1.1 这种做法确保了有效的灭活 ≥ 4日志 10 在不针对逆转录病毒抗原的啮齿动物衍生细胞系中制造的单克隆抗体或免疫球蛋白G（IgG）Fc融合蛋白的制造过程中，感染性啮齿动物逆转录病毒（即从10000减少到1个感染性啮齿动物逆转录病毒或去除99.99%的感染性啮齿动物逆转录病毒）的数量。啮齿动物逆转录病毒被用作这些蛋白质生产流中潜在存在的啮齿动物细胞基质内源性逆转录病毒样颗粒的模型。 1.2 本规程规定的参数为澄清、Triton X-100洗涤剂浓度、保持时间、pH值和灭活温度。 1.3 这种做法可以与与这种灭活机制正交的其他清除或灭活单元操作结合使用，以实现啮齿动物逆转录病毒的充分全过程清除或灭活。 1.4 该去污剂失活步骤在单克隆抗体或IgG-Fc融合蛋白的澄清、无细胞中间体上执行。 1.5 以国际单位制表示的数值应视为标准值。本标准不包括其他计量单位。 1.6 本标准并非旨在解决与其使用相关的所有安全问题（如有）。本标准的用户有责任在使用前制定适当的安全和健康实践，并确定监管限制的适用性。 ====意义和用途====== 3.1 啮齿动物来源的细胞系广泛用于生产生物制药药物，如单克隆抗体和Fc融合蛋白。这些细胞系含有编码内源性逆转录病毒样颗粒或内源性逆转录病毒的基因。尽管缺乏证据表明此类啮齿动物逆转录病毒与人类疾病之间存在关联，但人类疗法的潜在污染引起了生物制药药物的安全问题。此外，诸如病毒之类的不定因子可以从其他来源引入生物制药原料药制造过程，并且潜在的安全问题可以归因于这些潜在的未知因素。 由于这些原因，有效的病毒清除是将安全性测试和过程表征相结合的综合方法的一个重要方面，该方法可确保使用啮齿动物细胞系制造的生物制药产品的病毒安全性。 3.2 几十年来，溶剂/洗涤剂灭活已广泛用于灭活血浆衍生生物制药治疗中的包膜病毒 ( 1- 3. ) . 3. 使用Triton X-100或聚山梨酯80以及有机溶剂磷酸三（正丁基）酯（TNBP）的溶剂/洗涤剂系统已被用于通过破坏病毒包膜来灭活包膜病毒，从而降低包膜病毒附着并感染宿主细胞的能力 ( 4和 5. ) . 3.3 大多数单克隆抗体、重组蛋白和Fc融合蛋白的制造商都专注于在没有TNBP的情况下使用清洁剂Triton X-100或聚山梨酯80的病毒灭活方法 ( 6. ) ，这可能会干扰后续的生物处理步骤。在啮齿动物来源的细胞系中产生的单克隆抗体中已经证明了洗涤剂单独灭活逆转录病毒的能力 ( 6- 9 ) . 在2011年的一次研讨会上，专门讨论了生物加工中使用的病毒清除步骤 ( 7. ) ，来自一家公司的研究人员显示，在环境温度灭活>5 log的情况下，与0.2%Triton X-100孵育60分钟 10 X-MuLV在细胞培养基质中的四个单独单抗上的表达。 3.3.1 在2011年的同一研讨会上 ( 7. ) ，来自第二家公司的研究人员证实，在0.3%Triton X-100水平下，蛋白质浓度和脂质浓度对MuLV病毒灭活没有明显影响。此外，8种不同的单克隆抗体宿主细胞培养液（HCCF）在20°C下用0.3%Triton X-100处理60分钟， ≥ 4日志 10 在这些实验中，发现每种抗体都能灭活MuLV病毒。 3.4 Quertinmont公司 ( 8. ) 证明DNA水平、总蛋白浓度和脂质含量（超过1000微克/毫升）在0。 在使用单克隆抗体和Fc融合蛋白灭活60分钟后，使用HCCF的45%（w/v）Triton X-100洗涤剂灭活步骤对检测MuLV病毒没有统计学意义。此外，对三种不同生物药物浓度、总蛋白浓度、温度和Triton X-100浓度的单独分子进行了三次实验设计稳健性研究。这些研究表明，当Triton X-100浓度为 ≥ 0.2%，温度在15-25°C之间，保持时间为 ≥ 在HCCF中60分钟。 3.5 Blumel和Tounekti ( 9 ) 在所有时间点（0、5、30和60分钟），使用1.0%Triton X-100在60分钟的保持时间内，通过4个单克隆抗体[2个IgG和2个免疫球蛋白M（IgM）]显示MuLV完全失活。这15项研究的平均对数折减系数（LRF）为 ≥ 3.89对数 10 . 研究数据分析表明，较高水平的Triton X-100（1%）需要大量稀释以减轻MuLV指示细胞的细胞毒性。在这15项研究中测试的任何时间点均未发现可检测到的病毒，并且声称的LRF完全取决于这些研究中MuLV饲料的起始病毒滴度。 3.6 这种逆转录病毒失活的程度可能取决于某些反应参数，包括澄清、Triton X- 100浓度、保持时间、pH值和灭活温度。然而，按照本规程的规定，管理能够提供稳健有效的逆转录病毒灭活的参数，以及其他清除单元操作，可以确保有效的逆转录病毒灭活。 3.7 本规程包含了能够有效灭活逆转录病毒的参数，这些参数可用于特定病毒的病毒清除过程的模块化验证。

1.1 This practice assures effective inactivation of ≥ 4 log 10 of infectious rodent retrovirus (that is, reduction from 10 000 to 1 infectious rodent retrovirus or removal of 99.99 % of infectious rodent retroviruses) in the manufacturing processes of monoclonal antibodies or immunoglobulin G (IgG) Fc fusion proteins manufactured in rodent-derived cell lines that do not target retroviral antigens. Rodent retrovirus is used as a model for rodent cell substrate endogenous retrovirus-like particles potentially present in the production stream of these proteins. 1.2 The parameters specified for this practice are clarification, Triton X-100 detergent concentration, hold time, pH, and inactivation temperature. 1.3 This practice can be used in conjunction with other clearance or inactivation unit operations that are orthogonal to this inactivation mechanism to achieve sufficient total process clearance or inactivation of rodent retrovirus. 1.4 This detergent inactivation step is performed on a clarified, cell-free intermediate of the monoclonal antibody or IgG Fc fusion protein. 1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. 1.6 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 and health practices and determine the applicability of regulatory limitations prior to use. ====== Significance And Use ====== 3.1 Rodent-derived cell lines are widely used in the production of biopharmaceutical drugs such as mAbs and Fc fusion proteins. These cell lines have been shown to contain genes encoding endogenous retroviral-like particles or endogenous retrovirus. Despite the lack of evidence for an association between such rodent retroviruses and disease in humans, the potential contamination of human therapeutics raises safety concerns for biopharmaceutical drugs. Additionally, adventitious agents such as viruses can be introduced into a biopharmaceutical drug substance manufacturing process from other sources, and potential safety issues can be attributed to these potential unknowns. For these reasons, effective viral clearance is an essential aspect of an integrated approach combining safety testing and process characterization which ensures virus safety for biopharmaceutical drug products made using rodent cell lines. 