Method for establishing and maintaining online sterile environment of sterile tank

Abstract

The invention relates to the technical field of sterile tanks, and discloses an online sterile environment establishment and maintenance method for a sterile tank, which comprises the following steps: S1, tank body selection: adopting 316L stainless steel, and performing electrolytic polishing on the inner surface to ensure no dead angle, easy cleaning and corrosion resistance; s2, cleaning: performing programmed cleaning by using high-concentration alkali liquor, acid liquor and water for injection by rotating a cleaning ball to ensure that all organic and inorganic residues are removed; s3, sterilization: sterilizing the tank body and related pipelines through saturated pure steam; s4, sterile filtration: mounting a 0.22 mu m hydrophobic PTFE (Polytetrafluoroethylene) or PVDF (Polyvinylidene Fluoride) sterilization-grade air filter on the top of the tank for balancing the pressure in the tank during feeding, discharging or temperature change; according to the scheme, a plurality of sterile connection modes, inert gas coverage, phage prevention filtration and the like are adopted, so that different process requirements from traditional macromolecular biological medicines to high-activity cell and gene therapy products and the like can be flexibly met, and future product pipelines of enterprises are protected.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of aseptic tanks, and particularly relates to an online aseptic environment establishment and maintenance method for aseptic tanks. BACKGROUND

[0002] In the fields of biopharmaceuticals, high-end foods and aseptic preparation production, an aseptic tank is a core equipment for connecting upstream and downstream processes and realizing aseptic temporary storage and transfer of materials. The fundamental task of the aseptic tank is to provide a closed environment that can continuously maintain aseptic state in a production cycle to ensure the microbial safety of the final product. However, how to reliably 'establish' and long-term'maintain' the aseptic environment in the tank has always been a technical challenge faced by the industry.

[0003] At present, the industry usually carries out sterilization and maintenance (regarded as two relatively independent stages) for the aseptic tank. In the aseptic environment establishment stage, the traditional method seriously depends on steam sterilization. In the aseptic environment maintenance stage, the existing technology mostly adopts static and passive measures. The existing technology system regards cleaning, sterilization, maintenance and monitoring as a series of sequentially executed but mutually independent method sets, and lacks a whole-cycle and integrated method. Therefore, the application provides an online aseptic environment establishment and maintenance method for aseptic tanks. SUMMARY

[0004] In view of the above problems, the application provides an online aseptic environment establishment and maintenance method for aseptic tanks to overcome the defects of the prior art.

[0005] To achieve the above object, the application provides the following technical scheme: an online aseptic environment establishment and maintenance method for aseptic tanks, comprising the following steps: S1: tank body selection: 316L stainless steel is adopted, and the inner surface is electrolytically polished to ensure no dead angle, easy cleaning and corrosion resistance; S2: cleaning: a rotating cleaning ball is used to carry out programmed cleaning by using high-concentration alkali solution, acid solution and water for injection to ensure removal of all organic and inorganic residues; S3: sterilization: saturated pure steam is used to sterilize the tank body and related pipelines; S4: aseptic filtration: a 0.22 mu m hydrophobic PTFE or PVDF sterilization-grade air filter is installed on the top of the tank and used to balance the pressure in the tank when feeding, discharging or changing temperature, while blocking external microorganisms and particles. The bubble point pressure of the filter is greater than or equal to the bubble point specification value; S5: ventilation system: high-purity nitrogen is filled into the tank through another sterilization filter to maintain the positive pressure and inert environment of the material in the tank, and the dew point of the gas is required to be less than or equal to -40 DEG C to inhibit the growth of microorganisms; S6: Biological indicator challenge test: At the coldest point in the tank, place a bioindicator of Geobacillus stearothermophilus, after sterilization, culture, and should be sterile growth, the number of bioindicator spores: 1 x 106 CFU / piece, culture conditions: 55-60℃, time 7-14d; S7: Maintenance method: The following operation methods are required for long-term maintenance during the use of the sterile tank: Continuous positive pressure protection: Always keep the pressure in the tank slightly higher than atmospheric pressure. The pressure control system should use split-range control: when the pressure is lower than the set value, the sterile nitrogen gas inlet valve opens to supplement the pressure; when the pressure is higher than the set value, the breather valve is vented; Any operation that may cause pressure drop must be started in advance to ensure smooth pressure curve and avoid instantaneous negative pressure; Sterile gas quality assurance: The sterile gas after the breather needs to be regularly monitored for microorganisms, with a sampling volume of not less than 1 cubic meter, and the planktonic bacteria limit should meet the corresponding clean area (Class A) standard; The breather needs to be regularly tested for online integrity, or before and after each SIP. The filter cartridge should have an upper limit for the number of uses or cumulative SIP time, whichever comes first; Sterile material transfer: All material imports and exports must use sterile connections. Hard pipe connections recommend using double-seat valves with steam barriers for docking. Soft pipe connections must use validated sterile quick connectors, and the interface surface must be thoroughly disinfected with 70-75% alcohol before connection and ensured dry; The transfer process should preferentially use pressure transfer method (tank pressurization), and secondarily use sterile diaphragm pumps; ordinary centrifugal pumps that may generate negative pressure are strictly prohibited; Online sterile sampling: Before sampling, sterilize the sampling valve and short pipe with pure steam at least 121℃ for 30 minutes, and ensure that the condensate is discharged smoothly. After steam sterilization, the pipeline needs to be blown dry with sterile gas; During sampling, follow the "release-take-release" principle: first open the valve to discharge 3-5 times the dead volume of the pipeline, then use a sterile sample bottle to take enough sample, and finally discharge a small amount of material to flush the sampling port before closing the valve; Periodic re-sterilization and monitoring: Even without production, it is recommended to perform a short-term maintenance SIP on the sterile tank system every 48-72h, which involves passing pure steam to bring the system temperature to 121℃ and maintaining it for 5-10 minutes to kill possible slowly proliferating microorganisms.

