轉(zhuǎn)載：成品預(yù)裝培養(yǎng)基平皿實(shí)用技術(shù)手冊——培養(yǎng)基平皿個(gè)性化服務(wù)
發(fā)布時(shí)間：
2022-12-27
作者：
藥廠培養(yǎng)基廠家
個(gè)性化服務(wù)就是打破傳統(tǒng)的被動(dòng)服務(wù)模式，能夠充分利用各種資源優(yōu)勢，主動(dòng)開展以滿足客戶個(gè)性化需求為目的的全方位服務(wù)。我們知道對于抗生素企業(yè)和部分制劑廠家來說，僅僅按照國標(biāo)推薦的兩種培養(yǎng)基（TSA和SDA）來監(jiān)測潔凈區(qū)的微生物是不夠全面的，必要時(shí)可以加入適宜的中和劑，而中和劑培養(yǎng)基平皿就是根據(jù)客戶需要定制化的一種產(chǎn)品。
目前制藥企業(yè)環(huán)境微生物監(jiān)測存在兩大風(fēng)險(xiǎn)，也是很多企業(yè)容易忽視的問題。
風(fēng)險(xiǎn)之一就是藥物的API對個(gè)別微生物有不同程度的抑制或殺滅作用，特別是抗生素生產(chǎn)企業(yè)，潔凈室內(nèi)不可避免地混雜著一定量的抗生素粉塵，在環(huán)境監(jiān)控中所使用的培養(yǎng)基平皿，若不能有效消除抗生素粉塵對微生物生存和生長的環(huán)境影響因素，潔凈區(qū)環(huán)境監(jiān)測的結(jié)果可能出現(xiàn)“假陰性”的結(jié)果。所以要還原出潔凈室內(nèi)微生物的實(shí)際狀況，務(wù)必要消除抗生素對微生物的影響，目前采用的方法是水解取樣時(shí)落在平皿上的抗生素。如對于β-內(nèi)酰胺類抗生素，添加β-內(nèi)酰胺酶（即青霉素酶）；對于頭孢類抗生素，則添加頭孢菌素酶；對于培南類抗生素則需要添加金屬酶等。
如何在平皿中添加酶才能消除抗生素的殘留，目前主要依據(jù)：《中國藥典》2015年版三部附錄1105表2：“常見干擾物的中和劑或滅活方法”中所對應(yīng)的中和或滅活方法，然后根據(jù)抗生素生產(chǎn)企業(yè)的實(shí)際情況，收集在其生產(chǎn)環(huán)境中抗生素粉塵的量，最后做加酶實(shí)驗(yàn)得出最終的加酶量。為了保證加酶的均勻度，如TSA、SDA等必須在液體固化前的最低溫度時(shí)加入生物酶，而且要持續(xù)勻速攪拌，使酶與培養(yǎng)基充分混合均勻，再灌裝于空皿內(nèi)，這就注定了加酶的培養(yǎng)基平皿制作必須是一個(gè)工業(yè)化的生產(chǎn)過程。當(dāng)然確認(rèn)每塊平皿中加多少活性單位的酶量，是必須通過驗(yàn)證才可以得出的數(shù)據(jù)。
抗生素水解酶屬生物酶，其本質(zhì)是蛋白質(zhì)。酶的生物活性與溫度密切相關(guān)，酶活性最大的溫度與人的體溫接近，約37℃左右。例如頭孢菌素酶的穩(wěn)定性如下：
案例
（某生產(chǎn)頭孢菌素類藥企）
1
評估潔凈區(qū)環(huán)境監(jiān)測點(diǎn)
制藥企業(yè)在選擇潔凈區(qū)環(huán)境監(jiān)測點(diǎn)應(yīng)該通過風(fēng)險(xiǎn)評估確定監(jiān)測點(diǎn)。例如，在灌裝抗生素產(chǎn)品過程的無菌灌裝針位置，的確是無菌物料暴露的關(guān)鍵區(qū)域，但此監(jiān)測點(diǎn)也有大量抗生素粉塵沉降，所以選擇這個(gè)點(diǎn)作為關(guān)鍵點(diǎn)監(jiān)測細(xì)菌沉降意義并不大，如有監(jiān)測需要，可監(jiān)測霉菌和酵母菌；而選取稍微遠(yuǎn)離灌裝針的位置作為采樣點(diǎn)就比較科學(xué)，首先沉降的抗生素粉塵濃度稍低，其次這些位置往往極易受到微生物的污染。因此，抗生素生產(chǎn)環(huán)境監(jiān)測點(diǎn)的選擇更應(yīng)該關(guān)注的是“風(fēng)險(xiǎn)點(diǎn)”。這一點(diǎn)是有別于非抗生素產(chǎn)品生產(chǎn)時(shí)環(huán)境監(jiān)測的。
2
收集潔凈區(qū)環(huán)境中抗生素殘留粉塵量
企業(yè)在根據(jù)風(fēng)險(xiǎn)評估后確定的監(jiān)測點(diǎn)中選取幾個(gè)風(fēng)險(xiǎn)比較高的位置，在整個(gè)生產(chǎn)過程中放置潔凈的空皿（先稱好空皿質(zhì)量），并做好標(biāo)記。生產(chǎn)結(jié)束后，稱量每個(gè)皿的重量，得出每個(gè)皿中收集到的藥粉量并做好記錄。每個(gè)品種分別做3次。
3
加酶試驗(yàn)
