清洁验证之分析方法上中英文版PDA TR 49内容节选6.docx

1、清洁验证之分析方法上中英文版PDA TR 49内容节选6清洁验证之分析方法-上(中英文版)（PDA TR 49内容节选6）6.0 Analytical Methods分析方法It is essential to a cleaning validation program that the appropriate analytical methods are utilized.一个清洁验证程序使用适当的分析方法是非常必要的。Analytical methods must be appropriate in that they can adequately detect the residue(s

2、) of concern.分析方法必须适当，能充分检测到相关残留物。It is also important to understand what can be concluded from the analytical result (e.g., was the productnot removed or was the cleaning agent not removed?).对能从分析结果中推断出什么的理解也是非常重要的（比如：产品没有被去除或清洗剂没有被去除？） 。The results of testing will determine if the cleaning validat

3、ion cycle is acceptable or if it needs to beredeveloped.检测结果将决定清洁验证周期是否接受或者是否需要重新开发。Thus, it is important to have confidence in the results.因此，对结果的信任是非常重要的。This section discusses how to select the appropriate assay methods, detailed information on theapplicability and use of nonspecific assays and m

4、icrobial test methods,and assay method validation.本部分讨论怎样选择合适的分析方法及其适用性的详细信息，非特定分析和微生物测试方法的使用，和分析方法验证。本部分套乱怎样选择合适的含量分析方法，实用性的详细信息，非特定含量方法和微生物测试方法以及含量方法验证。6.1 Specific Analytical Methods6.1 特定分析方法Specific analytical methods are those which measure a certain residue in the presence of expectedinterfer

5、ences.特定分析方法是指用于测量存在预期干扰时某个残留的方法。In a cleaning process for biotechnology products where the specific analyte is the active protein, suchinterferences may include degradation products and related substances, excipients, cleaning agents andcleaning agent by-products.在生物技术产品的清洗过程中，特定分析物是活性蛋白，这样干扰可能包括降解

6、产品和相关物质，辅料，清洗剂和产品清洗剂。Examples of specific methods include HPLC, ELISA,SDS PAGE, and PCR.特定方法的例子包括 HPLC,ELISA,SDSPAGE,和 PCREach of these methods requires the use of an appropriate reference standard.这些方法每个都需要使用一个适当参考标准。In contrast, nonspecific analytical methods measure a general property, such as co

7、nductivity orTOC,which could be due to a variety of analytes or sources.相对而言， 非特定分析方法测量通用属性， 比如：电导率或者 TOC， 因为分析物或者来源的多样性。Selection of an analytical method will depend on the nature of the residue as it exists after the cleaningprocess.一个分析方法的选择将取决于清洁过后残留物的性质。Only if a protein (or other organic activ

8、e) is not degraded during the cleaning process (surviving hightemperatures and pH extremes in an aqueous environment, for example) does it make sense to use a specific analytical method for that active.只有当一种蛋白质（或者其他有机活性物质）在清洁过程中不能被降解（例如：在高温和极端 PH 的水环境下残存） ，使用那种物质的特定分析方法才有意义。The advantage of using a

9、specific analytical method in this situation is that it gives a precise measure ofthe major residue of concern the active itself.在这种情况下使用特定分析方法的优势是它可以给出主要相关残留-活性物本身的精确测量。If a specific analytical method for an active protein were utilized following a cleaning process which hasbeen demonstrated to den

10、ature (degrade) that active protein, it is likely that residues of the activeprotein would be non-detectable (i.e., not measurable) by that specific analytical method.如果一个活性蛋白质的特定分析方法在紧接着的清洁过程中被使用，这个清洁过程用来证明降解（变性）这个活性蛋白质。这个活性蛋白残留通过这个特定分析方法将很可能检测不到（不能被检测） 。Residues of that protein would be various de

11、graded fragments.那个蛋白质的残留可能讲解成多种片段。If the native protein were actually detected using a specific method for that protein, it is likely that therehad been a serious problem with the cleaning process, such as a clogged spray device causing a lack ofcoverage of that portion of the equipment surface.如果天