[0006] For long-term stored materials, a regular sterile sampling plan should be established, and samples should be subjected to full sterility testing for aerobic bacteria, anaerobic bacteria, fungi, and mycoplasma.

[0007] Preferably, the cleaning process in S2 is specifically as follows: Full coverage is achieved by rotating the cleaning ball, and the cleaning process is specifically as follows: Pre-flushing: flushing with WFI or purified water at room temperature or ≤45°C for 5-10 minutes to remove most visible residues; Alkaline cleaning: using 1.0-2.0% (w / v) sodium hydroxide solution, circulating cleaning at 75-85°C for 15-30 minutes to remove organic contaminants such as proteins and fats; Intermediate flushing: flushing with WFI until the outlet water conductivity is consistent with the inlet water to remove the alkali solution; Acid cleaning: using 0.8-1.5% (w / v) nitric acid solution, circulating cleaning at 60-70°C for 10-20 minutes to neutralize the residual alkali and remove inorganic salt scale; Final flushing: flushing with WFI at ≥80°C for at least 15 minutes until the outlet water conductivity is ≤1.3 μS / cm (25°C) and the TOC is ≤500 ppb; Online sterilization: the sterilization process must verify that the "coldest point" reaches the specified temperature and maintains it for a sufficient time, with parameters of 121.1°C and 45-90 minutes.

[0008] Preferably, the sterilization process in S3 is specifically as follows: drain the accumulated water in the tank and jacket, preheat the pipeline by introducing steam, open all condensate discharge valves to remove air and non-condensable gases, then introduce saturated pure steam into the tank, and perform wet heat sterilization of the tank body and related pipelines by pure steam. Industrial pure steam (non-process steam) must be used, with key quality indicators of dryness ≥0.9, non-condensable gas content ≤3.5%, and superheat ≤25°C. The steam quality standard is expanded to uniformly raise the temperature of the coldest point to 121°C, and then start timing after the coldest point temperature reaches 121°C, maintaining for 30-60 minutes. The temperature difference at each point in the tank should be ≤2°C, then slowly close the steam, introduce sterile compressed air or nitrogen to maintain positive pressure, and cool the tank body at the same time. The cooling rate needs to be controlled (such as ≤0.5°C / s) to prevent vacuum and tank body deformation.

[0009] Preferably, the specific methods for verifying and monitoring data of each key step of the sterile tank are as follows: .

[0010] Preferably, the methods for handling and maintaining abnormalities during the use of the sterile tank are as follows: Pressure abnormally drops: immediately start investigation, consider it as a major sterile risk event, check the integrity of the breather, valve sealing, and gasket status, and if necessary, isolate the material in the tank for evaluation and re-sterilize the system.

