Analytical Methods 分析方法 Itis essential to a cleaning validation program that the appropriate analyticalmethods are utilized. Analytical methods must be appropriate in that they canadequately detect and measure the residue(s) of concern. It is also importantto understand what can be concluded from the analytical result (e.g., was theproduct or cleaning agent measured and were the results acceptable?). Theresults of testing will determine if the cleaning cycle is acceptable or needsimprovement. This section discusses considerations in selecting the appropriatetest methods, including information on the applicability and use of bothchemical and microbial test methods, and test method validation. 在清洁验证中选择一个合适的分析方法是非常必要的，该适当的分析方法应能够充分检测相关残留物。理解可以从分析结果中得出什么结论也很重要（例如检测的是产品还是清洁剂，结果是否合格？），检测结果决定了清洁方法是否可行或需要进一步改进。本章讨论的内容是怎样选择一个合适的测试方法，包括适用性、化学和微生物测试方法的信息，以及测试方法的验证。 Theemphasis in this section will not be so much on describing the features andlimitations of methods (although that will be done to a limited extent), as itwill be on the thought process of deciding what information is obtainedand when a certain analytical method will be useful. Cleaning process understandingis the key to selecting the appropriate analytical method for various stages ofcleaning validation 本章的重点不是阐述分析方法的特性和局限性（尽管对此也有一定描述），而是判断获得了什么信息以及何时可采用某一特定分析方法的思考过程。对于清洁工艺的理解是在清洁验证不同阶段选择适当分析方法的关键。 Purposes of the Analytical Methods 分析方法的目的 Ina lifecycle approach to cleaning validation, different analytical methods maybe appropriate for evaluation of residues at the different stages of thecleaning validation lifecycle. The lifecycle stages of cleaning validation aredesign/development, qualification, and validation maintenance. Analytical methodsmay also be used as part of investigations during any lifecycle stage. It isimportant to consider and evaluate what information one wants to obtain andwhat information can be obtained from use of a given analytical procedure. 在一个生命周期的清洁验证方法里，在清洁验证的不同的阶段可能使用不同的分析方法来评价残留物。清洁验证的生命周期包括设计/开发、确认和验证维护。在生命周期中的任一阶段，分析方法也可以作为调查的一部分。考虑和评估希望以及能够从一个特定分析程序中获得什么信息是很重要的。 Forexample, in early development work, there may not be adequate information onthe nature of residues (e.g., is the active ingredient degraded?) and aspecific analytical method may not have been validated. However, nonspecificmethods may give a reasonably accurate picture of the overall effectiveness ofthe cleaning process for cleaning process development, even though thatnonspecific method may or may not be the analytical method chosen for thecleaning validation protocols. 例如，在早期的开发过程中，可能没有残留物的足够的信息（例如活性成分是否降解），而且一个特定的分析方法可能也没有经过验证。然而，在清洁工艺开发时，采用非专属性方法可能有助于较准确地了解清洁工艺的综合效果，即使这种非专属性方法可能是或不是最终清洁验证方案中选定的分析方法。 Anotherexample involves the selection of analytical methods for investigations. Forthe validation runs (qualification runs), it is usually preferred to have ananalytical method that can appropriately determine whether the target residue(e.g., the active ingredient) is at or below the predetermined acceptance limitfor that residue. But for an investigation into a deviation (nonconformance),in certain circumstances (such as with the use of a nonspecific method in avalidation protocol) it may be more important for the investigation to have ananalytical method that can qualitatively determine the nature of that residue(e.g., is it active ingredient, cleaning agent or excipient?). 另外一个例子是用于调查的分析方法的选择。在验证（确认）中，通常首选的分析方法应能够适当检测出目标残留物（例如活性成分）是否符合预定的可接受限度。但是，对于一个偏差（不符合）的调查，在特定的条件下（例如在验证方案中使用的是一个非专属性方法），采用一个能够定性检测出该残留物性质（例如，是活性成分、清洁剂还是辅料？）的分析方法则是更为重要的。 Itis important to emphasize that why an analytical method is being used iscritical for having a robust, science and risk-based approach to cleaningvalidation. Just because a method has been used in the past does notnecessarily mean it will be useful for a new application. 需要重点强调的是，理解为何选择一个分析方法是建立一个可靠的、科学的和基于风险的清洁验证方法的关键。仅仅因为是过去使用过的分析方法并不一定意味着它可以用于新的应用中。 