12、然的蛋白质真的用特定方法被检测到，那么清洁过程很有可能有一系列的问题，比如堵塞喷淋设备引起的设备表面喷淋覆盖不全面。In such a case, failure would also most likely be detected by a nonspecific method and/or by visualexamination.在这种情况下，失败也可以通过非特定方法或目视检察被检测到。Consequently, if a specific assay method is used, a nonspecific assay method is also required, unlesss

13、tudies prove that the product is not degraded by the cleaning process.因此，如果一个特定的含量分析方法被使用，一个非特定的分析方法也是需要的，除非研究证明清洗过程中产品没有被分解。In biotechnology cleaning validation, specific analytical techniques such as HPLC are more likely to beused for detergents, because the surfactants or other functional materia

14、ls in the detergents are not likelyto degrade in the cleaning process.在生物技术清洁验证中，特定分析技术比如 HPLC 更倾向用于清洗剂的检测，因为表面活性剂或者清洁剂中的功能材料在清洁过程中不可能分解。However, it should be noted that nonspecific methods can also be used for detergents and other cleaningagents.无论怎样，必须注意非特定方法也可以用于洗涤剂或者其他清洁剂6.2 Impact of Inactivati

15、on/Degradation of the Active6.2 活性物质的降解/ 灭活的影响Product inactivation means that the active protein is modified in some way such that it is no longer activeand may no longer be measurable by specific analytical methods for that native protein.产品灭活意味着活性蛋白被某些方式改变，这样这些蛋白不再有活性和不再被这种天然蛋白质的特定分析方法测量到。This mod

16、ification usually involves degradation of the active protein into smaller fragments, but may alsoinvolve a process in which larger molecules are formed.这个改变通常包括活性蛋白降解成小片段，但也可以包括蛋白大分子的形成过程。A key issue for process equipment cleaning in biotechnology manufacturing is the degradation ordeactivation of t

17、he active protein during the cleaning process.在生物技术产品生产中，工艺设备清洁的一个关键问题是活性蛋白在清洁过程中的降解或者钝化。This is a result of cleaning processes in biotechnology utilizing hot, aqueous,alkaline and acidic cleaningsolutions.这是生物技术产品的清洗过程使用热，水，碱和酸的清洗溶剂的结果。Under such conditions, it is well recognized that protein activ

18、es will degrade.在这种条件下，将要降解的蛋白质活性物是容易被识别的。This degradation affects several issues in the cleaning and cleaning validation process.这个降解物在清洁和清洁验证过程中的影响是多方面的。Because of the degradation, the residues of the active protein (which are actually now residues of thedegraded active protein) are more readily r

19、insed away during the rinsing step of the cleaning process.因为降解，活性蛋白的残留（实际上是活性蛋白降解物的残留）很容易在清洁过程中的淋洗这步时被淋洗掉。This is because the degraded fragments typically have a lower molecular weight and are potentially morepolar, both conditions leading to greater water solubility.这是因为降解的片段通常分子量小和极性更大，两种条件都导致更好的

20、水溶解性。A second consequence of the degradation is that it no longer is scientifically justified to have an analyticalmethod which is specific for the native protein.降解的第二个结果是使用一个特定的天然蛋白质分析方法不再是科学合理的。For this reason, a nonspecific method such as TOC or Total Protein is typically used to measure residue

21、s ofthe degraded active (as well as other organic molecules) in acleaning validation protocol.基于这个原因，在清洁验证方案中，一个非特定方法（如：TOC 和总蛋白）通常被用于测量降解活性物质（其他有机分子也一样）的残留。A third consequence is that limits in bulk biotechnology manufacturing are typically not appropriatelyestablished based on a “fraction of a dos

22、e” calculation of the native protein, since the residues aredegraded fragments.还有一个结果是生物原料生产中的限度通常不能基于天然蛋白“剂量分数”的计算建立，因为残留是降解片段。Since residues being sampled are residues of the degraded protein, it may also make more scientific senseto perform sampling recovery studies based on recovery of the degra

