PDA-确保微生物测试数据的完整性（二）
Microbiological Test Data – Assuring Data Integrity
微生物测试数据-确保数据的完整性
Edward Charles Tidswell and Tim Sandle
PDA Journal of Pharmaceutical Science and Technology 2017,
Access the most recent version at doi:10.5731/pdajpst.2017.00815
Microbiological Test Data – Assuring Data Integrity
微生物测试数据–确保数据的完整性（二）
QC Microbiological Tests QC微生物测试
Generally, the harmonized pharmacopoeial reference and referee microbiological tests methods are purposed for the assessment of a material in achieving the necessary microbiological quality attributes.
通常，协调的药典参考和裁判微生物检测方法是为了评估一种材料是否达到必要的微生物质量属性。
Test methods are growth-based culture methods that either determine the presence or absence of microorganisms or enumerate bioburden.
测试方法是基于生长的培养方法，可以确定微生物的存在或不存在，也可以检测生物负载。
Presence/absence tests include the test for specified microorganisms per USP (<62 Microbiological examination of non-sterile products: tests for specified microorganisms), EP (2.6.13 Microbiological examination of non-sterile products: test for specified micro-organisms)
and JP (4.05 Microbial limit test), USP <71>, EP 2.6.1, and JP 4.06; these are binary in nature relying upon the inspection of media for the physically visible signature of microbial presence.
存在/不存在检验包括USP规定的微生物检验(<62非无菌产品微生物检验:规定微生物检验)、EP(2.6.13非无菌产品微生物检验:规定微生物检验)USP <71>， EP 2.6.1, JP 4.06;它们本质上是二元的，依赖于对培养基的检查，以获得微生物存在的物理可见特征。
In contrast microbial enumeration tests per USP (<61> Microbiological examination of non- sterile products: microbial enumeration tests), EP (2.6.12 Microbiological examination of non- sterile products: microbial enumeration tests and JP (4.05 Microbial limit test) rely upon inspection of the test with an enumeration of discrete recovered colonies to make an assessment of the material.
相比之下微生物计数测试/ USP(< 61 >非无菌产品的微生物检查:微生物计数测试),EP(2.6.12非无菌产品的微生物检查:微生物计数测试和JP(4.05微生物限度测试)依靠检查测试计数的离散恢复菌落数的评估材料。
Similar non-compendial bioburden test methods may be used for the enumeration of microorganisms within process inputs (raw materials, additives, processing aids), process intermediates (including process steps such as intermediate hold) and environmental monitoring samples.
类似的非药典生物负载试验方法可用于工艺输入物(原料、添加剂、加工助剂)、工艺中间体(包括中间货舱等工艺步骤)和环境监测样品中微生物的计数。
The following sections briefly detail the pharmaceutical compendial sterility test, microbial enumeration test and non-compendial bioburden test methods discussing their constraints and the fundamental microbiological facts related to potential data integrity issues.
以下部分简要介绍了药物无菌试验、微生物计数试验和非药物生物标准试验方法，讨论了它们的限制条件以及与潜在数据完整性问题相关的基本微生物事实。
Sterility Test 无菌测试
In the harmonized compendial sterility test an observation is made by a trained professional microbiologist rather than a measurement made and recorded by an instrument.
在协调的无菌检查中，由受过训练的专业微生物学家进行观察，而不是由仪器进行测量和记录。
This evaluation may be prone to misinterpretation, inconsistency of discrimination and result in the erroneous conclusion of an article’s sterility.
这种评价可能容易产生误解，不一致的歧视，并在物品的无菌性导致错误的结论。
Errors can arise based on the need for the microbiologist to visually assess the presence or absence of turbidity.
由于微生物学家需要直观地评估浑浊度的存在与否，可能会出现错误。
This requires, in addition to pre-requests of good eyesight and a suitable light source, and understanding of the variations of microbial growth.
这需要，除了预先要求良好的视力和合适的光源，并了解微生物生长的变化。
Issues can arise where product residues react with the culture medium causing turbidity (a problem that affects the direct inoculation method more-so than the membrane filtration method).
当产品残留物与培养基发生反应导致浑浊时，就会出现问题(这个问题对直接接种法的影响比膜过滤法更大)。
The definition of turbidity is left up to the practitioner.
浑浊度的定义由从业者决定。
A potential issue with discerning turbidity and microbial growth is the presence of debris; this can originate from the elastomeric closures of product, media and rinse solution bottles.
鉴别浊度和微生物生长的一个潜在问题是是否存在碎屑;这可能是由于弹性密封件的产品，培养基和冲洗溶液瓶。
‘Sterility’ is a microbial quality attribute required of certain therapies (drugs, biological and
devices) determined by both the product and its route of administration.
