制药企业在清洁验证和清洁确认中的分析方法验证

制药企业在清洁验证和清洁确认中的分析方法验证
7.7Analytical Method Validation
分析方法验证
Thissection focuses on analytical method validation for “chemical”residues. Typically endotoxin methods are compendial methods and do not requireformal validation but require a confirmation for their application of use orsuitability. Microbial methods that are approved microbiology laboratory methodsdo not require additional method validation.
这一节我们关注“化学”残留的分析方法验证。

通常内毒素检验方法都是药典方法，不需要进行正式的验证，但需要确认其适用性。

经批准的微生物检验方法也不需要额外的方法验证。

7.7.1General Principles
一般原则
Sinceone key part of cleaning validation is setting residue limits and thenmeasuring (using an analytical method) the actual residues left on surfacesafter cleaning, it is critical that the analytical method be appropriatelyvalidated. Method validation is typically accomplished using the criteria inICH Q2(R1) (29). However, the types of assays listed in ICH Q2 donot explicitly cover cleaning validation methods. One approach is toessentially validate analytical methods, much like an “Assay”in ICH Q2, establishing accuracy, precision, specificity, linearity and range,with added determination of limit ofquantitation/ limit of detection (LOD/LOQ).LOD/LOQ must be below the acceptance limit for the sample, and ideally issignificantly below the acceptance limit so that the robustness of the cleaningprocess can be established. In addition to the ICH Q2 parameters,
samplestability as a function of storage conditions (time, temperature, vial forstorage, etc.) may be evaluated if there is a significant interval betweensampling and analysis. Specific methods should address possible interferencesfrom other species, such as cleaning agents, which might occur only in thecleaning process.
由于清洁验证的一个关键点就是确定残留限度，然后再测定（通过分析方法）清洁后残留在设备表面的残留物，很关键的一点就是分析方法应经过适当的验证。

通常方法验证应按照ICH Q2（R1）（29）进行。

然而，在ICHQ2中所列出的含量测定的验证并没有明确涵盖清洁验证中的分析方法。

一种方法是按照ICH Q2“含量”进行全面的验证，包括准确性、精密度、专属性、线性和范围，以及定量限（LOQ）/检测限（LOD）。

LOD/LOQ必须低于样品的可接受限度，而且最好是远低于可接受限度，以确保清洁程序的耐用性。

除了ICH Q2中规定的参数外，如果取样和检测时间间隔比较大时，应评估贮存条件（时间、温度、贮存的小瓶，等）对样品稳定性的影响。

专属性方法应关注可能只有在清洁工艺中使用到的其他物质（例如清洁剂）可能带来的干扰。

Incases where a nonspecific method is utilized, it is not necessary to compensatefor the lack of specificity by “other supportinganalytical procedures” (as suggeste d in ICHQ2). The reason for this is that for cleaning validation purposes, the limitvalue is not a target (as it is for a potency assay); rather the limit is avalue not to be exceeded. As long as these other species that contributeto the nonspecific response do so in a positive manner (thus increasing theresponse value), and as long as the total measured value is attributed to thetarget residue, such complementary methods suggested by ICH Q2 are notrequired. Furthermore, it is not required to correlate nonspecific methods
witha specific analytical method except to the extent that accuracy in the methodvalidation of the nonspecific method may be established using a known standardwhere the concentration or activity is established by a specific analyticalmethod. While Detection Limit and Quantitation Limit are not part of the “Assay”requirementin ICH Q2, it is critical that these values be at or below the pre-establishedlimit for the residue (otherwise it would not be possible to claim thatresidues were below predetermined limit values). However, it is not necessaryto drive detection or quantitation limits as low as possible; having detectionor quantitation limits of 10% or less of the residue limit in the analyticalsample is ideal (but not always possible) to establish the robustness of thecleaning process. Assay capability should take into account both thetarget/limit and the process capability, and provide relevant measurements forboth.
当采用非专属性方法时，没有必要采用“其他分析方法”（ICH Q2的建议）对缺少专属性进行补偿，这是由于对于清洁验证而言，限度值不是一个目标（如需要精确测定的含量），而是不能超出该限度值。

只要其他物质是增加非专属性响应值的，而且只要总的响应值都归结于目标残留物，那么ICH Q2中所建议的补充方法就是不需要的。

而且，也没有必要用一个专属性方法去关联非专属性方法，除了非专属性方法验证中准确性研究可能采用一个已知标准物质，而该标准物质的浓度或活性是采用一个专属性方法建立的。