Practical Considerations in Selecting Analytical Methods 选择分析方法的实际考虑 Inan ideal world, the best method for a given task could be chosen; in the realworld, selection of analytical methods may be limited by practical considerations.In many cases, it is important not that the analytical method be the best methodavailable but that it be adequate for the intended purpose.In selectinganalytical methods, one must consider readily available methodologies within agiven company. For example, it is not likely that a company will invest in anew analytical method if existing methods are adequate for the intendedpurpose. New methods may mean capital equipment purchases, training of analystsand maintenance of the equipment; the related costs should be weighed againstthe expected benefits. For example, total organic carbon (TOC) was not widelyconsidered for cleaning validation until TOC replaced the readily oxidizablesubstances pharmacopeial method, after which pharmaceutical companies werereadily familiar with and comfortable with the technology. 在理想的情况下，应选择最好的分析方法；实际上，分析方法的选择可能会受到实际情况的限制。许多情况下，更重要的不是选择的分析方法是最佳的，而是该方法能够满足预期的目的。在选择分析方法时，必须考虑一个特定公司现有的分析方法，例如，如果现有的分析方法能够满足检测目的，公司就不太可能再投入开发另一个新的分析方法。新的方法可能意味着设备采购、人员培训和设备维护投入，相关的费用需与预期的收益进行平衡。例如，直到TOC方法替代了药典方法中的易氧化物检验之后才被广泛应用于清洁验证中，之后制药公司也就很快地熟悉和接受了这项技术。 Onthe other hand, if a new analytical method is required because existingin-house methods are not adequate for the intended purpose, then that newmethod should be considered. These may be implemented by using contractanalytical laboratories or by bringing the new analytical methodology in-house.A decision on bringing the method in-house versus using a contract laboratorymay be based on business considerations. 另一方面，如果现在的内控方法不足以满足预期的目的，则需要采用一个新的分析方法，这些工作可以在合同实验室完成或建立新的内控分析方法，可基于商业上的考虑决定建立新的内控分析方法还是由合同实验室完成。 Specific vs. Nonspecific Analytical Methods for ValidationProtocols 验证方案中的专属性和非专属性方法对比 Specificanalytical methods are those which measure a certain residue in the presence ofexpected interferences. If the target analyte in a validation protocolis the active ingredient, such interferences may include degradation productsand related substances, excipients, cleaning agents and cleaning processby-products. Examples of specific methods include liquid chromatography(including HPLC,UPLC and TLC) and spectrophotometry (including UV, visible andinfrared). Each of these methods requires the use of an appropriate referencestandard. In contrast, nonspecific analytical methods measure a generalproperty, such as conductivity or TOC, which could be due to a variety ofanalytes or sources. 专属性分析方法是指那些在有预期干扰物存在的情况下仍可以检测特定残留物的方法。如果在验证方案中的目标分析物是活性成分，那些干扰物可能包括降解物和有关物质、辅料、清洁剂和清洁工艺副产物。专属性方法包括液相色谱法（包括HPLC、UPLC和TLC）和光谱法（包括紫外、可见和红外），这些方法都需要使用适宜的对照品。相反的，非专属性方法测量的是一种大致的性质，例如电导率和TOC，它可能源于多种分析物和不同来源。 Selectionof an analytical method may depend on the nature of the residue as it existsafter the cleaning process. Only if an active ingredient is not degraded duringthe cleaning process (e.g., surviving high temperatures and pH extremes in anaqueous environment) does it make sense to use a specific analytical method forthat active ingredient. If a specific analytical method for an activeingredient were utilized following a cleaning process that has beendemonstrated to degrade that active ingredient, it is likely that residues ofthe active ingredient would be nondetectable (i.e. not measurable) by thatspecific analytical method. In such a case, use of a specific analytical methodfor the degradant or use of a nonspecific method (such as TOC) may beconsidered for measuring residues in a validation protocol. Alternatively, iflimits are established for the degradation product of an active ingredient, thena specific analytical method for the degradant may be considered for use. 分析方法的选择可能取决于清洁工艺后存在的残留物的性质。只有在清洁过程中活性成分没有发生降解（例如，在经过高温和极端的pH之后），使用一个专属性的方法测定该活性成分才有意义；如果已知在清洁工艺中活性成分会发生降解，那么使用专属性方法检测活性成分时，可能就不会检测（测量）到活性成分。在这种情况下，验证方案中就应该考虑使用一个专属性测定降解物或采用一个非专属性方法（例如TOC）测量残留；如果限度是按照活性成分的降解物建立的，也可以考虑采用一个专属性分析方法测定降解物。 Itshould be recognized that the proper use of a nonspecific analytical method mayprovide a more robust demonstration of acceptable cleaning in a validation protocol,because it may have responses from species other than the target residue, yetthose responses must be assumed as due to the target residue (24).However, exceeding the residue limit using a nonspecific analytical methodprovides no information on the nature of the failure. The high analyticalresult may be due to responses from the active ingredient, the excipients, thecleaning agent, and/or a combination of those species. 应该认识到，在验证方案中，使用适宜的非专属性分析方法更能证明清洁方法的耐用性，因为除了目标残留物外其他成分也会有响应，而这种响应将被假设为源于目标残留物（24）。然而，当检测结果超出限度时，使用一个非专属性分析方法无法为失败提供任何信息，偏高的检测结果可能来源于活性成分、辅料、清洁剂和/或这些成分共同作用造成的响应值。 