Method for preparing and filling piperacillin sodium and tazobactam sodium for injection based on staged freeze-drying

Abstract

The invention discloses a staged freeze-drying-based preparation and filling method of piperacillin sodium and tazobactam sodium for injection, which comprises the following steps of: repeatedly freeze-drying piperacillin sodium and tazobactam sodium for injection in stages, and matching with a filling process of semi-plug pressing and plug pressing, so that the piperacillin sodium and tazobactam sodium for injection is uniformly freeze-dried to form loose blocks; and moreover, due to staged repeated freeze-drying, the product is in the form of loose blocks (the moisture in the upper part and the lower part is uniform, and the moisture is low but uniform although a little moisture exists), so that the product can be stored for a long time, the stable pH value is ensured, the quality guarantee period of the product is long, the product is not easy to degrade, the related substances are few, and the purity is high. The traditional Chinese medicine composition has the advantages that the curative effect is guaranteed, the occurrence probability of clinical adverse reactions is reduced, the product is loose blocks, the dissolution is quick in clinical use, the redissolution waiting time is shortened, and the application prospect is good.

Description

Technical Field

[0001] The present invention relates to the technical field of medical drugs, and in particular to a preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged freeze-drying. Background Art

[0002] Piperacillin sodium and tazobactam sodium compound preparations are generally prepared by direct packaging of sterile powders. They have a common defect that is poor preparation stability, high impurity content, and certain safety hazards. In piperacillin sodium and tazobactam sodium products, since most of its "related substances" impurities come from piperacillin sodium, the product impurities of piperacillin sodium raw materials determine the impurity level of the final product. The quality of piperacillin acid plays a very critical role in improving the antibacterial activity and safety of drugs during use. Therefore, the "A piperacillin sodium and tazobactam sodium preparation for injection and its preparation and filling method" disclosed in patent number "201510040537.5" is a piperacillin sodium and tazobactam sodium preparation for injection. The production of the piperacillin sodium and tazobactam sodium lyophilized preparation for injection generally uses piperacillin acid, tazobactam acid and sodium bicarbonate as reaction raw materials and auxiliary materials for production. After 24 months of storage, the prepared lyophilized preparation has significantly reduced content of piperacillin sodium and tazobactam sodium, and significantly reduced pH, and the stability of the product needs to be further improved. In the prior art, the patent number "201610027784" discloses a "method for preparing and filling piperacillin sodium and tazobactam sodium for injection". Before freeze-drying, the method adds "step (1) if the pH is lower than 6.3, sodium bicarbonate is added for adjustment; if the pH is higher than 6.6, citric acid is added for adjustment". By adding citric acid to form salt, the acidity stability of the product is improved, the decomposition of piperacillin sodium and tazobactam sodium is inhibited, and the storage stability of the product is improved. However, the following problems also exist:

[0003] (1) In the prior art, piperacillin sodium and tazobactam sodium are mixed to form a drug material without a filtration process (or a simple single-stage filtration), which affects the purity of the drug prepared for piperacillin sodium and tazobactam sodium injection;

[0004] (2) The conventional freeze-drying process currently used cannot completely remove the water for injection from the piperacillin sodium and tazobactam sodium raw powder, so that the pH value of the piperacillin sodium and tazobactam sodium for injection is significantly reduced during the subsequent storage process, affecting the stability. In addition, the prior art does not record the detailed filling process to ensure good sealing to achieve long-term storage of piperacillin sodium and tazobactam sodium for injection and ensure the efficacy;

[0005] (3) Since there is still water after freeze-drying in the prior art, the product is easily degraded and has low purity, which increases the probability of clinical adverse reactions.

[0006] How to solve the problems in the preparation process of the above-mentioned piperacillin sodium and tazobactam sodium for injection is a problem that needs to be solved at present. Summary of the invention

[0007] The purpose of the present invention is to overcome the problems in the preparation process of piperacillin sodium and tazobactam sodium for injection in the prior art. The preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization of the present invention makes the piperacillin sodium and tazobactam sodium for injection freeze-dried evenly in a loose block by repeatedly lyophilizing piperacillin sodium and tazobactam sodium for injection in stages, and cooperates with the filling process of semi-tamponade and tamponade, so that the freeze-dried piperacillin sodium and tazobactam sodium for injection is uniform and loose, ensuring that the product is quickly re-dissolved in clinical use, and because the product is a loose block (the upper and lower inside and outside moisture is uniform, although very little moisture exists, that is, the moisture is low but uniform) due to the repeated freeze-drying in stages, it is conducive to long-term storage, ensuring a stable pH value, a long shelf life of the product, the product is not easy to degrade, the related substances are few, the purity is high, and the probability of clinical adverse reactions is reduced while ensuring the efficacy, and the product is guaranteed to be a loose block, dissolve quickly in clinical use, and reduce the waiting time for re-dissolution, and has good application prospects.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] A preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization comprises the following steps:

[0010] Step (A), weighing

[0011] The raw and auxiliary materials are removed from their outer packaging, disinfected, and brought into the Class C clean area, and the raw and auxiliary materials are weighed according to the instructions. The raw and auxiliary materials include APIs and auxiliary materials. The APIs include piperacillin and tazobactam, and the auxiliary materials include edetate disodium, sodium bicarbonate, and citric acid;

[0012] Step (B), ingredients

[0013] (B1), add the injection water of the preparation volume into the 700L concentration tank, cool the injection water to 10-20°C and maintain it, take out 35% of the injection water into a stainless steel sealed barrel for standby use before adding the raw materials and excipients, and keep the remaining 65% of the injection water in the concentration tank;

[0014] (B2), taking the weighed citric acid and edetate disodium and adding them into the concentration preparation tank in sequence under static state, turning on the stirring at a speed of 30 Hz, stirring for 20 minutes to completely dissolve the citric acid and edetate disodium in the injection water to form a drug solution;

[0015] (B3), adding the weighed piperacillin under a stirring condition at a rotation speed of 15 Hz, and continuing to suspend the drug solution under a stirring condition at a rotation speed of 30 Hz for 5 minutes;

