1、Table of Contents 目录1 Introduction 简介 92 General Concepts 一般概念 92.1 Size Exclusion 尺寸排除 102.2 Other Retention Mechanisms 其他滞留方法 112.3 Virus Retention Probability of Reduction Factor (RF) 病毒滞留可能性的减少因素（RF ） 112.4 Virus Size/Retention Rating病毒大小/滞留量 112.5 Protein Size/Sieving/Passage Rating 蛋白质大小 /过滤/通
2、过量 113 Virus Filter Selection and Characterization 病毒选择和特性 123.1 Filter Construction 过滤器结构 123.2 Filter System Configurations 过滤系统配置 123.3 Particulates and Extractables 微粒和萃取物 133.4 Filter Compatibility 过滤兼容性 143.5 Protein Recovery (Adsorption/Retention/Biocompatibility) 蛋白质恢复（吸附/滞留/生物兼容性） 153.6 The
3、rmal Stress Resistance 热应力阻止 163.7 Hydraulic Stress Resistance 水应力阻力 163.8 Toxicity Testing 毒性试验 163.9 Viral/Phage Challenge Testing 病毒/噬菌体挑战试验 163.10 Cleaning/Sanitization/Sterilization 清理/消毒/灭菌 174 Physical and Mechanical Characteristics 物理力学特性 174.1 Filtration Rate and Clogging (Throughput) 过滤速度及
4、堵塞（通过量） 174.2 Fluid/Piping 液体/管道 174.3 Fluid/Filter 液体/过滤器 174.4 Physical and Structural Limitations 物理结构限制 174.5 Miscellaneous 多样性 175 Virus Filter Validation/Evaluation Studies 病毒过滤器/评估研究 175.1 Filter Properties 过滤器属性 185.2 Test Virus 测试病毒 185.2.1 Virus Selection 病毒选择 185.2.2 Virus Stock Preparati
5、on and Maintenance 病毒库制备和维护 195.2.3 Virus Spike 病毒峰值 205.3 Scaled-down Models 缩小模型 215.4 Operating Conditions for Validation, Revalidation, and Number of Runs 验证，再验证的运行条件和运行次数 215.5 Raw Materials and Equipment 原材料和设备 225.5.1 Virus Filtration Devices and Configurations 病毒过滤设备和配置 225.5.2 Integrity Tes
6、ting during Development 研发期间的完整性测试 225.5.3 Membrane Lot Selection 膜批选择 225.5.4 Feedstream 原料流 235.5.5 Filtration Conditions 过滤条件 235.6 Virus Assays and Assay Validation 病毒分析试验和分析验证 235.6.1 Assay Method Validation 试验方法验证 235.7 Establishing Representative Worst-case Process Conditions 确定代表性的最坏工艺条件 246
7、 Integrity Testing 完整性测试 256.1 Manufacturers Checklist 生产商检验单 266.2 Virus Retention Integrity Tests 病毒滞留完整性测试 276.2.1 Dextran Retention 葡聚糖滞留 276.2.2 Gold Particle Retention 金颗粒滞留 276.2.3 Gas-Liquid Porosimetry 气- 液测量仪 296.2.4 Manual Bubble Point or Leak Testing 人工起泡点或检漏测试 296.2.5 Manual Forward/Dif
8、fusive Flow 人工顺流/扩散流 296.2.6 Manual Pressure Hold/Decay 人工压力保持/下降 306.2.7 Automated Integrity Test Instruments for Gas Porosimetry-based Test Methods 基于气体测量仪测试法的自动完整性测试仪器 306.2.8 Liquid and Liquid-liquid Porosimetry 液体和液-液测量仪 316.3 Relationship between Integrity Tests and Virus/Phage Retention 完整性测试
9、和病毒/噬菌体截留之间的关系 316.4 Failure Analysis/Troubleshooting 故障分析/处理 317 Sterilization 灭菌 327.1 Steam Sterilization 蒸汽灭菌 327.2 Autoclave Sterilization 高压蒸汽灭菌 327.3 Sterilize-in-Place (SIP) 在线灭菌 327.4 Irradiation Sterilization 辐射灭菌 337.5 Gas Sterilization 气体灭菌 33APPENDIX A: Virus Retention and Protein Passa
10、ge Nomenclature Classification 附录A ：病毒截留和蛋白质通过的术语分类 34APPENDIX B: Large Virus Filter Experimental Protocol 附录B ：大分子病毒过滤器的试验方案 35B.1 Large Virus Filter Experimental Design 大分子病毒过滤器试验设计 35B.1.1 Strategy 策略 35B.1.2 Model Virus 模型病毒 35B.1.3 Model Proteins 模型蛋白质 35B.1.4 Buffer System 缓冲系统 35B.1.5 Scaled-
11、down Model Filters 按比例缩小的模型过滤器 35B.1.6 Operating Parameters 运行参数 36B.1.7 Set Point Values for Relevant Operating Parameters 相关运行参数的设定值 36B.1.8 Nomenclature Acceptance Criteria 命名验收标准 36B.2 Preparation of Bacteriophage Stocks 噬菌群制备 37B.2.1 Introduction 简介 37B.2.2 Equipment 设备 37B.2.3 Procedure 程序 38B
