Konz John O, Lee Ann L, Lewis John A, Sagar Sangeetha L
Biologics Development & Engineering and Live Viral Vectors, BioProcess R&D, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
Biotechnol Prog. 2005 Mar-Apr;21(2):466-72. doi: 10.1021/bp049644r.
The clearance of host cell DNA is a critical goal for purification process development for recombinant Ad5 (rAd5) based vaccines and gene therapy products. We have evaluated the clearance of DNA by a rAd5 purification process utilizing nuclease digestion, ultrafiltration, and anion exchange (AEX) chromatography and found residual host cell DNA to consistently reach a limiting value of about 100 pg/10(11) rAd5 particles. Characterization of the purified rAd5 product using serial AEX chromatography, hydroxyapatite chromatography, or nuclease treatment with and without particle disruption showed that the residual DNA was associated with virus particles. Using a variety of additional physical characterization methods, a population of rAd5 virus in an aggregated state was detected. Aggregation was eliminated using nonionic detergents to attenuate hydrophobic interactions and sodium chloride to attenuate electrostatic interactions. After implementation of these modifications, the process was able to consistently reduce host cell DNA to levels at or below 5 pg/10(11) rAd5 particles, suggesting that molecular interactions between cellular DNA and rAd5 are important determinants of process DNA clearance capability and that the co-purifying DNA was not encapsidated.
清除宿主细胞DNA是基于重组腺病毒5型（rAd5）的疫苗和基因治疗产品纯化工艺开发的关键目标。我们评估了一种利用核酸酶消化、超滤和阴离子交换（AEX）色谱的rAd5纯化工艺对DNA的清除效果，发现残留的宿主细胞DNA始终达到约100 pg/10¹¹个rAd5颗粒的极限值。使用连续AEX色谱、羟基磷灰石色谱或在有或无颗粒破坏情况下进行核酸酶处理对纯化的rAd5产品进行表征，结果表明残留DNA与病毒颗粒相关。使用多种其他物理表征方法，检测到一群处于聚集状态的rAd5病毒。使用非离子去污剂减弱疏水相互作用，使用氯化钠减弱静电相互作用，从而消除了聚集现象。实施这些改进后，该工艺能够持续将宿主细胞DNA降低到5 pg/10¹¹个rAd5颗粒或更低水平，这表明细胞DNA与rAd5之间的分子相互作用是工艺DNA清除能力的重要决定因素，并且共纯化的DNA未被包裹。
