Development of advective membrane technology for the purification of viruses and viral vectors

大多数工业生物工艺都寻求确保从其工艺流中消除病毒。因此，大量的工作已经进入病毒清除研究。然而，病毒本身可以作为疫苗接种剂、溶瘤治疗剂和基因递送载体是非常有用的产品。虽然有用，但缺乏关于最大限度地恢复大规模应用程序的“活动”病毒的信息。该资助申请是一名博士生和一名博士后研究员对两种病毒的捕获和纯化进行的为期三年的研究：一种假杆状病毒（一种经修饰的昆虫病毒）基因治疗载体和一种治疗性溶瘤Maraba病毒，该病毒在临床试验中显示出巨大的前景作为癌症治疗。这种捕获和纯化将使用构成Natrix分离公司基础的膜技术进行。核心业务，并承诺最大限度地减少下游处理步骤的数量，并确保恢复活跃的病毒。广泛的已知病毒及其独特的特性可能对开发用于病毒制剂的稳健通用下游策略构成重大挑战-特别是那些预定用于临床的病毒制剂。这些挑战包括病毒的数量、纯度和效力。为了尽量减少不同的属性，这项工作的重点是水泡性口炎病毒包膜糖蛋白G（VSV-G）假型杆状病毒，和VSV相关的病毒，马拉巴病毒。许多基于病毒的载体用VSV-G进行假型化以增加其效力。基于VSV-G的纯化策略，将允许使用Natrix技术平台纯化许多领先的治疗性候选病毒的综合策略。将同时审查两项主要战略。首先，与Natrix，肝素功能化的膜将用于亲和捕获病毒与水泡性口炎样糖蛋白。其次，将对Natrix离子交换膜进行评价。将评价两种策略对病毒回收率、纯度和效价的影响。

Most industrial bioprocesses seek to ensure the elimination of viruses from their process streams. As a result, a significant amount of work has gone into viral clearance research. However, viruses themselves can be very useful products as vaccination agents, oncolytic therapeutics and gene delivery vectors. Although useful, there is a dearth of information available on maximizing recovery of 'active' viruses for large scale applications. This grant application is for a three year study by a PhD student and a post-doctoral fellow on the capture and purification of two viruses: a pseudo-typed baculovirus (a modified insect virus) gene therapy vector, and a therapeutic oncolytic Maraba virus, which has shown tremendous promise in clinical trials as a treatment for cancer. This capture and purification will be done using membrane technologies, which form the basis of Natrix Separations Inc. core business, and that promise to minimize the number of downstream processing steps and ensure recovery of active virus. The wide array of known viruses and their distinct properties can pose major challenges in the development of robust universal downstream strategies for viral preparations - especially those destined for clinical use. These challenges include quantity, purity and potency of the viruses. To minimize the distinct properties, this work focuses on vesicular stomatitis virus envelope glycoprotein G (VSV-G) pseudo-typed baculovirus, and a VSV-related virus, the Maraba virus. Many virus-based vectors are pseudo-typed with VSV-G to increase their potency. Basing our purification strategies on the VSV-G, will allow a comprehensive strategy to purify a number of leading therapeutic viral candidates using the Natrix technology platform. Two major strategies will be examined in parallel. First, with Natrix, a heparin-functionalized membrane will be made for affinity capture of viruses with vesicular stomatitis-like glycoproteins. Second, Natrix ion exchange membranes will be evaluated. Both strategies will be evaluated for their effect on virus recovery, purity and potency.