1.1 此实践确保6.0日志 10 去除逆转录病毒（例如MuLV）。 1.2 本规程适用于单克隆抗体（mAb）、免疫球蛋白G（IgG）融合蛋白、重组蛋白或使用哺乳动物细胞系（例如中国仓鼠卵巢（CHO）、小鼠杂交瘤、小鼠骨髓瘤或人类胚胎肾（HEK）293）生产的其他蛋白。 1.3 该步骤在无细胞中间体上进行。 1.4 小型病毒截留过滤器对逆转录病毒的原木去除要求可与其他清除单元操作（例如，低pH值灭活或表面活性剂灭活病毒）结合使用，以确保对潜在病毒污染物进行充分的全过程清除，这将支持早期阶段（临床1期或2a期试验）监管备案。 1.5 根据PDA技术报告病毒过滤中的定义，通过过滤去除逆转录病毒的声明仅限于小型病毒滞留过滤器 ( 1. ) 2. 在本标准的上下文中。 1.6 以国际单位制表示的值应视为标准值。本标准不包括其他测量单位。 1.7 本标准并不旨在解决与其使用相关的所有安全问题（如有）。本标准的使用者有责任在使用前建立适当的安全、健康和环境实践，并确定监管限制的适用性。 1.8 本国际标准是根据世界贸易组织技术性贸易壁垒（TBT）委员会发布的《国际标准、指南和建议制定原则决定》中确立的国际公认标准化原则制定的。 =====意义和用途====== 3.1 哺乳动物细胞系广泛用于生产生物治疗剂，如单克隆抗体和其他重组蛋白。其中一些细胞系，如啮齿类动物细胞系，已知含有编码内源性逆转录病毒的基因- 类似颗粒或产生内源性逆转录病毒，但没有证据表明啮齿类逆转录病毒与人类疾病之间存在关联。不定病毒可以从其他来源引入到药物生产过程中，人类治疗方法的污染是一个安全问题 ( 3. ) . 3.2 病毒过滤是病毒清除平台中与低pH值或表面活性剂灭活等步骤正交的技术，传统上被认为是设计良好的病毒清除方法。大小排除已被证明是通过病毒截留过滤去除病毒的主要机制，也就是说，较大的病毒比细小病毒（如细小病毒）更容易被截留 ( 4. , 5. ) 大量的病毒滞留也被证明对工艺流体特性（如蛋白质类型、蛋白质浓度、pH值和离子强度）不敏感 ( 4. , 6. , 7. , 8. , 9 , 10 ) 相比之下，对于小型病毒来说，诸如流暂停和/或通量衰减等方面会影响清除 ( 4. , 6. , 11 ) . 3.3 大型病毒滞留过滤器或逆转录病毒过滤器用于去除较大的包膜病毒，如逆转录病毒或MuLV（80 nm至100nm），并且具有检测不到的大噬菌体PR772（64 nm至82 纳米） ( 1. ) 小型病毒截留过滤器或细小病毒过滤器旨在去除细小病毒，如MMV（18 nm至26 纳米） ( 1. ) 由于大小排除已被证明是病毒保留的机制，逆转录病毒（比细小病毒大三到四倍）应足够大，以通过设计用于去除细小病毒的所有小型病毒保留过滤器完全保留，滤液中的逆转录病毒水平无法检测。 3.4 包括过去20年数据在内的许多已发表的研究和综述表明，无论是大的还是小的病毒截留过滤器都能有效且一致地去除逆转录病毒。在1990年至2010年对监管提交的公开审查中，无论是大型还是小型病毒截留过滤器，都很少出现逆转录病毒突破。 然而，这些异常并未得到解决，可能归因于研究设计、实验伪影或对监管提交文件进行的荟萃分析的局限性 ( 12 ) 在向Paul Ehrlich研究所（PEI）提交的89份材料的摘要中，使用大型或小型病毒截留过滤器的过程显示，没有检测到来自大型病毒的任何传染性颗粒 ( 12 ) 对八家生物制药公司的病毒过滤结果进行的收集表明，在所有198项实验中，任何小型病毒截留过滤器都没有出现大的病毒突破 ( 7. ) 此外，最近对两个病毒清除测试机构20多年的小型病毒滞留过滤器实验的回顾显示，只有0。 61 % （2311次实验中的14次）对较大病毒进行的病毒过滤研究发现了可检测到的复制病毒 ( 10 ) 该手稿进一步表明，所有的阳性结果都不是由于整体式小型病毒截留过滤器的病毒突破，而是由于其他原因，包括病毒检测分析、过滤过程中病毒的雾化、飞溅、溢出或非整体式实验室规模过滤器的潜在使用。 3.5 来自删失数据集的模块化索赔的合理LRV水平，即所有观测值低于某一LRV水平可能难以估计 ( 13 ) ，并且几乎总是低估了实际值。来自删失数据的LRV近似值受病毒刺突体积、病毒刺突滴度、载量以及对产品基质的分析敏感性的影响，此外还受小病毒滞留过滤器性能的影响。在Mattila综述中，198个实验中未检测到大病毒，小型病毒截留过滤器显示Reo3（一种中等病毒）的平均清除率＞5.86 LRV±0.91（60 纳米至80 nm），表明对较大逆转录病毒MuLV的清除率至少相等或更好 ( 7. ) 对于根据审查值创建模块化索赔，Stuckey等人。 提出了一组删失数据中的最高LRV可用于模块化索赔 ( 6. ) 在这些实验中，掺有细小病毒和逆转录病毒的负载材料被用于挑战小型病毒截留过滤器。观察到细小病毒突破，但未检测到逆转录病毒突破，进一步支持基于大小排除的病毒保留机制和使用小型病毒保留过滤器的逆转录病毒保留的鲁棒性 ( 6. ) 此外，即使是设计用于保留更大病毒且平均孔径比小病毒保留过滤器大的大型病毒保留过滤器，也被分类为具有保留>6 log PR772的能力 nm至82 在病毒过滤研究中，纳米噬菌体常被用作逆转录病毒的替代品 ( 1. , 14 ) 。这些大型病毒过滤器已显示可清除PR772的>8个日志 ( 15 ) ，根据定义，清除超过6个日志 10 逆转录病毒的 ( 12 ) 这些已发表的数据共同支持了逆转录病毒（MuLV）的小病毒保留过滤器>6.0 LRV的模块化要求。 3.6 实施由该实践建立的小病毒滞留过滤的参数可以提供鲁棒的逆转录病毒去除，并且可以用作病毒过滤步骤的模块化逆转录病毒验证。结合其他清除单元操作（例如，层析和pH或表面活性剂灭活），可以实现充分的逆转录病毒清除 ( 3 ) .

1.1 This practice assures 6.0 log 10 removal of retrovirus (for example, MuLV). 1.2 This practice is applicable to monoclonal antibody (mAb), immunoglobulin G (IgG) fusion proteins, recombinant proteins, or other proteins produced using mammalian cell lines (for example, Chinese hamster ovary (CHO), murine hybridomas, murine myelomas, or human embryonic kidney (HEK) 293). 1.3 The step is performed on cell-free intermediates. 1.4 The log removal claim for retrovirus by small virus retentive filters can be used in conjunction with other clearance unit operations (for example, low pH inactivation, or inactivation of virus by surfactant) to assure sufficient total process clearance of potential virus contaminants, which would be supportive of early phase (clinical phase 1 or phase 2a trials) regulatory filings. 