Aseptic Process Validation In The Production Of Sterile Raw Materials

The aseptic production process is one of the most difficult process verifications in the preparation of sterile raw materials. Since many raw materials cannot be finally sterilized, the raw materials must be filtered, dried, pulverized, subpackaged, etc. as much as possible. Avoid microbial contamination. There are quite a lot of factors that affect the sterility of a product, such as production design and equipment layout, production environment conditions, pollution conditions of all production-related equipment and materials, personnel operation and hygiene conditions, etc., and each link has an impact on the quality of the final product. All count. In order to ensure the reliability and adaptability of the aseptic production process system, a series of verifications will be required to ensure the sterility of the product.

Aseptic process verification needs to solve many problems, such as the selection of simulated medium, the relevant requirements of aseptic process verification, the sterilization of equipment, how to achieve the purpose of full culture, aseptic tissue assurance and final result analysis, etc., any item If the problem is not handled properly, it will affect the final result.

Part 1 Selection Of Simulated Medium

Sterile raw materials are critical to the selection of the simulation medium, and the selected simulation medium should have the following characteristics:

No bacteriostatic effect. If the simulated medium has antibacterial effect, it will inhibit the growth of bacteria in the production system, so that bacteria cannot be cultured, resulting in false results. The simulated medium preferably has the effect of promoting bacterial growth. If a solid culture medium that promotes bacterial growth is selected for verification, the protection of the production environment is particularly important. Because, if some operations expose the medium, the dust particles of the medium will float to some places that are difficult to clean, such as near the air supply and return air outlets of the air conditioning system, creating conditions for the growth of bacteria there, so it should be There are measures to protect the dead corners that are not easy to clean from being polluted by the medium.

The simulated medium should also have good solubility. Because if the solubility is not good, the simulated medium suspended in the medium will make the medium turbid, which will affect the judgment of the result.

After the simulated medium is added to the medium, the quality of the medium should not be affected, and the pH does not change greatly, so as to adapt to the growth of most bacteria.

It is not corrosive to equipment, harmless to human body, and does not pollute the environment.

The choice of simulated medium needs to be determined according to the specific conditions of each company. At present, most manufacturers use medium filling tests to prove the reliability of their aseptic process. The medium used should have good solubility, high nutrient level, and be able to meet the growth of various microorganisms. Generally verified by growth promotion test. If the production process is under anaerobic conditions, the selected medium should be able to grow anaerobic microorganisms.

Part 2 Relevant Requirements For Aseptic Process Validation

Preparation Before Verification

Design And Validation Of Production Facilities

Manufacturing facilities should be designed to minimize potential contamination. For example, production facilities should be easy to clean and disinfect, the air in the open place of products or materials should have certain quality requirements, the control of temperature and humidity should be appropriate, the operators wearing sterile clothing should feel comfortable, and they can prevent contamination and cross-contamination, etc. Wait. Attention should be paid to the details of the design to reduce sources of contamination to ensure the implementation of aseptic production. Aseptic process validation is one of the more difficult validations to perform, and participants should be fully aware of facility design and validation, which are major factors for validation success.

Confirmation Of Public Works

Utilities (such as steam, compressed air, heating, cooling, water systems, etc.) that affect product quality shall be identified. Utilities are one of the guarantees for the normal implementation of production. If any one of the systems fails, it will cause potential or serious pollution to the product. There should be a maintenance plan for public works and it should be implemented according to the plan. The purpose is to prevent the system from malfunctioning and ensure the smooth progress of production, instead of repairing after a breakdown occurs. Public works that often break down and are repaired cannot guarantee aseptic production. .

Equipment And Process Validation

All equipment, aseptic environment, computer control system and production process shall be verified before the verification is carried out. Product exposure is one of the main reasons for the failure of aseptic process verification due to contamination. In order to prevent or reduce the probability of contamination, it is very important to provide laminar flow air to exposed products. Hundreds of laminar flow hoods or isolators are also sterilized The transfer of sterilized items from the sterilizer to the production line and the protection before entering the production line through necessary manual operations are also crucial, and operations in which personnel come into contact with equipment or materials should be avoided as much as possible. These should all be verified.

