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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8.1

Table 8.1: ICH Q2Validation Parameter Definitions [107]

表8.1 ICH Q2验证参数定义（107）

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 cellpadding="0">

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 low

levels (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

Disvantage 

8.1.1.4 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, and

LOQ 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）。