FDA警告信之10大微生物发现项精选案例分析，你的实验室中招了吗？

序言：在中国制药圈，FDA的警告信几乎是人人谈“信”色变。今天，雨花石精选了FDA警告信中关于微生物实验室的10大发现项案例，并进行了点评分析。各位看官对照自己的实验室看一看，你们中招了吗？
“合规”是每一个质量人经常挂在嘴边的词。那么如何才能算合规？有没有统一的标准？要回答这个问题，很多人都会把审计有没有严重的缺陷项作为一项重要的指标。而在众多的审计中，FDA的审计似乎是“严厉”的代名词。一旦FDA审计出现问题、拿到警告信，企业立即声名扫地。今天，笔者精选了FDA警告信中与微生物实验室相关的缺陷项，聊聊微生物实验室的合规问题。

  案例一
On August 22, 2012, an FDA investigator observed your microbiologist reading an environmental monitoring (personnel) plate. The microbiologist reported the result for that plate as zero; however, our FDA investigator observed one (1) colony forming unit (CFU) on the plate.  Your microbiologist corrected this observation on the form WI-MI-150-108-J Microbiology Laboratory after the FDA investigator pointed it out to him. Your firm did not take further action to investigate and determine the impact of inaccurate reporting of your microbiological plate readings on the release of your batches.


2012年8月22日，检查员观察了贵公司的微生物分析员读取环境监测（人员监控）平板。该分析员报告了所读取的平板长菌数为零。然而，我们的检察员注意到该平板上有1cfu的菌。在我们的检察员指出之后，贵公司的分析员在表格“WI-MI-150-108-J微生物实验室”上纠正了这一错误。你们没有采取更多的措施去调查你们的分析员报告结果不准确对放行批次的影响。
  点评：这个是最基本的合规问题，也是最低级的造假。在FDA检查官眼皮底下谎报结果，真不知道这位分析员心里怎么想的。


  案例二
On June 23, 2014, during the inspection of the QC Microbiology Laboratory, our investigators observed missing in-progress microbiological test plates for various finished drug products, in-process products, water, and media growth promotion samples. For example….


2014年6月23日，在QC微生物实验室检查过程中，我们的检查官发现正在培养中的成品、中控产品、水和培养基促生长的平板数量缺少。例如：…...
  点评：正在培养中的平板数量与实际数量对不上，这已经成为了不少公司的发现项。据说核对平板数量已经成为部分检查员的固定套路，真实性有待考证。


  案例三：
Poor aseptic techniques were observed of Microbiology Technician performing sterility testing on 9/19/2012.


2012年9月19日，检查员观察微生物技术进行无菌检查时，发现无菌操作技术不当。
  点评：对于无菌生产企业，FDA检查员非常关注无菌操作技术。因此，在微生物实验室检查时，很多检查员会全程观察无菌检测试验，过硬的无菌操作技术才是硬道理。


  案例四
Your environmental monitoring data is not reliable. This is a serious deviation, as your ability to detect microbial contamination in the manufacturing environment during aseptic processing is fundamentally compromised. This information is critical to monitor the acceptability of the environment that the sterile drug and its container-closure components are exposed to during processing, and assure that these conditions consistently safeguard product sterility.
你们的环境监控数据是不可靠的。这是一个非常严重的偏离，因为你们无法探测到无菌生产过程中生产环境的微生物污染。这一信息（环境监控结果）对于监控无菌药品及其容器-密封组件在无菌生产过程中所暴露的环境、以及对于确保这些条件能够持续保护产品的无菌性是极为重要的。


a) Your firm used dried/desiccated media agar plates for environmental monitoring testing used to support the release of batches. On November 15, 2011, you documented that 155 of a total of 247 media plates evaluated (more than 50%) were dried. The use of dried agar plates, which do not reliably support microbial growth, to recover microbial contamination is inadequate.
贵公司使用干燥/失水的琼脂培养基进行环境监控，并用于批次的放行。2011年11月15日，你们记录中评估的247个平板中有155个平板干燥失水（超过50%）。干燥的平板不能可靠的支持微生物的生长，使用其检测微生物污染是不合适的。


b) On November 14, 2011, the FDA investigator observed desiccated environmental monitoring plates in your incubators. However, your analysts only recorded the results as dried media but not the counts from the plates (if any). On this same day, the FDA investigator observed plate “(b)(4),” sampled on November 9, 2011, to have growth of 1 Colony Forming Unit (CFU). However, your firm documented the result of this plate’s reading as "SAUSEN MEDIUM", dry medium, and failed to report the microbial growth.
2011年11月4日，FDA检查员观察到你们的培养箱中有失水的环境监控平板。然而，你们的分析员只记录为平板干燥，而不记录平板上的微生物数量（如有）。同一天，FDA检查员观察到平板xxx（取样日期2011年11月9日）上有1cfu的微生物。然而，贵公司记录这个平板的读数结果仅为“SAUSEN MEDIUM”，干燥的培养基，但没有报告培养基上有微生物生长。