[0011] Respirator clogging or wetness: pressure differential increase indicates clogging, which requires timely replacement; respirator condensation (dew) failure requires ensuring that the inlet gas dew point is low enough, or using a respirator with an electric heating jacket.

[0012] Periodic revalidation: all critical components should be revalidated periodically, usually once a year, or after significant changes, deviations.

[0013] Preferably, sampling and valve system: Use a sterile diaphragm valve, whose dead volume should be close to zero; use a double seat valve + steam barrier design for the sampling valve to ensure that the sampling port itself can be sterilized online; Before sampling, use pure steam to sterilize the sampling valve and short tube in situ at 121°C for 30 minutes, or use a verified 70-75% isopropyl alcohol to wipe the sterile sampling diaphragm and maintain sufficient contact time.

[0014] Preferably, instrument and control system: All pressure and temperature sensors directly connected to the tank must use a flush diaphragm sanitary design and be able to withstand a complete SIP cycle.

[0015] F0 value calculation and recording is the core of SIP, the control system must be able to calculate and record the F0 value of the coldest point in the tank in real time (F0=∫10^((T-121.1) / 10)dt), and automatically end the heat preservation phase after reaching the set value (such as F0≥15 minutes).

[0016] Preferably, quantitative method for surface cleanliness verification: Sensitive detection of chemical residues: Method: high performance liquid chromatography (HPLC) or total organic carbon (TOC) analyzer.

[0017] Sampling: sampling of the final rinse water, or using a specific solvent (such as water / ethanol mixture) to wet a dust-free cloth on a specified area (such as 100 cm²), and then extracting and analyzing; Standard: the residual amount of a specific active ingredient should be <10 ppm or less than 1 / 1000 of the acceptable daily exposure (ADE), and the TOC increment should be <500 ppb; Microbial recovery rate verification: before performing surface microbial swabbing, the recovery rate of the neutralizing agent, swabbing method and culture conditions used should be verified, usually requiring a recovery rate of ≥50% to demonstrate the effectiveness of the detection method.

[0018] Preferably, dynamic data benchmark for environmental monitoring: Planktonic bacteria: use an active air sampler, with a sampling volume of 1 cubic meter. At the tank respirator outlet area under A-level background, the standard is <1 CFU / m³.

[0019] Sedimentation bacteria: using φ90mm petri dish, exposed for 4h, in the same area, standard <1 CFU / 4h·dish A numerical control pipe bender with automatic feeding mechanism.

[0020] Preferably, all environmental monitoring data should be entered into the statistical process control system, draw control chart, monitor whether it is in the controlled state, and early warning potential trend.

[0021] Compared with the prior art, the beneficial effects of the present application are: The present application adopts multiple sterile connection methods, inert gas coverage, phage filtration and the like, so that it can flexibly adapt to different process requirements from traditional macromolecular biological drugs to high-activity cell and gene therapy products, and protect the future product pipeline of enterprises; The present application can make the environment in the tank reach and maintain the highest sterile guarantee standard in the industry through the integration of multiple barriers such as online steam sterilization, redundant sterilization filtration and continuous positive pressure protection, and it has been verified that the system can stably maintain the sterile state for more than several weeks without material intervention, thereby fundamentally eliminating the risk of microbial contamination caused by the environment and providing an extremely reliable storage environment for sensitive materials; The present application greatly reduces the catastrophic quality cost caused by pollution, such as the scrapping of entire products and market recall, through the design concept of "first correct", and at the same time, the preventive maintenance strategy method based on data and the optimized utility consumption can effectively reduce unplanned downtime and resource waste; The present application is compatible with multiple sterile connection methods, inert gas coverage and anti-condensation filters, so that it can not only be applied to traditional pharmaceutical processes, but also flexibly adapt to the special requirements of advanced therapies such as high-activity products and cell therapy products for sterile environments. BRIEF DESCRIPTION OF DRAWINGS

[0022] The accompanying drawings are included to provide a further understanding of the present application, and constitute a part of the specification, which together with the embodiments of the present application, serve to explain the present application, and do not constitute a limitation on the present application.

[0023] In the drawings: Figure 1 The flow chart of the method for establishing and maintaining the online sterile environment of the sterile tank of the present application. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application; based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of the present application.

