1. Routine sterilization methods and their corresponding biological indicators
1.1 Hygrothermal sterilization and its corresponding biological indicators
Hygrothermal sterilization is to put sterilized articles in sterilizer and use high-pressure saturated steam or hot water spray to denature protein and nucleic acid in microbial cells to kill microorganisms. No matter what sterilization parameters (sterilization temperature and time) are used, the sterility assurance level of articles after sterilization must be ensured to be less than or equal to 10-6. The biological indicator used in this sterilization method is spores of Bacillus The killing degree of stearothermophilus atcc7953) is the most intuitive index to evaluate the effectiveness of a sterilization program. It requires that each piece (or bottle) or spore number of biological indicator should be 5 × 105 ~ 5 × 106, and the SAL of sterilized articles should be less than 10-6.
1.2 Dry heat sterilization and its corresponding biological indicators
Dry heat sterilization method refers to that the sterilized articles are placed in the dry heat sterilizer, tunnel oven and other dry heat sterilization equipment. It is a method to use dry and hot air to carbonize the whole microbial cell to kill microorganisms or remove heat source. No matter what sterilization parameters (sterilization temperature and time) are used in dry heat sterilization, it is necessary to ensure the sterility assurance level sal ≤ 10-6 after sterilization, and the spores of Bacillus subtilis atcc9372 used in this sterilization method It is required that the number of spores in each piece (or bottle) of biological indicator should be 5 × 105 ～ 5 × 106, and the SAL after sterilization should be less than 10-6. Dry heat sterilization was performed at 250 ℃ for 45 min. in addition to ensuring sal ≤ 10-6 after sterilization, at least 3 logarithmic units of endotoxin reduction after sterilization should be considered. The biological indicator used was Escherichia coli Endoxin CMCC (b) 44102) is the most intuitive index to evaluate the effectiveness of a sterilization program with heat source removal. Each bottle of biological indicator should contain no less than 1000 units of bacterial endotoxin. At least 3 log units of endotoxin were decreased after sterilization.
1.3 Radiation sterilization and its corresponding biological indicator
The most commonly used method is 60Co - γ - ray radiation sterilization. It is a method to kill microorganisms by placing sterilized articles in the electron beam generated by gamma ray electron accelerator for ionizing radiation. No matter what sterilization parameters (radiation dose) are used in radiation sterilization method, Sal after sterilization must be ensured to be ≤ 10-6. The biological indicator used in this sterilization method is the killing degree of spores of Bacillus pumilus atcc27142, which is the most intuitive index to evaluate the effectiveness of a sterilization program.
1.4 Filtration sterilization and its corresponding biological indicators
This method is based on the principle that bacteria cannot remove microorganisms in liquid or gas through dense porous filter materials. The sterility guarantee during filtration is related to the initial biological load of the filtered liquid and the logarithmic decline value of the filter, which means the filtration and sterilization efficiency of the filter. For the filter with pore diameter of 0.22 μ m, the LRV of every 1 cm2 effective filtration area is ≥ 7. The biological indicator was Pseudomonas diminuta (atcc19146). Since LRV method is not used in actual filtration and sterilization, integrity test should be conducted before and after each filtration to confirm the effectiveness and integrity of the filter in the process of sterilization and filtration.
1.5 Gas sterilization
It is a method of killing microorganisms by using gas formed by chemical disinfectant. The commonly used chemical disinfectants are formaldehyde fumigation, ethylene oxide, gaseous hydrogen peroxide, atomized hydrogen peroxide and ozone (O3). We know that no matter what kind of gas sterilization method is used, the corresponding biological indicator can be used to evaluate the sterilization method. The teaching of gas sterilization method is complicated, so I will introduce it separately;
1.5.1 Sterilization with formaldehyde fumigation gas
Formaldehyde fumigation sterilization due to its safety, residual and corrosive problems, although the 2015 edition of the Chinese Pharmacopoeia still exists, 2020 edition may be eliminated, but in Europe and the United States and other countries Pharmacopoeia has clearly prohibited the use of formaldehyde gas sterilization. Therefore, this paper will not introduce the biological indicator.
1.5.2 Ethylene oxide gas sterilization
The same as dry heat sterilization biological indicator Bacillus subtilis spores (atcc9372) was used.
1.5.3 Sterilization with gaseous hydrogen peroxide
The biological indicator of Bacillus stearothermophilus spores (atcc7953) was used.