Nothingin this Technical Report should be interpreted as saying that, as a generalprinciple, specific analytical methods should be used in preference tononspecific analytical methods. 本技术报告中的任何内容都不应理解为：一般应优先选择专属性方法而不是非专属性方法。 Regulatory Status of Specific and Nonspecific Methods 专属性和非专属性方法的法规状况 Bothspecific methods and nonspecific methods have been found acceptable byregulatory authorities. However, one must be careful not to misuse ananalytical method. For example, specific methods can be misused by failing torecognize the degradation of the active ingredient in the cleaning process, andnonspecific methods can be misused by failing to attribute the nonspecificresponse entirely to the residue of concern. 无论是专属性方法还是非专属性方法，监管部门都是可以接受的。然而，必须要注意不要误用分析方法。例如，如果不知道清洁过程中活性会发生降解，就可能错误地使用专属性方法；如果非专属性响应不完全源自目标残留物，则可能误用非专属性的方法。 TheU.S. FDA cleaning validation guidance states that one should “Determinethe specificity and sensitivity of the analytical method used to detectresiduals or contaminants”(20). While some have interpretedthis to mean that a specific analytical method should be used, a betterinterpretation is that irrespective of the type of method selected, make sureit is used appropriately. The European PIC/S recommendations state that “Theanalytical methods used to detect residuals or contaminants should be specificfor the substance to be assayed….”(22). This again has beeninterpreted to mean that only specific analytical methods should be used.However, it is not applied in that manner since nonspecific methods are widelyused by companies worldwide and have been accepted by the U.S. FDA and Europeanregulatory authorities. 美国FDA清洁验证指南要求“对于检测残留或污染物的分析方法，应该验证其专属性和灵敏度”（20）。因此有些人认为应使用专属性的分析方法，但更恰当的说法应该是，不管使用哪种类型的分析方法，要确保能够恰当地使用该方法。欧洲PIC/S建议“用于检测残留物或污染物的分析方法对于被分析物应该是专属的”。这再一次被解读为应该使用专属性的分析方法。但是实际并非如此，因为非专属性方法在世界上各公司已经广泛地应用并且被美国FDA和欧洲药监当局所接受。 Most Commonly Used Analytical Techniques 最常用的分析技术 Thefocus of this section is to discuss the most commonly used analyticalprocedures in pharmaceutical cleaning validation (25). The TaskForce believes it was more appropriate to focus on common uses of analyticalmethods, based on the stages of cleaning validation where they have beendemonstrated to provide relevant information. The features, benefits andlimitations of methods are often situational and are therefore not coveredhere. 这一节中我们所要讨论的焦点是清洁验证中最常用的分析方法（25）。专家组认为应基于清洁验证的各个阶段所提供的相关信息，选择常用的分析方法。方法的特性、优点和局限性经常因使用的条件而不同，因此本文不再赘述。 Additionalconsiderations in selecting methods are listed below: 选择方法还要考虑以下内容： •Availability of instrumentation 可用的仪器 •Speed of analysis 检测的速度 •Specificity of technique 检测技术的专属性 •Sampling limitations (including sampling solvents) 取样限制（包括取样溶剂） •Detection/quantitation limit 检测限/定量限 •Linearity of response 响应值的线性 •Online adaptability 能否在线检测 •Cost 费用 Mostapplications in pharmaceutical cleaning validation involve quantitation ofresidues over a validated range. However, in certain situations, pass-failtests, also known as “go-no go” testing, may be used toestablish that the residue is below the acceptance limit. Such testing may beused in qualification runs for clinical manufacture (where the effort to fullyvalidate an analytical method over a linear range may be costly) or for routinemonitoring and equipment release based on final rinse solvent testing. Apass-fail test generally does not demonstrate the robustness of the cleaningprocess unless the pass-fail point is significantly below the desiredacceptance limit. Since the transition point is a range, the range must beknown and its relationship to the limits must be established in the validation process.The actual result, although passing, could have been very close to failure andwith normal plus/minus variation it could actually represent a failed result. 在制药清洁验证最多的应用是在一已验证范围内定量测定残留物的量。然而，在一些特定的情况下，合格-不合格检测，也被称为“放行-不放行”检测，也可用来检测残留物是否在可接受限度之下。这种检测可以用于临床生产的确认批（如果在线性范围内进行全面验证则成本可能较高）或用于例行监测和设备放行（基于最终淋洗液的检测结果）。合格-不合格检测通常不能证明清洁工艺的耐用性，除非合格-不合格点远远低于所需的可接受限度。由于该转换点是一个范围，因此这个范围必须是已知的，并在验证过程中建立该范围与限度之间的关系。实际的结果，尽管是合格的，可能与不合格结果是非常接近的，考虑到结果的正负误差，可能实际上就已经是不合格的结果了。 Formore information on analytical method use in biotechnology manufacture, pleaseconsult PDA Technical Report No. 49, Points to Consider for BiotechnologyCleaning Validation (2). 对于生物制药企业的分析方法的更多信息请参考PDA技术报告第49号，《生物技术清洁验证要点》（2）。 