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  • 轉載:成品預裝培養基平皿實用技術手冊——培養基平皿個性化服務

    發布時間:

    2022-12-27

    作者:

    藥廠培養基廠家


    個性化服務就是打破傳統的被動服務模式,能夠充分利用各種資源優勢,主動開展以滿足客戶個性化需求為目的的全方位服務。我們知道對于抗生素企業和部分制劑廠家來說,僅僅按照國標推薦的兩種培養基(TSA和SDA)來監測潔凈區的微生物是不夠全面的,必要時可以加入適宜的中和劑,而中和劑培養基平皿就是根據客戶需要定制化的一種產品。
    目前制藥企業環境微生物監測存在兩大風險,也是很多企業容易忽視的問題。
    風險之一就是藥物的API對個別微生物有不同程度的抑制或殺滅作用,特別是抗生素生產企業,潔凈室內不可避免地混雜著一定量的抗生素粉塵,在環境監控中所使用的培養基平皿,若不能有效消除抗生素粉塵對微生物生存和生長的環境影響因素,潔凈區環境監測的結果可能出現“假陰性”的結果。所以要還原出潔凈室內微生物的實際狀況,務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落在平皿上的抗生素。如對于β-內酰胺類抗生素,添加β-內酰胺酶(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:《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”中所對應的中和或滅活方法,然后根據抗生素生產企業的實際情況,收集在其生產環境中抗生素粉塵的量,最后做加酶實驗得出最終的加酶量。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加入生物酶,而且要持續勻速攪拌,使酶與培養基充分混合均勻,再灌裝于空皿內,這就注定了加酶的培養基平皿制作必須是一個工業化的生產過程。當然確認每塊平皿中加多少活性單位的酶量,是必須通過驗證才可以得出的數據。
    抗生素水解酶屬生物酶,其本質是蛋白質。酶的生物活性與溫度密切相關,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定性如下:
    日水生物頭孢菌素酶的穩定性圖
    案例
    (某生產頭孢菌素類藥企)
    1
    評估潔凈區環境監測點
    制藥企業在選擇潔凈區環境監測點應該通過風險評估確定監測點。例如,在灌裝抗生素產品過程的無菌灌裝針位置,的確是無菌物料暴露的關鍵區域,但此監測點也有大量抗生素粉塵沉降,所以選擇這個點作為關鍵點監測細菌沉降意義并不大,如有監測需要,可監測霉菌和酵母菌;而選取稍微遠離灌裝針的位置作為采樣點就比較科學,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,抗生素生產環境監測點的選擇更應該關注的是“風險點”。這一點是有別于非抗生素產品生產時環境監測的。
    2
    收集潔凈區環境中抗生素殘留粉塵量
    企業在根據風險評估后確定的監測點中選取幾個風險比較高的位置,在整個生產過程中放置潔凈的空皿(先稱好空皿質量),并做好標記。生產結束后,稱量每個皿的重量,得出每個皿中收集到的藥粉量并做好記錄。每個品種分別做3次。
    3
    加酶試驗
    在獲取潔凈區監測點的抗生素殘留量之后,確定加酶的種類和加酶量。根據環境采樣點收集到最多殘留粉塵量的2-10倍來進行試驗,最終得出加酶量。微生物實驗室根據抗生素種類確定水解抗生素的酶的種類。根據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,再與不加酶的平皿做對照,最終選擇各種實驗細菌回收率都合格的平皿加酶量作為這個抗生素監測點的加酶種類和加酶量。例如,車間其中一個點收集到3.8mg/皿,取試管用15ml水將3.8mg粉塵溶解,然后加入酶進行中和,再加入微生物。若微生物促生長試驗能夠合格,即能測定出需要加多少酶量才能中和。務必注意,選擇的測試菌種必須是該抗生素抗菌譜內的敏感菌和環境常見菌。
    風險之二就是制藥企業潔凈環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,確保潔凈區微生物監測結果不出現“假陰性”。所以選擇合適的培養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。
    現在,國外制藥企業環境微生物監測時,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基(即TSAWLP)
    。選擇這樣的培養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在沒有弄清楚潔凈室消毒劑的種類和殘留量時就盲目采用卵磷脂吐溫胰蛋白胨大豆培養基(即TSAWLP)用來監測潔凈室表面微生物水平是不科學的。因為卵磷脂吐溫胰蛋白胨大豆培養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規定限度以內的。所以在沒有確認所使用的季銨鹽類消毒劑的殘留限度就盲目采用,同樣會產生“假陰性”結果。
    總之,設計一個潔凈室的微生物監測方案時,應該考慮上述兩大風險,從而可以真實還原制藥企業生產環境中可能存在的微生物的真實水平。
     
    每個生產企業都是一個個性化的個體,所以作為一個環境監控培養基平皿的生產企業,要使自己的產品體現客戶個性,必須了解客戶的“個性”。通過對企業生產建設、業務流程等模塊的重新思考和設計,以求為客戶創造高質量的個性化服務。
     

    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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