尽管ICH Q2中对于含量测定的验证并不需要确定检测限和定量限，关键是检测限/定量限应接近或低于设定的残留物限度（否则你很难说明测得的残留物是低于限度值的）。

然而，也没有必要让检出限或定量限过低，为了建立具有耐用性的清洁工艺，理想的检测限或定量限应不超过残留限度值的10%（但这并不总是可能的）。

含量测定应同时考虑目标值/限度和工艺的能力，并提供相应的测定结果。

Whenperforming carryover calculations it should be ensured that the analyticalmethods that will be used for cleaning validation are sensitive enough to meetthe acceptance criteria. To provide reliable results for carryovercalculations, the results should be equal to or above the LOQ. Results between theLOQ and the LOD typically show a higher-than-acceptable variation of theresults obtained and are typically reported as less than LOQ.
在进行残留计算时，应确保用于清洁验证的分析方法足够灵敏，以符合可接受标准。

为了获得可信的残留量，检测结果应等于或大于LOQ。

在LOQ和LOD之间的检测结果，通常具有过高误差，一般报告为低于LOQ。

Forcompanies that use a pass/fail analytical method for meeting cleaningvalidation limits, analytical method validation is less extensive. In such aprocedure, the only conclusion of the analytical procedure is whether theexperimental sample is less than or equal to or above the pass/fail value. Accuracyand precision are typically performed only at the residue limit but linearityand range are not performed. Note that in this case, the pass/fail valueselected should take into consideration any applicable correction factor due tothe sampling method recovering less than 100% from the surface. Such pass/failmethods do not allow collection of relevant data to support a processcapability determination to establish action or alert levels for routinemonitoring. Pass/fail analytical procedures are more likely to be used inmanufacture of early clinical trial materials where a cleaning verification modeis employed. However, such methods can also be used for qualification runs andfor routinemonitoring.
对于使用合格/不合格方法来进行清洁验证的公司，不需进行全面
的分析方法验证。

在该方法中，只需要报告样品的结果是低于或等于或高于合格/不合格限度值。

准确性和精密度的验证通常只需要在残留限度水平进行，不需要进行线性和范围的验证。

注意在这种情况下，合格/不合格限度值的选择应考虑从设备表面取样时的回收率低于100%时，需要进行适当的校正。

如果想通过收集相关数据建立警戒限或行动限进行日常监测以说明清洁工艺的能力，则不能使用这种合格/不合格的方法。

合格/不合格分析方法可能更多地用于需要进行清洁效果确认的早期临床产品的生产，然而这类方法也可以用于清洁确认和日常监测中。

Analyticalmethod validation protocols may only include validation of the residue insolutions. It may also include sampling recovery studies, although thosesampling recovery studies may be performed as separate studies apart from theanalytical method validation.Acceptability of variability of results forparameters, such as accuracy and precision for chemical methods at typicalresidue levels, are generally much broader than in a typical potency assay.Relative standard deviation (RSD) requirements of 15-20% are typical.
分析方法验证方案可以只包括残留物溶液的验证，也可以包括取样回收率研究，尽管取样回收率研究可以独立于分析方法验证而单独进行。

各验证项目（例如在一典型的残留物水平化学方法的准确性、精密度）的结果的变动范围一般要远宽于含量测定的要求，通常相对标准偏差（RSD）要求为15-20%。

7.7.2Compendial Methods
药典方法
Compendialmethods do not require separate analytical method validation provided thosemethods are used within the parameters in the compendia. For example, acompendial method
for endotoxin is generally appropriate for measuringendotoxin in final rinse water samples. However, suitability of use ofcompendial methods should be addressed.
在规定的参数范围内使用药典方法不需要再单独进行分析方法验证，例如使用药典测定内毒素的方法就可以用来测定最终淋洗水中的内毒素。

然而，应该说明使用药典方法的适用性。

Whenusing swab or rinse samples with a compendial analytical method, items thatshould be considered for suitability of use include the validated range,possible interferences from the cleaning process, possible interference fromthe swab, and recovery of residue from the swab (see Section 6.1.3).
当使用药典方法检测棉签擦拭或淋洗样品时，需要考虑方法的适用性，包括验证的范围、清洁工艺可能带来的干扰、棉签的干扰和从棉签回收残留物时的回收率（见6.1.3节）
Whenusing TOC in rinse-water samples (a compendial method), additional work shouldbe done to support its applicability for test samples where the TOC valuescould be above 500 ppb or where a linear range is to be established. Justperforming system suitability as specified in the USP requirement may not beadequate to demonstrate that the TOC analytical procedure could accuratelyanalyze samples at 1 ppm or 5 ppm. For that reason, analytical methodvalidation as for any other method should be considered. An additional reasonfor formal method validation for TOC in rinse-water samples is that the USPmethod is essentially set up as a pass/fail test, not as a quantitative assay.
使用TOC（一种药典方法）测定淋洗水取样时，如果TOC的结果可能大于500ppb或需要建立线性范围时，仍需要做一些额外的工作
以证明该TOC方法适用于测试样品。