Liquid Chromatography (LC) 液相色谱法（LC） LCincludes HPLC (High Performance Liquid Chromatography), UPLC (Ultra PerformanceLiquidChromatography, and TLC (Thin Layer Chromatography). All these methodsinvolve the separation of component by a chromatography procedure and then themeasurement of one or more separated species. For HPLC and UPLC, themeasurement is typically ultraviolet (UV) detectors, although other appropriatedetectors may be used based on the analyte of interest. 液相色谱法包括HPLC（高效液相色谱）、UPLC（超高效液相色谱）和TLC（薄层色谱）。所有这些方法都是通过色谱方法先将各组分分离，再测定各分离组分的量。对于HPLC法和UPLC法，通常都是采用紫外（UV）检测器进行的测定，根据待测物也可以选择其他适宜的检测器。 HPLCand UPLC methods are typically specific methods, which are widely used formeasurement of active ingredients in small molecule-manufacturing (both API anddrug product manufacturing). In many cases, HPLC/UPLC methods have beenpreviously developed as a potency assay method for the active ingredient, andonly need minor modification to make the method suitable for use as a methodfor residue determination in qualification runs. Those additional modificationsmay involve confirming that the useful range is suitable for residuedeterminations and that additional “expected interferences”that are present in the cleaning system do not interfere with measurement ofthe active ingredient. HPLC/UPLC methods may not be suitable for measuringresidues of an active ingredient if the active ingredient is degraded in thecleaning process, unless the chromatography conditions allow separation andmeasurement of degradants of interest. HPLC法和UPLC法通常为专属性方法，被广泛应用于小分子生产（包括API和制剂）中活性成分的测定。多数情况下，HPLC法/UPLC法已经被开发成活性成分的含量测定方法，只需要做小的修改即可用于确认时残留物的测定。这些额外的修改包括确认线性范围是否适用于残留物的测定，以及在清洁系统中存在的“预期的干扰物”是否会干扰活性成分的测定。如果活性成分在清洁过程中发生降解，那么HPLC法/UPLC法可能就不适用于测定活性成分的残留，除非该色谱条件可以将降解物分离并测定出来。 TLCmethods may be used for various stages for cleaning of small molecules. Forexample it may be used for design/development to confirm and characterizedegradation of the active. TLC methods may also be used for any investigation(at any stage of cleaning validation) to characterize residues. TLC方法可以应用于小分子化合物清洁的各个阶段。例如，它可以用于设计/开发阶段以确认和定性活性成分的降解情况。TLC方法也可以用于调查（在清洁验证的各个阶段）以定性残留物。 UltraViolet/Visible Spectrophotometry (UV/Vis) 紫外/可见分光光度法（UV/Vis） UV/Visinvolves measuring transmission/absorbance of a specified wavelength of lightby a solvent solution of the residue. It typically requires a chromophore in themolecule, although it is also possible to modify the residue to produce achromophore. For example, it is commonly used in small molecule manufacturing,particularly for API manufacturing where it is not necessary to separate it froma matrix to quantify the residue. Because of its simplicity, UV/Vis techniquesmay be used in the design/development, qualification and validation maintenancestages of cleaning validation as well as for any investigations. UV/Vis hasalso the possibility of being used in PAT applications for completion of thecleaning steps for small molecule API manufacturing (26). 紫外/可见分光光度法是通过在特定波长下测定残留物溶液的透过率/吸收值来进行测定的。它通常要求分子中存在一个生色基团，也可以通过衍生法使残留物产生一个生色基团。例如，紫外/可见分光光度法通常被小分子化合物生产企业特别是API生产企业所采用，它不需要将残留物分离出来进行定量。由于该方法比较简便，因此可以应用于清洁验证的设计/开发、确认和验证维护阶段，以及各种调查中。紫外/可见分光光度法在小分子API生产中也可以作为PAT技术判断清洁步骤的终点。 Total Organic Carbon (TOC) 总有机碳（TOC） TOCis applicable to any residue containing significant amounts of organic carbon.The TOC method is based on oxidizing the carbon present and measuring thecarbon dioxide produced. Oxidizing methods include UV, persulfate, andcombustion. Techniques for measuring the generated carbon dioxide includeconductivity, membrane-based conductivity and infrared. Both online and offlineapplications ofTOC are possible. TOC法可以用于含有大量有机碳的残留物的检测。TOC法是通过将样品中的碳氧化并测定所生产的二氧化碳的含量来完成的。氧化的方法包括紫外法、过硫酸法和燃烧法，检测产生的二氧化碳的技术包括电导率法、基于膜的电导率法和红外法。在线和离线的TOC都是可行的。 Foruse of TOC, the target residue must have adequate aqueous solubility for theintended purpose.The most common way of applying the TOC method to a cleaningvalidation testing strategy is to assume that all residues detected are due tothe target residue (24). In manufacturing situations, TOC iscommonly used for measuring residues if the target residue (e.g., the activeingredient) is degraded during the cleaning process. However, it may also beused in situations where the active is not degraded.The rationale for use ofTOC in such situations is ease of analytical method development and theworst-case assumptions inherent in TOC analysis. 