Departamento de Engenharia Química, Escola Politécnica da Universidade de São Paulo, Av. Prof. Luciano Gualberto, Trav. 3, Nº 380, São Paulo, SP, CEP 05508-900, Brazil.
CICS-UBI - Centro de Investigação de Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506, Covilhã, Portugal.
Mol Biol Rep. 2022 May;49(5):3893-3901. doi: 10.1007/s11033-022-07239-x. Epub 2022 Feb 18.
The production of nucleic acids (plasmid DNA or mRNA) in response to the development of new advanced vaccine platforms has greatly increased recently, mostly resulting from the pandemic situation. Due to the intended pharmaceutical use, nucleic acids preparations must fulfill all the required specifications in terms of purity and quality. Chromatography is a standard operation used to isolate these molecules from impurities, playing a central role in the manufacturing processes. However, the mechanism of nucleic acid adsorption in chromatographic resins is poorly understood, often leading to low adsorption capacities and a lack of specificity.
Here we investigated the adsorption of plasmid DNA and RNA molecules onto arginine-agarose, a resin with potential for large-scale application. Equilibrium batch studies were performed through pre-purified samples, using arginine-based ligands by varying the adsorption conditions in the pH value range from 6.0 to 9.0. Langmuir and Freundlich isotherm models were used to describe the adsorption equilibrium. The best fit for both nucleic acids was achieved using the Freundlich model. The correct choice of pH showed critical for controlling the efficacy of arginine-nucleic acid interaction, due to its influence on the nucleic acid structures. This type of analysis is necessary for the improvement of the selectivity and binding capacities of the resins used for plasmid DNA or mRNA purification.
The results presented here indicate that adsorption conditions can be tuned to enhance separation between pDNA and RNA, an important feature in the purification of nucleic acids for vaccine production.
随着新型先进疫苗平台的发展，核酸（质粒 DNA 或 mRNA）的生产最近大幅增加，这主要是由于大流行情况所致。由于预期的药物用途，核酸制剂必须在纯度和质量方面满足所有必需的规格。色谱法是一种用于从杂质中分离这些分子的标准操作，在制造过程中起着核心作用。然而，核酸在色谱树脂中的吸附机制理解甚少，这常常导致吸附容量低且缺乏特异性。
在这里，我们研究了质粒 DNA 和 RNA 分子在具有大规模应用潜力的精氨酸琼脂糖上的吸附。通过使用基于精氨酸的配体，通过在 pH 值为 6.0 至 9.0 的范围内改变吸附条件，进行了预纯化样品的平衡分批研究。使用 Langmuir 和 Freundlich 等温线模型来描述吸附平衡。对于两种核酸，最佳拟合均使用 Freundlich 模型。由于其对核酸结构的影响，正确选择 pH 值对于控制精氨酸-核酸相互作用的效果至关重要。这种类型的分析对于提高用于质粒 DNA 或 mRNA 纯化的树脂的选择性和结合能力是必要的。
这里呈现的结果表明，可以调整吸附条件以增强 pDNA 和 RNA 之间的分离，这是疫苗生产中核酸纯化的重要特征。