“无菌”是某些治疗(药物、生物和器械)所必需的微生物质量属性，由产品及其给药途径决定。
All injectable products (cutaneous, sub-cutaneous and parenteral), ophthalmic, and aqueous oral inhalation products are required to be sterile in the US market as per legislated Code of Federal Regulations 21 CFR 200.50 and 21 CFR 200.51 respectively.
根据联邦法规21 CFR 200.50和21 CFR 200.51的立法规定，所有可注射产品(皮肤、皮下和非肠道)、眼科和含水口服吸入产品在美国市场都必须无菌。
Sterility of a therapy can be defined as ‘Within the strictest definition of sterility, a specimen would be deemed sterile only when there is complete absence of viable microorganisms from it’ per USP (<1211> Sterilization and sterility assurance of compendial articles).
治疗的无菌性可以定义为“在无菌最严格的定义下，只有当样本中完全没有活菌时，才被视为无菌”。
This definition and similar definitions have long endured, to date adequately serving the patient population, regulatory authorities and manufacturing firms for the primary purpose of patient safety.
这一定义和类似的定义长期存在，迄今为止充分服务于患者群体、监管机构和生产企业，主要目的是确保患者安全。
This, regardless of significant scientific flaws (Tidswell, 2010; Tidswell & Bennett 2017), misalignment with contemporary microbiology, and the undisputable fact that it is not statistically or technically provable through product testing (sterility testing).
尽管存在重大的科学缺陷(Tidswell, 2010;(Tidswell & Bennett 2017)，与当代微生物学的不一致，以及一个不容置疑的事实，即它无法通过产品测试(无菌测试)在统计上或技术上证明。
The trinity of Pharm Europa, Japanese Pharmacopoeia and United States Pharmacopeia provide the precise harmonized test methodology for the referee sterility test per USP <71>, EP 2.6.1, and JP 4.06.
欧盟药典、日本药典和美国药典三位一体，为USP <71>、EP 2.6.1和JP 4.06的无菌鉴定提供了精确的统一检测方法。
One of the critical elements of the definition of sterility is the term ‘complete absence’ which infers that there is a complete and absolute empirical absence of microorganisms from any item which is purported to be sterile.
无菌定义的关键要素之一是“完全没有”一词，它推断任何声称无菌的物品中都完全没有微生物，而且绝对没有微生物。
Sutton (2011a) illustrates the inability of a test proving a statistical assurance of sterility of every item labeled as sterile within a manufactured batch.
Sutton (2011a)说明了无法通过检验来证明在生产批次中每一项标记为无菌的产品的无菌性的统计保证。
Despite the technical flaws of a test for sterility (Tidswell, 2010), and the statistical constraints, the definition and sterility test has utility in assuring patient safety and remains a legislated requirement.
尽管无菌检查存在技术缺陷(Tidswell, 2010)和统计限制，但无菌检查的定义和检查在确保患者安全方面具有实用价值，仍然是一项立法要求。
Current good manufacturing practice (CGMP) regulations are promulgated in The Code of Federal Regulations (CFR), Part 211 on Good Manufacturing Practices for Finished Pharmaceuticals. Legislative requirements governing sterility and sterility testing in 21 CFR Part 211 are supplemented in 21 CR Part 600-680.
现行的《药品生产质量管理规范》(CGMP)是在《联邦法规法典》(CFR)中颁布的，第211部分是关于成品药品生产质量管理规范的。21 CFR第211部分中有关无菌和无菌检测的立法要求在21 CR第600-680部分中得到了补充。
The requirement for the microbial quality attribute of sterility is referenced in 21 CFR Part 211.165(b) which states:
无菌微生物质量属性的要求见21 CFR第211.165(b)部分，其中规定:
“There shall be appropriate laboratory testing, as necessary, of each batch of drug product required to be free of objectionable microorganisms”
对每批要求不含有害微生物的药品，视需要进行适当的实验室检测。
Here, “objectionable” is any viable microorganism (within the current definition of sterility)
within a product labeled sterile.
在这里，“有害的”是任何活的微生物(在当前对无菌的定义范围内)。在标有无菌标签的产品内。
More specifically, 21 CFR Part 211.167(a) states that:
更具体地说，21 CFR第211.167(a)部分规定:
“For each batch of drug product purporting to be sterile and/or pyrogen-free, there shall be appropriate laboratory testing to determine conformance to such requirements.
“对于每批声称无菌和/或无热原的药物产品，应进行适当的实验室测试，以确定是否符合这些要求。
The test procedures shall be in writing and shall be followed”
测试程序应采用书面形式，并应遵循”
Notwithstanding an approved filing of parametric release (see USP <1222> Parametric release),
a test for sterility is always necessary.