c) Your environmental monitoring data for January 2009 through October 2011 contains documentation of only two action limit excursions in the Grade A manufacturing areas. In apparent contradiction, during an FDA visit to your microbiology laboratory on November 14, 2011, nine plates, collected as part of the environmental monitoring program from the Grade A manufacturing area were found inside an incubator in the microbiology laboratory with visible growth of microorganisms.
2009年10月至2011年，在A级区生产区，你们的环境监控数据仅有2次超出行动限。与之矛盾的是，2011年11月14日，一位FDA检查员检查你们的微生物实验室的时候，收集了部分在微生物实验室培养箱中正在培养的A级生产区的环境监控平板时，9块平板被观察到有微生物生长。


d) Your environmental sampling and testing program procedure is inadequate because it fails to adequately identify (e.g., with diagrams) the locations from which the surface samples are collected. In addition, you do not collect sufficient active viable air samples and dynamic non-viable particulate air samples from the critical area during manufacturing.
你们的环境监控程序是不充分的，因其不能识别（如：使用图表）表面微生物的取样位置。此外，在生产区的关键区域，你们没有采集足够的浮游菌样品和动态的粒子监控样品。


e) The agar level on surface contact plates (used for surface environmental and personnel monitoring sample testing) was below the rim of the plates creating the possibility that the agar would not have contact with the surface intended to be sampled.
接触平板（用于环境表面和人员监控）的琼脂高度低于平板的边缘，这使得琼脂在采样时可能没有接触到被取样位置的表面。


f) Your bioburden testing of the (b)(4) components is inadequate. Your firm lacks adequate controls to assure that the melted agar is sufficiently cool to prevent cell death of viable microorganisms. Specifically, your analyst determines by hand touch, without any instruments, the adequacy of the temperature of the melted agar medium used for the bioburden testing of the API, (b)(4) and (b)(4), before pouring the agar into the plates and mixing it with the samples.
你们对xxx组件的微生物负载的测试是不充分的。你们没有合适的控制措施确保融化的培养基充分冷却以防止微生物细胞的死亡。尤其，在把琼脂倒入到平板与样品混合前，你们的分析员靠手的接触而不是仪器确定融化的培养基的温度。
  点评：平板干燥、取样点位置不清晰、记录不准确、取样点数量不足、数据造假、检验过程培养基温度控制不当，这个环境监控体系的警告信够全面的。对于微生物的控制，每一个细节都是很重要的。


  案例五
For example: 例如b.   Sterility testing records for Anascorp® Bulk lots (b)(4) and Anascorp® Final Filled lots (b)(4) all show negative results for each day of the (b)(4) day incubation period. However, no analysts work on weekends and plates are only read Monday – Friday. An investigation revealed that microbiology personnel are instructed to record Saturday and Sunday results as negative if Monday’s results are negative as well. Anascorp®
半成品批次xxx和Anascorp®灌装批次xxx的无菌检查记录显示每一天的无菌培养结果均为阴性。然而，周末并没有分析员工作，平板仅在周一至周五观察。调查表明微生物人员被要求如果周一的结果也为阴性，则周六和周日的结果记录为阴性。


c.   Four different company personnel performed a secondary signoff on environmental monitoring records that had no documented results.
贵公司4个不同的人员在环境监控的二级复核上签了名，然而记录上结果记录部分却没有填写完整。
点评：这个有点尴尬，也是很多实验室面临的问题，即周末是否需要观察无菌检查结果。其实USP并没有规定无菌检查必须逐日观察（中国药典有此规定），因此只需真实记录观察日期的结果即可。然而，周六周日没有观察却记录为阴性，这就是数据完整性的问题了。记录复核部分，复核人员是检测结果放行的一道关键防线，不可掉以轻心，草率的签字，换来的是FDA对整个质量控制体系的质疑。


  案例六
Failure to follow and document laboratory controls at the time of performance; failure to document and explain any departures from laboratory procedures.
未实时记录；未记录和解释实验室程序的偏离。