12、.2.3.1 Plate Method 培养板的方法 38B.2.3.2 Broth Method 肉汤方法 38B.2.4 Crude Preparation 原料制备 38B.2.5 Pure Concentrate (Coetzee, et al. (25) 纯浓缩液 38B.3 Procedure For The Estimation of Bacteriophage Numbers 噬菌体数估算程序 39B.3.1 Introduction 简介 39B.3.2 Equipment 设备 39B.3.3 Procedure 程序 39B.3.3.1 Preparation of Ba
13、cteriophage Host 噬菌体寄主的制备 39B.3.3.2 Preparation of Dilution Tubes and Soft Agar for Use in Phage Assays 噬菌体分析用的稀释管和软琼脂制备 39B.3.3.3 Phage Assays 噬菌体分析 40APPENDIX C: FILTER VALIDATION RECOMMENDATIONS 附录C：过滤器验证建议 411 Introduction 简介Scope: This PDA Technical Report addresses virus removal filters that r
14、etain viruses by a size exclusion mechanism. It explains how they work, their selection, char-acterization, testing, and validation. This document should be considered as a guide; it is not intended to establish any mandatory or implied standard. 范围：此PDA技术报告陈述了病毒去除过滤器，该过滤器通过尺寸排除装置拦截病毒。本报告解释过滤器的工作原理、
15、选择、特征、测试及验证。本文件应作为指南；并不打算建立任何强制性或暗指的标准。Biotechnological and biological therapeutic products are often manufactured using materials of animal or human origin, including cultured primary or transformed cells, milk or other components from transgenic animals, natural extracts, and human or animal blood
16、 plasma components. These products are usually proteins that are manufactured by complex manufacturing processes. Although approved recombinant biotherapeutics have an excellent safety record, the risk of contamination by known or unknown pathogens exists (e.g., 1-10). 生物技术和生物医疗制品常使用动物或人体器官的原料制造，这些原
17、料包括培养的主要或改变的细胞、乳液或转基因动物成分、天然浸膏、人或动物的血浆成分。这些产品通常是蛋白质，通过复杂的制造工艺生产出来的。虽然经核准的重组体生物疗法拥有极好的安全记录，污染物风险还是存在已知或未知的病原体（见样例1-10）。Virus filtration is performed as part of a manufacturers overarching virus safety strategy. In this context, virus filtration (size-based removal) is a complement to virus inactivati
18、on, both of which contribute to virus clearance (e.g., 11-20). Implementation of virus clearance complements additional measures such as control over raw ma-terials and testing of cell culture or plasma feedstocks. Collectively, these measures form the framework of a virus safety strategy. 病毒过滤作为生产商
19、病毒安全策略的一部分执行。在这种环境中，病毒过滤（根据尺寸排除）是病毒失活的补充，两者都将促进病毒清理（见样例11-20）。执行病毒清理增加了额外测量，如原材料控制和细胞培养或血浆原料的测试。The risk of transmission of certain infectious pathogens cannot be completely mitigated by donor screening, vaccination of patients, a single virus inactivation step, or, in the case of cell-based product
20、s, virus testing of cell banks and raw materials. It is desirable to introduce additional robust steps in biotherapeutic purification processes to help reduce or eliminate viruses without compromising the molecular integrity of the products. Virus removal filters (often incorrectly termed “nanofilte
21、rs“) are specifically designed to remove viruses and other biomolecules from the product (protein) solution through a size exclusion mechanism. 转移特定传染病原体的风险无法通过供体过滤、患者接种、单个病毒失活步骤或者基于细胞的产品、细胞库和原材料的病毒测试完全缓解。将附加的稳定步骤引入生物疗法提纯工艺，以帮助减少或消除病毒，而不损害产品中分子的完整性。病毒移除过滤器（常被错误地叫做“纳米技术”）是通过尺寸排除装置移除病毒和产品（蛋白质）中其它生物分子溶
22、液。Commercially available virus removal filters have different membrane characteristics particular to each filter or filter type. These characteristics include membrane material, membrane surface graft characteristics, pore structure and pore size distribution, virus retention capacity, and protein t
23、ransmission and adsorption proper-ties. Virus filtration is conducted by either direct (also termed dead-end or normal) flow filtration (DFF) or tangential flow (also termed cross-flow) filtration (TFF). Selection of the mode of filtration (DFF or TFF) is usually based on the characteristics of the
