上：微生物实验室篇
1“陷阱”一：良好的记录规范
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1.For example: 例如
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。
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。
2.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。实验室对生产用API xxx的总菌数和控制菌进行监控。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。而在他初次看这本记录的时候，2014年7月10-13日的温度未记录。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。
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培养基的促生长试验平板，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。
分析：我们把记录规范从数据完整性中单独列出，因为记录规范是GMP的基本要求，是每一个从业人员的基本素质。案例中，周六、周日未进行观察本身并不是严重的偏离（中国药典对于无菌检测有逐日观察的要求），然而，倒签、做假记录则使企业的诚信全无，后果是灾难性的。记录一定要及时、准确，抱着侥幸心理做假记录是不可取的，说一个谎话要用十个谎话来圆。
2“陷阱”二：检测方法
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3.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。
分析：分析方法的准确性是检测结果可靠性的前提，此发现项中，检测方法的缺陷会导致手套的无菌性得不到保证，从而对于无菌生产带来潜在的污染风险。另外，分析方法未经过验证也是另外一个需要注意的问题。在最近几年的警告信中，有一些提到了产品检验的方法未经验证，或验证不充分。这个检验需要关注的基本问题，然而不止一封警告信均有此内容，表明我们还需要给予更多的重视。
3“陷阱”三：检验操作
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4.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：
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。
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。
5.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。
分析：对于微生物检测来说，检验操作是非常重要却容易被忽视的点。此案例中的滤膜和平板之间有气泡这一现象是典型的代表，很多企业都有类似问题。在其他警告信中，还有不同案例，如无菌检查阳性对照无菌生长等，这一情况有时也与检测操作的细节相关，对于抑菌性很强的样品，如果操作不当，抑菌成分去除不彻底，很容易导致阳性菌无法生长。
4关注点四：OOS调查
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6.You failed to adequately investigate the sterility failure of (b)(4) injection (lot (b)(4)). This test, performed in January 2017 as part of routine stability testing, reported Bacillus subtilis, Pseudomonas putida, and Pseudomonas entomophila growth. Microbiological growth was observed in both the (b)(4) and (b)(4) media canisters.
你们未能充分调查无菌检查失败。这一测试在2017年1月进行，作为日常稳定性研究的一部分，报告长出了枯草芽胞杆菌、恶臭假单胞菌和虫媒假单胞菌。微生物在xxx培养基和xxx培养基中均有生长。
According to your investigation, the most probable root cause was laboratory error. Specifically, your May 10, 2017, response states that an analyst failed to immerse sterility test sample vials and other materials in sporicidal solution before transferring them from the Grade C to the Grade B sterility testing room. Instead, the analyst performed a spray disinfection. You indicated that spraying with a sporicide will disinfect the top and sides of samples, but that the bottom of units might not be fully decontaminated.
根据你们的调查，最可能的原因是实验室错误。特别的是，在你们2017年5月10日的回复中，你们声称失败的原因是，分析员在将无菌测试样品和其他材料从C级区转移至B级无菌检查室时，未将其浸泡在杀孢子剂的溶液中。分析员而是使用了喷洒的消毒方式。你们声称杀孢子剂喷洒的方式仅会消毒样品的顶部和侧面，而底部可能未彻底消毒。
However, during the transfer step, the exterior of the units were spray-disinfected with the validated sporicidal disinfectant solution and held for a specified contact time. The units were also placed on a (b)(4), which is intended to facilitate exposure of the bottom of units to the sporicide. Further, following this transfer to the sterility testing room, the vials were exposed to an aggressive sporicidal agent two more times. These additional sporicidal disinfections were performed as part of the (b)(4) staging and transfer steps, and occured before the vials were used in the sterility test. The disinfections included exposure to a (b)(4) cycle in the sterility testing room for (b)(4), followed by a another spray disinfection with (b)(4). One or more extensive sporicidal disinfections, such as these, normally ensure suitability of samples for use in the sterility test.
然而，在转移步骤，容器的外部使用了经过验证的消毒剂进行了喷洒消毒，并且放置了足够的接触时间。这些容器也被放入了xxx中，此步的目的正是为了使得容器的底部暴露于杀孢子剂中。此外，在进入无菌室的转移步骤以后，这些容器又使用杀孢子剂进行了两次消毒。这些额外的杀孢子剂消毒是xxx放置和转移的一部分，并且是在容器用于无菌检查时进行。消毒包括将样品在无菌室暴露于xxx中，随后用另外一个消毒剂进行喷洒消毒。一个或者更多的深入的杀孢子剂消毒通常对于无菌测试样品的准备是合适的。
In addition:此外：
?The sterility test was performed using a (b)(4) filtration system. This (b)(4) testing system was used inside an ISO-5 closed restricted access barrier system (cRABS). Both provisions significantly minimize the potential for introduction of adventious contamination during a sterility test.
