Talking about the development and verification of sterilization process

1, two sterilization methods Steam-wet heat sterilization process development and application, two methods can be used: the overkill method (theoverkill method) and the bioburden method (the bioburden method). The bioburden method can also be called the survival probability method.

1.1 Overkill method The setting and application of the steam sterilization cycle in the industry generally uses the overkill method. The goal of using the overkill method is to ensure that a certain level of sterility assurance is achieved, regardless of the amount of actual load organisms and the heat resistance of the load. Assume that the amount of bioburden and the heat resistance value are set to the following levels:


D121=1 minutes Z=10°C

In order to achieve a sterility assurance level of SAL less than 10-6, NF = 10-6, using the above values, the FPHY and FBIO required to achieve the lethality can be calculated as follows:

F0 = D121 × (LogN0 - LogNF) = 12 minutes It is rarely found that naturally occurring microorganisms have a D121 value of more than 0.5 minutes. The amount of bioburden and the resistance assumed by the overkill method are higher than the actual ones. Most microorganisms have lower heat resistance and therefore provide a high degree of sterility assurance.

Even if the steam-wet heat sterilization process is the overkill method, it is still necessary to periodically monitor the initial bacteria of the sterilized product and periodically use the biological indicator test.

1.2 Bioburden method In general, the sterilization of heat-resistant products or articles cannot be used excessively. This requires that the sterilization cycle we establish must be able to properly kill the microbial load, but it does not lead to unacceptable product decomposition. The establishment of such a sterilization cycle depends on the amount of microorganisms and heat resistance on the product. Once the amount of microbial load and heat resistance are clearly defined, a sterilization cycle that achieves a SAL of less than 10-6 can be designed.

The actual bioburden of the products produced according to the CGMP specification should be very low, each package is about 1 to 100 CFU, and the product is given a heat shock of 80 to 100 ° C for 10 to 15 minutes, which can kill microorganisms with low heat resistance. E.g:

In the bioburden test of the product, it is measured:


D121=1 minutes Z=10°C

Then the sterility assurance level SAL is less than 10-6, NF=10-6. Using the above values, the FPHY and FBIO required to achieve the lethality can be calculated as follows:

F0=D121×(LogN0-LogNF)=8 minutes Bioburden method requires live test product viability count and heat resistance screening program. It is necessary to obtain sufficient data on bioburden and to establish historical data. The requirements for the number of bioburden screenings depend on the quality and changes of historical data, the type of product being sterilized, the type of production process and the type of sterilization process, and so on. If the production environment changes, additional monitoring of bioburden should be considered.

For the determination of the heat resistance of the bio-load, the bio-loaded product sample can be subjected to a gradual incremental exposure time under the recommended sterilization process, and then subjected to a sterility test to determine the amount of bacteria that survives during different exposure times, or The proportion of positive samples present. As an alternative, the heat tolerance of the bioburdened microorganism can be determined by isolation and propagation, followed by inoculation on the product or on a suitable carrier. However, it is important to note that reproduction can alter the resistance of the bioburden. In addition, the microbial heat resistance of conventional bioburdens should be measured.

In terms of bioburden, microbial strains having high heat resistance to moist heat include: Clostridiumsporogenes, Becillus coagulans, Bacillus subtilis, and Bacillus thermophilus.

The sterilization process based on the bioburden method requires frequent microbial screening to determine the amount and type of bioburden associated with the product. In routine production, representative product samples should be taken from each production facility and bioburden monitoring procedures designed to significantly affect the bioburden changes in product components and production, including the environment and production processes. Conduct an assessment.

2. Sterilization Process for Auxiliary Materials for Injection Production Part of the auxiliary materials used for injection production are in direct contact with the drug during the production process, and therefore sterilization is also required. They include: filters, rubber stoppers, hoses, garments, stainless steel vessels, filled mechanical parts, cleaning products, and the like. Inactivation is typically achieved by direct contact with saturated steam, which transfers heat through conduction or convection. A common method is to establish a standard period that provides the same level of Zuzu small sterilization assurance, plus a certain safety factor, regardless of the contents of the load.

For such items, the large obstacle to obtaining repeatable and predictable sterility assurance is the potential air in the item, so a suitable sterilization process is a pre-vacuum process followed by a saturated steam sterilization process. The effect of the pre-vacuum process is significantly better than the gravity displacement process.

For pre-vacuum sterilization procedures, handling of the load prior to the start of the sterilization process is important. If each vacuum is 0.1 atmospheres, each pulse will reduce the air in the sterilizer by 90% or 1 log unit. It can be increased to three vacuum pulses (steam injection above atmospheric pressure to prevent air from entering the sterilization chamber), leaving the load in a normal state. By this method, the efficiency of air removal is increased and the balance time is shortened.