[0016] (B3), slowly adding 95% of the weighed sodium bicarbonate under a stirring condition at a rotation speed of 20 Hz, and after the addition is completed, stirring for 40 minutes under a stirring condition at a rotation speed of 30 Hz to clarify the liquid;

[0017] (B4), at a speed of 15 Hz, continue to add the weighed tazobactam, stir for 30 minutes at a speed of 30 Hz, then continue to add the remaining 5% of the weighed sodium bicarbonate, continue to stir for 30 minutes, and confirm that the pH value of the solution is 5.9 to 6.3, indicating a complete reaction. If the pH value is not within the specified range, adjust it by adding sodium bicarbonate or citric acid;

[0018] Step (C), diluting and obtaining a filtered liquid

[0019] (C1), the drug solution is filtered through a 0.45 μm polyethersulfone filter into a 500 L dilution tank, wherein the filtration pressure is ≤0.2 MPa; the pressure difference between the upstream and downstream of the filter is ≤0.2 MPa, and the filter element is disposable;

[0020] (C2), add the 35% injection water in the stainless steel sealed barrel to the dilution tank, stir for 15 minutes at a speed of 30 Hz, and control the temperature of the dilution tank to between 5 and 10°C;

[0021] (C3), sampling and testing the intermediate from the dilution tank, determining the content by HPLC, and calculating the filling volume according to the product specifications;

[0022] (C4), the liquid in the dilution tank is filtered through a 0.22μm polyethersulfone filter in the liquid preparation room and a 0.22μm polyethersulfone filter in the liquid collection room to obtain a filtered liquid in a liquid storage closed barrel, the filtering temperature is 5-20°C; the filtering pressure is ≤0.2MPa; the filtering time is 0.5-3h / batch; the pressure difference between the upstream and downstream of the filter is ≤0.2MPa, and the filter element is disposable;

[0023] Step (D), respectively cleaning and sterilizing the borosilicate glass bottles, rubber stoppers, transporting devices, and potting aluminum caps used for filling the filtered drug solution;

[0024] Step (E), filling and half-stoppering

[0025] The filtered liquid medicine is tested according to the intermediate product standard of piperacillin sodium and tazobactam sodium for injection, and the content is determined by HPLC. The filling volume is calculated according to the product specifications, and the filtered liquid medicine is filled into a medium borosilicate glass bottle and half-stoppered to form a half-stoppered filling bottle, and the half-stoppered filling bottle is placed on a drop partition at a temperature of 5°C;

[0026] Step (F), freeze-drying and plugging in stages

[0027] (F1), pre-freezing stage: the temperature of the heat transfer oil in the half-stoppered bottles is gradually reduced to -50°C within 60 minutes and maintained for 120 minutes;

[0028] (F2), sublimation stage: sequentially, the temperature of the heat transfer oil is gradually raised to -25°C within 60 minutes, the temperature of the heat transfer oil is gradually raised from -25°C to 0°C within 120 minutes, the temperature of the heat transfer oil is gradually raised from 0°C to 25°C within 120 minutes, the temperature of the heat transfer oil is gradually raised from 25°C to 35°C within 90 minutes, and maintained for 420 minutes, and the vacuum is 30±5Pa;

[0029] (F3), desorption stage: the temperature of the heat transfer oil is gradually raised from 35°C to 38°C within 60 minutes, and then from 38°C to 48°C within 90 minutes, and maintained for 60 to 90 minutes, with a vacuum of 30Pa±5Pa;

[0030] (F4), close the aeration valve and continue to maintain for 2 hours, and the staged freeze-drying is completed;

[0031] (F5), for the half-stoppered filling bottles that have been freeze-dried in stages, the stoppering is completed after being filled with sterile nitrogen to form a stoppered filling bottle, and the re-pressurization pressure of the sterile nitrogen filled part is between -0.03 and -0.04 MPa;

[0032] Step (G), unpacking, capping, leak detection

[0033] (G1), after the freeze-drying is completed, the plugged filled bottles are unpacked in the filling room and enter the capping room via the conveyor belt. The capping speed is 20-50Hz. When testing the capping, first roll two circles and then stop the machine to check the tightness of the rolled aluminum cap and the edge of the aluminum cap to see if the capping is qualified. If the capping is qualified, start normal operation to achieve continuous aluminum capping of the plugged filled bottles to form aluminum capped filled bottles;

[0034] (G2), Leak detection of rolled aluminum cap filled bottles by color water method

[0035] (G21) Sampling: Random sampling is conducted at the beginning, middle and end stages of each batch of rolled aluminum cap filled bottles, with 10 bottles sampled at each stage;

[0036] (G22) Judgment standard: Take the sample and put it into a vacuum dryer filled with 0.1% methylene blue solution, immerse it completely, evacuate it to 30±2kPa, keep it for 30 minutes, and then return it to normal pressure; after another 30 minutes, take out the sample; rinse the surface of the bottle of the sample with water until there is no obvious color residue on the outer wall, which is qualified;

[0037] Step (H): Light inspection, labeling, packaging, and storage

[0038] The light inspector needs to check the illumination of the light inspection machine, which should be between 1000lx-1500lx. Take out each bottle of rolled aluminum cap filled bottle from the conveyor belt, place it 20 to 30 cm under the light, and conduct continuous light inspection manually. If defective products are found, take them out and place them in a red special box. Qualified rolled aluminum cap filled bottles are placed on another conveyor belt and sent to the outsourcing room via the conveyor belt for labeling, packaging and warehousing.

[0039] Preferably, in the preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization, in step (A), the weighing standard is the unit dosage required for each bottle of rolled aluminum cap filling bottle: piperacillin 2000 mg, tazobactam 250 mg, edetate disodium 0.5 mg, sodium bicarbonate 476.5 mg, citric acid 71.5 mg, and water for injection 8.5 ml.

[0040] Preferably, in the preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged freeze-drying, in step (B), during the batching process, the temperature of the concentrated preparation tank is controlled between 10 and 20°C; in step (C), during the diluted preparation process, the temperature of the diluted preparation tank is controlled between 5 and 10°C.

