How to add enzymes to a plate to eliminate it
Personalized service is to break the traditional passive service model, make full use of various resource advantages, and take the initiative to carry out a full range of services to meet the individual needs of customers. We know that for antibiotic companies and some preparation manufacturers, it is not comprehensive enough to monitor the microorganisms in the clean area only according to the two culture media (TSA and SDA) recommended by the national standard. If necessary, appropriate neutralizing agents can be added, and the neutralizing agent culture medium flat dish is a product customized according to customer needs.
At present, there are two major risks in environmental microbial monitoring in pharmaceutical companies, which are also easily overlooked by many companies.
One of the risks is that the API of the drug has different degrees of inhibition or killing effect on individual microorganisms, especially for antibiotic manufacturers, the clean room is inevitably mixed with a certain amount of antibiotic dust. If the culture medium flat dish used in environmental monitoring cannot effectively eliminate the environmental influence factors of antibiotic dust on the survival and growth of microorganisms, the results of environmental monitoring in the clean area may appear "false negative". Therefore, in order to restore the actual situation of microorganisms in the clean room, it is necessary to eliminate the impact of antibiotics on microorganisms. The current method is to hydrolyze the antibiotics that fall on the flat dish during sampling. For example, for beta-lactam antibiotics, beta-lactamase (ie, penicillinase) is added; for cephalosporins, cephalosporinase is added; for penem antibiotics, metalloenzymes need to be added.
How to add enzymes to a flat dish to eliminate antibiotic residues is currently mainly based on: Table 2 of the third appendix 1105 of the 2015 edition of the Chinese Pharmacopoeia: "Neutralizing agents or inactivation methods of common interfering substances". The corresponding neutralization or inactivation methods are then collected according to the actual situation of antibiotic manufacturers. The amount of antibiotic dust in their production environment is collected, and finally the final amount of enzyme is obtained by adding enzymes. In order to ensure the uniformity of enzyme addition, such as TSA, SDA, etc., biological enzymes must be added at the lowest temperature before the liquid solidifies, and constant stirring is required to make the enzyme andmediumMix it well and then fill it in an empty dish, which determines that the production of culture medium plates with enzymes must be an industrialized production process. Of course, confirming how many active units of enzymes are added to each plate is a data that can only be obtained through verification.
Antibiotic hydrolase is a biological enzyme whose essence is protein. The biological activity of enzymes is closely related to temperature, and the temperature at which enzyme activity is maximum is close to human body temperature, about 37 ° C. For example, the stability of cephalosporinase is as follows:
case
(A cephalosporin-producing pharmaceutical company)
1
Evaluate clean area environmental monitoring points
When selecting environmental monitoring points in clean areas, pharmaceutical companies should determine monitoring points through threat and risk assessment. For example, the aseptic filling needle position in the process of filling antibiotic products is indeed a key area where sterile materials are exposed, but this monitoring point also has a large amount of antibiotic dust deposition, so it is not significant to choose this point as a key point to monitor bacterial deposition. If there is a need for monitoring, mold and yeast can be monitored; and it is more scientific to choose a location slightly away from the filling needle as a sampling point. First, the concentration of settled antibiotic dust is slightly lower, and secondly, these locations are often highly susceptible to microbial contamination. Therefore, the choice of antibiotic production environment monitoring points should pay more attention to "risk points". This is different from environmental monitoring during the production of non-antibiotic products.
2
Collection of antibiotic residue dust in the clean area environment
The enterprise selects several high-risk locations among the monitoring points determined according to the threat and risk assessment, and places clean empty dishes throughout the production process (first weigh the quality of the empty dishes) and marks them well. After production, weigh the weight of each dish, obtain the amount of powder collected in each dish and make a record. Do 3 times for each variety.
3
enzyme test
After obtaining the residual amount of antibiotics at the monitoring point in the clean area, determine the type of enzyme added and the amount of enzyme added. According to the environmental sampling point to collect 2-10 times the maximum amount of residual dust for testing, and finally get the amount of enzyme added. The microbiology laboratory determines the type of enzyme for hydrolyzing antibiotics according to the type of antibiotic. According to the content of antibiotics, flat dishes with different enzyme contents are made, and the collection rate test is done by coating method. Then it is compared with the flat dishes without enzymes. Finally, the amount of enzyme added to the flat dishes with qualified collection rates of various experimental bacteria is selected as the type of enzyme added and the amount of enzyme added to the antibiotic monitoring point. For example, 3.8mg/dish is collected at one point in the workshop, and 3.8mg dust is dissolved with 15ml water in a test tube, and then enzymes are added for neutralization, and then microorganisms are added. If the microbial growth test is qualified, it can determine how much enzyme needs to be added to neutralize. It is important to note that the selected teststrainIt must be sensitive bacteria and common environmental bacteria in the antibiotic's antimicrobial spectrum.
The second risk is that pharmaceutical companies use disinfectants every day in a clean environment. How to determine that the residual disinfectants do not affect the growth of microorganisms and ensure that the microbial monitoring results in the clean area do not appear "false negative". Therefore, choosing the right culture medium flat dish can maximize the measurement of the microbial level on the surface of the clean room. Usually, the combination of lecithin and Tween-80 can eliminate the influence of quaternary amine compounds (benzalkonium bromide, benzalkonium chloride, etc.) disinfectants; the combination of lecithin, Tween-80 and L-histidine can eliminate the influence of aldehyde (formaldehyde, gluten, etc.) and phenol (phenol, resorcinol, etc.) disinfectants; sodium thiosulfate can eliminate the influence of halogen disinfectants (iodophor, iodine, etc.).
Nowadays, when foreign pharmaceutical companies monitor environmental microorganisms, most of the samples used for clean room surface are lecithin Tween tryptone soybean medium (ie TSAWLP). Choosing such a medium can effectively degrade the residues of disinfectants (mainly quaternary amine disinfectants) that may remain on the surface of the clean room. But at present, most pharmaceutical companies blindly use lecithin Tween tryptone soybean medium (ie TSAWLP) to monitor the level of microorganisms on the surface of the clean room without knowing the type and residue of the clean room disinfectant. Because lecithin Tween tryptone soybean medium contains lecithin +Tween-80It is quantitative, which means that the residues of the disinfectant that can be degraded must also be within a specified limit. Therefore, blindly using the used quaternary ammonium salt disinfectant without confirming the residue limit will also produce "false negative" results.
In short, when designing a clean room microbial monitoring program, the above two risks should be considered, so that the true level of microorganisms that may exist in the production environment of the pharmaceutical company can be truly restored.
Each production enterprise is a personalized individual, so as a manufacturer of environmental monitoring medium flat dishes, in order to make their products reflect the personality of customers, they must understand the "personality" of customers. Through the rethinking and design of the production and construction, business process and other modules of the enterprise, in order to create high-quality personalized services for customers.