无菌检查使用xxx过滤系统。这一过滤系统在ISO-5级的cRABS内被使用。这两个系统均显着的降低了无菌检查过程中引入外源污染的机会。
?No microbiological contamination was observed in the negative controls.
阴性对照无微生物生长。
?No aseptic breaches were observed during the sterility test.
无菌检查过程中未观察到无菌操作的偏离。
?Environmental monitoring data did not indicate that the sterility testing cRABS had a loss of control.
环境监测数据未显示无菌测试的cRABS失控。
?Other materials used in performing the sterility test were also subjected to additional sporicidal disinfections.
无菌检查使用的其他测试材料同样经过了额外的杀孢子剂消毒。
Your investigation was deficient in that it did not sufficiently address these factors and thoroughly investigate potential manufacturing root causes. Your manufacturing investigation substantively assessed environmental data for only the week before and the week after the product’s December 2015 manufacture date. It did not sufficiently address whether adverse trends or related incidents had occurred in the manufacturing area over a longer period and did not address the atypical findings of gram negative bacteria (e.g., Pseudomonas, spp.) earlier in the year in the production cRABS. Your review of environmental data was insufficient as it only addressed near term data trends and relied too heavily on cumulative contamination rates in assessing the potential routes of contamination in your manufacturing operation.
你们的调查是不充分的，未能调查生产过程中潜在的根本原因。你们的生产过程调查仅对2015年12月份生产的产品的前一周和后一周的环境数据进行了评估。未充分证实是否有异常趋势或相关的异常事件在此生产区域的更长时间内发生，也未调查生产区的cRABS内在当年更早的时候出现革兰氏阴性菌（如假单胞菌属）的异常事件。你们对环境数据的回顾是不充分的，因其仅回顾了短期的数据，且太过于依赖使用累积污染率来评估生产区的潜在污染途径。
7.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。
分析：微生物检测OOS调查一直是难点，因其被发现的时间远滞后于发生的时间，且微生物污染的偶然因素很多。很多企业在对检验过程进行调查时，一旦发现了检测的偏离，并揪住不放，有些企业的QC迫于压力，且自己在检测过程中确实有错，便“勇于”承担了错误，最终OOS认定为检测错误，结果无效。然而，OOS是否为无效结果不是仅凭某一现象就可判定的，需要有充分的证据证明检测异常和OOS结果之间的因果联系，否则就无法排除产品本身是否不符合无菌要求。对于无菌检查和培养基模拟灌装来说，这一点更加重要，关键的OOS需要进行溯源分析才能判定检测结果无效。另外，产品的影响范围也是不容忽视的问题，微生物OOS调查需要识别出所有可能受影响的产品，不可武断的仅对直接受影响的产品进行调查和处置。
5“陷阱”五：无菌操作规范
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8.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。
分析：对于微生物检验和控制来说，无菌操作技术是基本功，是从事微生物相关工作前必须培训的内容。欧盟新修订的GMP附录一草案新增了很多对于人员相关的要求，其中就有明确的关于无菌操作技术相关的要求。在GMP审计中，审计官也经常会在生产或检测的现场观察无菌操作规范。
6“陷阱”六：微生物实验室的数据完整性
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9.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。
10.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：….
11.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。
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。
12.Your firm failed to exercise appropriate controls over computer or related systems to assure that only authorized personnel can change master production and control records, or other records (21 CFR 211.68(b)).
贵公司的计算机或相关的系统无合适的控制，无法确保仅授权的人员可修改生产和控制主记录，或其他相关的记录。(21 CFR 211.68(b)).
Your Siemens computer-based BMS and NVPMS do not require passwords to access the network and servers. Your contractors’ access is uncontrolled. Responsibilities for system administrators are undefined.
你们基于电脑的西门子BMS系统和NVPMS系统（在线粒子监控系统）不需要密码即可进入网络和服务器。你们的承包商权限是不受控的。系统管理员的职责未被定义。
This violation is recurrent. On September 9, 2013, we cited your firm in Warning Letter 320-13-26 for failure to exercise appropriate controls over computer or related systems.
这个偏离是重复发生的，在2013年9月9日，我们引用了你们公司的警告信320-13-26，计算机和相关的系统无合适的控制。
分析：这里单独列出了一些与微生物实验室相关的数据完整性问题，这些问题在不同的企业反复发生。微生物的检测大多不具有电子数据，因此很容易产生数据完整性的风险。很多企业的员工会抱着侥幸心理，或迫于KPI的压力，故意伪造检验结果。然而，这些结果很多时候是与产品的质量直接相关的，一经发现，很容易产生严重的发现项。
本文虽然用了“陷阱”一次，罗列出微生物实验室容易中招之处，然而，通过分析我们不难看出，这些要求都是每个微生物实验室都应该做到的，也是可以做到的。希望各位同行引以为鉴。