[0025] The present application solves the problem that the prior art in the background art adopts static and passive measures, the prior art system regards cleaning, sterilization, maintenance, monitoring and other links as a series of sequentially executed but mutually independent method sets, and lacks a whole-cycle integrated method. Figure 1 An online sterile environment establishment and maintenance method for a sterile tank, S1: tank body selection: 316L stainless steel is adopted, the inner surface is electrolytic polished (Ra≤0.4 μm), and no dead angle, easy cleaning and corrosion resistance are ensured; S2: cleaning: through a rotating cleaning ball, high-concentration lye (such as 1-2M NaOH, 80°C), acid (such as 0.5-1M HNO3, 60°C) and water for injection (WFI, ≥80°C) are used for programmed cleaning to ensure removal of all organic and inorganic residues Full coverage is achieved through a rotating cleaning ball (a rotating cleaning ball that can rotate 360° is recommended, with an impact force of 8-15 meters / second); the specific steps and parameters of the cleaning program are as follows: pre-flushing: flushing with WFI or purified water at room temperature or ≤45°C for 5-10 minutes to remove most visible residues; alkali washing: using 1.0-2.0% (w / v) sodium hydroxide solution, circulating cleaning at 75-85°C for 15-30 minutes to remove organic contaminants such as proteins and fats; intermediate flushing: flushing with WFI until the outlet water conductivity is consistent with the inlet water (WFI≤5 μS / cm) to remove the lye; acid washing: using 0.8-1.5% (w / v) nitric acid solution, circulating cleaning at 60-70°C for 10-20 minutes to neutralize the alkali residues and remove inorganic salt scale; Final flushing: flushing with ≥80°C WFI for at least 15 minutes until the outlet water conductivity is ≤1.3 μS / cm (25°C) and TOC is ≤500 ppb; online sterilization: the sterilization program must verify that the “coldest point” (usually located at the condensate discharge port, instrument interface and other positions) reaches the specified temperature and maintains for a sufficient time, the parameters are: 121.1°C, 45-90min, the design of alkali washing→acid washing can specifically break most production residues, the flow rate is set to be ≥1.5m / s to ensure turbulent flow and obtain the best mechanical scouring force. The temperature is set to be ≥80°C to increase the chemical reaction rate and reduce the cleaning liquid viscosity, these parameters work together to ensure the consistency, reproducibility and high efficiency of the cleaning effect, and avoid sterilization failure or biofilm formation due to incomplete cleaning.

