培養(yǎng)基模擬灌裝試驗(yàn)討論
發(fā)布時間：
2022-12-24
作者：
培養(yǎng)基有哪幾個大廠家
1、2015版中國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。如果以往產(chǎn)品有過厭氧成長，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。厭氧菌要長也是在下層，但因有些瓶子不大，如2ml或5ml瓶子無法造出隔絕空氣層，因模擬陽性菌瓶也是不長的，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。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)會被殺死。另外，真菌相對細(xì)菌生長得慢，所以先培養(yǎng)真菌7天，后面還有7天的時間可繼續(xù)長。而細(xì)菌生長得快，一般3天就能看出大致，所以可以后培養(yǎng)。
第二個問題：培養(yǎng)基灌裝的培養(yǎng)溫度，可以在做完培養(yǎng)基的促成長試驗(yàn)后，選擇一個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，文章由四川蜀陽周珍字提供）
答：2010版藥品GMP指南：在培養(yǎng)基灌裝設(shè)計中，工藝條件的選擇應(yīng)選取合理的“最差條件”，用最差條件來對工藝流程、設(shè)備和整個體系進(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”考慮如下三個方面：
一是設(shè)施、設(shè)備、包材和輔料清潔、滅菌后存放時間：
二是灌裝人員及參數(shù)設(shè)定：
三是干擾項(xiàng)目的設(shè)計：
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)對上一階段的生產(chǎn)狀況進(jìn)行總結(jié)和趨勢分析，綜合考慮有代表性的活動及干預(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，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區(qū)分，同一批號不同板層的灌裝制品也需分開放置（小批號樣品一半正立放置、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；完整性測試（泄漏測試）失敗的樣品；文件中明確規(guī)定予以丟棄的中控測試樣品；設(shè)備自動剔除的樣品；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？
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)部審計報告），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，錄像的儲存時間等。
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級無法保證無菌性，如果可以模擬，還要A級監(jiān)測相關(guān)指標(biāo)做什么？一旦A級沒電，沒有層流保護(hù)，無菌狀態(tài)被破壞了，即使你無菌檢測是合格的，畢竟檢測只是僅有代表性，有其無菌檢測局限性。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級有UPS或22S內(nèi)迅速轉(zhuǎn)供電也是可以的。
日水培養(yǎng)基
1. In the 2015 edition of Chinese pharmacopoeia, the aseptic examination of aerobic bacteria and fungi was changed to tryptone soybean broth medium (TSB). Should the culture medium of simulated filling test be changed?
A: no change is required. Generally, broad-spectrum tryptone soybean broth medium (TSB), fungus, bacteria can grow. Products if ever had anaerobic growth, can use sulfur glycolic acid salt liquid medium (FTM) simulation of the filling to do simulation test in the same conditions, the anaerobic culture typically layered, upper used to isolate the lower air layer, leading to anaerobic bacteria in the upper is not long. Anaerobic bacteria to grow in the lower, but not because of some bottles, such as 2 ml and 5 ml bottle can't create isolation air layer, due to the simulation of positive bottle is not long, so, is not only make the anaerobic bacteria can also be difficult to survive, so most companies are not doing anaerobic culture medium filling simulation test.
2, guidelines for draft of training time at least 14 days, commonly used in the first 20 to 25 ℃ at least 7 days, and then continue in the range of 30-35 ℃ for 7 days. Also can use a single culture conditions of 20-35 ℃, but shall have the relevant data to prove that culture conditions conducive to the growth of microorganisms. One is whether first 30-35 ℃ after 20 to 25 ℃ training? The second is to use a single 20-35 ℃ training how to operate?
Answer: the first question: is the first low temperature fungi cultivation, and high temperature bacteria, for fear that some bacteria at low temperature to high temperature of 30-35 ℃ train would be killed. In addition, fungi grow more slowly than bacteria, so the fungus can be cultured for 7 days first, followed by another 7 days. And the bacteria grow very fast, generally can be seen in 3 days, so it can be cultured later.
The second question: the cultivation of the culture medium filling temperature, can be done in the medium to long after the test, select a 20-35 ℃ temperature for culture, but to control the temperature of the incubator in the plus or minus 2.5 ℃.
