol / L sterile magnesium chloride solution per 400 ml of 0.9% sterile sodium chloride solution; 2. The rinsing volume is 1000 ml, and the rinsing frequency is 10 times, with 100 ml rinses each time; 3. The shaking frequency is 12 times.

[0195] 5. Pass Rate Verification

[0196] To comprehensively evaluate the effectiveness of the improvement measures, the testing results of three batches of levofloxacin hydrochloride injection produced in January 2025 were statistically analyzed, including finished product, accelerated stability, and long-term stability (3-month stability) samples (9 batches of finished product from three sterilizers, 3 batches each of accelerated and long-term stability, totaling 6 batches, for a total of 15 batches). The statistical results are shown in the table below:

[0197] Table 8: Statistical Table of One-Time Pass Rate for Sterility Testing of Levofloxacin Hydrochloride Injection

[0198]

[0199]

[0200] In the same laboratory, with the same testing personnel and using the same equipment, 3 batches of 9 samples were tested before optimization, with a pass rate of 44.44%; after optimization, 3 batches of 15 samples were tested, with a pass rate of 100%.

[0201] Advantages: The sterility test pass rate of levofloxacin hydrochloride injection increased from 44.44% to 100%, significantly reducing quality risks, enhancing the product's market reputation and customer trust, and providing strong support for the company's long-term competitiveness. The optimized testing solutions developed during the research and development process provided experience for the prevention and resolution of similar problems in the future, and promoted the further improvement of the quality management system.