[0041] Preferably, in the preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization, the operation time of step (B) and step (C1)-step (C2) is no more than 8 hours; the operation time of step (C4) is no more than 10 hours, and the drug liquid in the dilution tank is filtered through a 0.22 μm polyethersulfone filter in the liquid preparation room and a 0.22 μm polyethersulfone filter in the liquid collection room into a liquid storage closed barrel to obtain a filtered drug liquid, and the 1000 ml filtrate in the front section after filtration is discarded.

[0042] Preferably, the preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization, step (D), respectively cleaning and sterilizing the borosilicate glass bottle, rubber stopper, transport device, and potting aluminum cap used for filtering and filling the liquid medicine, each including the following processes,

[0043] (D1), cleaning and sterilization of borosilicate glass bottles: including ultrasonic cleaning of bottles filled with circulating water, external spraying of circulating water, cleaning with purified water, blowing with clean compressed air, cleaning with injection water, blowing with clean compressed air, drying and sterilization, cooling, bottle removal, sampling and inspection, and standby. The drying and sterilization is set at 350°C and ≥5min; the speed of the ultrasonic cleaning machine is: 150 bottles / min for 20ml bottles; the storage of the borosilicate glass bottles after drying and sterilization (D1) shall not exceed 8 hours, and they shall be cleaned and sterilized again after 8 hours;

[0044] (D2) Cleaning and sterilization of rubber stoppers: including taking out rubber stoppers, adding materials, spraying, mixing, rinsing, checking for visible foreign matter, moist heat sterilization, dry sterilization, cooling, and removing stoppers. The sterilization temperature of dry sterilization is 121°C, and the sterilization time is greater than 30 minutes. The service life of the dry sterilized rubber stoppers should not exceed 24 hours;

[0045] (D3), cleaning and sterilization of transport equipment: including cleaning, sterilization, drying, cooling, and unloading of equipment in sequence. Dry heat sterilization or moist heat sterilization can be selected according to the different transport equipment. The effective period of dry heat sterilization is 24 hours, and the effective period of moist heat sterilization is 24 hours;

[0046] (D4), cleaning and sterilization of potted aluminum caps: including taking out the aluminum caps, framing, sterilizing at 120°C for 2 hours, cooling, and removing the aluminum caps. The potted aluminum caps after sterilization have a shelf life of 24 hours.

[0047] Preferably, the preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization, step (E), filling, half-stoppering, checking the filling amount and the quality of the half-stoppering and visible foreign matter every 30 minutes during the filling process and keeping records, the filtered drug solution should not exceed 10 hours from the start of sterile filtration to the start of filling; the storage time of the filtered drug solution in the liquid storage sealed barrel should not exceed 10 hours; the filling should be completed within 12 hours, and the room temperature should not exceed 8°C.

[0048] Preferably, in the preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization, in step (F), during the staged lyophilization and plugging process, the shutdown judgment standard is closing the box trap valve and the vacuum rise in the box is ≤0.5Pa / min.

[0049] Preferably, the preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged freeze-drying, (G1), whether the capping is qualified, use the thumb, middle finger and index finger to clamp the edge of the bottle cap, stand it upright in a triangle, and apply force evenly in opposite directions with both hands, and it is qualified if it cannot be twisted.

[0050] The beneficial effects of the present invention are as follows: the preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged freeze-drying of the present invention, through repeated freeze-drying of piperacillin sodium and tazobactam sodium for injection in stages, combined with a semi-tamponade and tamponade filling process, makes the piperacillin sodium and tazobactam sodium for injection freeze-dried uniformly into loose blocks, ensuring rapid reconstitution of the product in clinical use, and because the loose blocks are evenly dehydrated, the entire staged freeze-dried piperacillin sodium and tazobactam sodium for injection has uniform moisture content (very little moisture exists, that is, the moisture content is low but uniform), which is conducive to long-term storage, ensuring a stable pH value, and has the following characteristics:

[0051] (1) Three-stage filtration is adopted: dilution filtration: the drug liquid is filtered through a 0.45 μm polyethersulfone filter to a 500L dilution tank, and the filter element is disposable; filtration in the liquid preparation room: the drug liquid in the dilution tank is filtered through a 0.22 μm polyethersulfone filter in the liquid preparation room; filtration in the liquid collection room, the 0.22 μm polyethersulfone filter in the liquid collection room is filtered into a liquid storage closed barrel to obtain the filtered drug liquid. Through the three-stage filtration measures, the purity of piperacillin sodium and tazobactam sodium for injection is ensured, the related substances (total impurities) are reduced, and the efficacy of piperacillin sodium and tazobactam sodium for injection is greatly improved;

[0052] (2) Filling, half-stoppering, staged freeze-drying, and plugging are four steps carried out in sequence. The half-stoppered bottle is freeze-dried in stages before plugging. The staged freeze-drying is particularly critical, as shown below: in the pre-freezing stage, the filtered liquid in the half-stoppered bottle is a loose crystal structure that is conducive to the sublimation and discharge of internal moisture; in the sublimation stage, step-by-step heating is conducive to multiple sublimations, and the moisture is discharged more thoroughly; in the analysis stage, slow heating ensures that the product (filtered liquid in the half-stoppered bottle) is not thermally degraded, and the sublimated moisture in the half-stoppered bottle is discharged out of the bottle; the air injection valve is closed, and the process maintains a negative pressure state to isolate the air and prevent the product (filtered liquid in the half-stoppered bottle) from absorbing moisture in the air during storage. The above processes are very critical to achieve reliable and effective moisture discharge from the filtered liquid in the half-stoppered bottle without absorbing moisture in the air. Moreover, freeze-drying in stages after half-stoppering can ensure that the moisture in the product (filtered drug solution in the half-stoppered bottle) is drawn out of the bottle through the pores of the half-stopper after freeze-drying and sublimation, and then the stoppering process is carried out. The seal ensures the sterility of the drug (finished product) and also prevents the drug (finished product) from absorbing moisture in the air during storage.