1.5 Retrovirus removal claim by filtration is limited to small virus retentive filters, as defined in the PDA Technical Report Virus Filtration ( 1 ) 2 in the context of this standard. 1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. 1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. 1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee. ====== Significance And Use ====== 3.1 Mammalian cell lines are widely used in the production of biological therapeutics, such as monoclonal antibodies and other recombinant proteins. Some of these cell lines, like rodent cell lines, are known to contain genes encoding endogenous retroviral-like particles or produce endogenous retrovirus, but there is no evidence of an association between rodent retrovirus and disease in humans. Adventitious viruses can be introduced into a drug substance manufacturing process from other sources, and contamination of human therapeutics is a safety concern ( 3 ) . 3.2 Virus filtration, an orthogonal technology in a virus clearance platform to such steps as low pH or surfactant inactivation, has traditionally been accepted as a robust method for virus clearance when well designed. Size exclusion has been shown to be the primary mechanism of virus removal by virus retentive filtration, that is, larger viruses are more easily retained than smaller viruses such as parvoviruses ( 4 , 5 ) . Large virus retention has also been shown to be insensitive to process fluid characteristics such as protein type, protein concentration, pH, and ionic strength ( 4 , 6 , 7 , 8 , 9 , 10 ) . In contrast, for small viruses, aspects like flow pausing and/or flux decay can impact clearance ( 4 , 6 , 11 ) . 3.3 Large virus retentive filters, or retrovirus filters, are tested for removal of larger enveloped viruses like retrovirus or MuLV (80 nm to 100 nm) and have undetectable levels of the large bacteriophage PR772 (64 nm to 82 nm) ( 1 ) . Small virus retentive filters, or parvovirus filters, are designed to remove parvovirus, like MMV (18 nm to 26 nm) ( 1 ) . Since size exclusion has been demonstrated as the mechanism of virus retention, retroviruses, which are three to four times larger than parvoviruses, should be large enough to be completely retained, with undetectable levels of retrovirus in the filtrate, by all small virus retentive filters designed to remove parvovirus. 3.4 Numerous published studies and reviews encompassing data from the last 20 years have shown both large and small virus retentive filters are effective and consistent for removal of retrovirus. In published reviews of regulatory submissions from 1990 through 2010, rare occurrences of retrovirus breakthrough did occur across both large and small virus retentive filters. These anomalies, however, were not resolved and could be attributed to study design, experimental artifacts, or limitations of the meta-analyses performed on the regulatory submission ( 12 ) . In a summary of 89 submissions to Paul Ehrlich Institute (PEI), processes using either large or small virus