01. Does the fermentation tank need cleaning verification?

　　Experience factor:

　　Using microorganisms as production strains, or for producing bacteria, or for producing primary or secondary products, or for biotransformation, large-scale modern fermentation has been in China for decades, and there is no conclusion that the cleaning of fermentation tanks will have risks to the final products. Therefore, there is no unified verification specification for the cleaning of fermentation tanks, and the Countermeasures of various enterprises are different: some enterprises are strict Refer to the standards of pharmaceutical equipment for verification, while some enterprises do not.

　　Objective factors

　　There are other reasons:

　　1. The level of pharmaceutical equipment for preparing and washing tanks with tap water in open environment is different from that for purified water and water for injection in clean room, and the cost is not allowed to be verified according to the level of pharmaceutical equipment.

　　2. The raw materials for fermentation are complex, and most of the organic nitrogen sources come from agricultural products, which have been polluted by pesticides, industrial wastes and antibiotics. The risks brought by these factors are much greater than the residual pollution caused by tank cleaning. It is not helpful to pay attention to small risks regardless of large risks.

　　3. The fluctuation of fermentation results is affected by many factors, and it has never been heard that the cleaning residue of the tank is one of the reasons.

　　4. The fermentation medium needs high temperature sterilization, so it does not involve the risk that the cleaned tank is polluted by environmental microorganisms.

　　Do you want to verify?

　　Based on the above reasons, the discussion will not have a result. In fact, enterprises should decide whether they should carry out cleaning verification according to the characteristics of their products and the requirements of stakeholders.

　　02. Purpose and definition of cleaning verification

　　Cleaning purpose of fermentation tank:

　　On the one hand, it can reduce the risk of bacteria contamination by dealing with dead corner, on the other hand, it can prevent more residues from entering the medium or scaling on the equipment to form sterilization dead corner.

　　Concept of cleaning validation:

　　A documented process that demonstrates that the fermentor is cleaned in accordance with an approved cleaning procedure and that the cleaned fermentor does not significantly affect subsequent product quality.

　　It is required that the visible and invisible residues, including the residues of previous products or detergents, meet the specified cleaning limit requirements, and will not cause cross contamination to the products to be produced.

　　03. Misunderstandings in verification of fermentation tank cleaning

　　Transfer verification:

　　The cleaning of fermentation tank has different purposes, so it can be divided into different levels. For example, the cleaning after bacteria infection and the cleaning after production transfer are more complex and strict than the normal cleaning procedures.

　　Rigorous verification needs to set verification scheme according to different levels. Generally, one level of verification cannot replace other levels, unless the most stringent cleaning scheme is implemented regardless of level and cost.

　　If a workshop wants to transfer from cephalosporin to gentamicin, before the first tank of Gentamicin Fermentation, clean the fermentation tank according to the procedure of boiling the tank with alkali for 1 hour, and then clean it with purified water twice, and then:

　　1. The residual amount of cephalosporin can reach 0.03ug/ml after the detection of tank washing water;

　　2. The residue of tank wall wiping is less than 10ppm.

　　Meet the requirements, the end of the validation. You can find this article on the Internet. There are residual calculation methods in it. I suggest you read it.

　　This verification is aimed at the production transfer verification, but it is generalized as the cleaning verification of fermentation tank, because the cleaning procedure of normal fermentation tank has not been verified.

　　Treatment procedure after infection

　　The cleaning of the contaminated tank may be the last step. The more important work is to check the dead angle and leakage point, and the cleaning measure is to boil the tank with alkaline water. However, if the alkali water boiling tank is used as the bacteria contamination cleaning procedure for verification, it does not meet the requirements, because if the front dead corner is not handled well, the problem of pollution can not be solved for a long time.

　　Riboflavin as a validation index has a deviation in understanding：

　　There are some disputes about riboflavin as an indicator of cleaning validation, mainly because of the following reasons：

　　1. Riboflavin was detected by ultraviolet lamp, but not by naked eye.

　　2. There is no standard for riboflavin spraying concentration.

　　3. It has been verified in fat, so it is no longer necessary to use it as the cleanliness index of cleaning verification.

