【纯干货】清洁验证可接受限度-下（中英文版）（PDA TR 49内容节选4）

1、【纯干货】PDA TR49 生物制品清洁验证考虑要点（中英文版）节选1（1.0-2.0）
2、【纯干货】清洁程序的设计与开发（中英文版）（PDA TR 49内容节选）
3、【纯干货】清洁验证可接受限度（中英文版）（PDA TR 49内容节选3）
4.2 limits for Cleaning agentsLimits 
Limits  for cleaning agents will also depend on the stage of manufacturing (formulation/fill vs. bulk activemanufacture). Typical cleaning processes for biotechnology involve either a caustic wash followed by aphosphoric acid step, or an alkaline detergent followed by an acidic detergent. Each of these situationswill be handled separately.
4.2 清洁剂的限度
清洁剂的限度也取决于生产的各个阶段（制剂/分装与原液生产） 。典型的生物制药清洁程序包括强碱清洗以及下一步的磷酸物的清洗，或者是碱性清洁剂以及随后的酸性清洁剂。这些过程都是分开处理的。
4.2.1 limits for Commodity Chemicals
If only commodity chemicals such as sodium hydroxide and phosphoric acid are used for cleaning, it iscommon practice to set limits for these indirectly as a conductivity value. Preferably, an acceptable levelof sodium hydroxide or phosphoric acid is established based on toxicity carryover calculations or based onthe effects on process parameters. The conductivity limit is then set at a level equivalent to thatconcentration at a specified temperature. It is typically the case that the conductivity limit established in this way is well above the conductivity limit for WFI at the same temperature. Therefore, limits are basedon either WFI specifications or on a slightly higher value (such as 5 μS/cm). The rationale for this is that itis more stringent than the “scientific” calculation allows. Furthermore, in many cases, phosphate ionsand/or sodium ions may be part of any subsequently manufactured product. Therefore, carryover of smallamounts is not significant. It should also be noted that calculations based on toxicity of the commoditychemicals are extreme, since sodium hydroxide is not carried over into a final product as sodiumhydroxide, and phosphoric acid is not carried over into final product as phosphoric acid. If such chemicalswere carried over intact at high concentrations, process checks (such as a significant change in pH) wouldalso cause a non-conformance. Thus, the purpose is not to confirm compliance with WFI specifications,but rather to confirm low amounts of a cleaning agent. The rationale for allowing a conductivity limitslightly higher than the WFI limit is that the WFI limit applies to water in the recirculating WFI loop. Assoon as the water is taken out of that loop and passed through clean equipment (particularly throughspray devices where it can pick up carbon dioxide from the air), there is no expectation that it willnecessarily meet the WFI conductivity limit.
4.2.1 市售化学试剂的限度
如果只有市售化学试剂如氢氧化钠和磷酸用于清洁，那么通常的做法是用电导率的值为其设定间接的限度。更好的做法是，氢氧化钠或者磷酸的可接受水平建立在毒理学的污染量计算或者是工艺参数的效果之上。然后电导率限度的设定水平就等同于在特定温度下的那个浓度。这种情况下用这种方法设置电导率的限度，通常要好于在相同温度下注射用水的电导率限度。因此，限度是基于注射用水的标准或者是稍高于这个标准（例如 5 μ S/cm） 。这种做法的原因是它要比“科学”计算的限定值更加严格。此外，在许多情况下，磷离子/或钠离子可能是随后制造出来产品的一部分。因此，少量的污染量并不明显。还应指出的是，基于市售化学试剂的毒性计算是比较极端的，因为氢氧化钠并不会以氢氧化钠的形式污染到最终的产品中，磷酸也不会以磷酸的形式污染到最终产品的。如果这些化学物质以高浓度完整地污染下来，通过工艺检查（这样会引起 PH 的显著变化）也会发现这种不符合性。因此，目标并不要求与注射用水的标准一致，而是确认一个很低的清洁剂含量。而允许电导率限度略高于注射用水限度的原因是，注射用水的限度要求只是适用于管路中的循环注射用水。一旦从循环管路中取出水并通过清洁设备（尤其是通过喷淋装置时它可以从空气中摄取二氧化碳） ，不要期望它一定会满足 WFI 的电导率限度。
4.2.2 limits for Formulated Cleaning agents
