問：培養(yǎng)基模擬灌裝試驗(yàn)的8個(gè)問題討論
發(fā)布時(shí)間：
2022-12-24
作者：
青島有個(gè)賣培養(yǎng)基的
問：培養(yǎng)基模擬灌裝試驗(yàn)的8個(gè)問題討論
答：1、2015版中國(guó)藥典需氧菌和真菌無菌檢查改為胰酪胨大豆肉湯培養(yǎng)基（TSB），模擬灌裝試驗(yàn)的培養(yǎng)基要改嗎？
答：不需要改。一般選廣譜的胰酪胨大豆肉湯培養(yǎng)基（TSB），
真菌、細(xì)菌都能長(zhǎng)。如果以往產(chǎn)品有過厭氧成長(zhǎng)，可以用硫乙醇酸鹽液體培養(yǎng)基（FTM）模擬，同樣條件去做模擬灌裝試驗(yàn)，厭氧培養(yǎng)基一般會(huì)分層，上層用于隔絕下層的空氣層導(dǎo)致厭氧菌在上層不長(zhǎng)。厭氧菌要長(zhǎng)也是在下層，但因有些瓶子不大，如2ml或5ml瓶子無法造出隔絕空氣層，因模擬陽(yáng)性菌瓶也是不長(zhǎng)的，所以，既使有厭氧菌也難以存活，故大多企業(yè)是沒有做厭氧培養(yǎng)基的模擬灌裝試驗(yàn)。
2、指南征求稿中關(guān)于培養(yǎng)時(shí)間至少 14 天，一般采用先在 20-25℃最少培養(yǎng) 7 天，然后在30-35℃的范圍繼續(xù)培養(yǎng) 7 天。 也可采用單一的 20-35℃的培養(yǎng)條件，但應(yīng)有相關(guān)的數(shù)據(jù)證明培養(yǎng)條件有利于微生物的生長(zhǎng)。一是能否先30-35℃后20-25℃培養(yǎng)？二是采用單一的 20-35℃培養(yǎng)怎么操作？
答：第一個(gè)問題：就是先低溫真菌培養(yǎng)、再高溫細(xì)菌培養(yǎng)，因擔(dān)心有些低溫菌先高溫30-35℃培養(yǎng)會(huì)被殺死。另外，真菌相對(duì)細(xì)菌生長(zhǎng)得慢，所以先培養(yǎng)真菌7天，后面還有7天的時(shí)間可繼續(xù)長(zhǎng)。而細(xì)菌生長(zhǎng)得快，一般3天就能看出大致，所以可以后培養(yǎng)。
第二個(gè)問題：培養(yǎng)基灌裝的培養(yǎng)溫度，可以在做完培養(yǎng)基的促成長(zhǎng)試驗(yàn)后，選擇一個(gè)20-35℃的溫度進(jìn)行培養(yǎng)，但培養(yǎng)箱的溫度要控制在正負(fù)2.5℃。
3、什么是培養(yǎng)基模擬灌裝試驗(yàn)的“最差條件”？（轉(zhuǎn)摘自醫(yī)藥微學(xué)堂，文章由四川蜀陽(yáng)周珍字提供）
答：2010版藥品GMP指南：在培養(yǎng)基灌裝設(shè)計(jì)中，工藝條件的選擇應(yīng)選取合理的“最差條件”，用最差條件來對(duì)工藝流程、設(shè)備和整個(gè)體系進(jìn)行挑戰(zhàn)。如果在最差條件下能獲得好結(jié)果，說明在比最差情況要好的實(shí)際生產(chǎn)中，無菌保證的可靠性更有保證。“最差條件”考慮如下三個(gè)方面：
一是設(shè)施、設(shè)備、包材和輔料清潔、滅菌后存放時(shí)間；
二是灌裝人員及參數(shù)設(shè)定；
三是干擾項(xiàng)目的設(shè)計(jì)；
模擬實(shí)際生產(chǎn)過程中出現(xiàn)的各種類型的常規(guī)性干擾和非常規(guī)性干擾。在每半年的培養(yǎng)基灌裝前應(yīng)對(duì)上一階段的生產(chǎn)狀況進(jìn)行總結(jié)和趨勢(shì)分析，綜合考慮有代表性的活動(dòng)及干預(yù)，將其干擾類型與頻次列入方案中。從人、機(jī)、料、法、環(huán)、測(cè)各方面綜合考慮，組合出最具有代表的“最差條件”。
4、人員重大變更，是否需要進(jìn)行培養(yǎng)基模擬灌裝試驗(yàn)才能正式上崗？