[0053] (3) The borosilicate glass bottles, rubber stoppers, transfer equipment, and aluminum caps used for filling the filtered liquid are cleaned and sterilized separately to ensure product quality and prevent contamination during the filling process;

[0054] (4) Three quality control processes, namely capping, leak detection and light inspection, were set up to determine whether the finished product was qualified and to ensure the qualified rate of the finished product of piperacillin sodium and tazobactam sodium for injection based on staged freeze-drying. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The present invention is a process diagram of the preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization. DETAILED DESCRIPTION

[0056] The present invention will be further described below in conjunction with the accompanying drawings.

[0057] Example 1

[0058] like Figure 1 As shown, the preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization of the present invention comprises the following steps:

[0059] Step (A), weighing

[0060] The raw and auxiliary materials are removed from their outer packaging, disinfected, and brought into the Class C clean area, and the raw and auxiliary materials are weighed according to the instructions. The raw and auxiliary materials include APIs and auxiliary materials. The APIs include piperacillin and tazobactam, and the auxiliary materials include edetate disodium, sodium bicarbonate, and citric acid;

[0061] Step (B), ingredients

[0062] (B1), add the injection water of the preparation volume into the 700L concentration tank, cool the injection water to 10-20°C and maintain it, take out 35% of the injection water into a stainless steel sealed barrel for standby use before adding the raw materials and excipients, and keep the remaining 65% of the injection water in the concentration tank;

[0063] (B2), taking the weighed citric acid and edetate disodium and adding them into the concentration preparation tank in sequence under static state, turning on the stirring at a speed of 30 Hz, stirring for 20 minutes to completely dissolve the citric acid and edetate disodium in the injection water to form a drug solution;

[0064] (B3), adding the weighed piperacillin under a stirring condition at a rotation speed of 15 Hz, and continuing to suspend the drug solution under a stirring condition at a rotation speed of 30 Hz for 5 minutes;

[0065] (B3), slowly adding 95% of the weighed sodium bicarbonate under a stirring condition at a rotation speed of 20 Hz, and after the addition is completed, stirring for 40 minutes under a stirring condition at a rotation speed of 30 Hz to clarify the liquid;

[0066] (B4), at a rotation speed of 15 Hz, continue to add the weighed tazobactam, stir for 30 minutes at a rotation speed of 30 Hz, then continue to add the remaining 5% of the weighed sodium bicarbonate, continue to stir for 30 minutes, and confirm that the pH value of the drug solution is 5.9 to 6.3. The pH value in this range indicates that the reaction is complete and can ensure the quality stability of the pH value of the finished product (piperacillin sodium tazobactam sodium for injection based on staged lyophilization) during the storage period; if the pH value is not within the specified range, adjust it by adding sodium bicarbonate or citric acid;

[0067] Step (C), diluting and obtaining a filtered liquid

[0068] (C1), the drug solution is filtered through a 0.45 μm polyethersulfone filter into a 500 L dilution tank, wherein the filtration pressure is ≤0.2 MPa; the pressure difference between the upstream and downstream of the filter is ≤0.2 MPa, and the filter element is disposable;

[0069] (C2), add the 35% injection water in the stainless steel sealed barrel to the dilution tank, stir for 15 minutes at a speed of 30 Hz, and control the temperature of the dilution tank to between 5 and 10°C;

[0070] (C3) Take samples from the dilution tank to test the intermediate, determine the content by HPLC, and calculate the filling volume according to the product specifications.

[0071] (C4), the liquid in the dilution tank is filtered through a 0.22μm polyethersulfone filter in the liquid preparation room and a 0.22μm polyethersulfone filter in the liquid collection room to obtain a filtered liquid in a liquid storage closed barrel, the filtering temperature is 5-20°C; the filtering pressure is ≤0.2MPa; the filtering time is 0.5-3h / batch; the pressure difference between the upstream and downstream of the filter is ≤0.2MPa, and the filter element is disposable;

[0072] Step (D), respectively cleaning and sterilizing the borosilicate glass bottles, rubber stoppers, transporting devices, and potting aluminum caps used for filling the filtered drug solution;

[0073] Step (E), filling and half-stoppering

[0074] The filtered liquid medicine is tested according to the intermediate product standard of piperacillin sodium and tazobactam sodium for injection, the content is determined by HPLC, the filling volume is calculated according to the product specifications, and the filtered liquid medicine is filled into a medium borosilicate glass bottle and half-stoppered to form a half-stoppered filling bottle, and the half-stoppered filling bottle is placed on a drop partition at a temperature of 5°C;

[0075] Step (F), freeze-drying and plugging in stages

[0076] (F1), pre-freezing stage: the temperature of the heat transfer oil in the half-stoppered bottles is gradually reduced to -50°C within 60 minutes and maintained for 120 minutes;

[0077] (F2), sublimation stage: sequentially, the temperature of the heat transfer oil is gradually raised to -25°C within 60 minutes, the temperature of the heat transfer oil is gradually raised from -25°C to 0°C within 120 minutes, the temperature of the heat transfer oil is gradually raised from 0°C to 25°C within 120 minutes, the temperature of the heat transfer oil is gradually raised from 25°C to 35°C within 90 minutes, and maintained for 420 minutes, and the vacuum is 30±5Pa;

[0078] (F3), desorption stage: the temperature of the heat transfer oil is gradually raised from 35°C to 38°C within 60 minutes, and then from 38°C to 48°C within 90 minutes, and maintained for 60 to 90 minutes, with a vacuum of 30Pa±5Pa;

[0079] (F4), close the aeration valve, continue to maintain for 2 hours, and the staged freeze-drying is completed. Here, the repeated freeze-drying in stages makes the product freeze-dried evenly in the form of loose blocks. The product can be quickly re-dissolved in clinical use. Since the water is removed evenly from the loose blocks, the moisture of the entire freeze-dried product is uniform from top to bottom, inside and outside). Moreover, the freeze-dried product has uniform low moisture content, a long shelf life, is not easy to degrade, has less related substances, and has high purity. While ensuring the efficacy, the probability of clinical adverse reactions is reduced. The product is in the form of loose blocks, which dissolves quickly in clinical use, reducing the waiting time for re-dissolution.

