CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
N Biotechnol. 2020 Nov 25;59:1-9. doi: 10.1016/j.nbt.2020.04.003. Epub 2020 Jul 2.
New biotechnological strategies are being explored, aimed at rapid and economic manufacture of large quantities of DNA vaccines with the required purity for therapeutic applications, as well as their correct delivery as biopharmaceuticals to target cells. This report describes the purification of supercoiled (sc) HPV-16 E6/E7 plasmid DNA (pDNA) vaccine from a bacterial lysate, using an arginine-based monolith, presenting a spacer arm in its configuration. To enhance the performance of the purification process, monolith modification with the spacer arm can improve accessibility of the arginine ligand. By using a low NaCl concentration at pH 7.0, a condition to eliminate the RNA impurity directly in the flow through was established. The pH increase to 7.5 allowed the elimination of non-functional pDNA isoforms, the sc pDNA being recovered by increasing the ionic strength. As well as a binding capacity of 2.53 mg/mL obtained with a pre-purified sc pDNA sample, the column also purified sc pDNA from high lysate loading, with capacities above 1 mg/mL. Due to the sample displacement phenomena, non-functional pDNA isoforms were eliminated throughout column loading, favoring the degree of purity of final sc pDNA of 93.3%-98.5%. Thereafter, purified sc pDNA was successfully encapsulated into CaCO-gelatin nano-complexes. Delivery of the pDNA-carriers to THP-1 cells was assessed through pDNA cellular uptake evaluation and correct E6 expression was verified by mRNA and protein detection. A biotechnological platform was established for sc pDNA purification and delivery to dendritic cells, stimulating further in vivo studies.
正在探索新的生物技术策略，旨在快速、经济地制造大量具有治疗应用所需纯度的 DNA 疫苗，以及将其正确递送至靶细胞的生物制药。本报告描述了从细菌裂解物中纯化超螺旋（sc）HPV-16 E6/E7 质粒 DNA（pDNA）疫苗的方法，使用基于精氨酸的整体柱，其构型中带有间隔臂。为了提高纯化过程的性能，通过间隔臂修饰整体柱可以改善精氨酸配体的可及性。通过在 pH 7.0 下使用低 NaCl 浓度，可以建立直接在流通过程中消除 RNA 杂质的条件。pH 增加到 7.5 允许消除非功能性 pDNA 同种型，通过增加离子强度回收 sc pDNA。使用预纯化的 sc pDNA 样品获得 2.53mg/mL 的结合容量，该柱还可以从高裂解物负载中纯化 sc pDNA，容量超过 1mg/mL。由于样品置换现象，非功能性 pDNA 同种型在整个柱加载过程中被消除，有利于最终 sc pDNA 的纯度达到 93.3%-98.5%。此后，成功地将纯化的 sc pDNA 封装到 CaCO-明胶纳米复合物中。通过评估 pDNA 细胞摄取来评估 pDNA-载体向 THP-1 细胞的递呈，并通过 mRNA 和蛋白质检测验证正确的 E6 表达。建立了用于 sc pDNA 纯化和递送至树突状细胞的生物技术平台，刺激了进一步的体内研究。