在獲取潔凈區(qū)監(jiān)測點(diǎn)的抗生素殘留量之后，確定加酶的種類和加酶量。根據(jù)環(huán)境采樣點(diǎn)收集到最多殘留粉塵量的2-10倍來進(jìn)行試驗(yàn)，最終得出加酶量。微生物實(shí)驗(yàn)室根據(jù)抗生素種類確定水解抗生素的酶的種類。根據(jù)抗生素含量分別制做不同酶含量的平皿，采用涂布法，做回收率試驗(yàn)，再與不加酶的平皿做對照，最終選擇各種實(shí)驗(yàn)細(xì)菌回收率都合格的平皿加酶量作為這個(gè)抗生素監(jiān)測點(diǎn)的加酶種類和加酶量。例如，車間其中一個(gè)點(diǎn)收集到3.8mg/皿，取試管用15ml水將3.8mg粉塵溶解，然后加入酶進(jìn)行中和，再加入微生物。若微生物促生長試驗(yàn)?zāi)軌蚝细?，即能測定出需要加多少酶量才能中和。務(wù)必注意，選擇的測試菌種必須是該抗生素抗菌譜內(nèi)的敏感菌和環(huán)境常見菌。
風(fēng)險(xiǎn)之二就是制藥企業(yè)潔凈環(huán)境每天都在使用消毒劑，怎樣確定殘留的消毒劑沒有影響微生物的生長，確保潔凈區(qū)微生物監(jiān)測結(jié)果不出現(xiàn)“假陰性”。所以選擇合適的培養(yǎng)基平皿可以最大限度的衡量潔凈室表面的微生物水平。通常卵磷脂、吐溫-80組合后能夠消除季胺類化合物（苯扎溴銨、苯扎氯銨等）消毒劑的影響；卵磷脂、吐溫-80和L-組氨酸組合后能夠消除醛類（甲醛、戊二銓等）和酚類（苯酚、間苯二酚等）消毒劑的影響；硫代硫酸鈉能夠消除鹵素類消毒劑（碘伏、碘酒等）的影響等。
現(xiàn)在，國外制藥企業(yè)環(huán)境微生物監(jiān)測時(shí)，用于潔凈室表面取樣的大多為卵磷脂吐溫胰蛋白胨大豆培養(yǎng)基（即TSAWLP）
。選擇這樣的培養(yǎng)基就可以有效的降解可能殘留在潔凈室表面的消毒劑（主要是季胺類化合物消毒劑）殘留。但是目前絕大部分制藥企業(yè)在沒有弄清楚潔凈室消毒劑的種類和殘留量時(shí)就盲目采用卵磷脂吐溫胰蛋白胨大豆培養(yǎng)基（即TSAWLP）用來監(jiān)測潔凈室表面微生物水平是不科學(xué)的。因?yàn)槁蚜字聹匾鹊鞍纂舜蠖古囵B(yǎng)基中所含有的卵磷脂+吐溫-80是定量的，這就意味著能夠降解的消毒劑殘留必定也是在一個(gè)規(guī)定限度以內(nèi)的。所以在沒有確認(rèn)所使用的季銨鹽類消毒劑的殘留限度就盲目采用，同樣會產(chǎn)生“假陰性”結(jié)果。
總之，設(shè)計(jì)一個(gè)潔凈室的微生物監(jiān)測方案時(shí)，應(yīng)該考慮上述兩大風(fēng)險(xiǎn)，從而可以真實(shí)還原制藥企業(yè)生產(chǎn)環(huán)境中可能存在的微生物的真實(shí)水平。
每個(gè)生產(chǎn)企業(yè)都是一個(gè)個(gè)性化的個(gè)體，所以作為一個(gè)環(huán)境監(jiān)控培養(yǎng)基平皿的生產(chǎn)企業(yè)，要使自己的產(chǎn)品體現(xiàn)客戶個(gè)性，必須了解客戶的“個(gè)性”。通過對企業(yè)生產(chǎn)建設(shè)、業(yè)務(wù)流程等模塊的重新思考和設(shè)計(jì)，以求為客戶創(chuàng)造高質(zhì)量的個(gè)性化服務(wù)。
Practical technical manuals for prepackaged medium plates for finished products -- individualized service of Petri dishes
Personalized service is to break the traditional passive service mode, make full use of various resources advantages, and actively carry out the full range of service to meet the customer's personalized needs. We know that for antibiotic enterprises and some pharmaceutical manufacturers, it is not comprehensive to monitor the microbes in the clean area only according to the two medium (TSA and SDA) recommended by the national standard, and the suitable neutralizer is added when necessary, and the medium plate is a product customized according to the customer needs.