A typical gravity displacement procedure is based on the theory that the cold air in the chamber is heavier than the incoming steam and will settle to the bottom of the chamber. As the steam enters the chamber, air and condensate are discharged together from the steam trap at the bottom of the chamber. The success of the air removal process depends on the precise operation of the steam trap and the proper steam distribution. Steam is usually injected into the sterilizer chamber through a baffle or a perforated tube. If the steam increases too quickly or is not properly distributed, air pockets may form near the top of the load. If the steam increases too slowly, the air may be heated. It is mixed into the steam to make air removal more difficult. Gravity displacement removes air less efficiently than other methods and is not recommended for items that are difficult to remove air.

For the sterilization of porous/hard materials, the following operating parameters need to be determined (but not limited to this):

Description of parameters during the sterilization procedure phase Sterilization procedure The overall case temperature/pressure case temperature must not exceed or be significantly lower than the sterilization temperature of the sterilizer chamber. To control the temperature to avoid excessive heating or excessive cooling.

The monitoring probe recording probe must be independent and have a suitable size of recording paper to record all temperature and pressure changes of the sterilization procedure. The number, extent and duration of the vacuum pulse during the heating phase (if used)

Number, extent and duration of removal of positive pressure pulses from air used for sterilization of porous hard objects (if used)

For controlling the sterilization of the load chamber before the start of the sterilization process, the sterilization method for the saturated steam can be set according to the temperature and pressure changes. The alarm point sterilization stage sterilization time is an important control parameter for each sterilization procedure. The temperature during the sterilization phase is important for each sterilization procedure. The temperature fluctuations in the chamber during the sterilization phase are important for each sterilization procedure. The accumulation of control parameter values ​​for the sterilizer's own measurement probe, The small value of zui is favorable for the evaluation of the sterilization process. The cooling time can be selected by heating, vacuum, etc. to dry the load. The vacuum release rate can be used to protect the integrity of the package or filter if sprayed. When the water cools down, the quality of the spray water must be monitored. However, porous/hard materials are not suitable for cooling with spray water, and generally use vacuum or jacket to cool down. Such as filters, rubber stoppers, hoses, clothing, stainless steel vessels, filling machinery parts, cleaning supplies and so on.

For the sterilization loading method, you need to pay attention to the following points:

The type and structure of the load should be confirmed and documented;

The load cannot touch the inner wall of the sterilization chamber;

The contact between the surface of the metal box and the base should be reduced to a low degree, usually using a holed bracket and, if necessary, an adjustable bracket;

In order to facilitate air removal, condensate discharge and steam penetration, it is necessary to clearly determine the location of the load and record it;

• Large mass loads should be placed on the lower shelf in the sterilizer to minimize wetting water;

? Control the number of items in the sterilizer, if the size of the load can be changed, you need to determine the large load of zui small and zui, confirm that the reasonable insertion method of the intermediate load should include the zui hard-to-sterilize items in the low load of zui ;

• If the validation study indicates that the location of the item does not affect the sterilization effect, the form of loading may be variable;

The load record can be used as a reference for the operator.

4. Methods of sterilization verification The verification of the sterilization process and the related contents of the routine control include the confirmation of the sterilization equipment, the verification of the sterilization process, the suitability of the packaging, the loading test, the inspection of the air filter, the quality and management of the steam, Vacuum check and revalidation cycle and more.

4.1 Before steam sterilization verification is carried out, the following must be confirmed:

Quality assurance for steam sterilizer manufacturers,

Inspection, installation and performance verification of steam sterilizers,

Leak test of air filter installed in steam sterilizer,

Air flow rate or flow pattern test (if required),

Pressure or vacuum test,

Steam quality test (if required),

Challenging test of biological indicators (chemical indicators),

Verification of different loads,

Control of heating time (if necessary),

Temperature fluctuation range during the holding time,

Calibrating the sterilizer for heat distribution/thermal penetration test equipment,

The IQ/OQ/PQ solution has the ability to demonstrate all the content of IQ, OQ and PQ and to handle and correct deviations.