对于使用TOC法，目标残留物必须具有足够的水溶性。用于清洁验证的TOC法通常是将所有的检测到的残留物都假设成目标残留物。在生产过程中，如果目标残留物（例如活性成分）在清洁过程中会发生降解，则TOC法是常用的方法。然而，TOC法也可以用于那些活性分成不发生降解的情况，在这种情况下使用TOC法的理由是分析方法易于开发，而且在TOC检测中最差条件的假设是始终存在的。 TOCmay be used for all stages of cleaning validation, includingdesign/development, qualification and validation maintenance as well as forinvestigations. TOC也可以用于清洁验证的各个阶段，包括设计/开发、确认和验证维护，以及调查。 Conductivity 电导率 Conductivitymeasurement is a method to detect dissociated ionic substances in watersamples. For qualification protocols conductivity readings are expressed inmicro-Siemens/cm (μS/cm); for control andmonitoring of the cleaning solution, conductivity readings are expressed asmilliSiemens/cm(mS/cm). It is often used to measure cleaning agent residues(e.g., caustic or acidic agents) and to control cleaning agent concentration inautomated cleaning processes (e.g., CIP). Conductivity readings are highlyinfluenced by the sample temperature. Temperature adjustment of the sample,automated temperature compensation or a conductivity/concentration curve at aspecified temperature can be used to standardize the measurements. 电导率测定是通过测定水溶液样品中解离的离子物质来完成的。在确认方案中，电导率读数表示为微西门子/厘米（μS/cm）；用于清洁溶液的控制和监测时，电导率表示为毫西门子/厘米（mS/cm）。电导率法经常被应用于清洁剂残留（例如碱性或酸性试剂）的检测，以及在自动清洁工艺（例如CIP）中控制清洁剂的浓度。电导率的读数受样品温度的影响很大，样品温度的调整、自动温度补偿以及指定温度的电导率/浓度曲线可以用于测量结果的校正。 Toallow correlation of conductivity readings with concentrations of cleaningagent, a dilution curve (conductivity vs. concentration) should be established(at a relevant temperature) by conductivity measurements of different dilutionsin the relevant range near the acceptance value. 为了将电导率读数与清洁剂的浓度相关联，应建立特定温度下的稀释曲线（电导率对浓度），这可以通过测定特定温度下接近可接受标准的不同稀释浓度的电导率完成。 Conductivityis a nonspecific method that correlates linearly (within a defined range) tothe ion concentration in an aqueous sample. Analytical instruments are robustand can be used on the manufacturing floor by trained personnel. The methodcannot differentiate between different ions. Therefore, as with TOC, allconductivity results above the water baseline should be attributed to thecontaminantin question (e.g., the cleaning agent). 电导率是一种非专属性的方法，它在一定范围内与样品溶液的离子浓度线性相关。分析仪器耐用，而且接受过相应培训的人员可以在生产车间进行检测。该方法不会区分不同的离子，因此如同TOC一样，所有超过水的基准电导率的结果都是由于污染造成的（例如清洁剂）。 Conductivityis often a function of alkaline or acidic cleaning agent. Measuringconductivity is a good measure of the completion of rinsing, and therefore anindirect measure of good cleaning for routine monitoring of a cleaning process. 电导率通常说明了碱性或酸性清洁剂的存在，测量电导率是一种判定冲洗终点的好方法，因此对于清洁工艺的日常监控，测量电导率也是间接证明清洁效果的好方法。 Conductivitycan also be used for measuring residues of an ionic active ingredient, eitherin cases where the cleaning agent is water alone or in other cases involvingionic cleaning agents if all the conductivity response is attributed to theactive ingredient (even though some of the response maybe due to the cleaningagent). 电导也被用于测定离子型活性成分的残留，不管是单独以水为清洁剂，还是使用了离子型清洁剂，只是所有的电导率响应值都应作为活性成分的响应值（即使其中的一些响应值源自清洁剂）。 Organoleptic Evaluation 感观评价 “Organoleptic”evaluation includes visual inspection as well as other evaluations such assmell. Visual inspection is commonly used during all stages of cleaningvalidation, as it is a minimum requirement under GMPs for use of equipment formanufacture. Visual inspection is a nonspecific method in that the nature ofthe residue generally cannot be identified except by further analysis. “感观”评价包括目视和其他评价，例如嗅觉。目视检查通常在清洁验证的各个阶段都有使用，因为目视洁净是生产设备符合GMP的最低要求。目视检查是一种非专属性方法，除非做进一步分析，一般无法鉴定残留物的性质。 Trainingand a detailed documented procedure is required to ensure that “visuallyclean” from one operator to the next isconsistent. What one can visually see will vary with distance, angle, lighting,nature of surface, and inspector’s visual acuity. Someequipment surfaces (e.g., piping) are usually notaccessible for visualinspection. The use of optical equipment like mirrors, remote videoscopes, or borescopescan help to facilitate visual inspection. 需要进行培训并建立一个详细的文件，确保不同人员的“目视清洁”是一致的。目检结果受距离、角度、光线、表面的性质，以及检查人的视力影响会有很大不同。使用光学设备例如镜子、远程光纤视镜或管道镜可有助于目视检查。 