尽管有参数放行的批准文件(见USP <1222>参数放行)，无菌检查总是必要的。
Even under exceptional circumstances, for example 21 CFR Part 211.165 (a):
即使在特殊情况下，例如21 CFR第211.165 (a)部分:
“….Where sterility and/or pyrogen testing are conducted on specific batches of shortlived
radiopharmaceuticals, such batches may be released prior to completion of sterility and/or pyrogen testing, provided such testing is completed as soon as possible”
“….当对特定批次的短寿产品进行无菌和/或热原检测在完成无菌和/或热原测试之前，这些批次的放射性药物可能会被释放，只要这些测试尽快完成。”
In addition to legislating the application of a test of sterility for articles purporting to be sterile, the CFRs require any test applied to be fit for purpose. 21 CFR Part 211.165 (d):
除了立法规定对声称无菌的物品实施无菌测试外，CFRs还要求任何适用于目的的测试。21 CFR第211.165 (d)部分:
“Acceptance criteria for the sampling and testing conducted by the quality control unit shall be
to assure that batches of drug products meet each appropriate specification and appropriate statistical quality control criteria as a condition for their approval and release.
质量控制单位抽样检测的验收标准为确保药品批次符合每一项适当的规范和适当的统计质量控制标准，作为批准和放行的条件。
The statistical quality control criteria shall include appropriate acceptance levels and/or appropriate rejection levels”
统计质量控制标准应包括适当的验收水平和/或适当的拒收水平。
In terms of the harmonized referee sterility test this highlights that a firm’s procedures must
clearly distinguish what constitutes a passing from a failing test.
在统一的无菌检查方面，这强调了一个公司的程序必须清楚地区分什么是通过测试，什么是失败测试。
The sterility test is reliant upon a professional microbiologist’s acuity of vision coupled with an informed decision on if growth in the sterility test canister has occurred.
无菌测试依赖于专业微生物学家敏锐的视力，以及对无菌测试筒是否发生生长的知情决定。
Those criteria are fundamentally dependent upon an individual’s capability and competency (experience and expertise) in microbiology.
这些标准基本上取决于个人在微生物学方面的能力和能力(经验和专业知识)。
If we literally apply 21 CFR Part 211.165 (e) it reinforces that a firm must describe and establish the
discriminatory capability of the sterility test and therefore the microbiologist:
如果我们实际应用21 CFR第211.165 (e)部分，它加强了一个公司必须描述和建立无菌检查的鉴别能力，因此微生物学家:
“The accuracy, sensitivity, specificity, and reproducibility of test methods employed by the firm shall be established and documented.
公司所采用的检测方法的准确性、敏感性、特异性和重现性应予以确立并形成文件。
Such validation and documentation may be accomplished in accordance with 211.194(a)”
这种验证和文件编制可按照211.194(a)条进行。
Furthermore, 21 CFR Part 211.160 (b) is clear in its intent that for any test that the associated
instrumentation is calibrated, maintained and in a condition which will assure the consistency in the determination of if an article meets the requisite specification:
此外，21 CFR第211.160 (b)部分的意图很清楚，对于任何相关的测试对仪器进行校正、保养，并确保在确定某物品是否符合所需规格时保持一致:
“(4) The calibration of instruments, apparatus, gauges, and recording devices at suitable
intervals in accordance with an established written program containing specific directions, schedules, limits for accuracy and precision, and provisions for remedial action in the event accuracy and/or precision limits are not met.
(4)在适当的位置校准设备、仪器、仪表和记录设备不符合既定书面程序所规定的时间间隔，该书面程序包含特定的方向、时间表、准确度和精密度的限制，以及事件准确度和/或精密度限制的补救措施的规定。
Instruments, apparatus, gauges, and recording devices not meeting established specifications shall not be used”
不符合规定规格的设备、仪器、仪表和记录设备不得使用。
Interpreting this literally in terms of the sterility test; assuring a consistent deterministic
application of the referee method would require the calibration and maintenance of the microbiologists visual acuity and professional competency.
从无菌试验的角度来解释;确保一致的确定性应用裁判方法需要对微生物学家的视力和专业能力进行校准和维护。
The sterility test is widely recognized by industry and regulators as possessing two fundamental constraints.
无菌检查被业界和监管机构普遍认为具有两个基本限制。
Firstly, the limit of detection of the Sterility Test is greater than a single viable cell (Smith et al., 2010).