　　There is some truth in the above, but it can only be concluded that riboflavin is not an optimal index.

　　For manual cleaning, understanding deviation:

　　At present, there are three ways to clean fermentation tank, CIP, CIP + manual and pure manual.

　　The reason why the manual cleaning is unstable is that the operation union changes its operation habits when it knows that it needs to be evaluated, so that the verification results do not conform to the reality. Therefore, it is not necessary to verify the manual cleaning.

　　However, this is not the reason why manual cleaning can not be verified, and it is not necessary to inform the operator that the tank he washed is to be verified, and the verification is not necessarily carried out only once. In addition, the consistency of manual cleaning can be improved through personnel training and management measures.

　　04. Cleaning verification of normal fermentation tank

　　There will be risks after cleaning validation. Therefore, after the first validation, it is necessary to conduct periodic retrospective validation or process inspection, and revise the SOP according to the actual situation.

　　The following will be our normal fermentation tank cleaning verification points to share, in the future will share more detailed scheme, please pay attention.

　　Preparation before verification

　　Before the drafting of the verification scheme, it is necessary to evaluate the risk according to the product characteristics or confirm the corresponding documents in the drafting process. The key issues identified in the risk assessment and documentation are as follows:

　　1. Evaluate the effect of fermentation tank cleaning to meet the production needs.

　　2. Evaluate whether the cleaning procedure is stable and reproducible, evaluate the hardware required for verification, and confirm that it has been provided.

　　3. Evaluate the test data of the cleaning method and make sure the conclusion is correct.

　　4. Acceptable criteria for evaluating cleaning effectiveness.

　　5. To evaluate the operability of sampling methods: generally, there are two sampling methods: surface wiping sampling and tank bottom sampling.

　　6. To evaluate whether the detection method can achieve the required accuracy, and there is no degradation of detection index in the test process.

　　Verification and implementation of fermentation tank cleaning．

　　1. Cleaning full coverage, no cleaning dead angle.

　　Spray riboflavin on the inner surface of the equipment. First, let a group of personnel clean the fermentation tank according to the cleaning procedure, and then let another group of personnel observe with ultraviolet light, especially pay attention to the possible dead angle.

　　If there is a dead angle, confirm whether it is the problem of cleaning method specified in SOP or the problem of cleaning method implementation. If it is the problem of cleaning method, end the verification and revise the SOP again. If the cleaning personnel do not operate according to the SOP, operate again and then verify.

　　2. Residual inspection of tank wall

　　It is difficult to find a method to measure the residue for a new tank without fermentation. The enterprise can determine a substance that can be accurately detected according to the actual detection ability, and then spray it on different positions of the inner surface of the fermentation tank. After cleaning, the residue can be detected by the method of swabbing and sampling, with the limit of one thousandth of the residue.

　　3. Cleaning agent residue

　　For the residual cleaning agent, samples are taken for several times at different times when the last tank washing water is released, and the test results are required to meet the requirements. If the cleaning agent is water, this step can be omitted.

　　Background:

　　Because it is believed that the cleaning of fermentation tank will not bring risk to the quality of final product, at present, there is no unified standard for cleaning and verification of most facilities in microbial fermentation process, and the cleaning and verification Countermeasures of these facilities are also different for various enterprises: some enterprises strictly refer to the standards of pharmaceutical equipment for verification, while some enterprises do not.

　　In this paper, the purpose of microbial fermentation tank cleaning validation, cognitive errors and experience of validation methods are shared, hoping to help friends who are ready to do fermentation tank cleaning validation and fermentation process related facilities validation.

　　This paper discusses more based on experience, although the process has also consulted students and friends, I believe there will be less rigorous place, so sincerely look forward to peer correction, and share the experience to the message area, thank you.

　　Good news: if an enterprise or company needs to train its employees in advanced skills of fermentation data processing, including basic exhaust gas analysis, optimal design of culture medium, comprehensive analysis of batch records, etc., please make an appointment through the contact information given at the end of the article, and we will provide 4 hours of free on-site lectures.

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