2. Sterilization of clean area by atomized hydrogen peroxide and its corresponding biological indicator
For example, the hydrogen peroxide atomization sterilization method can be used to sterilize the microorganisms in the liquid sterilization area, such as the hydrogen peroxide atomization sterilization system. It can also be used for hydrogen peroxide transfer window.
No matter what sterilization parameters (hydrogen peroxide concentration and time) are used for atomizing hydrogen peroxide, the biological indicator used is not recorded in Chinese Pharmacopoeia (2015 Edition), but USP stipulates that the same sterilization parameters (atcc12980, not atcc7953) of Bacillus stearothermophilus spores sterilized by gaseous hydrogen peroxide are used.
The same as dry heat sterilization biological indicator Bacillus subtilis spores (atcc9372) was used.
3. Ozone gas sterilization
Ozone (O3) sterilization is an old and brand-new gas sterilization method. Ozone generator produces O3 through the principle of high-voltage discharge, which can inhibit, sterilize and dissolve microorganisms, which belongs to biochemical reaction. Ozone sterilizer is commonly used in pharmaceutical industry to sterilize articles and microorganisms in clean area.
3.1 Validation of ozone gas sterilization
Using sterilization parameters (ozone concentration is not less than 50g / m3; sterilization time is 40min), using the same biological indicator of dry heat sterilization of Bacillus subtilis spores (atcc9372), the SAL after sterilization is ≤ 10-6.
3.2 Validation of ozone sterilization for clean room gas
Ozone is used to sterilize the clean room by using the purification system of air conditioning unit as the carrier. The ozone generated by the ozone generator is sent to the whole clean room through air supply, and the ozone concentration is distributed to the whole clean room. According to the requirements of the disinfection technical specifications, when the ozone concentration reaches 20mg / m3, the effect is 30min, The sterilization rate of microorganisms in the clean room is more than 95%. Therefore, the concentration of ozone and the time to maintain the corresponding concentration are monitored. According to experience, it is very necessary to install ozone concentration monitor in return air section of air conditioning unit and monitor it from time to time. Due to the rapid decomposition of ozone (O3) itself, which is different from that of oxygen in gas sterilization of articles, it is very difficult to achieve the effect of killing spores by decomposing into oxygen quickly in the closed equipment in the clean room. Therefore, it is not possible to verify the ozone gas sterilization method for articles, which is the same as that of Bacillus subtilis spores (atcc9372). As a biological indicator, the SAL of articles after sterilization is ≤ 10-6. No matter what kind of biological indicator is used for ozone sterilization of clean room gas, the 2015 edition of Chinese Pharmacopoeia and the current USP are not recorded. According to the technical specifications and experience of disinfection, Staphylococcus aureus CMCC (b) 44112, gram positive bacteria and Escherichia coli were recommended CMCC (b) 44113) gram negative bacteria were used as biological indicators to evaluate the effectiveness of a sterilization program. The number of viable bacteria in each TSA plate of the biological indicator should be between 150 and 200 before sterilization, and the sterilization rate after ozone sterilization should be more than 95%, which meets the requirements. In addition to the above biological indicators, it should also be considered to isolate typical microorganisms found in environmental monitoring and sterility inspection as biological indicators for validation of ozone sterilization method for clean room gas.
4. Sealing verification of sterile preparation and its corresponding biological indicator
Appendix 1 of 2010 edition of "good manufacturing practice for pharmaceutical products" has clear requirements for the sealing verification of sterile preparations. This paper introduces the method of microbial invasion for sealing verification. Pseudomonas aeruginosa CMCC (b) 10104 was used as the biological indicator. In the normal production process, TSB medium was poured into a certain number of infusion bottles, penicillin bottles or amperometric bottles to replace the liquid medicine. After being pressed and capped, the samples were cultured at 30-35 ℃ for 14 hours, and the samples were tested by lamp. After that, the samples were completely immersed in the high concentration of Pseudomonas aeruginosa, and then cultured for 14 at 30-35 ℃. After that, the samples were cleaned and the lamp inspection was conducted to confirm the contamination of the samples.
5. Aseptic process simulation test and its corresponding biological indicator
Aseptic simulation test is to verify the effectiveness of aseptic safeguard measures in the production process by simulating the whole process of aseptic production process. In order to ensure the effectiveness of this test, it is very important to test the growth promoting ability and positive control of TSB medium used in the simulation test.
