Green Anthony P, Huang James J, Scott Miller O, Kierstead Timothy D, Beaupré Irene, Gao Guang-Ping, Wilson James M
Puresyn, Inc., Malvern, PA 19355, USA.
Hum Gene Ther. 2002 Nov 1;13(16):1921-34. doi: 10.1089/10430340260355338.
Recombinant adenovirus vectors continue to be the preferred vectors for many types of gene therapy. However, issues regarding production and safety as well as the development of a scalable process for these vectors remain a challenge. Additionally, any process must address the well-documented immune and toxicologic responses to these vectors. Some alternatives to classic CsCl-gradient purification based on column chromatography have been developed, but these first-generation processes are still limited in potential application. We report the development of a tandem column chromatography process incorporating two resins; anion-exchange and PolyFlo (Puresyn, Inc., Malvern, PA). PolyFlo is used in a novel manner as a polishing step to remove additional host and viral proteins not removed by the anion-exchange capture step. By using the beta-galactosidase reporter vector, H5.CMV-lacZ, the purity of the product is improved compared to the same vector purified by 2x CsCl or anion-exchange alone as determined by high-performance liquid chromatography (HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; silver stain), Western analysis, electron microscopy, and particle:infectious (VP:IU) unit ratio. The recovery over the entire process is significantly better than 2x CsCl and higher than other first-generation tandem chromatography processes. This new process is reproducible and scalable to 10(15) input viral particles per run. Furthermore, the purified adenovirus product remains intact after multiple freeze/thaw cycles and is stable at 4 degrees C, -20 degrees C, and -75 degrees C. The process described here permits purification of adenovirus particles at a high concentration at large scale without centrifugation.
重组腺病毒载体仍然是多种基因治疗类型的首选载体。然而，这些载体的生产和安全性问题以及可扩展生产工艺的开发仍然是一项挑战。此外，任何工艺都必须解决已被充分证明的针对这些载体的免疫和毒理学反应。已经开发了一些基于柱色谱法替代经典氯化铯梯度纯化的方法，但这些第一代工艺在潜在应用方面仍然有限。我们报告了一种结合两种树脂的串联柱色谱法的开发；阴离子交换树脂和PolyFlo（Puresyn公司，宾夕法尼亚州马尔文）。PolyFlo以一种新颖的方式用作精制步骤，以去除阴离子交换捕获步骤未去除的额外宿主和病毒蛋白。通过使用β-半乳糖苷酶报告载体H5.CMV-lacZ，与通过2倍氯化铯或单独阴离子交换纯化的相同载体相比，通过高效液相色谱（HPLC）、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE；银染）、蛋白质免疫印迹分析、电子显微镜以及颗粒：感染性（VP：IU）单位比测定，产品的纯度得到了提高。整个过程的回收率明显优于2倍氯化铯法，且高于其他第一代串联色谱法。这种新工艺具有可重复性，并且每次运行可扩展至10¹⁵个输入病毒颗粒。此外，纯化后的腺病毒产品在多次冻融循环后仍保持完整，并且在4℃、-20℃和-75℃下稳定。本文所述的工艺允许在不进行离心的情况下大规模高浓度纯化腺病毒颗粒。