3.2 Solvent/detergent inactivation has been widely used for decades to inactivate enveloped viruses in blood plasma derived biopharmaceutical therapies ( 1- 3 ) . 3 Solvent/detergent systems using the detergents Triton X-100 or Polysorbate 80 along with the organic solvent tri(n-butyl)phosphate (TNBP) have been used to inactivate enveloped viruses by disrupting the viral envelope thereby reducing the ability of the enveloped virus to attach to and then infect the host cell ( 4 and 5 ) . 3.3 Most manufacturers of mAbs, recombinant proteins, and Fc fusion proteins have focused on viral inactivation methods using the detergent Triton X-100 or Polysorbate 80 in the absence of TNBP ( 6 ) , which can interfere with subsequent bioprocessing steps. The ability of the detergents alone to inactivate retroviruses has been demonstrated in monoclonal antibodies produced in rodent-derived cell lines ( 6- 9 ) . At a 2011 workshop devoted to viral clearance steps used in bioprocessing ( 7 ) , investigators from one firm showed incubation with 0.2 % Triton X-100 for 60 min of hold time at ambient temperature inactivated >5 log 10 of X-MuLV across four separate mAbs in cell culture matrices. 3.3.1 At the same 2011 workshop ( 7 ) , investigators from a second firm confirmed that levels of protein concentration and lipid concentration had no observable effect on MuLV virus inactivation at levels of 0.3 % Triton X-100. Additionally, eight different monoclonal antibody Host Cell Culture Fluids (HCCF), were treated with 0.3 % Triton X-100 for a 60 minute hold time at 20°C. Effective inactivation, ≥ 4 log 10 of inactivation of MuLV virus, was seen for each antibody in these experiments. 3.4 Quertinmont ( 8 ) demonstrated that DNA level, total protein concentration, and lipid content (exceeding 1000 µg/mL) in a 0.45 % (w/v) Triton X-100 detergent inactivation step using HCCF were not statistically significant to the detection of MuLV virus following 60 minutes of inactivation using both monoclonal antibodies and Fc fusion proteins. Additionally, three Design of Experiment (DOE) robustness studies were carried out for three separate molecules varying biological drug concentration, total protein concentration, temperature, and Triton X-100 concentration. These studies demonstrated effective viral inactivation when Triton X-100 concentration is ≥ 0.2 %, temperature is between 15–25°C, and hold time is ≥ 60 minutes in HCCF. 3.5 Blumel and Tounekti ( 9 ) showed complete inactivation of MuLV across 4 mAbs [2 IgGs and 2 immunoglobulin M (IgMs)] for all time points (0, 5, 30, and 60 min) using 1.0 % Triton X-100 for a 60-minute hold time. The average log reduction factor (LRF) for these 15 studies was ≥ 3.89 log 10 . Analyses of the study data showed the higher level of Triton X-100 (1 %) necessitated a large dilution to mitigate cytotoxicity of the MuLV indicator cells. No detectable virus was seen at any of the time points tested across these 15 studies and the claimed LRF was completely dependent on the starting viral titer of the MuLV feed stock in these studies. 3.6 The extent of this retroviral inactivation could be dependent on certain reaction parameters including clarification, Triton X-100 concentration, hold time, pH, and inactivation temperature. However, managing parameters that give robust and effective retrovirus inactivation as specified by this practice, in conjunction with other clearance unit operations, can assure effective retroviral inactivation. 3.7 This practice incorporates parameters that give effective retrovirus inactivation, which can be used as modular validation of the viral clearance process for the specified viruses.