Department of Analytical Development, Biogen Inc., 225 Binney St., Cambridge, MA, 02142, United States.
Department of Analytical Development, Biogen Inc., 225 Binney St., Cambridge, MA, 02142, United States.
J Pharm Biomed Anal. 2018 Sep 10;159:477-482. doi: 10.1016/j.jpba.2018.07.022. Epub 2018 Jul 18.
For drug substances manufactured in cell lines, host cell DNA is a common contaminant and its level must be carefully monitored. While residual DNA assays have been developed for many production cell lines, a robust assay is unavailable for baby hamster kidney (BHK) cells. The lack of genomics data of Syrian hamster, the origin of BHK cells, makes it challenging to design primers and probes for PCR-based methods. In this paper, we identified intracisternal A-particle (IAP) as an efficient PCR target for BHK DNA. PCR against IAP has been tested with conventional qPCR as well as with the recently developed ddPCR method, both of which demonstrated good efficiency with purified BHK DNA. However, the ddPCR-based method is less prone to matrix interference and is significantly more accurate than qPCR when testing complex samples, including multiple process intermediates. This study not only established a robust assay for the detection of residual BHK DNA, but also evaluated the capability of ddPCR technology for a new application.
对于在细胞系中制造的药物物质，宿主细胞 DNA 是一种常见的污染物，必须仔细监测其水平。虽然已经为许多生产细胞系开发了残留 DNA 检测方法，但对于仓鼠肾细胞（BHK）细胞，还没有一种可靠的检测方法。由于缺乏 BHK 细胞的起源叙利亚仓鼠的基因组学数据，因此为基于 PCR 的方法设计引物和探针具有挑战性。在本文中，我们将内粒体 A 粒子（IAP）鉴定为 BHK DNA 的有效 PCR 靶标。已经使用常规 qPCR 以及最近开发的 ddPCR 方法对 IAP 进行了 PCR 检测，这两种方法都证明了用纯化的 BHK DNA 进行检测时具有良好的效率。然而，与 qPCR 相比，基于 ddPCR 的方法不易受到基质干扰，并且在测试复杂样品（包括多个过程中间体）时，准确性显著更高。这项研究不仅建立了一种用于检测残留 BHK DNA 的可靠方法，还评估了 ddPCR 技术在新应用中的能力。