Equipment Cleaning And Sterilization

The surface of the equipment contacting the simulated medium before aseptic process validation should be cleaned and sterilized, and recontamination after sterilization should be prevented. If the medicine produced has antibacterial or bactericidal properties, this kind of cleaning is particularly important. It must be confirmed that there is no antibacterial effect on microorganisms after cleaning, or that the antibacterial effect can be effectively eliminated. If the cleaning or sterilization is not thorough, the remaining drugs will contaminate the simulated medium or the microorganisms on the surface of the equipment will contaminate the simulated medium and the verification will fail. This cleaning and sterilization process should be validated to ensure thorough cleaning and successful sterilization.

Equipment sterilization is a key part of aseptic process verification. All process-related equipment must be sterilized. First, the sterilization of equipment must pass the sterilization verification, including the determination of the coldest point, temperature distribution and heat penetration, The challenge of biological indicators, finalize the temperature, time and exhaust point of sterilization. The type, source, concentration and D value of the biological indicator used should be confirmed.

Part 3 Organizational Assurance For Sterile Process Validation

In order to ensure the smooth execution of the aseptic process validation, an aseptic process validation executive team was established to: (1) provide human, financial and material support for the validation work; (2) finally decide on the handling of various affairs during the validation period; ( 3) Approve the verification scheme and plan and verification report; (4) Responsible for the work of each subgroup respectively.

Part 4 Aseptic Process Validation Process

The design of aseptic process validation should fully reflect the concept of QbD (Quality by Design), conform to the process as much as possible, and ensure the quality of the product to the greatest extent.

1 Confirmation Of Standard Operating Procedures And Personnel Training

Standard operating procedure (SOP) is a written program that guides operators to perform certain operations, is the operator’s operating standard, and is the key to the success of production operations. Standard operating procedures should be able to provide sufficient guidance to the operator to complete the operation.

Standard operating procedures should be written by technical staff, reviewed by department heads, and approved by the head of quality assurance department. Train operators on the approved standard operating procedures so that they can truly understand and master them proficiently. The originals of standard operating procedures and training records are stored in the data management department, and copies are stored at the operation site. Before the verification, it should be confirmed that each operation has a standard to avoid causing loopholes in management, and it should be confirmed that this operation has been trained and that the operator has mastered it proficiently.

Before the verification, all operators and quality management personnel should be trained on various operations related to the verification (according to the verification plan and SOP), so as to avoid that the relevant personnel do not understand or even know how to do it during the verification process, and ensure the smooth implementation of the verification. If more than 20% of aseptic operators are newly added or replaced in the production workshop, the aseptic process verification should be done again to ensure that the aseptic operation can be carried out smoothly.

2 Instrument Calibration

Instrument calibration should be carried out by qualified personnel or units in accordance with the SOP approved by the company, and comply with national traceable standards. The calibration frequency of the instrument should at least meet the national regulations, and be adjusted according to the frequency of use and be stricter than this standard. The higher the frequency of use, the more severe the wear of the meter, and the higher the frequency of calibration should be. The data obtained are not valid due to the use of an uncalibrated meter.

3 Process Conditions

The production process of sterile raw materials has its own characteristics: there are many types of equipment, complex equipment, and long pipelines, and different production processes have different characteristics, and solid or liquid simulation media are selected according to their respective characteristics. Although the internal exposure of the equipment and the exposure of materials and products have been minimized, it is inevitable that some equipment will communicate with the outside environment, or the materials will be exposed to the environment, and these openings should have respirators or class 100 laminar flow For air protection, it is better to use an isolator for protection. During verification, there should be operations that simulate exposure to these materials.

Some production processes have crystallization operations, which makes the simulation process somewhat difficult, because there is a phase change in the crystallization process, and it should be considered whether this change will affect the growth of microorganisms. The best way to do this is to choose two simulants that react during crystallization to form another simulant. It may be very difficult to choose such a medium. For the sake of simplicity, the same liquid simulation medium can also be used to simulate the crystallization process and flow through all the pipelines to add to the crystallization tank. If there is an operation of adding seeds in the crystallization process, such operation should also be performed in the simulation process. Seed crystals can be replaced by simulated media. In order to make the crystal size uniform, some crystallization processes have a cooling process, and the temperature should not be lowered during the simulation process, because microorganisms are not easy to grow at low temperatures.

The temperature during the drying process generally inhibits the growth of microorganisms, or even kills the microorganisms. During the simulation process, room temperature should be maintained to avoid the failure of the verification due to the high temperature that kills the bacteria. Freeze-drying is also used to remove moisture, and the choice of simulated freeze-drying parameters should not inhibit the growth of bacteria.

