EVALUATION OF PERT ASSAYS IN BIOLOGICAL PRODUCTS

生物制品中 PERT 检测的评估

• 批准号: 6293788
• 负责人: KE E STEIN
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6293788
• 关键词: Retroviridae disease; biohazard control; biological products; bioreactors; diagnosis procedure safety; monoclonal antibody; polymerase chain reaction; transmission electron microscopy

Monoclonal antibody (mAb) cell lines produce endogenous retroviruses (ERV), thus validation of retrovirus removal by a mAb manufacturing process is a requirement for licensure and clinical IND use for mAb products. Validation of retrovirus removal typically involves spike/removal studies of chromatography and other steps of the manufacturing process. The amount of retrovirus in antibody bulk harvest samples is typically quantitated by transmission electron microscopy (TEM) and in samples from validation studies of processing steps by infectivity assays (Mus dunni). Although many steps in downstream processing have been shown repeatedly to be robust (protein A and anion exchange chromatography, solvent/detergent inactivation, viresolve filtration), current policy requires measurement of ERV for each antibody-producing cell line at the bulk harvest stage and clearance validation studies for multiple downstream processing steps. Currently employed clearance validation studies are limited, however, by the feasible load of spiking infectious ERV and by assay sensitivity. Given the cost and review time expended, improvements in the way viral clearance/inactivation is measured are highly desirable. Recently developed, PCR-based reverse transcriptase (PBRT, PERT) assays, may be able to improve these studies. PBRT assays are about one million fold more sensitive than conventional reverse trascriptase (RT) assays and because they measure RT activity, not infectious viruses, PBRT has a detection limit 4 to 6 orders of magnitude lower than TEM and Mus dunni, and are more rapid than infectivity assays. In theory, PBRT assays could replace the current assays used in validation studies, possibly lowering the number of steps that need to be validated or even allowing final product testing only. We propose to perform a small scale production of a model mAb, using steps commonly employed by industry in mAb manufacture and measure virus clearance of these steps using a modified PBRT assay. Several PERT assays are currently in place at DVP/OVRR, including a fluorescent probe PBRT assay. Our strategy is to produce the model mAb using an FPLC system currently in place in DMA, purifying the mAb using protein G, anion exchange and hydrophobic interaction columns. RT levels in column loads, washes and eluates will be measured by PBRT to determine ERV clearance. It is possible that we may be able to demonstrate total clearance by individual steps. If it is the case that all or almost all of the ERVs are removed by the first step (protein G), spike/removal studies can be performed on subsequent steps. PBRT will be compared to TEM and infectivity assays to demonstrate the relative advantage of PBRT assays to measure ERV in bulk harvests and validation studies.

单抗细胞系产生内源性 逆转录病毒(ERV)，从而验证通过一种 MAB制造工艺是获得许可和 单抗产品的临床应用。逆转录病毒的验证 移除通常涉及尖峰/移除研究 制造过程中的层析等步骤。 批量采集的抗体样本中的逆转录病毒数量为 通常由透射电子显微镜进行定量 (TM)和来自加工验证研究的样品中 感染性试验(Mus Dunni)。虽然有很多步骤 在下游加工中已经被反复证明是 Robust(蛋白A和阴离子交换层析， 溶剂/洗涤剂灭活，visolve过滤)，电流 政策要求为每个产生抗体的人测量ERV 大量收获阶段的细胞系和清除验证 研究了多个下游加工步骤。目前 然而，受雇的许可验证研究受到以下因素的限制 传染性呼吸道合胞病毒的适宜载量及检测方法 敏感度。考虑到花费的成本和审查时间， 改进了病毒清除/灭活的方式 有分寸是非常可取的。 新近开发的基于聚合酶链式反应的逆转录酶 (PBRT，PERT)检测，或许能够改进这些研究。 PBRT检测的灵敏度大约是 传统的逆转录酶(RT)检测和因为它们 测量RT活性，而不是传染性病毒，PBRT具有 检测下限比透射电子显微镜低4到6个数量级 Mus Dunni，而且比传染性检测更快。在……里面 理论上，PBRT检测可以取代目前使用的 验证研究，可能会减少 需要进行验证，甚至允许进行最终产品测试 只有这样。 我们建议进行一个模型的小规模生产 单抗，使用业界在单抗中常用的步骤 制造和测量这些步骤的病毒清除 改良PBRT法。几种PERT检测目前正在进行中 放置在DVP/OVRR处，包括荧光探针PBRT分析。 我们的策略是使用FPLC系统生产单抗模型 目前在DMA中，使用蛋白G纯化mAb， 阴离子交换柱和疏水作用柱。RT水平 在柱负荷中，洗脱液和洗脱液将通过PBRT进行测量 以确定ERV的通过率。有可能我们可能是 能够通过单独的步骤演示整个清关过程。如果 情况是，所有或几乎所有ERV都被移除 通过第一步(蛋白G)，尖峰/移除研究可以 在后续步骤中执行。我们将把PBRT与瞬变电磁进行比较 以及传染性测试，以证明其相对优势 用PBRT检测散装收获中的ERV及其验证 学习。