Thevisual inspection procedure should specify how operators are to deal withvisual observations.Visual inspection may find four different types of visualobservations: residue, surface anomalies, foreign object and water. Residue isthe main concern which would constitute a visual failure when one isassessing the acceptability of a cleaning cycle. A sample of the residue shouldbe collected for further testing, if possible, to assist in the investigationof the cause. Typically, surface anomalies and foreign objects are notconsidered visual inspection failures for cleaning validation purposes, butmust be further investigated and corrected, as applicable. Surface anomaliesshould be noted and a “suitability for use”assessment should be performed to remediate any issue(s) found. Rouge is themost common type of surface anomaly discovered during visual inspection; rougeis generally considered a preventive maintenance problem, not a cleaning processproblem. Foreign objects and their removal should be documented. Also, how theforeign object came to be in the equipment should be investigated.Sometimes adistinction is made between absence of water pooling (“freedrained equipment”)and the absence of any visiblewater droplets (“dry equipment”).Particularly for water pooling, the observation should be documented, the causeinvestigated, and the impact on issues such as visual examination and bioburdenproliferation on storage should be addressed. 目视检查程序应当明确检查者怎样处理观察结果。目视检查可以发现四种不同类型结果：残留物、表面异常、异物和水。残留物是主要关注点，当评估清洁行程是否合格时，发现残留物就可能意味着目视检查结果的失败。如果可能的话，应收集样品残留做进一步检测，以协助调查可能的原因。通常，在清洁验证中出现表面异常和异物时，不认为目检结果不合格，但必须进行进一步调查和纠正。应记录表面异常情况，评估“是否适合使用”，并纠正发现的各种问题。红锈在目视检查中是一种最常见的表面异常，它往往被认为是一种预防性维护问题而不是清洁工艺问题。异物及其去除应被记录下来。同样，异物是怎么进入到设备当中也要进行调查。有时应区分无积水（“自排水设备”）和无可视水滴（“干燥设备”），特别是对于积水，应记录观察结果，调查原因，并说明其对目视检查以及贮存过程中的微生物增长的影响。 Allequipment surfaces should be visually inspected if possible. Visual inspectionmay not be performed on the interior of lines and tubing (although outlets maybe inspected) on equipment where disassembly of the equipment is not practicalor possible, or where inspection of the equipment could potentially bedangerous to the inspector (e.g., entry into a confined space). 如果可能的话，所有的设备表面被应该进行目检。当无法或不宜拆卸设备时，或者对设备进行目检可能造成人员伤害（如，进入限制性空间）时，可能无法对安装在该设备上的管线和管道内部（尽管可以对出口处进行观察）进行目检。 Atraining program should be developed for visual inspection. Inspectorstypically should be trained and/or requalified on an established basis. Ifvisual inspection is not possible on an area of concern, it is important toensure that other sampling methods (such as rinse sampling) can adequatelydetect potential residues of concern. 应建立目检培训计划，检查人员应按照要求接受培训和/或进行资格确认。如果对于一些区域无法进行目检，则应确保他取样方法（例如冲洗取样）能够充分地检测出潜在的目标残留物。 Smellas an organoleptic method is generally only used if an unusual smell occursduring sampling of the equipment, which would suggest the need for aninvestigation. 作为一种感观方法，嗅觉检查通常只用于设备取样时有一种特殊气味，应对此进行调查。 Other Useful Analytical Techniques 其他有用的分析技术 Beloware other techniques which may be useful for various stages of cleaningvalidation. 以下其他技术可能用于清洁验证的不同阶段。 pH pHis a measure of the hydrogen ion concentration. It can be used as a monitoringprocess check, particularly when equipment is stored wet in a preservativesolution (typically acid or base). pH can also be used to verify qualitativelythe presence of the correct cleaning solution. pH can be used to complementconductivity measurements. However, pH is less useful than conductivity for measuringresidues of alkaline or acidic cleaning solutions because pH has a logarithmicrelation with hydrogenion concentration, whereas conductivity has a direct,linear relationship with ions. Furthermore, there is not necessarily a directcorrelation of conductivity and pH, particularly for neutralized cleaning agents. pH是一种测量氢离子浓度的技术，它可以作为工艺监测手段，特殊是当设备贮存在防腐溶液中（通常为酸性或碱性）时。pH法也可以用于定性确认是否存在正确的清洁溶液。pH法也可以作为电导率测量的补充，然而在测量碱性或酸性清洁剂残留时，pH法不如比电导率法，因为pH值与氢离子浓度呈对数关系，而电导率与离子浓度是直接线性相关的。此外，也没有必要将电导率与pH值直接关联，特别是对于中性清洁剂。 InfraRed (IR) 红外光谱法（IR） Thisincludes both FTIR (Fourier Transform InfraRed) and NIR (Near InfraRed). Thesetechniques are most useful in an investigation where there is a need toidentify organic residues that may be present. FTIR has also been combined witha fiber-optic probe for direct quantitative measurement of residues on surfacesfor qualification protocols (27). 红外光谱法包括FTIR（傅立叶变换红外光谱法）和NIR（近红外光谱法）。当调查中需要对可能存在的有机残留进行鉴别时，这些技术最为有用。确认过程中，可采用FTIR联合光纤探头直接测定设备表面残留物的量。 