只是按照USP要求进行系统适用性测定，不能充分证明TOC方法可以准确测量浓度为1ppm或5ppm 的样品。

因此，可以考虑其他方法和方法验证。

另一个使用TOC测定淋洗水样品需要进行正式方法验证的原因是USP方法实质是一种合格/不合格的测试方法，而不是一个定量的含量测定方法。

7.7.3Visual Inspection
目视检查
Methodvalidation in this case is actually the determination of a quantitative “visuallimit” where visual examination is the sole sampling/analytical method and “visually clean” is used as the sole acceptance criterion for thegiven residue in the absence of swab or rinse sampling for that residue.If visual examination is used to supplement swab or rinse sampling, suchdetermination of a visual limit is not required. A visual limit underspecified viewing conditions can be determined by spiking coupons of the equipmentsurface materials with solutions of the residue at different levels (in μg/cm2)and having a panel of trained observers determine the lowest level at whichresidues are clearly visible across the spiked surface. The significance ofsuch a visual limit is that if equipment surfaces are determined to be visuallyclean under the same (or more stringent) viewing conditions in a cleaningvalidation protocol, the level of the residue is below the visual limit.Appropriate viewing conditions include distance, lighting and angle. The visuallimit depends on the nature of the residue as well as the natureof the surface(e.g., stainless steel vs. PTFE).
对于目视检查，方法验证实际是建立一个定量的“可视限度”，而目视检查是唯一的取样/分析方法，在没有棉签和淋洗取样的情况下，“目视清洁”是目标残留物的唯一的可接受标准。

如果目视检查只是
作为棉签/淋洗取样的补充，则不需确定目视限度。

特定观察条件下的目视限度可以通过将不同浓度的（μg/cm2）残留物溶液涂布在材质试样表面，然后让接受过培训的观察者来确定材质试样表面上有明显可见残留的最低残留水平。

这种目视限度的意义在于，在清洁验证方案相同的（或更严格的）观察条件下设备表面如果是目视清洁的，则说明残留物的水平是在目检限度之下。

适宜的观察条件包括距离、光线和观察角度。

目视限度取决于残留物的性质和设备的性质（例如，不锈钢或PTFE）。

7.7.4Bioburden Methods
生物负载法
Approvedand qualified microbiological lab procedures do not require additional methodvalidation for use in cleaning validation programs. However, suitability foruse of such methods in the presence of cleaning process chemicals should beaddressed (30).
在清洁验证中，经过批准和确认的微生物检验方法不需要进行额外的方法验证。

然而，应当说明存在化学清洁剂时使用这些方法的适用性。

7.7.5Transfer to another Laboratory and Use of Contract Laboratories
方法转移到另一个实验室和合同实验室的使用
Otherlaboratories (other than the laboratory that originally validated a method) canbe utilized to perform an analytical method for cleaning validation purposes.In such cases, a method transfer protocol should be established and executed todetermine that the other laboratory can suitably analyze samples using thatmethod. If a method is developed by a contract laboratory and qualification runsamples are analyzed by
that contract laboratory, then no transfer protocol isrequired. It is preferable that analytical method validation protocol bereviewed and approved by the pharmaceutical company prior to execution of thatprotocol. Care should be used in the transfer protocol to first determine whetherthe measurements between the two laboratories are practically significantbefore any determination of statistical significance is performed (31).If an analytical method has been developed and validated previously bythe contract laboratory, then the pharmaceutical company should review that protocoland the final report to determine the acceptability of the method for its (new)intended use as well as perform an audit of the contract laboratory.
其他实验室（即不是原来进行方法验证的实验室）也可以进行清洁验证的相关检测，此时，应当建立一个方法转移方案，验证其他实验室能够采用该方法进行样品检测。

如果方法是由合同实验室开发的，并且样品检测也在合同实验室进行，此时就不需要进行方法转移了。

方法验证方案在执行前应得到药品生产企业的审核和批准。

在方法转移方案的执行中应注意的是，在进行显著性差异判断前，首先要看看不同实验室之间测定的结果是否存在较大差异（31）。

如果分析方法之前是在合同实验室开发和验证的，药品生产企业应审核验证方案和最终的报告，以确定该方法是否适用于使用目的，并进行合同实验室审计。