3. What is the "worst condition" for simulated filling test of medium? (translated from the medical micro-school, the article was provided by zhou zhen, shu Yang, sichuan province)
A: 2010 version of the drug GMP guidelines: in the design of media fills, the choice of technological condition selection should be the reasonable conditions of "worst", in the worst condition to the technological process, equipment and the whole system. If good results can be obtained under the worst conditions, the reliability of aseptic guarantee is more guaranteed in the actual production which is better than the worst case. The "worst condition" considers the following three aspects:
First, the storage time after facilities, equipment, packaging materials and auxiliary materials are cleaned and sterilized:
Ii. Filling personnel and parameter setting:
Third, interference project design:
Simulation of the actual production process in various types of regular interference and non-conventional interference. On every six months before the media fills, should a stage summarize production status and trend analysis, considering the activities of the representative and intervention, to type and frequency of the interference on the plan. According to the comprehensive consideration of human, machine, material, method, ring and measurement, the most representative "worst condition" is combined.
4. Major personnel changes. Do you need to carry out the simulated filling test of culture medium before you can be officially employed?
A: article 47 of the aseptic drugs appendix: the first validation of the simulated filling test of the culture medium, three consecutive qualified tests shall be conducted for each shift. After major changes in the air purification system, equipment, production technology and personnel, the simulated filling test of culture medium shall be repeated. The simulated filling test of culture medium shall normally be carried out once every half a year according to the production process, at least one batch at a time. Significant changes, certainly need to medium filling simulation experiment was carried out, only participated in filling simulation test personnel, is allowed to enter to the core filling area for normal production operations.
5. After the completion of the simulated filling of the culture medium, should all the cylinder be inverted or only partially inverted during the culture?
A: as long as the culture medium is in contact with all contact surfaces of the cylinder, whether upright, inverted or flat. Industry there are three kinds of practice: one is the medium filling simulation over, the cultivation of the normal, does not need to be inverted, medium during the bottle come into contact with any surface to be medium, it is ok to flip a few times; The second is to fully contact the culture medium with the cylinder and the rubber stopper, and then half of the vertical culture and half of the vertical culture; Third, the culture medium was fully exposed to the cylinder and the rubber stopper of xilin. The culture medium was incubated at 20-25 degrees for 7 days and inverted at 30-35 degrees for 7 days. Guidelines for draft, is the first one is to cultivate, to reverse the filling simulation products, light wave so that the medium contact all inner surface, and is set to cultivate, began to develop should not be reversed after interference, etc. We are a mixture of the second and third, such as filling products to distinguish between different batches according to first, the same batch number plate layer filling products also need to separate different place (small batch samples half is placed, placed upside down), load transfer in the basket, first place 20 -- 25 ℃ to cultivate, 7 days for visual inspection, will be made into a handstand, inverted into are set, will buy 30-35 ℃ for 7 days.
6. How to determine which filling samples do not need to be cultured?
A: including damaged samples that affect the sealing performance; Failure of integrity test (leak test) samples; Intermediate control test samples that are clearly specified in the document to be discarded; Samples automatically removed by the equipment; It is clearly stipulated in the document that samples should be removed manually after each interference (the provisions should be detailed and clear and need to prove their operability and stability credibility). In addition, the removed samples should be recorded to indicate the reason for the discard.
7. Is it necessary to conduct video recording in the filling test of medium? How long does it take to keep the video?
A: not necessarily. Video is an optional auxiliary means of investigation and training. If video recording is used, it will only be used as information within the company (such as internal audit reports) and will not be required to present it to inspectors. The company shall specify in the documents the circumstances under which the video shall be recorded, the storage time of the video, etc.
8. Can the power failure be simulated in the media simulated filling test?
A: no. After power failure, AB level in the core filling area cannot guarantee asepsis. If it can be simulated, what should A level monitor do? Once grade A is out of power and there is no laminar flow protection, the aseptic state is destroyed. Even if your aseptic test is qualified, after all, the test is only representative and has its limitation of aseptic testing. The simulated power failure in the media simulation filling test cannot be used as the excuse for product release behavior. It is also possible to consider the quick power transfer within A UPS or 22S.