Lee Micky Fu Xiang, Chan Eng Seng, Tan Wen Siang, Tam Kam Chiu, Tey Beng Ti
Chemical Engineering Discipline, School of Engineering, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor, Malaysia.
Chemical Engineering Discipline, School of Engineering, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor, Malaysia; Advanced Engineering Platform, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor, Malaysia.
J Chromatogr A. 2016 May 6;1445:1-9. doi: 10.1016/j.chroma.2016.03.066. Epub 2016 Mar 24.
Purification of virus-like particles (VLPs) in bind-and-elute mode has reached a bottleneck. Negative chromatography has emerged as the alternative solution; however, benchmark of negative chromatography media and their respective optimized conditions are absent. Hence, this study was carried out to compare the performance of different negative chromatography media for the purification of hepatitis B VLPs (HB-VLPs) from clarified Escherichia coli feedstock. The modified anion exchange media, core-shell adsorbents (InertShell and InertLayer 1000) and polymer grafted adsorbents (SQ) were compared. The results of chromatography from packed bed column of core-shell adsorbents showed that there is a trade-off between the purity and recovery of HB-VLPs in the flowthrough fraction due to the shell thickness. Atomic force microscopic analysis revealed funnel-shaped pore channels in the shell layer which may contribute to the entrapment of HB-VLPs. A longer residence time at a lower feed flow rate (0.5ml/min) improved slightly the HB-VLPs purity in all modified adsorbents, but the recovery in InertShell reduced substantially. The preheat-treatment is not recommended for the negative chromatography as the thermal-induced co-aggregation of HCPs and HB-VLPs would flow along with HB-VLPs and thus reduced the HB-VLPs purity in the flowthrough. Further reduction in the feedstock concentration enhanced the purity of HB-VLPs especially in InertLayer 1000 but reduced substantially the recovery of HB-VLPs. In general, the polymer grafted adsorbent, SQ, performed better than the core-shell adsorbents in handling a higher feedstock concentration.
以结合-洗脱模式纯化病毒样颗粒（VLPs）已达到瓶颈。阴离子交换色谱法已成为替代解决方案；然而，缺乏阴离子交换色谱介质及其各自优化条件的基准。因此，本研究旨在比较不同阴离子交换色谱介质从澄清的大肠杆菌原料中纯化乙型肝炎病毒样颗粒（HB-VLPs）的性能。比较了改性阴离子交换介质、核壳吸附剂（InertShell和InertLayer 1000）和聚合物接枝吸附剂（SQ）。核壳吸附剂填充床柱的色谱结果表明，由于壳层厚度，HB-VLPs在流穿级分中的纯度和回收率之间存在权衡。原子力显微镜分析揭示了壳层中呈漏斗状的孔道，这可能导致HB-VLPs的截留。在较低的进料流速（0.5ml/min）下延长停留时间，略微提高了所有改性吸附剂中HB-VLPs的纯度，但InertShell中的回收率大幅降低。不建议对阴离子交换色谱进行预热处理，因为热诱导的宿主细胞蛋白（HCPs）和HB-VLPs共聚集体会与HB-VLPs一起流出，从而降低流穿液中HB-VLPs的纯度。进一步降低原料浓度可提高HB-VLPs的纯度，尤其是在InertLayer 1000中，但会大幅降低HB-VLPs的回收率。总体而言，聚合物接枝吸附剂SQ在处理较高原料浓度方面比核壳吸附剂表现更好。