During the inspection of your microbiology laboratory, our investigators observed multiple examples of your firm’s practice of back-dating and falsifying laboratory data. This laboratory monitors the quality of (b)(4) used in the manufacture of APIs for total plate count as well as the absence of objectionable organisms. Without contemporaneous and accurate data, there is no way for you to ensure that your APIs meet specifications for the absence of objectionable microorganisms.
在微生物实验室检查期间，我们的检查员观察到很多倒签和实验室数据造假的行为。实验室检测生产用API xxx的总菌数和控制菌的质量。不实时记录和数据不准确，你们无法确保你们的API符合控制菌的标准。


"Temperature Record" logbooks in microbiology laboratory
微生物实验室的“温度记录”记录本


On July 14, 2014, our investigator noticed that the daily record in the 2-8°C refrigerator #(b)(4) temperature logbook had only been completed up to July 9, 2014. When the investigator requested the logbook later that day, he observed that the logbook had been completed up to July 13, 2014. The entries for July 10–13, 2014, were not present when the investigator initially reviewed the log. When questioned by the investigator, the laboratory analyst responsible for performing these entries stated three times that she had documented the newly-completed temperature values at the time of performance. The same analyst’s supervisor later admitted to directing the analyst to fill out the logbook after the fact. The investigator also observed another analyst actively backdating/back-filling the “Temperature Record” logbook for refrigerator #(b)(4) during the inspection.
2014年7月14日，我们的检查员注意到2-8°C冰箱的每天的温度记录仅记录到2014年7月9日。当检查员在当天再次要求看这本记录的时候，他发现这本温度记录本已经记录到了2014年7月13日。在他初次看这本 记录的时候，2014年7月10-13日的记录并未出现。当检查员询问时，负责记录的分析员3次均陈述她是实时记录温度数据的。这位分析员的主管随后承认了其要求分析员在事实后填写记录。检查员还发现另一位分析员主动在检查期间倒签冰箱的温度记录。


(b)(4) Sample Data 取样日期
During the inspection, investigators visually examined the (b)(4) quality and media growth promotion samples (plates) currently in incubation, and compared them with the QC documentation for those samples purported to be in progress (incubation). Your (b)(4) sampling records showed that 45 (b)(4) quality samples had been prepared and incubated on July 9, 2014 ((b)(4), total viable aerobic count) and were in process. During the inspection, three of these plates were not in the incubator, although your (b)(4) sampling logbook recorded the presence of these three plates. QC worksheets for these three plates showed that documentation for the sample preparation and incubation had been created, even though the plates were not actually tested.
在检查期间，检查员目视检查了正在培养的xxx培养基的促生长实验平板，并且将其与QC记录样品的记录进行比对。你们的xxx取样记录表明xxx样品已制备并在2014年7月9日开始进行培养（总需氧菌）并正处于培养过程中。在检查中，其中的3块平板不在培养箱中，尽管你们xxx的取样记录本表明这3块平板应在培养箱中。这3块平板的QC检验记录显示样品的制备和培养的文件已经产生，然而这些样品实际上并未被检测。


Your management informed the investigators that one microbial plate had been found. However, upon inspection of this plate, the investigator noted that the handwriting was different from all the other microbial plates. After questioning, your microbiologist admitted that the microbial plate was re-created (falsified) to appear as if the sample was complete.
你们的管理层告诉检查员其中一块平板已经找到。然后，在检查这块平板时，检查员注意到这块平板的笔迹与其他平板的笔迹不一样。经过询问，你们的微生物分析员承认这块平板是造假的，以便看起来样品检测完成了。


In the 20-25°C and 30-35°C incubation chambers, our investigator reviewed documentation for 117 growth promotion samples. Only 74 samples were in the chambers; 43 were missing. According to your firm’s response, the plates were missing because, during the inspection, you were moving the microbiology laboratory from the (b)(4) floor to the (b)(4) floor. No one mentioned the laboratory move during the inspection.
在20-25°C 和30-35°C的培养箱中，我们的检查员审核了117块培养基促生长的样品，只有74个样品在培养箱中，43块缺失。根据你们公司的回复，这些平板缺失是因为你们在检查期间把平板从xxx楼移到了xxx楼。没有人在审计期间提到实验室移动了平板。


  点评：造假被当面揭穿，求这家公司的心理阴影面积。面对GMP审计，不管有什么样的应对策略，所有问题的回答都应以实事求是为基础。检查员怀疑公司造假，并且找到证据，警告信是无法避免的。