[0202] Although the present invention has been described above with reference to embodiments, various modifications can be made and components can be replaced with equivalents without departing from the scope of the invention. In particular, as long as there is no structural conflict, the features in the disclosed embodiments can be combined with each other in any manner. The lack of an exhaustive description of these combinations in this specification is merely for the sake of brevity and resource conservation. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A method for improving the sterility test pass rate of levofloxacin hydrochloride injection, characterized in that, The specific steps are as follows: S100, Preparation: Prepare 2.5 mol / L sterile magnesium chloride solution: S101: Rinse beakers, glass rods, volumetric flasks, and wide-mouth bottles with purified water; S102: First, weigh 50.8g of analytical grade magnesium chloride and add it to a beaker. Then, add an appropriate amount of purified water and stir with a glass rod until completely dissolved. S103: First, use a glass rod to guide the flow and transfer the S102 solution to a volumetric flask. Then, rinse the beaker and the glass rod used for guiding the flow with purified water. Transfer the rinsing solution into the volumetric flask and make up to the target volume with purified water. After mixing thoroughly, transfer the solution to a wide-mouth bottle. S104: Slightly unscrew the cap of the wide-mouth bottle containing 2.5 mol / L magnesium chloride in S103, and sterilize it using an XG1.G type pulsating vacuum sterilizer; S105: First, take out the wide-mouth bottle containing 2.5 mol / L magnesium chloride that was sterilized in S104, and tighten the cap. Then, observe whether the color change of the color bar on the sterilization indicator card is consistent with the standard chart. S106: First, seal the wide-mouth bottle containing 2.5mol / L sterile magnesium chloride that meets the requirements after sterilization in S105. Then, label the preparation date and expiration date and store it in a refrigerator for later use. S200, Sample Testing Process: S201: Consumables and samples used in the testing process are transferred to the clean area through a transfer window, and during the transfer, air shower and disinfection are performed based on the fan and ultraviolet lamp disinfection at the transfer window; S202: Wipe the clean workbench with 75% ethanol; S203: After spraying or wiping the petri dishes for disinfection, transfer them to the clean workbench disinfected in S202. Place three petri dishes in the order of left, center, and right to monitor the settling bacteria throughout the process and mark the petri dishes. Then, open the lids of the placed petri dishes one by one to expose the surface of the culture medium to the air. After all sampling is completed, invert the petri dishes in the incubator for incubation. Then, after sampling the petri dishes prepared with tryptic soy agar medium, incubate them in the incubator. After sampling the petri dishes prepared with Sabouraud dextrose agar medium, incubate them in the incubator. S204: First, light an alcohol lamp in the clean workbench where the settling bacteria are monitored in S203. Then, disinfect the tools that were passed through the transfer window in S201 by spraying or wiping them with disinfectant before moving them into the clean workbench. S205: First, spray or wipe the drain tank with the outer packaging of the package that was passed through the transfer window to disinfect it, and then transfer it to the clean workbench. After removing the outer packaging of the drain tank, place it on the bacteria collection instrument. Then, place the drain outlet of the drain pipe in the water bucket on the clean workbench floor after each use for cleaning and disinfection. After that, take the sample to be tested, rinsing solution, and culture medium out of the trolley, spray or wipe them, and then place them on the clean workbench. S206: First, spray or wipe the pH 7.0 sterile sodium chloride-peptone buffer solution or 0.9% sterile sodium chloride solution and the self-prepared 2.5 mol / L sterile magnesium chloride solution for disinfection, and then transfer them to the clean workbench. Next, remove the bottle cap under laminar flow, and use the outer flame of the alcohol lamp in the clean workbench to ignite the outer bottle mouth of the pH 7.0 sterile sodium chloride-peptone buffer solution or 0.9% sterile sodium chloride solution and the self-prepared 2.5 mol / L sterile magnesium chloride solution. S207: Using a sterilized 30ml syringe, draw 16ml of 2.5mol / L sterile magnesium chloride solution from the mouth of the S206 flask and add it to 400ml of 0.9% sterile sodium chloride solution or pH 7.0 sterile sodium chloride-peptone buffer solution from the mouth of the S206 flask. Mix well and use it as a diluent for dissolving the test sample. S208: Take a sterilized 30ml syringe, draw 20ml of 2.5mol / L sterile magnesium chloride solution from the end of the S206 flask, and add it to 400ml of 0.9% sterile sodium chloride solution or pH 7.0 sterile sodium chloride-peptone buffer solution from the end of the S206 flask. Mix well and use it as a rinsing solution for rinsing the filter membrane. S209: Transfer the test sample, after being sprayed or wiped for disinfection, to a clean workbench; S210: Take 30 test samples from S209 and transfer them to the clean bench. Use a syringe to draw 10 samples at a time and add them to the 400ml test sample dissolving diluent prepared in S207. Shake thoroughly until the solution is homogeneous. Mix well and use it as the test solution for later use. Prepare 3 parallel solutions. S211: Transfer the tryptic soy peptone liquid culture medium and thioglycolate fluid culture medium, which have been disinfected by spraying or wiping, into a clean workbench; S212: Turn on the sterilizer, disinfect the well-packaged sterilizer by spraying or wiping it, and transfer it to the clean workbench. Check the integrity of the sterilizer packaging bag, then open the packaging bag and write the product name, batch number, and test date on the three tubes of the sterilizer. S213: First, insert the base of the bacterial collector from the clean bench into the drain hole of the bacterial collector. Then, install the bacterial collector hose onto the peristaltic pump of the bacterial collector. After that, adjust the pump speed of the bacterial collector to 180 rpm / min to draw 50 ml of the rinsing solution prepared in S208 into each culture vessel to wet the filter membrane. S214: Take 3 portions of the test solution prepared in S210 and filter them in 1 portion per tube of each bacterial collector. The amount of sample filtered by each filter membrane is equivalent to the total amount of 10 test samples. After filtering all the test solution, rinse the filter membrane with the rinsing solution prepared in S208. The amount of rinsing solution used for each filter membrane is 1000ml, 100ml each time, and rinse 10 times. Also, hold the bacterial collector horizontally with both hands and rotate and shake it 12 times to achieve rinsing and shaking at the same time. S215: After the liquid in the collector is completely drained, stop the peristaltic pump of the collector with the collector tubing installed, and use the two clamps passed through the transfer window to clamp the upper tubing at the liquid inlet of the two filter cups respectively, and plug the bottom of the collector with the stopper. Then turn on the peristaltic pump that was just turned off, hold the culture medium bottle in your left hand and ignite it over a flame, and pull out the needle of the collector that has been drained of liquid with your right hand and insert it into the culture medium bottle. Invert the culture medium bottle and add tryptic soy peptone liquid culture medium to the 100ml mark on the cup. S216: Use a disposable syringe to draw 5 ml of the self-prepared 2.5 mol / L sterile magnesium chloride solution and add it to S215 tryptic soybean liquid medium; S217: First, place the bottle of tryptic soy broth culture medium containing 2.5 mol / L sterile magnesium chloride solution in S216 upright. After emptying the culture medium in the collection tube, stop the peristaltic pump started by S215 using the foot switch. Next, take another clamp and clamp the upper end of the tubing at the inlet of the filter cup containing tryptic soy broth culture medium. Then, remove the clamps from S215 that clamp the upper end of the tubing at the inlet of the two filter cups. Finally, hold the culture medium bottle in your left hand and ignite it over a flame. With your right hand, remove the needles from the collection tubes that have been emptied by S215 and insert them into the thioglycolate fluid culture medium bottle. Invert the culture medium bottle and add 100 ml of thioglycolate fluid culture medium to each of the other two filter cups. S218: Use a disposable syringe to draw 5 ml of the self-prepared 2.5 mol / L sterile magnesium chloride solution and add it to two portions of thioglycolate fluid culture medium in S217. One portion is used as the test sample and the other portion is used as the positive control. S219: First, use the two clips removed from S217 to clamp the upper end of the tubing at the inlet of the filter cup containing thioglycolate fluid culture medium. Then, take a pair of sterile scissors passed through the transfer window and cut the tubing 3-4 cm above the clips clamping the tubing of the filter cup containing thioglycolate fluid culture medium. Finally, insert the tail end into the exhaust port at the top of the filter cup. S220: Remove all tubing from the peristaltic pump of the bacterial collector used on the device; S221, negative control: Take another new collection tube and replace the test solution with an equal volume of diluent. The other steps are the same as adding thioglycolate fluid medium. Add tryptic soy peptone liquid medium to another tube. S222: Place the test sample and negative control collection container into the transfer window and incubate the thioglycolate fluid medium tubes and tryptic soy peptone liquid medium used for the test sample and negative control examination. S223: Transfer the thioglycolate fluid culture medium tube used for the positive control to the positive test room, add 1 ml of Staphylococcus aureus (not more than 100 CFU), and then incubate in a positive incubator. S224: Daily, remove the test sample, negative control group, and positive control group from the incubator to observe whether bacteria grow. S225, Result Interpretation: Positive control tubes should show good growth within 5 days of incubation; negative control tubes should not show bacterial growth, otherwise the experiment is invalid; if all test tubes are clear, or although they appear turbid but are confirmed to have no bacterial growth, the test samples are judged to meet the requirements; if any test tube is turbid and is confirmed to have bacterial growth, the test samples are judged to not meet the requirements.

