清洁验证分析方法的专属性、准确度、精密度要求！

翻译：刘校对：流浪的沙子GMP办公室翻译组组织翻译，欢迎加入GMP办公室翻译组QQ群参与更多指南翻译！译自2020年8月份发布的《ISPE基准指南：清洁验证生命周期–应用，方法和控制（ISPE Baseline Guide: Cleaning Validation Lifecycle: Applications, Methods, and Controls）》8 Analytical and Biological Assay Methods清洁验证分析和生物分析方法Testing cleaning validation samples is critical to provide an accuraterepresentation of the level of cleanliness of equipment both after cleaning(API or formulation; cleaning agent and bioburden as necessary) and before use(bioburden). API residue is typically tested, as it may be the high hazardcomponent in the formulation. Any test method used must be validated for thesample being taken [21]. The primary consideration for the test method is thatit is sensitive to levels lower than the cleaning limit for the analyte ofinterest. If the test method is not sensitive enough to test residue levels lowerthan the cleaning limit, the method sensitivity must be enhanced, a differenttest method must be employed, or themanufacturing equipment must be dedicated [17].清洁验证样品的检测对于对于提供设备清洁(API或制剂、清洁剂、生物负荷)后和使用前（生物负荷）的清洁水平的准确表征是非常重要的。通常测试API残留，因为它可能是配方中的高危害成分。所用的任何测试方法都必须经过取样验证[21]。测试方法的主要考虑是它对低于相关分析物的清洗极限的水平敏感。如果测试方法不够灵敏，无法进行现场测试任何用于测试目标样品的方法均应被验证，检测方法首要考虑的是其灵敏度应低于被检测物的清洁限度。如果试验方法灵敏度不够，无法检测低于清洁限度的残留物水平，则方法灵敏度必须提高，或者采用其他方法，或生产设备专用。A specific method (e.g., HPLC) ispreferred by regulatory agencies [17], but a non-specific method (e.g., TOC)can be acceptable. Bioburden testing isoften conducted by compendial methods as a general test, with individualspecies identified asnecessary.监管机构通常首选专属性的方法（如HPLC)，但非专属性的方法（如TOC)也可能可以接受，生物负荷检测经常采用药典方法，必要时进行菌种鉴定。The analytical performancecharacteristics, or validation parameters, should conform to those defined byICH Q2 [107]: accuracy, precision, specificity, LOD, LOQ, linearity, range,robustness, and recovery (see Section 8.1). Definitions of the parameters areshown in Table 8.1.分析性能特征或验证参数应符合ICHQ2（107）的要求，包括准确度、精密度、专属性、LOD、LOQ、线性、范围、耐用性和回收率（见8.1章），这些参数的定义见表8.1。8.1 Analytical Methods分析方法8.1.1 Validation Parameters验证参数Validation parameters are describedin Table 8.1.验证参数的描述见表8.1Table 8.1: ICH Q2Validation Parameter Definitions [107]表8.1 ICH Q2验证参数定义（107）Parameter参数Definition定义Accuracy准确度“Expresses the closeness of agreement between the value which is  accepted either as a conventional true value or an accepted reference value  and the value found”表示真实值或认可的参考值与测量值之间的相近程度。Precision精密度“Expresses the  closeness of agreement (degree of scatter) between a series of measurements  obtained from multiple sampling of the same homogeneous sample under the  prescribed conditions是指规定条件下对均质样品多次取样进行一系列检测结果的接近程度（离散程度）· Repeatability –  the precision under the same operating conditions over a short interval of  time. Repeatability is also termed intra-assay precision.重复性——指在同样的操作条件下，在较短时间间隔的精密度：也称为间隙测量精密度（Intra-assay precision）· Intermediate  Precision – within-laboratories variations: different days, differentanalysts, different  equipment, etc.中间精密度——是指实验室内部条件改变，如不同日，不同试验分析者，不同仪器等情况下的精密度。。· Reproducibility –  expresses the precision between laboratories (collaborative studies, usually applied to standardization of  methodology)”重现性是指不同试验室之间的精密度（合作研究，通常用于方法学的标准化）。Specificity/Selectivity专属性/选择性“Specificity is the  ability to assess unequivocally the analyte in the presence ofcomponents which may  be expected to be present.”