  案例七
Your firm failed to establish and document the accuracy, sensitivity, specificity and reproducibility of test methods employed by the firm (21 C.F.R. 211.165(e)).
贵公司未建立和文件化具有足够准确度、灵敏度和重现性的方法。


c. During a walkthrough of your microbiology laboratory on September 8, 2014, the investigator observed that sterile gloves intended for use in the manufacture of sterile products were partially immersed in (b)(4) medium. The investigator found that the fingers of the gloves were not immersed; the “Testing Procedure for Sterile (b)(4) Gloves” SOP lacked a requirement for full immersion of gloves in sterility test media; and the test method was not validated.
2014年9月8日，在微生物实验室现场检查中，检查员发现用于无菌生产的手套只有部分被浸泡在培养基中。检查员发现手套的手指部分未浸没在培养基中，“无菌手套检测程序”未规定手套须完全浸没在无菌检查培养基中，且该无菌检查方法未经验证。


  点评：典型的细节决定成败。看似不经意的错误，其对手套的无菌保证、进而对产品的无菌保证均会产生影响。


  案例八
You did not adequately investigate media fill and sterility test failures. These failures indicate that there is a lack of adequate sterility assurance in your manufacturing facility.
贵公司的培养基模拟灌装和无菌检查失败的调查不彻底。这些失败表明贵公司的生产设施缺乏足够的无菌保证能力。


b.    Sterility Test Positive Investigations
无菌检查阳性的调查
You also did not thoroughly investigate sterility test positives. For example, your investigation of  a sterility test failure for (b)(4) injection (batch (b)(4)) did not adequately assess the hazards in the aseptic manufacturing operation that led to the sterility failure. You also did not determine whether other batches made on the same production line were affected.  
贵公司未彻底调查无菌检测阳性。例如，你们对无菌注射液（批次为xxx）的无菌检查阳性调查未充分评估无菌生产操作中导致无菌失败的危害因素。贵公司也未确定同一生产线上其他批次的产品是否受影响。


In addition, you invalidated multiple sterility test positive results obtained during batch release testing. However, we note that your firm uses a sterility test (b)(4) as well as a sterility testing kit that minimizes potential for adventitious contamination that could cause false positives.
此外，很多批放行的无菌检测阳性被判定为检测结果无效。然而，我们注意到贵公司使用无菌检查xxx和无菌检查试剂盒，这可以降低偶然因素导致的假阳性结果。


  点评：微生物OOS调查不彻底的典型案例。要判定无菌检查结果需要有充分的证据，同时，在调查中，不能仅限于直接受影响批次的调查，还需要评估对其他批次的影响。


  案例九
During inspection of the QC microbiology testing laboratory, our investigators observed:
在QC微生物实验室的检查中，检查员发现：


A.  No growth on the positive control plate for media used to test microbiological (b)(4) samples. When a positive control fails to yield growth, test results cannot be considered valid due to the potential for false negatives.
微生物检查样品xxx的阳性对照平板上无菌生长。当阳性对照上不能生长微生物时，测试结果不能被认为是有效的，因为这会导致潜在的假阴性结果。


B.  Desiccation of a contact media plate used during environmental monitoring of the sterility testing area. Desiccated, cracked, or otherwise damaged (b)(4)  compromises microbial growth promotion and accurate enumeration, and can lead to artificially low microbiological counts and false negatives. Using deficient media compromises the validity of your microbiological test results.
无菌检查区域使用干燥失水的接触碟进行环境监控。失水、破裂或其他xxx因素会损害微生物促生长能力和计数的准确性，这会导致人为的微生物计数结果偏低和假阴性结果。使用缺陷的培养基损害了微生物检测结果的有效性。


Also, you did not appear to routinely identify (i.e., to species level) bacterial and fungal isolates recovered during environmental monitoring of your aseptic processing room.
贵公司也未对无菌操作间环境监控中所生长的微生物进行常规鉴定（如种的水平的鉴定）。


C.  Air bubbles between filtration (b)(4)  and (b)(4)  plates in 13 out of (b)(4)  microbiological (b)(4)  system sampling plates. Inadequate contact between the filter (b)(4)  and the (b)(4)  plate may compromise recovery.
xxx系统的微生物取样检测平板中，xxx个中的13个平板滤膜和平板之间有气泡。滤膜和培养基接触不充分会影响微生物的检出。


  点评：培养基干裂、滤膜和平板之间有气泡，同样是看似不经意的错误，同样，FDA很生气，后果很严重。微生物检测结果是无菌保证不可或缺的一部分，这个都不能保证，产品的无菌性如何保证？


  案例十
Failure to record activities at the time they are performed.未实时记录检验活动，During the inspection, we observed that you did not have worksheets for recording microbial test results and that you failed to contemporaneously document microbial limits test results for (b)(4) API batch (b)(4).
在检查中，检查员发现贵公司没有测试记录用于记录检测结果，这会导致你们的API测试结果不能实时记录。


  点评：数据完整性不是理化实验室的“专利”，微生物实验室同样需要关注数据完整性的问题。

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