Establishment and Verification of a Capillary Electrophoresis-laser Induced Fluorescence Method for Assessing Linear mRNA Integrity in Gene Therapy Vaccines

doi:10.19586/j.2095-2341.2025.0083

Current Biotechnology ›› 2025, Vol. 15 ›› Issue (6): 1064-1070.DOI: 10.19586/j.2095-2341.2025.0083

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Establishment and Verification of a Capillary Electrophoresis-laser Induced Fluorescence Method for Assessing Linear mRNA Integrity in Gene Therapy Vaccines

Wenyan CUI¹(), Yue YANG¹, Wenzhong CHEN¹^,², Yan ZUO¹, Liangliang CHEN¹, Xiaodong HU¹

^1.Nanjing Geneleap Biotechnology Co. ，Ltd. ，Nanjing 210000，China
^2.State Key Laboratory of Advanced Drug Delivery and Release Systems，Shandong Luye Pharmaceutical Co. ，Ltd. ，Shandong Yantai 264003，China

Received:2025-07-17 Accepted:2025-09-15 Online:2025-11-25 Published:2026-01-04

Abstract

Abstract:

A linear mRNA integrity capillary electrophoresis-laser induced fluorescence （CE-LIF） method for gene therapy vaccines has been established and validated， in order to provide a reliable detection method for characterization research， quality control and process stability monitoring of gene therapy vaccine products. Linear mRNA integrity was analyzed using CE-LIF， with an injection time of 3 s， a voltage of -2 kV， a separation voltage of 9 kV and an analysis time of 35 min. Using sample GL2327-4-120A， the method was validated for specificity， precision， accuracy， linearity， limit of quantitation （LOQ） and limit of detection （LOD）. The results showed that the resolution between peaks in a mixture of GL2327-4-120A and GL2327-4-0A was 2.86. For six replicate analyses of sample， the relative standard deviation （RSD） of the main peak migration time was 0.05%， and the RSD of the corrected peak area was 0.32%. The recovery rate of the peak area of the GL2327-4-120A samples spiked with the test sample was within the range of 90% to 115%. The mRNA concentration showed a good linear relationship within the range of 0.30~60.0 μg·mL^-1， and the linear regression equation was y = 217 601x - 216 294， R2 = 0.994 9. The LOQ was determined to be 0.30 μg·mL^-1 and the limit of detection （LOD） was 0.06 μg·mL^-1. The established CE-LIF method has good specificity， precision， and accuracy， and can be used to analyze the linear mRNA integrity in gene therapy vaccines.

Key words: gene therapy, mRNA vaccine, capillary electrophoresis, mRNA integrity, method validation, quality control

摘要：

建立基因治疗疫苗线性mRNA完整度毛细管电泳激光诱导荧光（capillary electrophoresis laser induced fluorescence，CE-LIF）检测方法，并进行方法验证，以期为基因治疗疫苗产品的表征研究、质量控制及生产工艺稳定性的监控提供可靠的检测方式。采用激光诱导荧光模式进行线性mRNA完整度分析，进样时间设为3 s，电压为-2 kV，分离电压为9 kV，分析时间为35 min。以GL2327-4-120A为待测样品，验证方法的专属性、精密度、准确性、线性范围、定量限（limit of quantitation，LOQ）及检测限（limit of detection，LOD）。结果发现，GL2327-4-120A与GL2327-4-0A混合溶液样品分离度为2.86；6次重复检测待测样品，主峰迁移时间的相对标准偏差（relative standard deviation，RSD）为0.05%、校正峰面积的RSD为0.32%；待测加标GL2327-4-0A样品峰面积的回收率均在90%~115%范围内；mRNA浓度在0.30~60.0 μg·mL^-1范围内，呈现良好的线性关系，线性回归方程为y= 217 601x-216 294，R²=0.994 9；LOQ为0.30 μg·mL^-1；LOD为0.06 μg·mL^-1。建立的CE-LIF检测方法具有良好的专属性、精密度及准确性，可用于基因治疗疫苗线性mRNA完整度的分析。

关键词: 基因治疗, mRNA疫苗, 毛细管电泳法, mRNA完整度, 方法验证, 质量控制

CLC Number:

Q812

Wenyan CUI, Yue YANG, Wenzhong CHEN, Yan ZUO, Liangliang CHEN, Xiaodong HU. Establishment and Verification of a Capillary Electrophoresis-laser Induced Fluorescence Method for Assessing Linear mRNA Integrity in Gene Therapy Vaccines[J]. Current Biotechnology, 2025, 15(6): 1064-1070.

崔文燕, 杨悦, 陈文忠, 左彦, 陈靓靓, 胡晓冬. 基因治疗疫苗线性mRNA完整度毛细管电泳激光诱导荧光检测方法的建立及验证[J]. 生物技术进展, 2025, 15(6): 1064-1070.

Figures/Tables 8

Fig. 1 Capillary effective length optimization electropherograms

Fig. 2 Separation buffer optimization electropherograms

Fig. 3 Sieving matrix optimization electropherograms

Fig. 4 HEC concentration optimization in sieving matrix electropherograms

Fig. 5 Verification for specificity

Fig. 6 Linear regression curve for GL2327-4-120A

Fig. 7 mRNA mixed samples capillary gel electrophoresis electropherograms

Fig. 8 Capillary gel electrophoresis electropherograms of 6 pilot process samples

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Establishment and Verification of a Capillary Electrophoresis-laser Induced Fluorescence Method for Assessing Linear mRNA Integrity in Gene Therapy Vaccines

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