[0080] (F5), for the half-stoppered filling bottles that have been freeze-dried in stages, the stoppering is completed after being filled with sterile nitrogen to form a stoppered filling bottle, and the re-pressurization pressure of the sterile nitrogen filled part is between -0.03 and -0.04 MPa;

[0081] Step (G), unpacking, capping, leak detection

[0082] (G1), after the freeze-drying is completed, the plugged filled bottles are unpacked in the filling room and enter the capping room via the conveyor belt. The capping speed is 20-50Hz. When testing the capping, first roll two circles and then stop the machine to check the tightness of the rolled aluminum cap and the edge of the aluminum cap to see if the capping is qualified. If the capping is qualified, start normal operation to achieve continuous aluminum capping of the plugged filled bottles to form aluminum capped filled bottles;

[0083] (G2), Leak detection of rolled aluminum cap filled bottles by color water method

[0084] (G21) Sampling: Random sampling is conducted at the beginning, middle and end stages of each batch of rolled aluminum cap filled bottles, with 10 bottles sampled at each stage;

[0085] (G22) Judgment standard: Take the sample and put it into a vacuum dryer filled with 0.1% methylene blue solution, immerse it completely, and evacuate it to 30±2kPa. After maintaining it for 30 minutes, return it to normal pressure; after maintaining it for another 30 minutes, take out the sample; rinse the surface of the bottle of the sample with water until there is no obvious color residue on the outer wall, which means it is qualified, because the white powder medicine in the bottle is dyed blue by the methylene blue solution;

[0086] Step (H): Light inspection, labeling, packaging, and storage

[0087] Light inspection personnel need to check the illumination of the light inspection machine, which should be between 1000lx-1500lx. Take out each bottle of rolled aluminum cap filled bottle from the conveyor belt, place it under the light for 20-30cm, and conduct continuous light inspection manually. If defective products are found, take them out and place them in a red special box.

[0088] Qualified rolled aluminum cap filled bottles are placed on another conveyor belt and sent to the outsourcing room via the conveyor belt for labeling, packaging and storage.

[0089] Preferably, in the preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization, in step (A), the weighing standard is the unit dosage required for each bottle of rolled aluminum cap filling bottle: piperacillin 2000 mg, tazobactam 250 mg, edetate disodium 0.5 mg, sodium bicarbonate 476.5 mg, citric acid 71.5 mg, and water for injection 8.5 ml.

[0090] Preferably, in the preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged freeze-drying, in step (B), during the batching process, the temperature of the concentrated preparation tank is controlled between 10 and 20°C; in step (C), during the diluted preparation process, the temperature of the diluted preparation tank is controlled between 5 and 10°C.

[0091] Preferably, in the preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization, the operation time of step (B) and step (C1)-step (C2) is no more than 8 hours; the operation time of step (C4) is no more than 10 hours, and the drug liquid in the dilution tank is filtered through a 0.22 μm polyethersulfone filter in the liquid preparation room and a 0.22 μm polyethersulfone filter in the liquid collection room into a liquid storage closed barrel to obtain a filtered drug liquid, and the 1000 ml filtrate in the front section after filtration is discarded.

[0092] Preferably, the preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization, step (D), respectively cleaning and sterilizing the borosilicate glass bottle, rubber stopper, transport device, and potting aluminum cap used for filtering and filling the liquid medicine, each including the following processes,

[0093] (D1), cleaning and sterilization of borosilicate glass bottles: including ultrasonic cleaning of bottles filled with circulating water, external spraying of circulating water, cleaning with purified water, blowing with clean compressed air, cleaning with injection water, blowing with clean compressed air, drying and sterilization, cooling, bottle removal, sampling and inspection, and standby. The drying and sterilization is set at 350°C and ≥5min; the speed of the ultrasonic cleaning machine is: 150 bottles / min for 20ml bottles; the storage of the borosilicate glass bottles after drying and sterilization (D1) shall not exceed 8 hours, and they shall be cleaned and sterilized again after 8 hours;

[0094] (D2) Cleaning and sterilization of rubber stoppers: including taking out rubber stoppers, adding materials, spraying, mixing, rinsing, checking for visible foreign matter, moist heat sterilization, dry sterilization, cooling, and removing stoppers. The sterilization temperature of dry sterilization is 121°C, and the sterilization time is greater than 30 minutes. The service life of the dry sterilized rubber stoppers should not exceed 24 hours;

[0095] (D3), cleaning and sterilization of transport equipment: including cleaning, sterilization, drying, cooling, and unloading of equipment in sequence. Dry heat sterilization or moist heat sterilization can be selected according to the different transport equipment. The effective period of dry heat sterilization is 24 hours, and the effective period of moist heat sterilization is 24 hours;

[0096] (D4), cleaning and sterilization of potted aluminum caps: including taking out the aluminum caps, framing, sterilizing at 120°C for 2 hours, cooling, and removing the aluminum caps. The potted aluminum caps after sterilization have a shelf life of 24 hours.

[0097] Preferably, the preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization, step (E), filling, half-stoppering, checking the filling amount and the quality of the half-stoppering and visible foreign matter every 30 minutes during the filling process and keeping records, the filtered drug solution should not exceed 10 hours from the start of sterile filtration to the start of filling; the storage time of the filtered drug solution in the liquid storage sealed barrel should not exceed 10 hours; the filling should be completed within 12 hours, and the room temperature should not exceed 8°C.

[0098] Preferably, in the preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization, in step (F), during the staged lyophilization and plugging process, the shutdown judgment standard is closing the box trap valve and the vacuum rise in the box is ≤0.5Pa / min.

[0099] Preferably, the preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged freeze-drying, (G1), whether the capping is qualified, use the thumb, middle finger and index finger to clamp the edge of the bottle cap, stand it upright in a triangle, and apply force evenly in opposite directions with both hands, and it is qualified if it cannot be twisted.