For formulation/fill manufacturing, limits for residues of formulated cleaning agents (which may contain avariety of organic components in addition to inorganic hydroxide) are typically set based on a carryovercalculation using the short-term toxicity (LD 50 ) data for that formulated cleaner. Such toxicity informationmay be supplied by the cleaning agent manufacturer or may be calculated using worst-case assumptionsbased on an analysis of the formulation components. A typical calculation is given in Example 3 of Section15.0 of the Appendix. For the determination of the limit in the next drug product, a default value for theformulated cleaning agent may also be used if it is more stringent than the carryover calculation. Thatdefault value is typically 10 ppm formulated cleaning agent. Note that the only difference between acarryover calculation for the active and a carryover calculation for the formulated cleaning agent is howthe limit in the subsequent product is calculated – one uses a fraction of a dose and one uses a fraction ofthe LD 50 . Once that calculation is performed, subsequent calculations for cleaning agents use the sameformulas as for actives to determine the limit per swab or the limit per analytical sample.For manufacturing of bulk actives, some of the same issues discussed for limits of actives also apply tocleaning agents. That is, a carryover calculation considering the total shared surface area results inextremely low limits, typically not measurable. However, the formulated detergents typically used may beremoved by various purification processes (such as ultrafiltration or size exclusion chromatography) based on molecular weight. As with limits for actives, toxicity calculations may be applicable for downstreamprocesses following the purification steps or for the last downstream process vessel.
4.2.2 处方清洁剂的限度
对于制剂/分装制造，处方清洁剂（可能在无机氢氧化物中添加很多有机组分）残留量的限度通常是依据其短期毒性（LD50）数据而计算污染量的。这种毒性资料可提供的清洁剂制造商或可使用的计算制订成分分析的基础上最坏情况的假设。这种毒理学的资料可由制造商提供或使用基于处方成分分析得出的最差情况而计算。在附录的 15.0 章节中给出的第三个例子是一个典型的计算。为了确定下一个药物产品中的限度值，如果处方清洁剂的默认值比污染量计算的值更加严格，也可以采用。处方清洁剂的默认值一般为 10ppm。请注意活性物污染量计算与处方清洁剂污染量计算的差异在于对后续产品的污染限度计算方法不同，一个使用的是剂量比率，而另一个用的是半致死率 LD50 比率。一旦完成计算，随后的清洁剂计算使用与活性物同样的公式，来确定每个拭子或每个分析样品的限度。对于原液生产，为活性物限度而讨论的一些问题也同样适用于清洁剂。也就是说，考虑到整个共享面积的污染量计算值是极低的，通常难以测量。但是，使用的处方洗涤剂通常在分离提纯等过程中可根据分子量去除（例如超滤或者是分子排阻色谱法） 。就像活性成分的限度一样，毒性计算可能适用于下游纯化后的步骤或下游工艺的最后一个容器。
4.3 Bioburden limits
In considering bioburden limits following cleaning, it is not expected that the cleaning process itselfresults in sterile equipment. However, even if the process equipment is steamed in place or autoclavedprior to manufacture of the next product, it is typically the practice to evaluate bioburden to establishthat the subsequent process is not overly challenged. Achievement of typical bioburden limits fornon-sterile manufacturing (1-2 CFU/cm 2 for surface sampling methods) is considered more than adequate.For rinse sampling, some companies will utilize typical WFI values (10 CFU/100 mL), while others willutilize a value of either 100 CFU/100 mL or 1,000 CFU/100 mL. The rationale for the higher limit is thatthe equipment will be subsequently sterilized. Furthermore, the WFI value is the value for the WFI in therecirculating loop; once it is removed from that loop and placed in clean equipment, there is notnecessarily an expectation that it will still meet the WFI value.