In short, try to make the process parameters consistent with the actual situation during the verification process, but in order to make the microorganisms grow well, the selected process parameters are allowed to be inconsistent with the actual situation, but these parameters should not be significantly different from the production process. The subpackage dosage of sterile raw materials is generally large. In this case, the subpackage speed should be slowed down during the subpackage process to increase the possibility of product exposure and operate under the worst conditions.

When conducting verification, the worst conditions should be set according to the abnormal conditions that have occurred in the production process or the possible abnormal conditions that can be expected to investigate whether the aseptic properties of the product will be affected when the abnormal conditions occur during the production process.

4 Operating Time Limit

In the production of APIs, each operation should have an operating time limit to ensure that the sterilized items are protected from microbial contamination or product degradation. The longer the operating time frame, the greater the chance of product contamination.

During the verification of aseptic process, the operation time limit should be adjusted to the maximum, and the operation should be performed under the worst condition. If the verification result is reliable at this time, then the operation performed under this time is of course also reliable. Especially the storage time of sterilized items from sterilization to use is more important. When verifying, the sterilized items should be kept for at least the specified storage time, such verification results are more convincing. Each step of operation should extend or even exceed the operation time limit, which will make the total process time very long, but such verification results can better ensure the sterility state during normal production.

5 Environmental Monitoring

The quality of the environment is very important in the verification process, and it should be operated in a harsh environment, but it should not be so bad that the product will be contaminated. Generally, the method of increasing the number of operators is used to deteriorate the environment. The environment should be monitored to evaluate the verification process, and all operations exposed to product should also monitor the environment in the area. Environmental monitoring includes the following items: temperature, humidity, dust particles, sedimentation bacteria, planktonic bacteria, personnel and surface microorganisms in the sterile room.

Settling bacteria and planktonic bacteria are tested with petri dishes, and the medium is nutrient agar or other suitable medium. Petri dishes should be placed in the worst spots in the history of microbiological monitoring. During the verification process, the frequency of sampling should be increased, and more than 2 sampling culture dishes should be placed in the subpackage area for continuous exposure sampling. The exposure time should be the whole process of subpackage, but should not exceed 4h, otherwise the nutrient agar will dry out and Not enough nutrients are provided so that bacteria cannot grow. If the process time exceeds 4 hours, a new Petri dish should be placed for monitoring. Nutrient agar should be tested for microbial growth promotion before use to ensure the adaptability of nutrient agar to microbial growth.

The method is as follows: select an appropriate amount of nutrient agar medium (this amount is statistically significant), and inoculate 10-100 CFU of Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Candida albicans (Candidaalbicans), Aspergillus niger (Aspergillus niger) and the detected bacteria in the environment. Bacteria were placed in an environment of (32.5±2.5)°C for no more than 3 days, and fungi were placed in an environment of (22.5±2.5)°C for no more than 5 days. The inoculated microorganisms grew well. To test planktonic bacteria, gap-agar impact sampling method, centrifugal sampling method, liquid impact sampling method and membrane filtration method can be used. Generally speaking, planktonic bacteria can better reflect the quality of the environment than sedimentary bacteria. Microbiological testing of surfaces can be done by swabbing or Rodac plates. The swab method is used for microbial testing of irregular surfaces, and the recovery rate should be measured before the swab method is used to test the surface microorganisms. There are many factors that affect the recovery rate, including the type and quantity of microorganisms. Different operators will get different recovery rates when testing. The main reason is the amount of force when wiping. Each test operator has its own recovery rate value. When testing recovery, do not select fragile microorganisms as these will die when swabbed, giving unreliable results. It is more convenient to use the Rodac dish to test the degree of microbial contamination on the regular surface, but this test will leave nutrients on the tested surface, which should be cleaned, otherwise it will be a big pollution. Online monitoring of dust particles is used as much as possible. The number of sampling points and sampling volume meet the requirements of current regulations. The monitoring results should prove that the air quality in the key areas where the product is exposed has reached the air quality standard of a 100-class clean area.

6 Simulation And Full Culture Of The Process

Since the general raw material drug production process goes through the crystallization process, phase transition will occur during it, so it is impossible to simulate the aseptic process validation completely according to the original actual process.