专属性是指可能存在某些组分时，对被分析物准确可靠测定的能力。Detection Limit (LOD)检测限“The lowest amount  of analyte in a sample which can be detected but not necessarily quantitated as an exact value”样品中的被分析物能够被检测到的最低量，但不一定要准确定量。Quantitation Limit (LOQ)定量限“The lowest amount  of analyte in a sample which can be quantitatively determined with  suitable precision and accuracy”是指在合适的准确性和精密度下，能够定量测定样品中被分析物的最低量。Linearity线性“The ability (within  a given range) to obtain test results which are directly proportional to the  concentration (amount) of analyte in the sample”是指在给定的范围内检测结果与样品中被分析物的浓度（量）成比例关系的能力。Range范围“The interval between the upper  and lower concentration (amounts) of analyte in the sample (including these  concentrations) for which it has been demonstratedthat the analytical procedure has  a suitable level of precision, accuracy and linearity”是指样品中被分析物的较高浓度(量)和较低浓度(量)的一个区间，并已证实在此区间内，该方法具有合适的准确性、精密度和线性。Robustness耐用性“A measure of its capacity to  remain unaffected by small, but deliberate variations in method parameters  and provides an indication of its reliability during normal usage”是指试验参数适当地发生细小改变时，测量保持不受影响的能力，可用于说明正常使用时的可靠性。Ruggedness重现性The degree of reproducibility of  test results obtained by the analysis of the same samples under a variety of  normal test conditions such as different laboratories, analysts, instruments,  reagents lots, or days是指样品在正常的测试条件下在不同试验室，分析员、分析仪器、试剂批号、日期所获得分析结果的重现程度8.1.1.1 Accuracy准确度Accuracy determines the closeness of test results to the true value acrossthe range [107].准确度决定了测试结果在一定范围内与真实值的接近程度Samples extracted into solutions aremeasured and compared to standard solutions at comparable concentrations to determine accuracy. For cleaning validation samples,accuracy is measured through recovery of samples from equipment surfaces and extraction ofthe recovered samples into a testing solution. Accuracy should be establishedusing a sufficient number of data points. One example is three recovery levelsin triplicate, for a total of nine recoveries (see Chapter 7). Accuracy isreported as % recovery of the amount of analyte in the recovered samples measured against the amount of analyte spiked onto thesample recovery surface.测定提取溶液中的样品并与同等浓度的标准溶液进行比较，以确定准确度。对于清洁验证样品，准确度是通过从设备表面回收样品并将回收样品提取到测试溶液中进行测量，建立准确度应使用足够数量的数据。一种方式是三个回收水平，每个水平三份，总共九个回收样（见第7章）。准确度报告为回收样品中被测物相比取样表面上所涂布的量的%回收率。Accuracy should be determined aroundthe ARL, which is the point around which accurate data is most essential.However, for a relatively safe product, the ARL could be relatively high. Forexample:准确度应在可接受残留限度上测定，在可接受限度附近得到准确数据是最重要的。然而，对于相对安全的产品，可接受残留限度可能相对较高。例如:· Typical accuracy at 75%, 100%, and125% of ARL典型的准确度在可接受残留限度的75%、100%和125%· If ARL > 100 µg/25 cm2 swab, perform recovery around 100 µg/25 cm2如果可接受残留限度> 100 µg/25 cm2 ,则在 100 µg/25 cm2左右进行回收测试· Advisable to extend linearity upto ARL to understand possible method limitations建议将线性扩展到可接受残留限度以了解可能的方法限制· Advisable to extend accuracy downto method LOQ and one to three levels in between, which is where most data should be建议将准确度向下延伸到检测限，采用介于两者之间的一到三个级别，大多数数据都在该区间8.1.1.2 Precision精密度Precision determines the degree of agreement among individual test resultsapplied repeatedly to multiple samples from a homogeneous sample [107]. It is ameasure of the combined variability of the sample recovery, the sample extraction, and the samplemeasurement. Precision may be considered at three levels: repeatability, IntermediatePrecision (IP), and reproducibility. All the parameters of the recovery process(see Chapter 7) can affect the recoveryof the sample. The key is to control sample parameter variability as muchas possible.