[0026] S3: Sterilization: Sterilize the tank body and related pipelines by saturated pure steam; Pure steam quality: dryness value ≥ 0.9, non-condensable gas ≤ 3.5%, superheat degree ≤ 25℃, F0 value ≥ 15 minutes (F0 value is the sterilization time at equivalent 121℃, which is the core acceptance standard), continuous and unobstructed condensate discharge 115-125℃ for at least 30-90 minutes, using pure steam (not process steam) can avoid the pollution risk of boiler additives to the product. Real-time calculation and record F0 value, change the sterilization effect from a qualitative concept of "temperature x time" to a cumulative and quantitative lethality index. This allows flexible adjustment of temperature and time (such as using 115°C for a longer time) to adapt to heat-sensitive equipment while providing irrefutable physical evidence while ensuring the same sterilization effect; S4: Sterile filtration: Install a 0.22μm hydrophobic PTFE or PVDF sterilization-grade air filter on the tank top to balance the pressure inside the tank when feeding, discharging or changing temperature, while blocking external microorganisms and particles, filter bubble point pressure ≥ bubble point specification value (usually PTFE 0.22μm filter > 3.5bar); S5: Ventilation system: Fill high-purity nitrogen (or other inert gases such as ) into the tank through another 0.22μm sterilization filter to maintain a positive pressure (usually 0.2-0.5bar) and inert environment in the tank, with a gas dew point requirement of ≤-40℃ to inhibit microbial growth; S6: Biological indicator challenge test: Place a thermophilic fat Bacillus stearothermophilus biological indicator at the coldest point in the tank (such as near the exhaust port or blowdown valve), incubate after sterilization, and there should be no sterile growth. The number of biological indicator spores is 1x106 CFU / piece, and the incubation conditions are 55-60℃ for 7-14 days. This method directly and objectively proves that the sterilization process can kill standard microorganisms with high resistance, which is more convincing than simple theoretical calculation or physical parameter recording. It provides biological end-point evidence for the validation of SIP program, which is the most recognized ultimate proof method by regulatory agencies; S7: Maintenance method: The following operation methods are required for long-term maintenance of the sterile tank during use: Continuous positive pressure protection: Always maintain the tank pressure (0.1-0.3bar) slightly higher than atmospheric pressure, and the pressure control system should use split control: when the pressure is lower than the set value, the sterile nitrogen gas inlet valve opens to supplement the pressure; when the pressure is higher than the set value, the breather valve is discharged to reduce the pressure; Before any operation that may cause pressure drop (such as discharging, cooling), the sterile gas supplement program must be started in advance to ensure smooth pressure curve and avoid instantaneous negative pressure; This is a simple, passive and extremely reliable physical barrier. As long as the pressure inside the tank (e.g. 0.2 bar) is maintained higher than the environment, any potential leak direction is from inside to outside, thus physically preventing the intrusion of unfiltered external air. Using an inert gas such as nitrogen to cover, it also prevents the product from oxidation at the same time, a two-in-one solution; Sterile gas quality assurance: Sterile gas after the respirator needs to be monitored regularly (such as every batch or every week), the sampling volume is not less than 1 cubic meter, and the planktonic bacteria limit should meet the corresponding clean area (A level) standard; The respirator needs to be tested regularly (such as WIT), or before and after each SIP. The filter core should have an upper limit of the number of uses or the cumulative SIP time (such as no more than 50 SIP cycles or 1 year), whichever comes first; Sterile material transfer: All material imports and exports must use sterile connections. Hard pipe connections recommend using double-seat valves with steam barriers for docking, and soft pipe connections must use validated sterile quick connectors. Before connecting, the interface surface must be thoroughly disinfected with 70-75% alcohol and ensured dry; The transfer process prefers to use pressure transfer method (pressurize the tank), and the second choice is sterile diaphragm pump; Ordinary centrifugal pumps that may produce negative pressure are strictly prohibited; Online sterile sampling: Before sampling, sterilize the sampling valve and short pipe with pure steam at least 121℃ for 30 minutes online, and ensure that the condensate is discharged smoothly. After steam sterilization, the pipeline must be blown dry with sterile gas [+] Sterilization after treatment expansion; Follow the "release-take-release" principle when sampling: first open the valve to discharge 3-5 times the dead volume of the pipeline material, then use a sterile sample bottle to take enough sample, and finally discharge a small amount of material to flush the sampling port before closing the valve; Periodic re-sterilization and monitoring: Even if there is no production, it is recommended to perform a short-term holding SIP on the sterile tank system every 48-72 hours, that is, to pass pure steam to make the system temperature reach 121℃ and maintain for 5-10 minutes, in order to kill the microorganisms that may slowly proliferate.

[0027] For long-term stored materials, a regular sterile sampling plan should be established (such as once a month), and the sample should be subjected to aerobic bacteria, anaerobic bacteria, fungi and mycoplasma full-item sterility test [+] Detection item expansion.

[0028] The specific procedure for sterilization in S3 is as follows: drain the water in the tank and the jacket, preheat the pipeline with steam, open all the condensate discharge valves to remove air and non-condensable gases, then introduce saturated pure steam into the tank to sterilize the tank body and related pipelines with wet heat. Industrial pure steam (non-process steam) must be used, and the key quality indicators are: dryness ≥ 0.9, non-condensable gas content ≤ 3.5%, and superheat degree ≤ 25°C. The steam quality standard is expanded to make the temperature at the coldest point uniformly rise to 121°C, and then the timer starts when the temperature at the coldest point reaches 121°C, maintaining for 30-60 minutes. The temperature difference at each point in the tank should be ≤ 2°C, and then the steam is slowly turned off and sterile compressed air or nitrogen is introduced to maintain positive pressure while cooling the tank body. The cooling rate needs to be controlled (e.g., ≤ 0.5°C / s) to prevent vacuum and tank body deformation.

[0029] The specific method for verifying the results of each key step of the sterile tank and monitoring data is as follows:

[0030] The method for handling abnormalities and maintenance during the use of the sterile tank is as follows: Abnormal pressure drop: immediately initiate an investigation, consider it a major sterility risk event, check the integrity of the breather, valve sealing, gasket condition, and if necessary, isolate the material in the tank for evaluation and re-sterilize the system.