答：無菌藥品附錄中第47條：培養(yǎng)基模擬灌裝試驗(yàn)的首次驗(yàn)證，每班次應(yīng)當(dāng)連續(xù)進(jìn)行3次合格試驗(yàn)?？諝鈨艋到y(tǒng)、設(shè)備、生產(chǎn)工藝及人員重大變更后，應(yīng)當(dāng)重復(fù)進(jìn)行培養(yǎng)基模擬灌裝試驗(yàn)。培養(yǎng)基模擬灌裝試驗(yàn)通常應(yīng)當(dāng)按照生產(chǎn)工藝每班次半年進(jìn)行1次，每次至少一批。人員重大變更，肯定需要重新進(jìn)行培養(yǎng)基模擬灌裝試驗(yàn)，只有參加過模擬灌裝試驗(yàn)的人員，才可以進(jìn)入到核心灌裝區(qū)進(jìn)行正常產(chǎn)品生產(chǎn)的操作。
5、在培養(yǎng)基模擬灌裝結(jié)束后，在培養(yǎng)時(shí)，是否所有的西林瓶都要倒置培養(yǎng)，還是只需要部分的倒置培養(yǎng)即可？
答：只要培養(yǎng)基與西林瓶所有接觸面接觸過就可以，無論正立、倒立或平放。行內(nèi)有三種做法：一是培養(yǎng)基模擬灌裝結(jié)束，正常的培養(yǎng)，不需要倒置，培養(yǎng)基培養(yǎng)期間，瓶子里的任何表面都要被培養(yǎng)基接觸到，翻轉(zhuǎn)幾次就可以了；二是將培養(yǎng)液與西林瓶和膠塞充分接觸，然后一半倒立培養(yǎng)、一半正立培養(yǎng)； 三是將培養(yǎng)液與西林瓶和膠塞充分接觸，在20-25度正立培養(yǎng)7天，30-35度倒立培養(yǎng)7天。指南征求稿中是第一種，就是培養(yǎng)前，對(duì)模擬灌裝產(chǎn)品進(jìn)行顛倒、輕搖以使培養(yǎng)基接觸所有內(nèi)表面，再正立培養(yǎng)，開始培養(yǎng)后不應(yīng)再進(jìn)行反轉(zhuǎn)等干擾。我們是第二種和第三種的混合，如先將灌裝制品按不同批號(hào)區(qū)分，同一批號(hào)不同板層的灌裝制品也需分開放置（小批號(hào)樣品一半正立放置、一半倒立放置），裝入中轉(zhuǎn)筐中，先置20-25℃培養(yǎng)7天，進(jìn)行目檢，將正立轉(zhuǎn)成倒立，將倒立轉(zhuǎn)成正立，將再置30-35℃培養(yǎng)7天。
6、如何判斷哪些灌裝樣品不需要進(jìn)行培養(yǎng)？
答：包括破損，影響到密封性的樣品；完整性測(cè)試（泄漏測(cè)試）失敗的樣品；文件中明確規(guī)定予以丟棄的中控測(cè)試樣品；設(shè)備自動(dòng)剔除的樣品；文件中明確規(guī)定，每次干擾后應(yīng)予手工剔除的樣品（規(guī)定應(yīng)詳細(xì)、明確，并需證明其可操作性和穩(wěn)定可信性）。另外，剔除的樣品應(yīng)進(jìn)行記錄注明丟棄原因。
7、在培養(yǎng)基灌裝試驗(yàn)中，是否必須進(jìn)行錄像？錄像的保存期需要多長(zhǎng)？
答：不是必需的，錄像僅是一種可選的輔助調(diào)查和培訓(xùn)手段。如果采用了錄像措施，也僅作為公司內(nèi)部的資料（如同內(nèi)部審計(jì)報(bào)告），不必向檢查員出示。公司應(yīng)有文件規(guī)定，什么情況下要進(jìn)行錄像，錄像的儲(chǔ)存時(shí)間等。
8、是否可在培養(yǎng)基模擬灌裝試驗(yàn)中模擬停電的狀況？
答：不允許。停電后，核心灌裝區(qū)的AB級(jí)無法保證無菌性，如果可以模擬，還要A級(jí)監(jiān)測(cè)相關(guān)指標(biāo)做什么？一旦A級(jí)沒電，沒有層流保護(hù)，無菌狀態(tài)被破壞了，即使你無菌檢測(cè)是合格的，畢竟檢測(cè)只是僅有代表性，有其無菌檢測(cè)局限性。不能以培養(yǎng)基模擬灌裝試驗(yàn)中模擬停電為產(chǎn)品放行為借口，可考慮A級(jí)有UPS或22S內(nèi)迅速轉(zhuǎn)供電也是可以的。
Discussion on 8 problems of simulated filling experiment in culture medium
The 1 and 2015 edition of sterility test of aerobic bacteria and fungi in Chinese pharmacopoeia is changed to tryptone soy broth medium (TSB).