5.1 Growth promoting ability of TSB medium and corresponding biological indicators
The growth promoting ability of TSB medium should be tested according to the method of Chinese Pharmacopoeia (2015 Edition) before aseptic process simulation test and after 14 days culture. The biological indicators (strains) used include Candida albicans CMCC (f) 98001, Bacillus subtilis CMCC (b) 63501, Aspergillus niger CMCC (f) 98003, Staphylococcus aureus CMCC (b) 26003, Clostridium sporogenes CMCC (b) 64941.
In addition to the above five biological indicators, the typical microorganisms found in environmental monitoring and sterility inspection should also be considered in the actual sterilization of clean room gas with ozone. In this experiment, the number of inoculated microorganisms was less than 100 CFU / infusion bottle, penicillin bottle or ampere bottle. To prove that the medium can support the growth of microorganisms.
5.2 TSB medium positive test and corresponding biological indicator
The positive test of TSB medium should be carried out according to the method of Chinese Pharmacopoeia (2015 Edition) after aseptic process simulation test. The biological indicators (strains) used include Candida albicans CMCC (f) 98001 and Bacillus subtilis CMCC (b) 63501.
In addition to the above two biological indicators, the growth promoting ability test of the same medium should also consider the addition of typical microorganisms found in environmental monitoring and sterility test. In this experiment, the number of inoculated microorganisms was less than 100 CFU / infusion bottle, penicillin bottle or ampere bottle. It was further proved that the medium could support the growth of microorganisms at 20-25 ℃ for 7 days.
6. Application of commonly used biological indicators in validation
Serial number biological indicator name number application
1. Spores of Bacillus stearothermophilus atcc7953 validation of moist heat sterilization method and validation of gaseous hydrogen peroxide
2. Spores of Bacillus subtilis atcc9372 dry heat sterilization method validation of ethylene oxide gas sterilization method validation of ozone gas sterilization method
3. Verification of spores of Bacillus pumilus atcc27142 60Co - γ - ray irradiation sterilization
4. Validation of filtration method for Pseudomonas diminuta atcc19146
5. Verification of atomized hydrogen peroxide by spores of Bacillus subtilis atcc12980
6. Staphylococcus aureus CMCC (b) 44112 validation of ozone sterilization in clean room
7. Escherichia coli CMCC (b) 44113
6. Pseudomonas aeruginosa CMCC (b) 10104 simulation test of aseptic process for sealing validation of sterile preparation
7. Aseptic process simulation test of Candida albicans CMCC (f) 98001
8. Bacillus subtilis CMCC (b) 63501
Aspergillus niger CMCC (f) 98003
Staphylococcus aureus CMCC (b) 26003
Clostridium sporogenes CMCC (b) 64941
12. Escherichia coli endoxin CMCC (b) 44102 validation of dry heat sterilization method
7. Terminology
(1) (f): it means that it is a biological indicator; it is a mould; B: it is a biological indicator and it is a bacterium
(2) CMCC: China Medical bacteria preservation and Management Center
(3) ATCC: American standard strain collection and Management Center
(4) Sal: sterility assurance level
(5) CFU: bacterial colony count
(6) TSB: tryptone soybean liquid medium
8. Conclusion
(1) For different validation methods in pharmaceutical process, the suitable biological indicator should be selected, so that the verification results can be reliable. Conventional sterilization method and its corresponding biological indicator can be found in Chinese Pharmacopoeia (2015 Edition); atomized hydrogen peroxide sterilization of clean area and its corresponding biological indicator are not recorded in 2015 edition of Chinese Pharmacopoeia, but USP can be found; the biological indicator selected for ozone gas sterilization in clean room is based on the technical specification of disinfection and the summary of actual work, which is only for reference; The sealing validation and aseptic process simulation test of aseptic preparation and their corresponding biological indicators are references to other literatures and only for reference.
（2） The biological indicator belongs to the special standard. If it is commercialized, please pay attention to the following conditions: it should be carried out under a certain procedure. Before preparation, the characteristics of microorganisms must be understood. The standard strain should be cultured in appropriate medium. The number of spores in the culture should be as large as possible. The biological indicator contains a certain number of spores, and these spores must be attached to the filter paper In this way, the validation results are reliable.
(3) Positive control and negative control tests should be carried out for the biological indicators selected in each pharmaceutical process validation.
(4) Two different biological indicators used for hydrogen peroxide gas sterilization are Bacillus stearothermophilus spores, but the number is different. Atomized hydrogen peroxide gas sterilization atcc12980, not gaseous hydrogen peroxide sterilization using atcc7953.
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