[0031] Breather blockage or dampness: increased pressure difference indicates blockage, which needs to be replaced in time. Breather condensation (condensation) will fail, so ensure that the dew point of the incoming gas is low enough, or use a breather with an electric heating jacket.

[0032] Periodic revalidation: all critical components (such as filters, gasket materials) should be revalidated periodically, usually once a year, or after a major change or deviation.

[0033] Sampling and valve system: Use sterile diaphragm valves with a dead volume close to zero. The sampling valve is recommended to use a double-seat valve + steam barrier design to ensure that the sampling port itself can be sterilized online. Before sampling, sterilize the sampling valve and short tube with pure steam at least 121°C for 30 minutes, or use a verified 70-75% isopropyl alcohol to wipe the sterile sampling diaphragm and maintain sufficient contact time (≥ 1 minute) Dynamic data benchmark for environmental monitoring: Planktonic bacteria: use an active air sampler, sampling volume 1 cubic meter, in the tank breather outlet area under A-level background, standard <1 CFU / m³.

[0034] Sediment bacteria: use φ90mm petri dishes, expose for 4h, in the same area, standard <1 CFU / 4h·dish.

[0035] Data Trend Analysis: All environmental monitoring data shall be entered into a statistical process control (SPC) system, control charts (e.g. I-MR charts) shall be plotted to monitor whether the process is in control and to alert potential trends.

[0036] Instrumentation and Control System: All pressure and temperature sensors directly connected to the tank shall be sanitary design with flush diaphragm and able to withstand the complete SIP cycle.

[0037] F0 value calculation and recording is the core of SIP, the control system must be able to calculate and record the F0 value of the coldest point in the tank in real time (F0 = ∫10^((T-121.1) / 10)dt), and automatically end the heat preservation phase after reaching the set value (e.g. F0≥15 minutes) [+] Key control logic expansion.

[0038] Through multiple dimensions of end point judgment such as conductivity, TOC, microbial limit, surface wiping, etc., to ensure that the cleaning effect is measurable and verifiable. This directly supports the requirements of cross-contamination control and cleaning verification.

[0039] Quantitative method for surface cleanliness verification: Sensitive detection of chemical residues: Method: High performance liquid chromatography (HPLC) or total organic carbon (TOC) analyzer.

[0040] Sampling: Sampling of final rinse water, or using a specific solvent (such as water / ethanol mixture) to wet a dust-free cloth to wipe a specified area (such as 100 cm²), and then extract and analyze; Standard: The residual amount of specific active ingredients should be <10 ppm or less than 1 / 1000 of the acceptable daily exposure (ADE), and the TOC increment should be <500 ppb; Microbial recovery rate verification: Before performing surface microbial wiping, the recovery rate of the neutralizing agent, wiping method and culture conditions used for microorganisms should be verified, usually requiring a recovery rate of ≥50% to prove the effectiveness of the detection method.

[0041] Through the target of SAL≤ , the quantitative sterilization standard of F0≥15 minutes and the BI challenge test, the highest level of sterility assurance is provided for product quality, which is not only a mandatory requirement of international regulations (FDA, EMA, PIC / S), but also the fundamental to prevent product contamination and ensure the safety of patients / consumers.