Light Microscopy 光学显微法 Lightmicroscopy, including Scanning Electron Microscopy (SEM), is a method ofidentifying contaminants on equipment surfaces. In many cases, conventionallight microscopy and SEM can be combined with other analytical techniques, suchas x-ray diffraction, mass spectrometry, and nuclearmagnetic resonance (NMR).Microscopic techniques alone may identify the physical nature of a residue butnot the chemical nature. One of the practical applications of microscopy is inthe evaluation and identification of unknown contaminants on new or usedequipment. These techniques are especially valuable in the evaluation ofresidues in an investigation. 光学显微法包括扫描电镜法（SEM），它是一种可以鉴定设备表面污染物的方法。大多情况下，传统的光学显微法和SEM可以与其他分析技术联合使用，例如X-射线衍射、质谱和核磁共振（NMR）。单独的显微技术可以鉴定残留物的物理性质，但不能鉴定其化学结构，显微法的一个实际的应用是用于评估和鉴定一个新的或旧的设备中的未知污染物，这些技术对于在调查中评估残留物是特别有价值的。 Titrations 滴定法 Titrationis another simple analytical method that is often overlooked even though itmight provide valuable information in the proper cleaning situation. Titrationsmay be specific (orthophosphate ions) or nonspecific (e.g., for all anionicsurfactants). This method is more likely to be used for alkaline or acidiccleaning agent analysis in qualification runs. 滴定法是另一种简单但经常被人忽视的方法，尽管其在适宜的清洁情况下它可以提供有价值的信息。滴定法可能是专属性（磷酸盐）或非专属性（例如，对于所有的阴离子表面活性剂）方法，本方法可能更多地用于确认过程中碱性或酸性清洁剂的检测。 Gravimetric Analysis 重量分析法 Gravimetricanalysis can be useful for design/development studies and for qualificationruns. It is most commonly used for determining residues in small-molecule APIsynthesis where a larger volume of a solvent rinse or solvent reflux isevaporated to dryness. 重量分析法可以用于设计/开发和确认中。它多应用于测定小分子API合成过程中的残留物，通过将大量冲洗溶剂或回流溶剂蒸干而测得。 Enzyme Linked Immunosorbant Assay (ELISA) 酶联免疫分析法（ELISA） AnELISA assay is an antigen-antibody type reaction involving the use of specificchemicals developed especially for the residue involved. Its use is generallylimited to biotechnology and biologics manufacture where it can be used in thedesign/development stage to confirm degradation of the active ingredient and inany investigations. 酶联免疫分析法是一种抗原-抗体反应，使用特定的化合物测定残留物。酶联免疫分析法通常局限于生物技术和生物制品生产中，可以用于设计/开发阶段以确认活性成分的降解，以及任何调查中。 Capillary Zone Electrophoresis (CZE) 毛细管区带电泳（CZE） Alsoknown as capillary electrophoresis (CE), this technique separates residues bycharge and frictional forces in an electrical field. Detection is usually witha fluorescence detector. CZE has been applied mostly in the biotechnologyindustry for active ingredients and degraded active ingredients where it can beused in design/development and qualification stages as well as ininvestigations. 毛细管区带电泳又被称为毛细管电泳，这项技术是依靠电场里的电荷和摩擦力将残留物进行分离的，通常采用荧光检测器进行检测。CZE主要被应用于生物技术工业中，在设计/开发及确认阶段检测活性成分及其降解物，以及用于调查。   Atomic Absorption (AA) and Inductively Coupled Plasma (ICP) 原子吸收法（AA）和电感耦合等离子体 Bothof these techniques can be used for measuring metals in solution, where themetal is part of a formulation or for unknown residues, such as suspectedrouge. 这两种技术都可以用于溶液中的金属，这种金属可以是是处方中的一部分或未知残留物（例如疑似红锈）。 7.5.9Ion Mobility Spectrometry (IMS) 离子迁移光谱（IMS） Thistechnique is a type of mass spectrometry which only provides information on thetime of flight of the analyzed species. It has been promoted for its shortanalysis time (a few minutes). It may have more application for routinemonitoring and release. 这项技术是质谱的一种类型，它只提供被分析样本的飞行时间。由于其分析时间较短（几分钟）而被提倡，可以更多地用于日常监测和放行。 7.6Microbial Test Methods 微生物检验方法 The1993 U.S. FDA cleaning validation guidance states that “Controlof the bioburden through adequate cleaning and storage of equipment isimportant to ensure that subsequent sterilization or sanitization proceduresachieve the necessary assurance of sterility”(20). The PIC/Srecommendations call for “the validation ofcleaning procedures for the removal of contaminants associated with the previousproducts, residues of cleaning agents as well as the control of potentialmicrobial contaminants”(22). Control of microbialresidues is thus an important part of cleaning validation. Microbial residues includebioburden and endotoxin. Typically bioburden sampling and analysis is performedduring cleaning validation protocols unless there is a documented science-andrisk-rationale for omitting such sampling and analysis. Science-and risk-basedrationales for excluding microbiological testing in protocols may includemanufacturing considerations, such as all solvent processing for small moleculeAPI manufacture, use of a final alcohol rinse for oral dose drug products, useof subsequent sterilization cycles, and/or demonstration of adequate microbialcontrol in sufficiently similar cleaning processes. 