清洁验证可接受限度（中英文版）（PDA TR 49内容节选3）
4.0 Acceptance Limits Cleaning
validation is performed to demonstrate the effectiveness and consistency of a cleaningprocedure. The rationale for lecting limits for product residues, cleaning agents and microbialcontamination, as well as any other process components, should be logically bad on the materials thatimpact the manufacturing process and the safety and purity of the product. The acceptable limits forcleaning manufacturing systems and components should be “practical, achievable and verifiable.” (8)Limits for cleaning validation generally contain some measure related to the active protein (or othermajor component of interest), some measure related to the cleaning agent, some measure related tobioburden levels, some measure related to endotoxin levels, and a requirement that the equipment bevisually clean. In addition, if there are any specific toxicity concerns related to the active protein or otherprocess components (for example, cytotoxicity, allergenicity, or reproductive hazards), the manufacturer’stoxicology or pharmacology groups may determine if a modification of li
mits is required, or whether theu of dedicated equipment is needed.In the discussion that follows, issues for limits are considered bad on the nature of the residue and onthe stage of manufacturing (e.g., bulk active vs. formulation/fill). Manufacturing stages include bulk activemanufacturing (all steps resulting in the bulk active drug substance) and formulation/ fill (formulation ofthe bulk active into a finished drug product and primary packaging of that drug product). Bulkmanufacturing is further divided into upstream process steps (all process steps through harvesting) anddownstream process steps (purification and following steps).
4.0 可接受限度
清洁验证的目的是为了证明清洁程序的高效性与一致性。选择产品残留、清洁剂、微生物污染，以及其他任何工艺成分的限度，逻辑上应该基于物料对生产流程和产品的安全性和纯度的影响而定。清洁生产系统和成分的可接受限度应该“切合实际、可达到、可证实”。清洁验证的限度一般包含与活性蛋白 （或者其他的主要物质成分） 相关分析、 与清洁剂相关的分析、与生物负载水平相关的分析、与内毒素水平相关的分析，以及设备目检清洁
要求。另外，如果存在任何与活性蛋白或者其他工艺组分相关的特别毒性物质（例如细胞毒性、变应原性、或者生殖危害物） ，那么生产商的毒理学或生理学团队就需要确定是否有必要改变限度，或者是否需要使用专用的设备。在接下来的讨论中，限度的考虑应该基于自然残留和生产的各个阶段（例如，原液 vs.配制/分装） 。生产阶段包括主要原液生产（所有的步骤是为了获取活性药物）和配制/分装（将原液加入到成品的制剂以及药品物质的初级包装） 。原液的生产进一步分为上游生产步骤（收获药物前的所有步骤）和下游生产步骤（药物纯化以及后续步骤） 。
4.1 key Issues in limits for activesBiotechnology cleaning process often involve a change of the active molecule itlf, which is commonlya protein. Proteins typically are degraded to some extent by the cleaning process commonly ud inbiotechnology manufacturing. The most important mechanism for degradation is summarized below.In alkaline solutions, such as hot, aqueous solutions containing sodium or potassium hydroxide, proteinsmay hydrolyze to soluble oligomers or free amino acids. Ester groups on actives may be hydrolyzed to analcohol and a fatty acid. A common example of this is saponification of fats and oils to glycerol and fattyacid anions.Sodium hypochlorite is som
etimes ud inbiotechnology cleaning.Asacleaningagent,itisparticularly effectivein removing denatured protein residues from surfaces. It is a reactive oxidizer which will degrade proteinsin a more random manner to smaller fragments. A general concern with sodium hypochlorite u is its49 © Inc. 25xpossible deleterious effect on stainless steel components. Therefore, it is critical that the rin cyclefollowing the u of sodium hypochlorite is adequate enough to remove any residual chloride ion beforeadding the subquent acid wash.Proteins will hydrolyze at a high pH. The parameters of time and temperature have a significant influenceon protein hydrolysis. Therefore, the higher the temperature and pH, the more extensive proteinhydrolysis will occur. Becau the protein is typically degraded into smaller fragments and thofragments tend to be more polar, they are likely to be more water soluble and more readily removed fromequipment surfaces during the washing and rinsing process. A cond effect after protein exposure tohigh pH solutions is a possible irreversible, significant decrea of biological activity due to hydrolysis.
4.1 活性物限度的关键问题生物技术的清洁过程经常涉及到活性分子本身的改变，因为这
些活性物质通常是蛋白质。在生物制品制造通常应用的清洁过程中，蛋白质都会在一定程度上发生降解。下面就总结一下最主要的降解机制。在碱性溶液中，尤其是高温，溶液中含有氢氧化钠和氢氧化钾，蛋白质就会水解成可溶的寡聚物或者自由氨基酸。活性物质上的酯旨基团会水解为一个醇和一个脂肪酸，一个最为普遍的例子就是将脂肪和油脂转化为甘油和脂肪酸的皂化反应。次氯酸钠有时会用于生物技术清洁， 作为一种清洁剂， 它能非常有效地从表面去除变性蛋白质残留。
它是一种活泼的氧化剂，它能够使蛋白质更随机地降解为更小的片段。使用次氯酸钠时通常关注其对不锈钢成分的危害作用。因此， 关键的是， 使用次氯酸钠后的漂洗循环能将氯离子残留冲洗干净，然后再进行酸清洁。在高 pH 下蛋白会水解，控制时间和温度参数都会在很大程度上影响蛋白质的水解。因此，pH和温度越高， 蛋白就越容易发生水解。因为蛋白通常降解为更为小的片段， 这些小片段趋向于高度极化，他们更加易于溶于水，也更加容易通过清洁、漂洗的过程从设备表面去除。蛋白质暴露于高 pH 的第二个效果就是，水解导致其生物学活性的下降可能是一个不可逆的、显著的过程。
Degradation of the active can be demonstrated in a laboratory study by exposing the acti
ve to thecleaning solution under simulated cleaning conditions (or less stringent conditions) and performinganalytical and/or biochemical tests on the resultant mixture.For the reasons, in most cas biotechnology manufacturers do not directly t limits for and directlymeasure the active in cleaning validation. Becau of the degradation of the active, no active proteinshould remain after completion of the cleaning process. It is for that reason that analytical methods likeTOC (e Section 6.0) are ud for the detection of protein residues (or their fragments). If a nonspecificmethod like TOC is ud for the correlation to residues of the active, it should be noted that the “real”value of protein residues after cleaning may be significantly lower, as TOC measures all sources of organiccarbon (and not just residues from the active protein).