From the preparation of raw materials, the culture medium is used as the simulated medium, and after several steps of sterile filtration, crystallization, filtration, and washing, in order to ensure that all the medium used is used for sterility testing, it is necessary to take measures to measure the excess simulated medium. Culture to ensure the integrity of the medium used. The following steps of drying, pulverizing and subpackaging are carried out according to the production process. In order to investigate the sterility of each step, sampling should be carried out at each stage to simulate the sampling during the production process, and do not deliberately increase the sampling points. The samples taken should also be cultured to achieve the purpose of comprehensive culture. In order to ensure the sterility of sampling, sterilize the sampling port. If steam sterilization cannot be used, sporicide can be used to sterilize, but before sampling, make sure that the sporicide residue is not bacteriostatic.

During the production process, there may be heating and cooling processes. At this time, attention should be paid to the sensitivity of the simulated medium to temperature. Can be tested in the laboratory.

7 Sample Culture

Negative control: During the verification process, a certain number of packaging containers containing medium are randomly selected as negative controls.

Positive control: During the verification process, a certain number of representative packaging containers containing simulated media were randomly taken out, and 1/3 of the five standard strains with a concentration of <100 CFU and the detected bacteria in the environment were inoculated. After inoculation, the bacteria were inoculated at (32.5 Cultivate at ±2.5)°C for 48-72 hours, molds and yeasts at (22.5±2.5)°C for 5 days, and the inoculated microorganisms grow obviously in the inoculated packaging container, which is used as a positive control.

Sample culture: fill the subpackaged product with culture medium at the operation site, then seal it, and check the tightness. All products should be cultured at (22.5±2.5)°C and (32.5±2.5)°C for 7 days respectively, for a total of 14 days, and the microbial growth of all samples should be checked every day. If contamination is found, the batch number and packaging container number should be indicated, and the sealing condition should be checked at the same time. If damaged, state the reason. Contaminated samples should be microbiologically identified to identify species.

Part 5 Result Evaluation And Revalidation

The acceptance standard of filling less than 5000 units in the preparation is 0 pollution, but in the production of sterile raw materials, the packaging unit is large, and the total number of sub-packaging containers obtained is generally small, which makes the sampling test and result calculation lose statistical significance , and because the production batch is fixed, all products should be cultured, and all negative and positive controls must be qualified. If the negative control or positive control is unqualified, the aseptic process verification fails and needs to be repeated. If a container is found to have grown bacteria, it is considered that there is a factor of bacterial contamination in the aseptic production process, and the production should be stopped immediately, the cause of the contamination should be investigated, and the verification should be carried out after taking measures.

Before the new production line is put into production, aseptic process verification is required, and the production line can only be put into use after passing the verification. The production line in operation should perform aseptic process verification twice a year, and each person who may enter the clean area or be in contact with production participates in the aseptic process verification at least once a year. For the overhaul of the factory building, the replacement of major equipment, and the new factory building, at least 3 consecutive cycles of aseptic process verification should be carried out. If it is a periodic re-validation, if no contamination of the product occurs during the two verifications, it is sufficient to carry out the verification of one operation cycle; if the verification is passed, it proves that the system can maintain sterility stably; If the verification fails, the cause should be investigated in detail and the hidden dangers should be eliminated, and then the verification should be repeated for at least 3 consecutive cycles. Only when all the verifications are passed can the aseptic production process be considered reliable.

If the product has been contaminated with bacteria during the two verifications, after investigation and bacterial identification, it is determined that the cause of the contamination is caused by factors that can be controlled, or the cause of the contamination is relatively minor, and the production can continue after correcting these mistakes; if the investigation is carried out If the identification of bacteria and bacteria cannot confirm the cause of the infection, the aseptic process verification should be carried out on the aseptic production system to confirm which links cause the system to be infected, and the aseptic risk investigation and evaluation of the products produced during this period should be carried out.

Part 6 Verification Report

After the verification is completed, a verification report should be drafted to make a brief summary of the above contents, mainly including the following contents: verification topic, verification purpose, participating verification departments and personnel, personnel training, adopted process parameters and operating time limit, selected simulation Media and media, interruptions in the simulation process, deviations and their investigation, culture conditions and final results, the final report should be approved by the head of corporate quality. Only when the final results are qualified and the final report is approved, the enterprise’s sterile API production can start (or continue).