[0100] Among them, the content determination by HPLC recorded in step (C4) and step (E) is a technology widely used in the field of chemical analysis, which is a high performance liquid chromatography method for determining the content of a substance.

[0101] According to the preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization of the present invention, combined with the comparison table of preparation examples of piperacillin sodium and tazobactam sodium for injection (using different preparation processes) in Table 1 below, the ingredients (the same as in Example 1) are as follows:

[0102] The weighing standard is the unit dosage required for each rolled aluminum cap filling bottle: piperacillin 2000 mg, tazobactam 250 mg, edetate disodium 0.5 mg, sodium bicarbonate 476.5 mg, citric acid 71.5 mg, water for injection 8.5 ml;

[0103] The above-described embodiment 1 includes three-stage filtration in dilution, liquid preparation room, and liquid collection room, semi-plugging, staged freeze-drying, plugging preparation, and filling processes;

[0104] Example 2, using the same process parameters as Example 1, two-stage filtration between dilution and preparation, half-plugging, staged freeze-drying, plugging preparation and filling process, this Example 2 does not involve filtration between liquid collection;

[0105] Example 3, using the same process parameters as Example 1, dilution preparation, two-stage filtration between liquid collection, half-plug pressing, staged freeze drying, plug pressing preparation, and filling process, this Example 3 does not involve filtration between liquid preparation;

[0106] Comparative Example 1: Single-stage filtration during dilution (consistent with the dilution filtration parameters of Example 1), combined with conventional freeze-drying (the existing freeze-drying process is mainly the sublimation stage of Example 1, that is, the temperature of the heat transfer oil is gradually increased from -25°C to 0°C within 120 minutes, and then the temperature of the heat transfer oil is gradually increased from 0°C to 25°C within 120 minutes, and then the temperature of the heat transfer oil is gradually increased from 25°C to 35°C within 90 minutes, and maintained for 420 minutes, and the vacuum is 30±5Pa), filling with a disposable tampon (a traditional disposable tampon, a conventional technical means of those skilled in the art);

[0107] Comparative Example 2: Single-stage filtration between the preparation liquids only (the filtration parameters between the preparation liquids are consistent with those in Example 1), combined with conventional freeze-drying (the existing freeze-drying process is mainly the sublimation stage of Example 1, that is, the temperature of the heat transfer oil is gradually increased from -25°C to 0°C within 120 minutes, and then the temperature of the heat transfer oil is gradually increased from 0°C to 25°C within 120 minutes, and then the temperature of the heat transfer oil is gradually increased from 25°C to 35°C within 90 minutes, and maintained for 420 minutes, and the vacuum is 30±5Pa), and a one-time tampon is filled (a traditional one-time tampon is a conventional technical means of those skilled in the art);

[0108] Comparative Example 3: Single-stage filtration in the liquid collection room only (consistent with the filtration parameters in the liquid collection room in Example 1), combined with conventional freeze-drying (the existing freeze-drying process is mainly the sublimation stage of Example 1, that is, the temperature of the heat transfer oil is gradually controlled from -25°C to 0°C within 120 minutes, and then the temperature of the heat transfer oil is gradually controlled from 0°C to 25°C within 120 minutes, and then the temperature of the heat transfer oil is gradually controlled from 25°C to 35°C within 90 minutes, and maintained for 420 minutes, and the vacuum is 30±5Pa), filling with a disposable tampon (a traditional disposable tampon, a conventional technical means of those skilled in the art).

[0109] Table 1 Comparison table of preparation examples of piperacillin sodium and tazobactam sodium for injection

[0110]

[0111]

[0112] By comparing the test data analysis of different processes for the preparation and filling of piperacillin sodium and tazobactam sodium for injection in Table 1 (the test results of various parameters are obtained by conventional drug testing methods), it is clearly obtained that in Example 1, three-stage filtration is performed in the dilution, liquid preparation and liquid collection rooms, and the total impurities of the relevant substances can be controlled at 0.6, thereby verifying that the impurity removal effect of filtering in the dilution, liquid preparation and liquid collection rooms is the best; and, through the process of half-plugging, staged freeze-drying and plugging of the present invention, the moisture content is controlled at 0.4, In particular, by using the staged freeze-drying process of the present invention, the moisture content of the finished product is far lower than that of Comparative Examples 1, 2, and 3, and the moisture content of the entire staged freeze-dried piperacillin sodium and tazobactam sodium for injection can be uniform (very little moisture exists, that is, the moisture content is low but uniform), which is conducive to long-term storage, ensures a stable pH value, and makes the freeze-dried piperacillin sodium and tazobactam sodium for injection uniformly present as loose blocks, ensures that the product is quickly re-dissolved in clinical use, has low moisture content and less degradation of related substances, reduces the occurrence of clinical adverse reactions, and ensures the efficacy.

[0113] In summary, the preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization of the present invention, through repeated staged lyophilization of piperacillin sodium and tazobactam sodium for injection, combined with the filling process of semi-tamponade and tamponade, makes the piperacillin sodium and tazobactam sodium for injection lyophilized uniformly into loose blocks, ensuring that the product is quickly reconstituted in clinical use, and because the water is removed uniformly in the loose blocks, the entire staged lyophilized piperacillin sodium and tazobactam sodium for injection has uniform water content on the top, bottom, inside and outside (very little water exists, that is, the water content is low but uniform), which is conducive to long-term storage, ensuring a stable pH value, and has the following characteristics:

[0114] (1) Three-stage filtration is adopted: dilution filtration: the drug liquid is filtered through a 0.45 μm polyethersulfone filter to a 500L dilution tank, and the filter element is disposable; filtration in the liquid preparation room: the drug liquid in the dilution tank is filtered through a 0.22 μm polyethersulfone filter in the liquid preparation room; filtration in the liquid collection room, the 0.22 μm polyethersulfone filter in the liquid collection room is filtered into a liquid storage closed barrel to obtain the filtered drug liquid. Through the three-stage filtration measures, the purity of piperacillin sodium and tazobactam sodium for injection is ensured, the related substances (total impurities) are reduced, and the efficacy of piperacillin sodium and tazobactam sodium for injection is greatly improved;