2. The method for improving the sterility test pass rate of levofloxacin hydrochloride injection according to claim 1, characterized in that, The XG1.G type pulsed vacuum sterilizer in S104 operates at a temperature of 121°C for 20 minutes, and the program is in liquid mode.

3. The method for improving the sterility test pass rate of levofloxacin hydrochloride injection according to claim 1, characterized in that, The temperature of the refrigerator in S106 is 2-8℃.

4. The method for improving the sterility test pass rate of levofloxacin hydrochloride injection according to claim 1, characterized in that, The air shower time in S201 shall not be less than 2 minutes, and the disinfection time shall not be less than 30 minutes.

5. The method for improving the sterility test pass rate of levofloxacin hydrochloride injection according to claim 1, characterized in that, The monitoring time for settling bacteria in S203 is 0.5-4 hours. The culture time for culture dishes prepared with tryptic soy peptone agar medium in S203 is not less than 3 days, and the culture temperature of the incubator is 30-35℃. The culture time for culture dishes prepared with Sabouraud dextrose agar medium in S203 is not less than 5 days, and the culture temperature of the incubator is 20-25℃.

6. The method for improving the sterility test pass rate of levofloxacin hydrochloride injection according to claim 1, characterized in that, In step S214, during rinsing, the drain tank is lifted 3-5cm, and during shaking, it rotates every 2 revolutions per second.

7. The method for improving the sterility test pass rate of levofloxacin hydrochloride injection according to claim 1, characterized in that, The S222 thioglycolate fluid culture medium tubes are cultured at a temperature of 30-35℃ for a time of not less than 14 days, and the tryptic soy peptone liquid culture medium is cultured at a temperature of 20-25℃ for a time of not less than 14 days.

8. The method for improving the sterility test pass rate of levofloxacin hydrochloride injection according to claim 1, characterized in that, The incubation temperature in the incubator in S223 is 30-35℃.

Images