Abstract:

The rapid development of multivalent mRNA therapeutic vaccines against human papillomavirus （HPV） has raised higher requirements for the quality control of long-chain nucleic acids molecules. Currently， Pharmacopoeiaof the People’s Republic of China and the United States Phanmacopoeia lack specific analytical conditions for separation of relevant substances in biological produds， with limited industry reports available. To establish a purity method for long-chain mRNA， the chromatographic conditions were systematically optimized using a poly-A-deficient impurity reference standard to determine optimal separation. The method was validated for linearity， limit of quantitation （LOQ）， limit of detection （LOD）， specificity， and precision. Optimal separation was achieved using a Thermo DNA Pac RP column （100 mm×2.1 mm， 4 μm） with a mobile phase of 400 mmol·L^-1 triethylamine acetic acid（TEAA）-methanol under gradient elution， at a flow rate of 0.4 mL·min^-1， column temperature of 80 ℃， and detection wavelength of 260 nm. Under these conditions， resolution between the main mRNA peak and the known impurity peak exceeded 1.5， and the LOQ and LOD were 4.0 μg·mL^-1 and 0.8 μg·mL^-1， respectively. Excellent linearity （R2=0.999 6） was demonstrated over the range of 4.0 μg·mL^-1 to 2 000.0 μg·mL^-1. Repeatability and intermediate precision both showed relative standard deviation （RSD）1.0%. This optimized and validated ion-pairing reversed-phase ultra performance liquid chromatography （IP-RP-HPLC） method significantly improves the resolution for separating long-chain nucleic acid related substances， providing reliable data for mRNA vaccine process development， quality control， and batch consistency studies.

Key words: gene therapy, mRNA vaccine, mRNA related substances, quality control, ion-pair reversed-phase ultra performance liquid chromatography

摘要：

人乳头瘤病毒（human papillomavirus，HPV）多价mRNA治疗型疫苗的快速发展，对长链核酸分子的质量控制提出了更高的分离要求。目前，《中华人民共和国药典》（简称 《中国药典》）与《美国药典》对于生物制品中有关物质的分离并没有给出具体的分析条件，行业相关报道也甚少。为了建立适用于长链mRNA的纯度分离方法，对色谱分离条件进行系统的优化与筛选，利用Poly A缺失的杂质对照品确定最佳分离工作条件，并对该方法进行了线性范围、定量限、检测限、专属性、精密度等验证。结果显示，采用Thermo DNA Pac RP色谱柱（100 mm×2.1 mm，4 μm），以400 mmol·L^-1 三乙胺乙酸（triethylamine acetic acid, TEAA）离子对试剂溶液-甲醇为流动相梯度洗脱，流速0.4 mL·min^-1，柱温80 ℃，检测波长260 nm。在选定的色谱条件下，mRNA主峰与对照品杂质色谱峰之间的分离度1.5，定量限（limit of quantitation，LOQ）为4.0 μg·mL^-1，检测限（limit of detection，LOD）为0.8 μg·mL^-1。mRNA原液质量浓度在4.0~2 000.0 μg·mL^-1范围内线性关系良好（R²=0.999 6），重复性与中间精密度试验相对标准偏差（relative standard deviation, RSD）均1.0%。对长链mRNA疫苗离子对反相高效液相色谱法进行了优化开发与系统的验证，旨在提升长链核酸有关物质的分离分辨率，并为mRNA疫苗的工艺开发、质量控制和批次间的质量一致性研究提供可靠的数据支撑。

关键词: 基因治疗, mRNA疫苗, mRNA有关物质, 质量控制, 离子对反相色谱法