Use of a quantitative product-enhanced reverse transcriptase assay to monitor retrovirus levels in mAb cell-culture and downstream processing
Use of a quantitative product-enhanced reverse transcriptase assay to monitor retrovirus levels in mAb cell-culture and downstream processing
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DOI：
10.1021/bp000153q
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发表时间：
2001-01-01
影响因子：
2.9
通讯作者：
Stein, KE
中科院分区：
文献类型：
--
作者：
Brorson, K;Swann, PG;Stein, KE
用于生产单抗(MAb)的小鼠杂交瘤细胞产生内源性C型逆转录病毒颗粒。监管机构要求证明供人类使用的单抗不含逆转录病毒，并具有足够的安全边际。这通常是通过小规模进行的验证研究来实现的，以证明制造过程能够去除或灭活几种不同的模型病毒，包括小鼠逆转录病毒。在这份报告中，我们评估了TaqMan荧光5‘-核酸酶产物增强型逆转录酶(TM-PERT)试验在测量细胞培养样本中逆转录酶(RT)活性和通过加工步骤模型去除RT的实用性。实验室规模的细胞培养收获中所含的RT活性水平(10(8)-10(13)PU/毫升)大大高于TM-PERT分析的检测限(类似于10(6)PU/毫升)。来自细胞培养的RT活性的性质是复杂的，但在澄清的mAb收获中，大部分RT活性似乎包含在大分子量病毒颗粒中。在实验室规模的层析运行中，含有单抗的洗脱液中存在足够的RT活性，以准确计算其对数(10)还原值(LRV)，通常在每一步2至4对数(10)之间。由三个串联柱组成的模型纯化方案纯化的单抗含有接近检测下限的残存RT活性。结果表明，TM-PERT法具有定量、灵敏度高、可在短时间内对大量样品进行分析等优点，非常适合于研究和优化反转录病毒在纯化过程中的清除。
Murine hybridoma cells used in the production of monoclonal antibodies (mAb's) produce endogenous type C retrovirus particles. Regulatory agencies require a demonstration that mAb's intended for human use are free of retrovirus with an adequate margin of safety. This is usually achieved by validation studies, performed at small scale, to demonstrate that the manufacturing process is capable of removing or inactivating several different model viruses, including a murine retrovirus. In this report, we assess the utility of the TaqMan fluorogenic 5'-nuclease Product-Enhanced Reverse Transcriptase (TM-PERT) assay for measuring reverse transcriptase (RT) activity in cell-culture samples and RT removal by models of processing steps. The levels of RT activity contained in laboratory-scale cell-culture harvests (10(8)-10(13) pU/ mL) were substantially above the detection limit of the TM-PERT assay (similar to 10(6) pU/ mL). The nature of the RT activity from cell culture was complex, but the bulk of RT activity in clarified mAb harvests appears to be contained in large molecular weight viral particles. In laboratory-scale chromatographic runs, sufficient RT activity was present in mAb-containing eluates to accurately calculate its log(10) reduction value (LRV), typically between 2 and 4 log(10) per step. Monoclonal antibody purified using a model purification scheme consisting of three serial columns contained some residual RT activity near the limit of detection. The data indicate that the TM-PERT assay, because it is quantitative and highly sensitive and can be used to analyze a large number of samples in a short period, is ideally suited to investigate and optimize retrovirus clearance in purification processes.