[0115] (2) Filling, half-stoppering, staged freeze-drying, and plugging are four steps carried out in sequence. The half-stoppered bottle is freeze-dried in stages before plugging. The staged freeze-drying is particularly critical, as shown below: in the pre-freezing stage, the filtered liquid in the half-stoppered bottle is a loose crystal structure that is conducive to the sublimation and discharge of internal moisture; in the sublimation stage, step-by-step heating is conducive to multiple sublimations, and the moisture is discharged more thoroughly; in the analysis stage, slow heating ensures that the product (filtered liquid in the half-stoppered bottle) is not thermally degraded, and the sublimated moisture in the half-stoppered bottle is discharged out of the bottle; the air injection valve is closed, and the process maintains a negative pressure state to isolate the air and prevent the product (filtered liquid in the half-stoppered bottle) from absorbing moisture in the air during storage. The above processes are very critical to achieve reliable and effective moisture discharge from the filtered liquid in the half-stoppered bottle without absorbing moisture in the air. Moreover, freeze-drying in stages after half-stoppering can ensure that the moisture in the product (filtered drug solution in the half-stoppered bottle) is drawn out of the bottle through the pores of the half-stopper after freeze-drying and sublimation, and then the stoppering process is carried out. The seal ensures the sterility of the drug (finished product) and also prevents the drug (finished product) from absorbing moisture in the air during storage.

[0116] (3) The borosilicate glass bottles, rubber stoppers, transfer equipment, and aluminum caps used for filling the filtered liquid are cleaned and sterilized separately to ensure product quality and prevent contamination during the filling process;

[0117] (4) Three quality control processes, namely capping, leak detection and light inspection, were set up to determine whether the finished product was qualified and to ensure the qualified rate of the finished product of piperacillin sodium and tazobactam sodium for injection based on staged freeze-drying.

[0118] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged freeze-drying, characterized in that: The following steps are included: Step (A), weighing The raw and auxiliary materials are removed from their outer packaging, disinfected, and brought into the Class C clean area, and the raw and auxiliary materials are weighed according to the instructions. The raw and auxiliary materials include APIs and auxiliary materials. The APIs include piperacillin and tazobactam, and the auxiliary materials include edetate disodium, sodium bicarbonate, and citric acid; Step (B), Ingredients (B1), add the injection water of the preparation volume into the 700L concentrated preparation tank, cool the injection water to 10-20℃ and keep it, take out 35% of the injection water into a stainless steel sealed barrel for standby before adding the raw materials and excipients, and keep the remaining 65% of the injection water in the concentrated preparation tank; (B2), add the weighed citric acid and edetate disodium to the concentrated preparation tank in sequence under static state, start stirring at a speed of 30 Hz, and stir for 20 minutes to completely dissolve the citric acid and edetate disodium in the injection water to form a drug solution; (B3), under a stirring condition of 15 Hz, add the weighed piperacillin, and continue stirring under a stirring condition of 30 Hz for 5 minutes to suspend the drug solution; (B3), slowly add 95% of the weighed sodium bicarbonate under a stirring condition at a rotation speed of 20 Hz. After the addition is completed, stir for 40 minutes under a stirring condition at a rotation speed of 30 Hz to clarify the liquid; (B4), continue to add the weighed tazobactam at a speed of 15 Hz, stir for 30 minutes at a speed of 30 Hz, then continue to add the remaining 5% of the weighed sodium bicarbonate, continue stirring for 30 minutes, and confirm that the pH value of the solution is 5.9-6.3 to indicate complete reaction. If the pH value is not within the specified range, adjust it by adding sodium bicarbonate or citric acid; Step (C), diluting and obtaining a filtered solution (C1), the drug solution is filtered through a 0.45μm polyethersulfone filter into a 500L dilution tank, wherein the filtration pressure is ≤0.2MPa; the pressure difference between the upstream and downstream of the filter is ≤0.2MPa, and the filter element is disposable; (C2), add 35% injection water in the stainless steel sealed barrel to the dilution tank, stir for 15 minutes at a speed of 30 Hz, and control the temperature of the dilution tank to between 5 and 10 °C; (C3), take samples from the dilution tank to test the intermediate, determine the content by HPLC, and calculate the filling volume according to the product specifications; (C4), the liquid in the dilution tank is filtered through a 0.22μm polyethersulfone filter in the liquid preparation room and a 0.22μm polyethersulfone filter in the liquid collection room to obtain a filtered liquid in a liquid storage closed barrel, the filtering temperature: 5-20℃; filtering pressure: ≤0.2MPa; filtering time: 0.5-3h / batch; the pressure difference between the upstream and downstream of the filter: ≤0.2MPa, the filter element is disposable; Step (D), respectively cleaning and sterilizing the borosilicate glass bottles, rubber stoppers, transporting devices, and potting aluminum caps used for filling the filtered drug solution; Step (E), filling and half-stoppering The filtered liquid medicine is tested according to the intermediate product standard of piperacillin sodium and tazobactam sodium for injection, and the content is determined by HPLC. The filling volume is calculated according to the product specifications, and the filtered liquid medicine is filled into a medium borosilicate glass bottle and half-stoppered to form a half-stoppered filling bottle, and the half-stoppered filling bottle is placed on a drop partition at a temperature of 5°C; Step (F), freeze-drying and plugging in stages (F1), pre-freezing stage: control the temperature of the heat transfer oil in the half-stoppered bottles to gradually drop to -50°C within 60 minutes and keep it for 120 minutes; (F2), sublimation stage: the temperature of the heat transfer oil is gradually raised to -25°C within 60 minutes, the temperature of the heat transfer oil is gradually raised from -25°C to 0°C within 120 minutes, the temperature of the heat transfer oil is gradually raised from 0°C to 25°C within 120 minutes, the temperature of the heat transfer oil is gradually raised from 25°C to 35°C within 90 minutes, and maintained for 420 minutes, with a vacuum of 30±5Pa; (F3), decomposition stage: gradually increase the temperature of the heat transfer oil from 35°C to 38°C within 60 minutes, and then gradually increase the temperature of the heat transfer oil from 38°C to 48°C within 90 minutes, and maintain it for 60 minutes to 90 minutes, with a vacuum of 30Pa±5Pa; (F4), close the aeration valve and continue to maintain for 2 hours, and the staged freeze-drying is completed; (F5), for the half-stoppered filling bottles that are freeze-dried in stages, the stoppering is completed after filling with sterile nitrogen to form a stoppered filling bottle. The re-pressurization pressure of the sterile nitrogen filling part is between -0.03 and -0.04 MPa; Step (G): unpacking, capping, leak detection (G1), after freeze drying, the plugged filled bottles are unpacked in the filling room and enter the capping room via the conveyor belt. The capping speed is 20-50Hz. When testing the capping, first roll two circles and then stop the machine to check the tightness of the rolled aluminum cap and the edge of the aluminum cap to see if the capping is qualified. If the capping is qualified, start normal operation to achieve continuous aluminum capping of the plugged filled bottles to form aluminum capped filled bottles; (G2), Leak detection of rolled aluminum cap filled bottles by color water method (G21) Sampling: Random sampling is conducted at the beginning, middle and end stages of each batch of rolled aluminum cap filled bottles, with 10 bottles sampled at each stage; (G22) Judgment standard: Take the sample and put it into a vacuum dryer filled with 0.1% methylene blue solution, immerse it completely, and evacuate it to 30±2kPa. After maintaining it for 30 minutes, return it to normal pressure. After maintaining it for another 30 minutes, take out the sample; rinse the surface of the sample bottle with water until there is no obvious color residue on the outer wall, which is qualified; Step (H): Light inspection, labeling, packaging, and storage The light inspector needs to check the illumination of the light inspection machine, which should be between 1000lx-1500lx. Take out each bottle of rolled aluminum cap filled bottle from the conveyor belt, place it 20 to 30 cm under the light, and conduct continuous light inspection manually. If defective products are found, take them out and place them in a red special box; qualified rolled aluminum cap filled bottles are placed on another conveyor belt and sent to the outsourcing room via the conveyor belt for labeling, packaging and warehousing.