23、ded fragments.因为残留取样是降解蛋白的残留，所以执行基于降解片段回收率的取样回收研究是更加科学有意义的。However, assuming an increase in solubility for degraded proteins, sampling recovery studies on thenative protein will typically be a worst case as compared to recovery of degraded fragments.假如降解蛋白的溶解性增加，天然蛋白的取样回收研究与降解片段的回收相比，其通常作为最差情况。While

24、 it is assumed in almost all cases that the active proteins or other large organic molecules producedin biotechnology manufacturing are readily degraded in hot, aqueous alkaline conditions, it is desirable todemonstrate this with a laboratory study.几乎所有情况下，活性蛋白或者大型有机分子在生物生产中，在热，水，碱条件下非常容易降解，这只是个假设，因

25、此需要在实验室中证明。In such a simple “beaker” study, the bulk active protein is exposed to the conditions of the cleaningprocess, including cleaning agent concentration,temperature and time.在这样一个简单的“烧杯”研究，活性蛋白原料暴露在清洗过程的条件下，包括清洗剂浓度，温度和时间。At the end of that exposure time, the pH is neutralized, and the tempera

26、ture is reduced.在暴露时间的最后，PH 是中性的，温度是降低的。The resultant solution is then analyzed for the active protein by the specific analytical procedure (such as ELISA, HPLC or a bioactivity assay).最终溶液被特定分析程序分析其活性蛋白质（例如：ELISA,HPLC 或者生物活性含量）In such a procedure, the ratio of protein to cleaning solution should re

27、present the same ratio present during cleaning, or a worst-case ratio (a worst-case is a higher ratio of protein to cleaning solution).在这个过程中，清洁溶液的蛋白质比例应等于清洁过程中蛋白质的比例，或者最差情况蛋白质的比例（最差情况是一个更高蛋白比例的清洁溶液） 。The assay methodology for such studies must be appropriate and valid.这个研究的含量方法学必须是合适的和经验证的。Care nee

28、ds to be exercised in performing such a study to ensure that the chemicals in the cleaningsolution do not interfere with the analytical procedure.为了确保清洁溶液中化学成分不被分析程序干扰，在进行这个研究过程中应特别注意。This can be addressed by having adequate controls, such as adding the active to a solution of theneutralized cleanin

29、g solution at ambient temperature.这可以用充分控制来解决，如：向日常温度的中性清洗溶液加入活性物质。If chemicals in the cleaning solution interfere with the specific analytical procedure, another option is toremove them by diafiltration.如果清洗溶液的化学成分被干扰特定分析程序，另一个选择是通过透析过滤去除它们。If it is just the surfactants in the cleaning agent that i

30、nterfere, another option is to perform thedegradation study with just the equivalent amount of alkali present in the cleaning solution.如果它只是清洗剂中的表面活性剂的干扰，另一个选择是用与清洗溶液中等量的碱进行降解研究。Note that in many cases, cleaning in a biotechnology facility utilizes alkaline cleaning agents followed byan acidic clean

31、ing solution.注意在许多情况下，生物技术设施的清洁碱清洁剂在使用酸清洁溶液之后使用。Current evidence suggests that it is the alkaline portion that is most effective in degrading active proteins.现有的证据证明含碱的部分对降解活性蛋白非常有效。Companies may choose to perform a degradation study only with the alkaline agent and not pursue degradation studies wi

32、th the acidic solution unless the alkaline cleaning agent alone is inadequate for degradation.公司可以选择只使用含碱试剂进行降解研究，不追求使用酸溶液进行降解研究除非单独使用含碱清洁剂对降解不充分。6.3 Nonspecific Analytical Methods6.3 非特定分析方法6.3.1 Total Organic Carbon (TOC)6.3.1 总有机碳Most of the compounds used in biotechnology processes are of organic nature.在生物技术过程中使用的大部分成分是有机物。TOC can detect organic carbon with a good sensitivity in the sub-ppm range;TOC 在亚于 PPM 范围内可以非常灵敏的检测有机碳。however this sensitivity may still not be adequate