At present, there are two risks in the monitoring of environmental microorganism in pharmaceutical enterprises.
One of the risks is the inhibition or killing effect of API on individual microorganisms in different degrees. In particular, antibiotic production enterprises, the clean room is inevitably mixed with a certain amount of antibiotic dust, and the medium plate used in environmental monitoring can not effectively eliminate the antibiotic dust for the survival and survival of microorganisms. Environmental impact factors may result in "false negative" results in environmental monitoring of clean areas. Therefore, to restore the actual condition of the microorganism in the clean room, it is necessary to eliminate the effect of antibiotics on the microorganism. The method currently used is to hydrolyze the antibiotics that fall on the plate of the culture base. For example, beta lactam antibiotics are added to beta lactamase (penicinase), cephalosporins are added to cephalosporins, and metal enzymes are needed for Penan antibiotics.
How to add enzymes in the culture of base plates to eliminate the residues of antibiotics is mainly based on the three appendix 1105 Table 2 of the 2015 edition of the Chinese Pharmacopoeia: neutralization or inactivation method corresponding to "neutralizer or inactivation method of common interferents", and then according to the actual situation of antibiotic production enterprises, collected in its production The amount of antibiotic dust in the environment was finally determined by adding enzyme experiments. In order to ensure the homogeneity of the added enzyme, such as TSA, SDA and so on, the medium must be added to the enzyme at the lowest temperature before the curing of the liquid, and it must be stirred at a constant speed to make the enzyme and the medium fully mixed and evenly mixed in the empty culture dish, which is doomed to be an industrialized production. Process. Of course, confirming the amount of enzyme added in each dish is the data that must be verified through validation.