2. The preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization according to claim 1, characterized in that: Step (A), the weighing standard is the unit dosage required for each bottle of rolled aluminum cap filling bottle: piperacillin 2000 mg, tazobactam 250 mg, edetate disodium 0.5 mg, sodium bicarbonate 476.5 mg, citric acid 71.5 mg, and water for injection 8.5 ml.

3. The preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization according to claim 1, characterized in that: In step (B), during the batching process, the temperature of the concentrated batching tank is controlled between 10 and 20°C; in step (C), during the diluted batching process, the temperature of the diluted batching tank is controlled between 5 and 10°C.

4. The preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization according to claim 1, characterized in that: The operation time of step (B) and step (C1)-step (C2) shall not exceed 8 hours; the operation time of step (C4) shall not exceed 10 hours, and the liquid in the dilution tank shall be filtered through a 0.22 μm polyethersulfone filter in the liquid preparation room and a 0.22 μm polyethersulfone filter in the liquid collection room to a liquid storage sealed barrel to obtain a filtered liquid, and the 1000 ml filtrate in the front section after filtration shall be discarded.

5. The preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization according to claim 1, characterized in that: Step (D) is to clean and sterilize the borosilicate glass bottles, rubber stoppers, transport equipment and aluminum caps used for filtering the liquid medicine, respectively, including the following processes: (D1), cleaning and sterilization of medium borosilicate glass bottles: including ultrasonic cleaning of bottles filled with circulating water, external spraying of circulating water, cleaning with purified water, blowing with clean compressed air, cleaning with injection water, blowing with clean compressed air, drying and sterilization, cooling, bottle removal, sampling and inspection, and standby. The drying and sterilization is set at 350°C and ≥5min; the speed of the ultrasonic cleaning machine is: 150 bottles / min for 20ml bottles; the medium borosilicate glass bottles after drying and sterilization in (D1) shall not be stored for more than 8 hours, and shall be cleaned and sterilized again after more than 8 hours; (D2), cleaning and sterilization of rubber stoppers: including taking out rubber stoppers, adding materials, spraying, mixing, rinsing, checking for visible foreign matter, moist heat sterilization, dry sterilization, cooling, and removing stoppers, wherein the sterilization temperature of dry sterilization is 121°C, the sterilization time is greater than 30 minutes, and the service life of the dry sterilized rubber stoppers shall not exceed 24 hours; (D3) Cleaning and sterilization of transport equipment: including cleaning, sterilization, drying, cooling, and unloading of equipment in sequence. Dry heat sterilization or moist heat sterilization can be selected according to the different transport equipment. The effective period of dry heat sterilization is 24 hours, and the effective period of moist heat sterilization is 24 hours; (D4), cleaning and sterilization of potted aluminum caps: including taking out the aluminum caps, framing, sterilizing at 120°C for 2 hours, cooling, and removing the aluminum caps. The potted aluminum caps after sterilization have a shelf life of 24 hours.

6. The preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization according to claim 1, characterized in that: Step (E), filling and half-stoppering. During the filling process, check the filling volume and the quality of the half-stoppering, and any visible foreign matter every 30 minutes and keep records. The filtered liquid should not exceed 10 hours from the start of aseptic filtration to the start of filling. The storage time of the filtered liquid in the sealed storage barrel should not exceed 10 hours. The time from the start of filling to the end of filling should be 12 hours, and the room temperature should not exceed 8°C.

7. The preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization according to claim 1, characterized in that: In step (F), during the freeze-drying and plugging process in stages, the shutdown judgment standard is closing the box trap valve and the vacuum in the box rising by ≤0.5Pa / min.

8. The preparation and filling method of piperacillin sodium and tazobactam sodium for injection based on staged lyophilization according to claim 1, characterized in that: (G1) Check whether the capping is qualified. Hold the edge of the bottle cap with your thumb, middle finger and index finger, forming a triangle and holding it upright. Apply force evenly in opposite directions with both hands. It is qualified if the cap cannot be twisted.

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