精密度是测定从一个均质样本中重复取出多个样品，每个样品的测试结果之间的一致程度【107】，它是对样品回收率、样品提取和样品测量的综合变异性的一种度量。精密度可以考虑三个层次：重复性、中间精密度(IP)和重现性。恢复过程的所有参数(见第7章)都会影响样品的恢复。关键是尽可能控制样本参数的可变性。回收过程的所有参数（见7章）均能影响样品的回收，尽可能控制样品参数的变化非常重要。During swab sample methodvalidation, a sufficient number of recovery samples should be run to have astatistically significant RF of a somewhat variable test. The samples used foraccuracy determination are also used for precision.在擦拭样品方法验证过程中，应运行足够数量的回收样品，以便得到一个某种程度上的变量测试具有统计学意义的RF（回收因子）值。用于准确度测定的样品也用于精密度测定。There is no regulatory acceptancecriterion for precision for cleaning samples. Variability measured as % RSD isoften used with a limit of ≤ 15% for swab samples.对于清洁样品的精密度没有官方可接收标准。对于擦拭样品，以%RSD来衡量的变异性通常使用限度为≤15%。PrameterDefinition8.1.1.3 Specificity专属性Specificity determines the abilityto assess the analyte in the presence of components expected to be present inthe sample matrix [107]. For cleaning samples, the expected components are theother ingredients of the formulation, potentialdegradants, and the cleaning agent.专属性决定了在样品中存在预期成分的情况下评估分析物的能力[107]。对于清洁样品，预期的成分是配方的其他成分、潜在降解物和清洁剂。Specificity is the capability of amethod to separate known degradants identified during product development and previously developed selective product assays. Thestability of the analyte in alkaline or acidic solutions should be reviewed todetermine the risk of degradation during the cleaning process. Although theability to detect degradants is necessary, the risk of their presence should be low. Degradants arestructurally related to the analyte and removed to the same extent as theanalyte. Since they are a low level to begin with, the risk of their presencein a post-cleaning sample should beminimal.专属性是区分产品开发过程中识别的已知降解物和之前开发的选择性产品测试物的能力。应确认分析物在碱性或酸性溶液中的稳定性，以确定在清洁过程中降解的风险。虽然存在降解物的风险应该很低，但检测降解物的能力还是必要的。降解物与被分析物的结构类似，并与被分析物同等程度被去除。由于它们一开始就处于低水平，因此它们在清洁后样品中存在的风险很小。An exception to this approach iswhere the analyte is known to degrade in the cleaning solution. This approachcan be leveraged to eliminate allanalyte from the residue and then the primary degradant(s) should be monitored,its pharmacological activity determined, and appropriate cleaning limitsestablished as necessary.此方法的一个例外是已知分析物在清洁溶液中降解。此方法可用于从残留物中消除所有分析物，然后监测主要降解物，确定其药理活性，并在必要时建立适当的清洁限度。A specific method (e.g., HPLC)should be able to distinguish the specific analyte of interest from the othermatrix components. A non-specific method (e.g., TOC) is not able to distinguishbetween the analyte of interest and other sample matrix components. Therefore,when using TOC, all measured residue has to be assumed to be the analyte ofinterest. HPLC and TOC generally have comparable sensitivity, so eitherapproach is acceptable if it can measure to levels lower than the ARL. Runtimes are also comparable but each technology can use shortened analysis times.专属的分析方法（如HPLC)应能从其他多个成分中分辨出特定的目标物，非专属的分析方法（如TOC）不能分辨目标物和其他同样的多个成分。