活性物的降解可以在实验室中验证，通过将活性物质暴露在模拟清洁条件（或者强度更低的条件）的清洁溶液中，然后对生成的混合物进行分析和/或者生物化学的测试。基于这些原因，在大多数情况下生物技术制造商在清洁验证流程中并不直接设置活性限度，或者直接检测活性。由于活性物质的降解，在清洁过程完成后，应该不会有活性物质的残留。正是出于这个原因，人们用像 TOC{总有机碳（Total organic carbon）}的分析方法（见第
6.0 节）检测蛋白质的残留物（或片段）。如像 TOC 的非特异性的方法被用于活性物质残留的检测等相关使用， 应该指出，清洁后真正的蛋白质残留是非常低的，因为 TOC 检测的是所有的有机碳源（并不仅仅是来源于活性蛋白的残余） 。
4.1.1 establishing limits for actives in Formulation and Final FillIn biotechnology formulation/fill manufacturing, limits for protein actives are typically t using acarryover calculation (often called MAC, or Maximum Allowable Carryover) in the same way as for smallmolecule cleaning validation. Though the product is degraded (as discusd above), the calculations arebad on active product. This is assumed to reprent a worst-ca approach if the cleaning method udin formulation/fill results in degradation of the protein active to fragments. Such calculations may berevid bad on degradation considerations.This method only applies when the therapeutic daily do is known. For products dod chronically, atypical calculation allows no more than 1/1000 of the minimum daily therapeutic do of an active in themaximum daily do of the subquent manufactured product; the factor of 1/1000 may be modifieddepending on the specifics of the situation. In addition, if that calculation allows more than 10 ppm of theactive protein i
n the subquent drug product, a limit of 10 ppm active protein in the next drug productmay be utilized. Similar criteria are included as examples in the both the U.S. FDA(8) and PIC/S guidance documents. (9)Limits per surface area can then be calculated bad on the minimum batch size of the next drug productand the shared surface area. Limits in swab and/or rin samples can then be calculated using thesampling parameters.When this method is ud for tting limits, the limit for the active is calculated. It can then be convertedto appropriate units for the analytical procedure to be utilized. For example, if the analytical procedure isTOC, the limit calculated for the active is converted to TOC bad on the TOC content (percentage) of theactive.
An example carryover calculation for formulation/fill is given as Example 1 in Section 15.0 of theAppendix.It should be noted that limits bad on carryover calculations are one example of a “science-bad”method of tting limits. Some companies choo to t limits bad on more stringent criteria, such asthe WFI TOC specification of 500 ppb TOC. Such an approach is acceptable, but should only be ud if itcan be demonstrated t
hat the WFI TOC specification is more stringent than the TOC result, as determinedby a carryover calculation.
4.1.1 在制剂和最终分装中建立活性物限度在生物技术产品的制剂和分装生产中，蛋白质活性限度的设定是用污染（传递）量计算（通常称为 MAC，或最大可允许的传递量） ，跟小分子清洁验证使用同样的方法。虽然产品降解（如上所述） ，计算是基于活性产品的量。如果在制剂/分装所用的清洁方法导致了活性蛋白降解为小分子片段， 该方法就代表了最差的情况。考虑到降解作用，这个计算可以进行修正。只有当每日治疗剂量是已知的，才能应用这种方法。对于长期使用的产品，一个典型的方法是，在随后制造最大活性日治疗剂量的产品中，允许其携带不超过最小活性日治疗剂量的 1/1000；1/1000 的因子可能根据特殊情况而修订。此外，如果计算允许在随后的药物产品中活性蛋白残留量超过 10ppm，可以使用在下一批次的药物产品中，10 ppm 活性药物蛋白的限度。类似的标准在包括美国 FDA（8）和 PIC / S 的指导文件中是有例子的。然后单位面积的限度可以基于下一个最小批量的药物生产所共用的设备面积上计算出来。擦拭或冲洗样品中的限度可以通过采样的参数计算出来。当使用这个方法设定限度时，活性物的限制可以计算出来。然后它可以转换为分析方法适当的单位而被利用。例如，如果分析方法是 TOC，活性物质
的限度可以通过转换为 TOC，即基于 TOC 在活性物质的含量（百分比）而计算出来。在文章的附录的第15 个章节中有一个关于制剂/分装污染量计算的例子。应该指出的是，基于污染量（传递量）计算的限度是设置限度的 “科学根据的”方法之一。一些公司选择基于更严厉的标准设定限度，例如用 WFI TOC 值500ppb 作为标准。虽然这种方式是可以接受的，但是只有证明 WFI TOC 标准要比污染量计算的 TOC 结果更加严格，才可以使用这种方法。