Antibiotic hydrolase is a biological enzyme, and its essence is protein. Enzyme activity is closely related to temperature. The maximum temperature of enzyme activity is close to human body temperature, about 37 degrees Celsius. For example, the stability of cephalosporins is as follows:
case
(a production of cephalosporins)
One
Assessment of environmental monitoring points in clean area
Pharmaceutical enterprises should set up monitoring points through risk assessment when choosing clean zone environmental monitoring points. For example, the position of aseptic filling needle in the process of filling antibiotic products is indeed the key area for the exposure of aseptic materials, but the monitoring points also have a large number of antibiotic dust settlement, so it is not significant to select this point as a key point to monitor bacterial settlement. It is more scientific to keep the location of the needle as a sampling point. First, the concentration of antibiotic dust is slightly lower, and the next location is often easily contaminated by microbes. Therefore, the choice of environmental monitoring points for antibiotics production should pay more attention to "risk points". This is different from the environmental monitoring during the production of non antibiotic products.
Two
Collection of antibiotic residue in the environment of clean area
The enterprise selects several high risk positions in the monitoring point determined after the risk assessment, and puts the clean empty dish in the whole production process (first called the empty dish quality) and marks it well. After the production is finished, weigh the weight of each dish, draw the amount of powder collected in each dish and record well. Each variety is done 3 times, respectively.
Three
Enzyme addition test
After obtaining the antibiotic residues in the monitoring area, the type of enzyme added and the amount of enzyme added were determined. According to the environmental sampling point collected up to 2-10 times the maximum residual dust volume to carry out the test, the final amount of enzyme added. According to the type of antibiotics, microbiology laboratory determines the types of enzymes that hydrolyze antibiotics. According to the content of different enzyme content, the plate was prepared by coating method, and the recovery rate was tested, and then compared with the unenzyme culture medium dish. For example, one point in the workshop was collected from 3.8mg/ dish, and the test tube was dissolved with 15ml water to dissolve 3.8mg dust, then added enzyme to neutralize, then added microorganism. If the microbial growth test is acceptable, it is possible to determine the amount of enzyme needed to neutralize. It is important to note that the selected strains must be sensitive bacteria and common environmental bacteria in the antimicrobial spectrum of the antibiotic.
The two of the risk is the use of disinfectants in the clean environment of pharmaceutical companies every day. How to determine the residual disinfectant does not affect the growth of microbes and ensure that the results of microbiological monitoring in the clean area do not appear "false negative". Therefore, choosing the right culture medium can maximize the measurement of the microbial level on the surface of the clean room. Usually, the combination of lecithin and Twain -80 can eliminate the effects of quaternary amine compounds (benzalkonium bromide, benzalkonium chloride and other) disinfectants. The combination of lecithin, Twain -80 and L- histidine can eliminate the effects of aldehydes (formaldehyde, amyl two, etc.) and phenols (phenol, Resorcinol, etc.); sodium thiosulfate can be eliminated. The effects of halogen disinfectants, such as iodophor, iodine, etc.
At present, most of the culture medium used for the surface sampling of clean rooms in the environmental microbiological monitoring of foreign pharmaceutical companies is lecithin Twain tryptone soybean culture medium (TSAWLP). The selection of such a medium can effectively degrade the residues of disinfectants (mainly Ji Anlei compound disinfectants) that may remain on the cleanroom surface. But at present, it is unscientific to use the lecithin Twain tryptone soybean culture medium (TSAWLP) to monitor the surface microorganism level of the clean room when most pharmaceutical companies do not know the species and residual amount of the disinfectant in the clean room. Because the lecithin + Twain -80 contained in the lecithin Twain tryptone soybean medium is quantitative, which means that the residue of the degrading disinfectant must be within a specified limit. Therefore, the residual limit of quaternary ammonium disinfectants used is not blindly adopted, and the false negative results will also occur.
In a word, when designing a microbiological monitoring scheme for a clean room, the above two risks should be taken into consideration, which can truly restore the true level of the possible microorganisms in the production environment of the pharmaceutical enterprise.
Each production enterprise is a personalized individual, so as an environment monitoring and training base plate production enterprises, to make their own products to reflect the customer's personality, we must understand the "personality" of the customer. Through the reconsideration and design of production and construction, business processes and other modules, we will create high-quality personalized services for our customers.
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