首先，无菌试验的检测极限大于单个活细胞(Smith et al.， 2010)。
Physiological, genotypic and phenotypic variability of microorganisms (Sutton, 2011b) essentially ensure the sterility test will only ever recover (culture) a fraction of potential contaminants;
微生物的生理、基因型和表型变异(Sutton, 2011b)从本质上保证了无菌试验只能恢复(培养)部分潜在污染物;
some microorganisms will be unculturable due to the limitations of the medium (Vartoukian et al., 2010) others unculturable due to their physiological prerogative (Postgate, 1969; Barer and Harwood 1999; Kell et al., 1998, Kell and Young, 2000).
：一些微生物由于培养基的限制而无法培养(Vartoukian et al.， 2010)，另一些微生物由于其生理特权而无法培养(Postgate, 1969;Barer and Harwood 1999;Kell et al.， 1998, Kell and Young, 2000)。
Secondly, the Sterility Tests cannot be applied on a meaningful statistical basis.
其次，无菌检查不能在有意义的统计基础上应用。
Odlaug (1984) used equation 1 (below) to demonstrating the probability of the test succumbing to a Type II error – a false negative, that is to say failing to identify non-sterility (Brown and Gilbert, 1977).
Odlaug(1984)用下面的公式1来证明测试服从于于II型错误率——假阴性，也就是说不能识别非无菌性(Brown and Gilbert, 1977)。
Equation 1.方程1
P = 1-e-λ
Where, 式中
P is the probability of failing the sterility test (a positive result) P为无菌试验失败的概率(阳性结果)
e = 2.7182818
λ = likelihood of a contaminated unit 受污染单位的可能性
Using equation 1, and assuming at least one microorganism is required to cause non-sterility,
sampling 20 units (n = 20) within a batch of items, the probability of detection of a non-sterile unit are summarized in Table 1 below.
利用公式1，假设至少需要一种微生物才能导致非无菌，在一批物品中抽样20个单位(n = 20)，非无菌单位的检测概率汇总如下表1所示。
Obviously where all units are contaminated (100% per first column Table 1) the probability of a positive sterility test i.e. identifying non-sterility is 100%.
显然，在所有单位都受到污染的情况下(表1第一栏100%)，无菌试验呈阳性，即鉴定为非无菌的可能性为100%。
Concomitantly per Table 1, when the frequency of contaminated units in a batch is 1% there is only an 18% probability of detecting non-sterility with a positive sterility test.
与此同时，根据表1，当批次中污染单位的频率为1%时，用阳性无菌试验检测非无菌的概率只有18%。
Table 1. Probabilities of a Positive and Negative Sterility Test With Varying Frequencies of Contaminated Units in a Batch (Odlaug, 1984)
表1批次中不同频率污染单位的阳性和阴性无菌试验的概率(Odlaug, 1984)
|
Frequency of contaminated Unit受污染单位的频率 |
Probability of a Positive Sterility Test无菌测试阳性的概率 |
Probability of a Negative Sterility Test无菌测试阴性的概率 |
|
1.0 (100%) |
1.0 (100%) |
0 |
|
0.1 (10%) |
0.86 (86%) |
0.14 (14%) |
|
0.01 (1%) |
0.18 (18%) |
0.82 (82%) |
|
0.001 (0.1%) |
0.02 (2%) |
0.98 (98%) |
The insufficiency of the sterility test in assuring sterility is remarkable and recognized by
regulators and industry alike as only capable of detecting gross contamination events (Téllez et al. 2006; FDA Guidance For Industry. Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice, 2004; van Doorne et al.,1998; Stevens-Riley, 2015).
无菌检查在保证无菌方面的不足是值得注意和认识的监管机构和行业一样，只能检测总体污染事件(Tellez et al. 2006;FDA工业指南。无菌处理生产的无菌药品-现行良好生产规范)。
The contributory value of the sterility test to assure microbial quality, reduce patient risk, and maximize patient safety is likely insignificant to the sum aggregate of design and controls of manufacture.
无菌检查对保证微生物质量、降低患者风险和最大限度地提高患者安全性的贡献价值，可能与设计和生产控制的总和无关。
Indeed, deploying additional microbiologists to visually check for growth in a sterility test is does not guarantee microbial growth in the test will be observed and does little to improve our odds in assuring sterility.
事实上，在无菌测试中部署额外的微生物学家来目测生长并不能保证在测试中观察到微生物的生长，也无助于提高我们确保无菌的几率。
There are a number of means of aiding the retirement of data integrity issues associated with sterility tests (see later).
有许多方法可以帮助解决与无菌测试相关的数据完整性问题。
（未完待续）
上节回顾：PDA-确保微生物测试数据的完整性（一）
下节预告：PDA-确保微生物测试数据的完整性（三）
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