1993年美国FDA清洁验证指南中指出“通过充分地清洁和设备存放以控制生物负载，这对确保随后的灭菌和消毒程序达到必要的无菌保证水平是非常重要的”（20）。PIC/S指南要求“清洁程序的验证内容应包括去除前一产品、清洁剂的残留所带来的污染，以及潜在微生物污染的控制”（22）。因此微生物残留的控制也是清洁验证的一个重要部分。微生物残留包括生物负载和内毒素。通常清洁验证方案中应包含生物负载取样和检测，除非有科学的和基于风险的书面理由表明不需要进行，这可能包括生产上的考虑，例如小分子API生产中所有使用溶剂的工艺、口服制剂生产中使用乙醇进行最终淋洗、清洁之后进行灭菌，和/或有证据表明类似的清洁工艺能够对微生物有充分的控制。   Endotoxin 内毒素 Typically,endotoxin testing is performed for cleaning validation runs if the next producthas endotoxin specifications. Endotoxin analytical methods are typicallycompendial methods. Science-and risk-based rationales for excluding endotoxintesting in protocols may include manufacturing considerations, such as allsolvent processing for small-molecule API manufacture, use of a validated endotoxinreduction step, and/or demonstration of adequate endotoxin control insufficiently similar cleaning processes. 通常如果下一个产品质量标准中有内毒素检查项的话，应在清洁验证中进行内毒素检测。内毒素分析方法通常是药典方法。验证方案中不进行内毒素检测需要科学的和基于风险的理由，这可包括生产上的考虑，例如小分子API生产中所有的使用溶剂的工艺，有经验证可以减少细菌内毒素的操作步骤，和/或有证据表明类似的清洁工艺能够充分控制内毒素。   Bioburden 生物负载 Testingof bioburden is done through rinse-water sampling, swab sampling and contactplate sampling. Rinse-water sampling typically involves membrane filtration,placement of the membrane on an appropriate agar, incubation, and a count ofCFUs. The main rationale for rinse-water sampling for bioburden is that itprovides an overall picture of equipment cleanliness. Also, bioburden testing ofrinse water is typically already a qualified method for testing water systemsfor bioburden. The biggest weakness of rinse-water sampling and membranefiltration is that the full range of the acceptance criteria is not able to beutilized. For example, if 100 ml of rinse water is used for testing withanacceptance criteria of 100 CFU/mL. The typical number of colonies that can becounted is 300 beforeToo Numerous To Count (TNTC) is achieved; this only allowsan acceptance criterion of 3 CFU/mL before failing to demonstrate that theacceptance criterion is met. In most situations this is not an issue; it mayresult in the need to test smaller sample volumes (or diluted samples). Analternative is to perform spread-plate or pour-plate microbiological analyses. 生物负载的检测可以采用淋洗水取样、棉签擦试取样和接触碟取样。淋洗水取样后一般采用薄膜过滤法过滤，再将膜贴在适宜的琼脂培养基上，培养、计数。淋洗水取样检测生物负载的优点是它可以提供设备清洁状况的全景信息。淋洗水生物负载检测通常采用水系统微生物的检测中已确认的方法。淋洗水取样/薄膜过滤检测的最大缺点是没有一个普遍适用的可接受标准。例如，如果取100ml淋洗水用于检测，可接受标准为100CFU/ml，而通常可以计数的菌落数应不超过300，这就要求如果符合计数标准要求的话样品的可接受标准应为3CFU/ml。在大多数情况下这并不是问题，可以通过降低样品的取样体积（或稀释样品）来解决。也可以是采用涂布平板阀或倒平板法进行微生物检验。 Twomethods for directly measuring on surfaces are swab and contact plate. For swabsamples, the swab can be desorbed and a count made by a pour-plate orspread-plate method. Contact plates are directly incubated and enumerated. Thebiggest concern with contact plates and swab procedures is potentially exposingproduct contact surfaces to an unknown media or buffer solution from swabs; thusacceptable removal of this media or buffer solution should be demonstratedbefore manufacturing can occur. Another concern is that contact plates requireflat surfaces. 还有两种可以直接测量表面上生物负载的方法是棉签擦试法和接触碟法。对于棉签擦试法，应将棉签上的菌释放出来，通过涂布法或倾注法进行计数。接触碟法则可直接培养并计数。接触碟法和棉签擦试法最大的问题是可能将产品接触表面与未知培养基或棉签中的缓冲溶液相接触，因此在生产前应确认培养基或缓冲溶液是否已清除。接触碟法的另一个问题是需要设备表面是平整的。 Mostcompanies use analytical techniques for bioburden involving incubation in anappropriate medium and counting of CFUs. Such a procedure has the disadvantageof only providing a number for CFUs and not individual cells. Sampling andprocessing of the test sample may affect the reported number of CFUs due todisruption of aggregated cells. In addition, while it is common to report bioburdencounts below 20 CFU as quantifiable numbers, it is recognized that enumerationbelow 20CFU is not scientifically established. Another alternative is to userapid instrumental microbiological procedures. PDA Technical Report 33, Evaluation,Validation and Implementation of New MicrobiologicalTesting Methods shouldbe consulted for a discussion of rapid methods (28). 大多数公司生物负载检测，需要在适宜的培养基中培养并计数。这种方法的缺点是只能提供菌落数而不是单个细胞数量。由于可能会破坏细胞，取样和检验可能影响报告的计数结果。另外，虽然生物负载报告结果通常小于20CFU，但一般说来计数结果如果低于20CFU，则说明你建立的方法是不科学的。另外一种方法是使用快速仪器法检测微生物，PDA技术报告33《新微生物检验方法的评价、验证和执行》可以作为讨论该快速检验方法的参考。