Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Austria.
Austrian Centre of Industrial Biotechnology, Vienna, Austria.
Vaccine. 2019 Nov 8;37(47):7070-7080. doi: 10.1016/j.vaccine.2019.07.001. Epub 2019 Jul 9.
Polymer-grafted chromatography media, especially ion exchangers, are high performance materials for protein purification. However, due to the pore size limitation, conventional chromatography beads are usually not considered for the downstream processing of large biomolecules such as virus-like particles (VLPs). Contrariwise, since the outer surface of the chromatography beads provides satisfactory binding capacity for VLPs and impurities of smaller size can bind inside of the beads, conventional porous beads should be considered for VLP capture and purification. We used HIV-1 gag VLPs with a diameter of 100-200 nm as a model to demonstrate that polymer-grafted anion exchangers are suitable for the purification of bionanoparticles. The equilibrium binding capacity was 1 × 10 part/mL resin. Moderate salt concentration up to 100 mM NaCl did not affect binding, allowing direct loading of cell culture supernatant onto the column for purification. Dynamic binding capacity at 10% breakthrough, when loading cell culture supernatant, was approximately 6 × 10 part/mL column; only 1-log lower than for monoliths. Endonuclease treatment of the cell culture supernatant did not increase the dynamic binding capacity, suggesting that dsDNA does not compete for the binding sites of VLPs. Nevertheless, due to simultaneous elution of particles and dsDNA, endonuclease treatment is required to reduce dsDNA contamination in the product. Proteomic analysis revealed that HIV-1 gag VLPs contain different host cell proteins in their cargo. This cargo is composed of conserved proteins and other proteins that vary from one particle population to another, as well as from batch to batch. This process allowed the separation of different particle populations. HIV-1 gag VLPs were directly captured and purified from cell culture supernatant with a total particle recovery in the elution of about 35%. Columns packed with beads can be scaled to practically any dimension and therefore a tailored design of the process is possible.
聚合物接枝色谱介质，特别是离子交换剂，是蛋白质纯化的高性能材料。然而，由于孔径限制，传统的色谱珠通常不被认为是用于下游处理大生物分子（如病毒样颗粒（VLPs））的材料。相反，由于色谱珠的外表面为 VLPs 提供了令人满意的结合能力，并且较小尺寸的杂质可以结合到珠内，因此对于 VLP 的捕获和纯化，应该考虑使用传统的多孔珠。我们使用直径为 100-200nm 的 HIV-1 gag VLPs 作为模型，证明了聚合物接枝阴离子交换剂适合于生物纳米颗粒的纯化。平衡结合容量为 1×10 个颗粒/mL 树脂。适度的盐浓度（高达 100mM NaCl）不会影响结合，允许直接将细胞培养上清液加载到柱上进行纯化。在 10%穿透时的动态结合容量，当加载细胞培养上清液时，约为 6×10 个颗粒/mL 柱；仅比整体柱低 1 个对数级。细胞培养上清液的内切核酸酶处理并未增加动态结合容量，这表明 dsDNA 不会与 VLPs 的结合位点竞争。尽管如此，由于颗粒和 dsDNA 的同时洗脱，需要内切核酸酶处理以降低产物中的 dsDNA 污染。蛋白质组学分析表明，HIV-1 gag VLPs 在其货物中包含不同的宿主细胞蛋白。这种货物由保守蛋白和其他从一个颗粒群体到另一个颗粒群体以及从一批到另一批变化的蛋白组成。该过程允许不同颗粒群体的分离。HIV-1 gag VLPs 可以直接从细胞培养上清液中捕获和纯化，洗脱中大约有 35%的总颗粒回收率。用珠子填充的柱子可以扩展到几乎任何尺寸，因此可以对工艺进行定制设计。