4.3 微生物限度
考虑到清洁后微生物的限度，不要预期清洁过程本身会导致设备的无菌结果。然而，即使如果设备进行在位灭菌或者是在下一产品生产前灭菌，通常的做法是评估微生物负载来确保随后的生产工艺不会过度挑战。只要达到一般的非无菌生产微生物限度标准（1-2CFU/cm2 表面取样方法）就已经是足够的。对于冲洗取样，一些公司将利用典型 WFI 的值（10 CFU/100 毫升） ，而其他公司将利用 100CFU/100 毫升或 1,000 CFU/100 毫升的值。设定较高的限度原因是随后设备将进行灭菌。此外，WFI 的值是在管路中循环的注射用水值，一旦它从循环管路中取出并加到设备中时，并不要期望它能必然符合原有的注射用水值。
4.4 endotoxin limits
Endotoxin carryover to the final product is more of a concern after the various endotoxin removal steps.In those cases, endotoxin is typically measured only in the final rinse water, and limits are set at thetypical WFI limit of 0.25 EU/mL. Prior to the endotoxin removal steps for cell culture processes, it can beexpected that rinse samples should meet the WFI limit. For bacterial fermentation withE. coli (a gram-negative bacterium which will produce large amounts of endotoxin in the washing step),meeting the WFI limits following cleaning at the fermentation and harvesting steps may not be achievable.For that reason, some companies may not set endotoxin limits for cleaning for those steps, while otherswill set endotoxin limits but at a higher value, such as 5-25 EU/mL. Achieving such values indicates ameasure of control in the cleaning process, and any possible carryover at those levels should be addressed by subsequent endotoxin removal process steps.
4.4 内毒素限度
在内毒素经各个去除步骤后，内毒素对最终产品的污染量是更值得关注的。在这种情况下，内毒素通常只在最后的冲洗水测定，限度设定通常是注射用水标准 0.25EU/ml。在细胞培养过程的内毒素去除步骤之前，冲洗水取样预期应该符合注射用水限度。对于用大肠杆菌进行的细菌发酵（革兰氏阴性细菌， 在冲洗过程中能够生产大量的内毒素） ， 在发酵和收获步骤后的清洗符合注射用水限度，是不可能完成的，这种情形下，其它公司在更高的水平设置内毒素限度，例如 5-25 EU/mL。实现这一水平表明清洁工艺得到控制，任何在这种水平上的可能污染量应在随后的内毒素清除工序中评价。
4.5 Visual Clean Criterion
The visual appearance of production surfaces is a direct measurement that verifies removal of residuals.Visual appearance is not a quantitative method but is very useful to directly verify that productionsurfaces are clean. Visual appearance is easy to perform provided there is ready access to the criticalsurfaces. It verifies the cleanliness of a significant area of production equipment. Literature indicates thatlow levels of residues (if present) are visible and can be detected. As used in biotechnology manufacturing,a visual clean criterion is typically used with swab and/or rinse testing for residues for cleaning validationprotocols.
4.5 视觉清洁
标准产品表面的外观目测是核实残留物去除的一种直接衡量手段。外观目测也不是一种定量的方法，但是对于直接检查产品表面清洁非常有用。只要有路径可以到达关键的表面，外观目测很容易完成。它能确认生产设备重要区域的洁净程度。文献表明少量的残留 （如果存在） 是可见的和可被察觉的。如同在生物制药中所使用的， 目测清洁标准能典型地用做清洁验证方案中的擦拭和/或冲液中残留物的测试。
4.6 modifying limits
Manufacturers may establish action and/or alert levels on validated processes as part of routinemonitoring. Those values are typically more stringent than the pass/fail limits in the validation protocol.Based on process capability showing consistently low values and the ability to maintain those values,manufacturers may perform a risk assessment and consider the use of more stringent limits for futurevalidation protocols.
4.6 修改限度
制造商可能会设立行动和/或警戒限度， 作为日常工艺验证监测的一部分。这些值通常比验证方案中的合格/失败限度更加严格。依据工艺能力显示连续的低水平和维持这些水平的能力， 制造商可以进行风险评估并考虑为将来的验证方案采用更加严格的限度。

       文章来源：允咨生物GMP学苑

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