24、solution to be filtered or process issues such as ease of use, ease of validation, and economics. 商用病毒移除过滤器对每种过滤器或过滤器类型都有不同的膜特性。这些特性包括膜材料、膜表面移植物特性、孔结构和孔径分布、病毒滞留能力以及蛋白质转移和吸附功能。病毒过滤器管理通过直流(也叫做终端或正常)过滤器 (DFF)或分流过滤器（TFF）。过滤器模式的选择（DFF 或TFF）通常根据过滤溶液的特性或工艺问题，如便于使用、便于验证且经济。2 General Concepts 一般概念Virus filte
25、rs are cast polymeric membranes with a complex internal porous structure. Solutions pass through this pore network while particles carried along by the fluid may be retained on the membrane surface or penetrate some distance into the membrane structure. Retention that occurs because particles are to
26、o large to pass through a pore is referred to as sieving or size exclusion. Traditional asymmetric surface “skinned“ ultrafiltration membranes that contain pinhole defects, while still suitable for protein concentration, may not provide consistent high clearance for large viruses (21). 病毒过滤器使用复杂的内部多
27、孔结构消除高分子膜。溶液穿过该孔状网，而液体携带的微粒会滞留在膜的表面或穿透至膜的结构中。发生滞留是因为微粒太大而无法穿过气孔，这就是所谓的筛分或尺寸排除。传统的非对称表面“表层”超滤膜包含小孔缺陷，但仍然适用于蛋白质浓度，可能不会持续提供大分子病毒的高清除度（21）。Smaller particles capable of passing through a pore may also be captured by adsorption to membrane pore surfaces. Retention by adsorption may involve a variety of di
28、fferent forces at different adsorption sites within the membrane structure. Adsorption of viruses, when it occurs, is a secondary phenomenon, while size-based exclusion is considered to be the main mechanism of clearance by filtration. 更小的可能通过小孔的微粒也会被膜孔表面吸附。吸附滞留包括膜的结构中多种不同吸附点的吸附力量。发生滞留时，病毒的吸附是一个次要现象
29、，而根据尺寸排除被认为是通过过滤器清理的主要方法。Terminology 术语Minimum Exposure Time: The shortest period for which a treatment step will be maintained. 最小暴露时间：维持处理步骤的最短时间Nonspecific Model Virus: A virus used for characterization of viral clearance of the process when the purpose is to characterize the capacity of the manu
30、facturing process to remove and/or inactivate viruses in general, i.e., to characterize the robustness of the purification process. 非特定病毒模型：一般来说，当用来描述生产工艺病毒移除和/或灭活的能力时，病毒具有工艺病毒清理的特性，即描述提纯工艺的稳定性。Process Characterization of Viral Clearance: Viral clearance studies in which nonspecific model viruses ar
31、e used to assess the robustness of the manufacturing process to remove and/or inactivate viruses. 病毒清理的工艺特性：在病毒清理研究中，非特定病毒模型用来评估生产工艺的稳定性。Process Evaluation Studies of Viral Clearance: Viral clearance studies in which relevant and/or specific “model“ viruses are used to determine the ability of the m
32、anufacturing process to remove and/or inactivate these viruses. 病毒清理的工艺评估研究：在病毒清理研究中，相关和/或特定“模型”病毒用来确定生产工艺移除和/或使病毒灭活的能力。Relevant Virus: Virus used in process evaluation studies that either is the identified virus, or of the same species as the virus, known to or likely to contaminate the cell substr
33、ate or any other reagents or materials used in the production process. 相关病毒：用于工业评估研究的病毒要么是已识别的病毒，要么是同类病毒，已知或可能污染细胞培养基或任何其它反应物或用于生产工艺的原料。Specific Model Virus: Virus that is closely related to the known or suspected virus (same genus or family), having similar physical and chemical properties as those
34、 of the observed or suspected virus. 特定病毒模型：病毒与已知或可疑病毒（同宗）有密切关联，作为那些被观测或可疑病毒，病毒拥有类似的物化特性。Viral Clearance: Elimination of target virus by removal of viral particles or inactivation of viral infectivity. 病毒清除：通过病毒微粒的移除或使病毒传染性失活排除目标病毒。Viral Inactivation: Reduction of virus infectivity caused by chemica