retentive filters showed no detection of any infectious particles from large viruses ( 12 ) . A collection of viral filtration results across eight biopharmaceutical companies showed no large virus breakthrough across any small virus retentive filter for all 198 experiments reviewed ( 7 ) . Additionally, a recent review of 20 plus years of small virus retentive filter experiments from two viral clearance testing houses showed only 0.61 % (14 out of 2311 experiments) viral filtration studies performed with larger viruses had detectable, replicated virus ( 10 ) . This manuscript further suggests that all positive results were not due to viral breakthrough of integral small virus retentive filters, but rather to other causes that included virus detection assay, aerosolization of virus during filtration, splashes, spills, or potential use of non-integral laboratory scale filters. 3.5 The level of justifiable LRV for a modular claim from a censored dataset, that is, all observations are below a certain LRV level, can be difficult to estimate ( 13 ) , and is almost always an underestimation of an actual value. LRV approximations from censored data are influenced by viral spike volume, viral spike titer, load volume, and assay sensitivities to product matrices, in addition to small virus retentive filter performance. In the Mattila review, where no large virus was detected in 198 experiments, small virus retentive filters showed an average clearance >5.86 LRV ±0.91 for Reo3, a medium-sized virus (60 nm to 80 nm), suggesting at least equal or better clearance for the larger retrovirus, MuLV ( 7 ) . For creation of modular claim from censored values, Stuckey, et al. proposed that the highest LRV in set of censored data could be used for a modular claim ( 6 ) . In these experiments, load material spiked with both parvovirus and retrovirus was used to challenge small virus retentive filters. Parvovirus breakthrough was observed but no retrovirus breakthrough was detected, further supporting both a size exclusion-based mechanism of virus retention and the robustness of retrovirus retention using small virus retentive filters ( 6 ) . Additionally, even large virus retentive filters, designed to retain larger viruses and having on average larger pore sizes than small virus retentive filters, are classified as having the ability to retain >6 logs of PR772, a 64 nm to 82 nm bacteriophage often used as a surrogate for retrovirus, in virus filtration studies ( 1 , 14 ) . These large virus filters have been shown to clear >8 logs of PR772 ( 15 ) , and by definition clear more than 6 log 10 of retrovirus ( 12 ) . These published data, collectively, support a modular claim for small virus retentive filters of >6.0 LRV for retrovirus (MuLV). 3.6 Implementing parameters of small virus retentive filtration established by this practice can provide robust retrovirus removal and can be used as a modular retrovirus validation of the virus filtration step. In conjunction with other clearance unit operations (for example, chromatography and inactivation by pH or surfactants), sufficient overall retrovirus clearance can be achieved ( 3 ) .