Nold Natalie M, Kriz Seth A, Waldack Sheridan, James Grace, Colling Trisha, Sarvari Taravat, Sharma Vaishali, Pohkrel Alexis, Burghardt Ethan, Joshi Pratik U, Heldt Caryn L
Department of Chemical Engineering, Michigan Technological University, USA; Health Research Institute, Michigan Technological University, USA.
Department of Chemical Engineering, Michigan Technological University, USA.
J Chromatogr A. 2025 May 10;1748:465866. doi: 10.1016/j.chroma.2025.465866. Epub 2025 Mar 13.
Virus-based vaccines and therapies require a purification method that is both cost-effective and easily scalable. An aqueous two-phase system (ATPS) consisting of polyethylene glycol (PEG) and citrate salt has been proven to deliver high virus recoveries along with high impurity removal. However, these systems often place the virus into a viscous PEG-rich phase or at the two-phase interface, leading to difficulties in subsequent downstream processes. This study explored a second ATPS to extract the virus product back into the citrate-rich phase by changing the chemical conditions, a required step for future application of ATPS in industrial processes. ATPS performance was tested as a function of phase component concentration, phase component volume ratios, PEG molecular weight, salt type, pH, and glycine addition to identify the most impactful parameters for the extraction of non-enveloped porcine parvovirus (PPV). By shifting the pH, lowering phase component concentrations, and increasing the volume ratio of the citrate-rich phase between the first and second ATPS steps, 66 % of infectious PPV was recovered with 2.0 logs of host cell protein removal and 1.0 logs of host cell DNA removal. Using a PEG molecular weight of 8 kDa enabled a pH shift between the first and second ATPS steps without precipitation. Glycine addition during the first step of ATPS and phosphate salt use during the second step of ATPS did not significantly increase the overall recovery. In future studies, the optimized process will be implemented for multiple viral vector types and continuously to demonstrate continuous and low-cost viral vector manufacturing.
基于病毒的疫苗和疗法需要一种具有成本效益且易于扩展的纯化方法。由聚乙二醇（PEG）和柠檬酸盐组成的双水相系统（ATPS）已被证明能实现高病毒回收率以及高杂质去除率。然而，这些系统常常将病毒置于富含PEG的粘性相中或两相界面处，导致后续下游工艺出现困难。本研究探索了第二种ATPS，通过改变化学条件将病毒产物提取回富含柠檬酸盐的相中，这是ATPS未来在工业过程中应用所需的一个步骤。测试了ATPS的性能，将其作为相组分浓度、相组分体积比、PEG分子量、盐类型、pH值和甘氨酸添加量的函数，以确定对非包膜猪细小病毒（PPV）提取影响最大的参数。通过在第一步和第二步ATPS之间改变pH值、降低相组分浓度以及增加富含柠檬酸盐相的体积比，回收了66%的感染性PPV，同时去除了2.0个对数级的宿主细胞蛋白和1.0个对数级的宿主细胞DNA。使用8 kDa的PEG分子量可在第一步和第二步ATPS之间实现pH值变化而不产生沉淀。在ATPS的第一步添加甘氨酸以及在第二步使用磷酸盐盐并没有显著提高总体回收率。在未来的研究中，将对多种病毒载体类型实施优化工艺，并持续进行以证明连续且低成本的病毒载体生产。