35、l or physical modification. 病毒失活：化学或物理变性造成的病毒传染性减少。Virus Removal: Physical separation of virus particles from the intended product. 病毒移除：来自目标产品的病毒微粒的物理分离。From ICH Q5A Viral Safety Evaluation of Biotechnology Products Derived From Cell Lines of Human or Animal Origin 来自 ICH Q5A人员或动物细胞线产生的生物技术产品的病毒安全性
36、评估A typical virus filter provides consistent, high virus clearance for viruses larger than the filter rating. For viruses with sizes below the filter rating, clearance may be de-pendent on fluid and process conditions, especially for small viruses significantly below the filters rating. 典型的病毒过滤器为病毒提
37、供比过滤值更大的一致的高病毒清Figure 1. Depiction of the log reduction factor (LRV) as a function of virus size. Especially for filters designed to retain larger viruses, retention will be high and reproducible for larger viruses, case-specific and less predictable for intermediate sized viruses, and negligible fo
38、r the smallest of viruses. 图表1. 对数减少系数（LRV）的描述作为病毒大小功能的。尤其对设计用于较大病毒的过滤器，滞留较高，且较大病毒可再生，中等大小病毒根据情况确定且可预测性较小，最小病毒可忽略。除率。对小于过滤值的病毒可以采用液体工艺条件清理，特别是比过滤值小很多的病毒。The lack of sensitivity of virus retention by a membrane to changes in the virus type (aside from size), feed solution, and operating conditions is
39、 referred to as the robustness of filter retention. Designing a reliable process requires the identification of the significant parameters of the process that can provide consistent assurance of virus retention. Parameters that can influence retention include virus size, membrane thickness, number o
40、f filter layers, and filter pore size distribution. Other factors, such as feedstream pH and ionic strength, can also influence retention, but to a much lesser degree (22). 由滤膜上病毒滞留的敏感度缺乏导致的病毒类型（除了尺寸）、供给溶液和操作条件的变化指的是过滤器滞留的稳定性。可靠工艺的设计需要识别工艺的重要参数，这些参数能够提供病毒滞留的持续保证性。能够影响滞留的参数包括病毒大小、膜厚度、过滤层数量以及过滤器孔径分布。其
41、它因素也会影响滞留，如原料流的pH和离子强度，但是影响程度要小得多。Virus filter retention properties may change during the course of filtration. Protein aggregates may deposit on the surface of the filter during the filtration process. For TFF, protein adsorption can add an additional layer on the surface of the membrane and reduce
42、 the effective pore size of the filter. The degree of protein polarization acting as a secondary virus separation layer can vary during startup and under the changing conditions of concentration and diafiltration processing. For DFF, protein aggregates depositing within the depth of a virus filter c
43、an also change the flow patterns within the membrane structure, potentially increasing the effective pore size distribution by blocking the smaller pores. In both cases, process fluid flow relative to the initial or H2O flux is lower. 病毒过滤滞留的特性可能在过滤期间有所改变。蛋白质聚合物可能沉积在过滤器表面。对于TFF，蛋白质吸附作用会增加膜表的附加层，并减小过
44、滤器的有效孔径。在启动期间以及浓度和过滤工艺改变的条件下，蛋白质极化度的等级作为次要的病毒离层可能发生变化。对于DFF，病菌过滤器的深度内沉积的蛋白质聚合物也可能改变膜结构内部的流型，通过阻滞更小的气孔来增加有效孔径分布的可能性。在这两种情况下，与原始细胞或水溶剂有关的工艺液体流动较低。Spiking studies are commonly used to characterize the ability of a virus filter to retain a particular virus under specified conditions. In these studies, s
45、caled-down virus filter systems are used, and the feedstream is spiked with high titer viruses. 峰值研究一般用于描述病毒过滤器在特定条件下滞留微粒的能力。在这些研究中，使用缩小比例的病毒过滤系统，且用高浓度病毒阻止原料流。2.1 Size Exclusion 尺寸排除As described above, viruses are carried along by the fluid as it approaches and passes through the porous membrane structure. These viruses may encounter