Answer: no need to change. Generally, the broad spectrum peptone soybean broth culture medium (TSB) is widely selected, and both fungi and bacteria can grow. If the previous product has been growing anaerobic, it can be simulated with FTM. The same conditions are simulated filling tests. The anaerobic medium is generally stratified and the upper layer is used to isolate the lower layer of air to cause the anaerobic bacteria in the upper layer. Anaerobes are also in the lower layer, but because some bottles are not large, such as 2ml or 5ml bottles can not create an isolated air layer, because the simulated positive bacteria are not long, so, even the anaerobic bacteria are difficult to survive, so most enterprises are not the anaerobic medium of simulated filling test.
2. In the guide draft, the training time is at least 14 days. It is generally adopted at least 7 days at 20-25 degrees C, and then continued to cultivate for 7 days at the range of 30-35. A single 20-35 degree culture condition can also be used, but the relevant data should prove that the culture conditions are beneficial to the growth of microorganism. First, can we first cultivate it at 20-25 C after 30-35? The two is how to operate with a single 20-35 degree culture.
A: the first problem is: low temperature fungal culture and high temperature bacteria culture. Because some low temperature bacteria can be cultured at high temperature for 30-35 degrees, they will be killed. In addition, fungi grow slower than bacteria, so they can grow fungi for 7 days, and 7 days later. And bacteria grow fast, generally 3 days can see roughly, so it can be cultured later.
The second question: the culture temperature of the culture medium can be selected at a temperature of 20-35 degrees centigrade after the growth test of the medium, but the temperature of the incubator should be controlled at positive and negative 2.5 degrees C.
3. What is the "worst condition" for simulating the filling test? (extracted from the medicine micro school, the article is provided by the word "Zhou Yang" written by Shu Yang, Sichuan).
Answer: 2010 edition of drug GMP Guide: in the culture foundation filling design, the selection of the technological conditions should select the reasonable "worst condition", and the process, equipment and the whole system should be challenged with the worst conditions. If the best results are obtained under the worst conditions, the reliability of aseptic assurance is more assured in actual production than in the worst case. The "worst condition" is considered as the following three aspects:
First, the storage time of facilities, equipment, packaging materials and accessories after cleaning and sterilization.
Two is the filling personnel and parameter setting;
The three is the design of the interfering project.
Simulation of various types of conventional interference and unconventional interference in the actual production process. The production status of the last stage should be summed up and trend analysis before the half year of the culture medium, and representative activities and interventions are considered, and the types and frequency of interference are included in the scheme. Considering the various aspects of human, machine, material, law, loop and measurement, the most representative "worst condition" is formed.
4. Is there any need to carry out the media simulation filling test to make the official change?
Answer: forty-seventh of the appendix of aseptic drugs: the first validation of the media simulation filling test, and 3 qualified tests for each shift. After major changes in air purification systems, equipment, production processes and personnel, repeated simulation tests should be carried out. The media simulation filling test should normally be conducted 1 times a year, at least once a year, according to the production process. The important change of personnel is that it is necessary to re carry out the simulated filling test of the medium. Only those who have participated in the simulated filling test can enter the operation of the normal production in the core filling area.
5. In the end of the media simulation filling, should all the bottles be placed upside down when they are cultured, or do they only need part of the inverted culture?
Answer: as long as the medium is contacted with all the contact surfaces of the vial, it can be upright, upside down or flat. There are three ways of doing it. One is the end of the culture based simulation filling, normal cultivation, no need for inversion. During the culture medium, any surface in the bottle should be touched by the culture medium and turned over a few times. The two is to fully contact the culture liquid with the Xilin bottle and glue plug, and then half stand upside down and half upright culture. Three is to contact the culture fluid with the vial and the rubber stopper, and to maintain the upright culture for 7 days at 20-25 degrees, and for 30-35 days to stand for 7 days. In the guide draft, it is the first kind in the guide draft, that is, before the cultivation, the simulated filling products are reversed and rocked to make the medium contact all the inner surfaces, and then the culture should be upright, and the inversion should not be carried out again after the training. We are the mixture of second and third kinds, for example, the filling products are distinguished by different batch numbers first, and the filling products of the same batch number different plate layers should be placed separately (the small batch sample is placed half upright and half inverted), loaded into the basket, first set up at 20-25 degrees C for 7 days, and the eye check will be carried out, will turn the upright to the inverted stand, will be turned upright to inverted stand. Turn upside down into upright position, and then reposition for 30-35 days for 7 days.
6, how to determine which filling samples do not need to be cultured?
Answer: the sample that is damaged and affected to the seal; the failure sample of the integrity test (leakage test); the document clearly stipulates the discarded sample of the control test; the automatic rejection of the sample; the document clearly stipulates that the sample should be removed by hand after each interference (the provisions should be detailed, clear and necessary. Its operability and stability credibility). In addition, the rejection sample