Claims

1. A method for establishing and maintaining an online aseptic environment in an aseptic tank, characterized in that: Includes the following steps: S1: Tank Selection: Made of 316L stainless steel, with electrolytic polishing of the inner surface to ensure no dead corners, easy cleaning, and corrosion resistance; S2: Cleaning: A programmed cleaning process using high-concentration alkaline solutions, acid solutions, and water for injection is performed by rotating the cleaning ball to ensure the removal of all organic and inorganic residues. S3: Sterilization: Sterilize the tank and related pipelines using saturated pure steam; S4: Aseptic filtration: A 0.22μm hydrophobic PTFE or PVDF sterilization-grade air filter is installed on the top of the tank to balance the pressure inside the tank during feeding, discharging or temperature changes, while blocking external microorganisms and particles. The filter bubble point pressure is ≥ bubble point specification value. S5: Ventilation system: High-purity nitrogen is introduced into the tank through another sterilization filter to maintain positive pressure and inert environment of materials inside the tank. The gas dew point is required to be ≤-40℃ to inhibit the growth of microorganisms. S6: Biological indicator challenge test: Place the thermophilic Bacillus stearothermophilus biological indicator at the coldest point in the container, sterilize and incubate. It should grow aseptically. Biological indicator spore count: 1x106 CFU / tablet. Incubation conditions: 55-60℃, time: 7-14 days. S7: Maintenance Method: The following operating methods are required for long-term maintenance of the aseptic tank during use: Continuous positive pressure protection: Always maintain the pressure inside the tank slightly above atmospheric pressure. The pressure control system should use split-range control: when the pressure is lower than the set value, the sterile nitrogen inlet valve opens to replenish pressure; when the pressure is higher than the set value, the pressure is released through the respirator exhaust valve. Before any operation that may cause a pressure drop, the sterile gas replenishment procedure must be started in advance to ensure a smooth pressure curve and avoid instantaneous negative pressure. Aseptic gas quality assurance: The sterile gas after the respirator needs to be monitored for microorganisms regularly, with a sampling volume of not less than 1 cubic meter. The limit of airborne bacteria should meet the corresponding clean area (Grade A) standard. Respirators require periodic online integrity testing, or testing before and after each SIP (Self-Installation Processing). Filter cartridges should have a maximum number of uses or a maximum cumulative SIP time, whichever comes first. Aseptic material transfer: All material inlets and outlets must be aseptically connected. For rigid pipe connections, it is recommended to use a double-seat valve with a vapor barrier. For flexible hose connections, validated aseptic quick-connect fittings must be used. Before connection, the interface surfaces must be thoroughly disinfected with 70-75% alcohol and ensured to be dry. The pressure transfer method (in-tank pressurization) should be used first during the transfer process, followed by a sterile diaphragm pump; the use of ordinary centrifugal pumps that may generate negative pressure is strictly prohibited. Online aseptic sampling: Before sampling, sterilize the sampling valve and short pipe online at at least 121°C for 30 minutes with pure steam, and ensure that the condensate drains smoothly. After steam sterilization, the pipeline must be dried with sterile gas. When sampling, follow the "release-take-release" principle: first open the valve to discharge 3-5 times the dead volume of the pipeline, then use a sterile sample bottle to collect a sufficient amount of sample, and finally discharge a small amount of material to rinse the sampling port before closing the valve. Periodic re-sterilization and monitoring: Even when there is no production, it is recommended to perform a short-term maintenance SIP on the aseptic tank system every 48-72 hours, which involves introducing pure steam to raise the system temperature to 121°C and maintaining it for 5-10 minutes to kill any microorganisms that may be slowly multiplying. For materials stored for a long period of time, a regular aseptic sampling plan should be established, and samples should be subjected to a full range of aseptic tests for aerobic bacteria, anaerobic bacteria, fungi and mycoplasma.

2. The method for establishing and maintaining an online aseptic environment for an aseptic tank according to claim 1, characterized in that: The cleaning process described in S2 is as follows: Full coverage is achieved through rotating cleaning balls, and the cleaning procedure can be expanded with specific steps and parameters: Pre-rinse: Rinse with WFI or purified water at room temperature or ≤45℃ for 5-10 minutes to remove most visible residues; Alkaline washing: Use a 1.0-2.0% (w / v) sodium hydroxide solution to circulate and wash for 15-30 minutes at 75-85℃ to remove organic dirt such as protein and fat; Intermediate rinsing: Rinse with WFI until the conductivity of the outlet water is the same as that of the inlet water to remove alkaline solution; Pickling: Use a 0.8-1.5% (w / v) nitric acid solution to circulate and clean for 10-20 minutes at 60-70℃ to neutralize the alkaline residue and remove inorganic salt scale; Final rinse: Rinse with WFI at ≥80℃ for at least 15 minutes until the outlet water conductivity is ≤1.3μS / cm (25℃) and TOC is ≤500ppb; Online sterilization: The sterilization procedure must verify that the "coldest point" reaches the specified temperature and is maintained for a sufficient time. The parameters are: 121.1℃, 45-90min.