因此，当使用TOC时，所有测得的残留都被当成目标物。HPLC和TOC通常有相当的灵敏度，因此如果能检测到低于可接受清洁残留限度的值，则两种方法都是可接受的。运行次数也是相当的，但每种方法均应使用较短的分析时间。Table 8.2: Advantagesand Disadvantages of Specific and Non-Specific Methods 专属和非专属分析方法的优缺点Test Method分析方法Advantage优势Disadvantage劣势Specific (e.g., HPLC)专属分析方法（如HPLC)· Separates analyte of interest  from matrix components从其他多个成分中分辨出特殊目标物· Can be tested in a lower volume  to increase sensitivity能在较低体积下进行测试，以提高灵敏度· Not as variable as TOC不像TOC那般变异性· Not limited by swab solvent不受擦拭溶剂限制· Newer instruments have shorter run times新型的分析仪器可缩短运行时间· Longer method development  timeline较长的分析方法开发时间· Individual injections can be lengthy单个进样时间可能较长Non-Specific (e.g., TOC)非专属分析方法（如TOC）· Shorter method development  timeline较短的分析方法开发时间· Newer instruments have shorter run times新型的分析仪器可缩短运行时间· All instrument response is assumed to be the analyte of interest仪器上的所有响应都被当成目标分析物· Generally requires a greater dilution factor, which could limit  sensitivity通常需要更大的稀释因子，这可能限制灵敏度· Variable response, particularly at lowlevels (ppb range)响应变异性大，特别是在低存在水平时（ppb级）· Swab solvent limited to aqueous擦拭溶剂仅限于水Disvantage8.1.1.4 LOQ/LOD定量限/检测限LOQ (also known as QuantitationLimit) is the lowest amount of analyte that can be determined with acceptableaccuracy and precision under the stated experimental conditions, while LOD(also known as Detection Limit) is the lowest amount of analyte that can bedetected under the stated experimental conditions [107].LOQ（也被称为定量限）是指在规定的实验条件下和可接受的准确度和精密度下能被确定的最低分析物的量，LOD（也被称为检测限）是指在规定的实验条件下能被检测到的最低分析物的量。8.1.1.5 Linearity线性Linearity is the ability of an assayto elicit a direct and proportional response to changes in analyteconcentration [107].线性是指含量对于分析物浓度变化产生直接和成比例响应的能力。8.1.1.6 Range范围Range is the interval between the upper and lower levels of analyte thathas been demonstrated to be determined with a suitable level of precision,accuracy, and linearity [107].范围是指样品中被分析物的较高浓度(量)和较低浓度(量)的一个区间，已证实在此区间内，该方法具有合适的准确性、精密度和线性。8.1.1.7 Robustness耐用性Robustness is the measure of thecapacity to remain unaffected by small but deliberate variations in proceduralparameters, for example, changes to [107]:耐用性是指试验参数适当地发生细小改变时，检测能力仍然不受影响，例如，下列变化：Mobile phase流动相Column temperature柱温Wavelength波长Injection volume进样体积Sample solvent样品溶剂8.1.1.8 IntermediatePrecision (IP)/Ruggedness/Reproducibility中间精密度（IP)/重现性/重复性Ruggedness is the degree of reproducibility of test results obtained by theanalysis of the same samples under a variety of normal test conditions such as different instruments,analysts (called IP), laboratories (called reproducibility), reagents lots, or days [107].是指样品在正常的测试条件下在不同分析仪器，分析员（称为IP）、试验室（称为重复性）、试剂批号、日期所获得分析结果的重现程度8.1.1.9 SolutionStability (Sample/Standard)溶液稳定性（样品/标准品）Solution stability is the measure ofhow long standard and samples can be retained and continue to demonstrate consistent detector response.溶液稳定性是指衡量标准品和样品的保存时间并持续表现出一致的检测器响应的能力。Standard solution标准溶液Sample solution after extraction提取后的样品溶液Sample before extraction (swab)提取前的样品（擦拭）Dried sample on the coupon干燥样品8.1.2 Methodology方法学There are a variety of methodsavailable to test cleaning validation samples. There are specific and non-specific methods. A specific method can distinguish the analyte of interestfrom other components of the sample matrix. A non-specific method measures theanalyte of interest along with other components of the matrix that elicit aresponse from the test method. The method of choice is based on the mostappropriate for the specific circumstances, first considering specific methodsfollowed by non-specific methods. All test methods require validation.