4.2 In general: verification (IQ/OQ/PQ) must contain:

4.2.1 The tests of the tested sterilization equipment and public systems must include:

1.1 Evaluation of the supplier of sterilization equipment;

1.2 volume, vacuum, pressure and leakage rate of the sterilization equipment;

1.3 What is used as a sterilization medium (such as steam, high-pressure air, superheated water, etc.) and can be proved effective;

1.4 It is safe for the sterilizing equipment to maintain the pressure and temperature between the interlayer, the interlayer and the air chamber;

1.5 What type of filter is used? How long does it take to conduct a challenging test?

1.6 What type of monitoring and control sensors are used? How to correct? Does it comply with the relevant regulations?

1.7 If a steam splitter is installed in the sterilization equipment, the risk of system operation safety must be eliminated;

1.8 time, temperature, pressure, pressure deceleration rate control (if necessary);

1.9 air quality, water quality control and alarms (if necessary);

Whether changes in all cases above 1.10 are necessary for evaluation of revalidation;

4.2.2 Verification of the sterilization process must include:

2.1 No-load heat distribution study 2.2 Thermal penetration study each loading method of each loading container;

The number of runs in each mode;

Is the cold spot for each method determined?

2.3 The influence of heating and cooling rate on production process and products;

2.4 The effect of spray cooling rate on the products to be sterilized;

2.5 Whether cooling water can effectively control microorganisms;

2.6 Does the temperature measurement system provide print data for each measurement point;

2.7 Whether the temperature measurement system is corrected before and after each operation;

2.8 Whether the calibration of the temperature measurement system complies with the relevant regulations;

2.9 Use of biological indicators:

Types of;


Density and D value;

Is the bioburden method used? Is the initial bacteria tested for the sterilized product?

If an accident occurs (positive), how to deal with it;

2.10 How to deal with the heat penetration deviation;

2.11 If necessary, provide characteristics and identification of contaminating bacteria (microbial load); alarm of microbial load and limits of measures taken; identification, heat resistance and stability of biological indicators; comparison of heat resistance of contaminating bacteria and biological indicators Microbial challenge test to prove that in the short sterilization cycle of zui, under the condition of zui (such as the product is in the position where the biological indicator is difficult to kill or it is a product that is difficult to sterilize or both) );

2.12 Risk analysis and management of excessive killing;

2.13 The effect of the drying procedure after sterilization on the sterilization effect;

2.14 study changes in sterilization processes resulting from changes in product tolerance;

2.15 Overall evaluation and guidance of the loading capacity of Zui Jia sterilization effect;

2.16 Training for verification and related projects 4.2.3 For the verification of the sterilization process, the following provisions shall be met:

1. The temperature fluctuation during the holding time should be within ±0.5°C~±1.0°C (the change of ambient temperature should be as small as possible);

2. The pressure fluctuation during the holding time should be within ±5Kpa (if necessary);

3, sterilization process verification includes zui small load, saturated load and typical load three; or use zui large load, zui small load and mixed load compatibility study;

4, the limit zui low temperature and zui large value difference is not more than 3K;

5, the deviation of the equivalent sterilization time is not more than 15s (the sterilization equipment for Zui is not more than 30s);

6, the air filter flow rate should be no more than 0.13Kpa / min (if necessary);

7, air removal rate inspection (if necessary, suitable for porous / hard materials);

8, the arrangement of biological indicators;

9, if necessary, the initial bacteria (microbial load) of the sterilized article;

10. The cold spot is used to control the temperature; that is, the standard sterilization time is calculated after confirming that all the measuring points reach the sterilization temperature.

5, related documents formed by sterilization verification 5.1 related documents The following is a classification of the relevant verification documents for the steam sterilizer or steam-wet heat sterilization process, which can be screened according to the actual situation, but must be evaluated whether the relevant content is complete :

Sterilizer manufacturer and its equipment information;

Installation report of the sterilizer;

First-time sterilization, all operations and control documents, including calibration of instruments, valves, alarms, and related facilities;

Product safety tolerance study report vacuum and pressure assurance measures verification leak rate test report Steam, water, compressed air and other auxiliary system quality test report including cold spot, load distribution, heat distribution, heat penetration and other project research IQ, OQ, PQ schemes and reports summarize the verification results and risk analysis, and present a complete operational record of the loading guidance report (also can be a table)

Maintenance procedures and records, including any changes in preventive maintenance, conditional maintenance, etc., any failure report processing training records, and performance evaluation report re-verification cycle provisions. Other 5.2 verification documents essential sterilization parameters and characteristics of sterilized items Confirmed vacuum (leakage rate)

Initial bacteria control for steam sterilization (number of bioburden of articles before sterilization)

Safety tolerance values ​​for water, air filters, etc. in steam sterilization. Limitation of overkills. Challenges. Revalidation cycle. Development of new sterilization processes. Verification of implementation steps, risk analysis, and verification reports.

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