3. The method for establishing and maintaining an online aseptic environment in an aseptic tank according to claim 1, characterized in that: The specific sterilization process in S3 is as follows: drain the water inside the tank and jacket, introduce steam preheating pipeline, open all condensate drain valves to remove air and non-condensable gases, and then introduce saturated pure steam into the tank to perform moist heat sterilization on the tank and related pipelines. Industrial pure steam (non-process steam) must be used, and its key quality indicators are: dryness ≥ 0.9, non-condensable gas content ≤ 3.5%, superheat ≤ 25℃[+] steam quality standard expansion, so that the coldest point temperature is uniformly raised to 121℃, and then start timing after the coldest point temperature reaches 121℃, maintain for 30-60 minutes, the temperature difference between all points in the tank should be ≤ 2℃, and then slowly shut off the steam, introduce sterile compressed air or nitrogen to maintain positive pressure, and cool the tank at the same time. The cooling rate needs to be controlled (e.g., ≤ 0.5℃ / second) to prevent vacuum and tank deformation.

4. The method for establishing and maintaining an online aseptic environment in an aseptic tank according to claim 1, characterized in that: Specific methods for verifying and monitoring data of key steps in aseptic tank production:

5. The method for establishing and maintaining an online aseptic environment for an aseptic tank according to claim 1, characterized in that: Methods for handling and maintaining aseptic tanks during use: Abnormal pressure drop: Initiate an investigation immediately, treat it as a major aseptic risk event, check the integrity of the respirator, valve seals, and gasket condition, and if necessary, assess the isolation of the contents of the tank and resterilize the system. The respirator is blocked or wet: An increased pressure differential indicates a blockage and the respirator needs to be replaced in time. Condensation on the respirator will cause it to fail. It is necessary to ensure that the dew point of the intake gas is low enough, or to use a respirator with an electric heating mantle. Periodic revalidation: All critical components should be revalidated regularly, usually once a year, or after a major change or deviation.

6. The method for establishing and maintaining an online aseptic environment in an aseptic tank according to claim 1, characterized in that: Sampling and valve system: The sterile diaphragm valve should be used, and its dead space volume should be close to zero. The sampling valve is recommended to use a double-seat valve + steam barrier design to ensure that the sampling port itself can be sterilized online. Before sampling, the sampling valve and short tube must be sterilized in place with pure steam at at least 121°C for 30 minutes, or the sterile sampling diaphragm must be wiped with validated 70-75% isopropanol and kept in contact for a sufficient time.

7. The method for establishing and maintaining an online aseptic environment for an aseptic tank according to claim 1, characterized in that: Instrumentation and Control Systems: All pressure and temperature sensors directly connected to the tank must be of flush diaphragm hygienic design and be able to withstand a complete SIP cycle. The calculation and recording of F0 value is the core of SIP. The control system must be able to calculate and record the F0 value of the coldest point in the tank in real time (F0=∫10^((T-121.1) / 10)dt), and automatically end the heat preservation stage after reaching the set value (e.g., F0≥15 minutes).

8. The method for establishing and maintaining an online aseptic environment for an aseptic tank according to claim 1, characterized in that: Quantitative methods for verifying surface cleanliness: Sensitive detection of chemical residues: Methods: High performance liquid chromatography (HPLC) or total organic carbon (TOC) analyzer; Sampling: Take a sample from the final rinse water, or wipe a specified area (e.g., 100 cm²) with a lint-free cloth moistened with a specific solvent (e.g., a water / ethanol mixture), and then extract and analyze; Standard: The residual amount of a specific active ingredient should be <10 ppm or less than 1 / 1000 of the acceptable daily exposure (ADE), and the TOC increment should be <500 ppb; Microbial recovery rate validation: Before wiping surfaces for microorganisms, it is necessary to validate the recovery rate of the neutralizing agent, wiping method and culture conditions used for microorganisms. Generally, a recovery rate of ≥50% is required to prove the effectiveness of the detection method.

9. The method for establishing and maintaining an online aseptic environment for an aseptic tank according to claim 6, characterized in that: Dynamic data benchmarks for environmental monitoring: Airborne bacteria: Using an active air sampler, the sampling volume is 1 cubic meter. In the tank respirator outlet area under a Class A background, the standard is <1 CFU / m³; Settling bacteria: Use φ90mm petri dishes, expose for 4 hours, in the same area, the standard is <1 CFU / 4h·dish.

10. Data trend analysis: All environmental monitoring data should be entered into the statistical process control system, control charts should be drawn, the control status should be monitored, and potential trends should be warned.

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