有各种方法用于测试清洁验证样品，专属性的和非专属性的，专属性的方法能从样品的其他成分中分辨出目标分析物，非专属性的方法目标分析物与测试方法中产生响应的其他多个成分一道被测试，方法的选择基于最适宜的具体情况，首先考虑专属方法，其次是非专属方法。所有测试方法应被验证。8.1.2.1 Specific TestMethods专属检测方法HPLC is the most commonly usedspecific test method for cleaning validation. The use of HPLC for testingcleaning validation samples is well established and can address all validationparameters. HPLC is a chromatographic method that passes a sample through a packed column and separates the analyte ofinterest from the other components of the sample. An HPLC method can separate the residue ofinterest from the components of the formulation as well as the detergent. Awell-designed HPLC recovery study can demonstrate accuracy, precision,linearity, range, LOD, andLOQ in a single run.HPLC是清洁验证中最常的专属检测方法，HPLC用于检测清洁验证样品是公认的，能解决所有的验证参数。HPLC是一种色谱方法，该方法通过将样品流过装填好的柱子将目标分析物从样品的其他成分中分离出来。一个HPLC方法能从制剂的多成分及清洁剂中分离出目标残留物，一个设计良好的HPLC方法的回收率研究能证明准确度、精密度、范围、检测限和定量限。Typically, HPLC methods for assayare designed to quantitate levels down to 0.1% of the API, making sensitivitywell below most calculated ARLs. However, the sensitivity of HPLC is dependenton the chemical structure of the residue of interest and the detector, which can quantitate the residue.通常，HPLC分析方法被设计用于定量0.1%的API水平，使灵敏度远低于大多数计算的可接受清洁残留水平。然而，HPLC的灵敏度依赖于目标残留物的化学结构和检测器。HPLC assay methods can be lengthy,30 to 40 min per injection, which could be an issue for quick turnaround of samples.HPLC分析方法可能时间较长，每次进样为30到40分钟，可能是快速样品周转的一个问题。Use of other specific test methodssuch as GC, MS, or IR can be used in certain situations and are subject to the same validation parameters.使用其他的专属检测方法，如GC,MS或IR可用于某种特定条件下并且遵从于同样的验证参数。8.1.2.2 Non-SpecificTest Methods非专属的检测方法TOC is the most commonly usednon-specific test method for cleaning validation. TOC analysis involves theoxidation of carbon and the detection of the resulting carbon dioxide produced.Sensitivity is down to the ppm or ppb level. A well-designed TOC recovery studycan also demonstrate accuracy, precision, linearity, range, LOD, and LOQ in a single run.TOC是清洁验证中最常用的非专属性检测方法，TOC分析包含了碳的氧化和产生的二氧化碳的检测。灵敏度可低至ppm或ppb，一个设计良好的TOC回收率研究也能证明准确度、精密度、线性、范围、检测限和定量限。In a TOC analysis, all organiccarbon is detected; therefore, any residue detected must be considered theresidue of interest. Residues for TOC analysis must dissolve in water. This canlimit the effective linear range of the residue assay.在一个TOC的分析中，所有的有机碳均能被检测，因此，检测到的残留一定会被认为是目标残留，用于TOC分析的残留必需是溶于水的，这就限制了残留物含量的有效线性范围。Use of other non-specific testmethods such as pH or conductivity can be used in certain situations andrequire only instrument calibration.非专属检测方法如pH或电导率能用于某种特定条件下，只需要仪器校准。Use of other non-specific methods(e.g. pH, conductivity) can be applied in certain situations if a strongscientific justification is provided to ensure it is appropriate and providesthe necessary controls for an effective cleaning process.在某些特定条件下，如果提供了有力的科学依据以确保其适用，并为有效的清洁过程提供必要的控制，则可使用其他非专属的方法（例如pH值、电导率）。

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