mRNA technology demonstrated remarkable success in the development of COVID-19 vaccines and has also achieved significant breakthroughs in preventive vaccines, cancer treatment, protein replacement therapy, and gene editing. Currently, approximately 70% of global mRNA projects in preclinical and clinical stages target diseases beyond COVID-19—31% focus on cancer, and 9% on other genetic and immune disorders. This shift indicates that non-vaccine applications will drive the future development of mRNA technologies.

To ensure product quality and regulatory compliance, the USP’s 3rd edition of Analytical Procedures for Quality of mRNA Vaccines and Therapeutics and the CDE’s Guidelines for the Development of COVID-19 Preventive Vaccines (Trial) have established standardized methods and acceptance criteria for mRNA quality assessment. Notably, both documents specify Capillary Gel Electrophoresis (CGE) as the preferred method for evaluating plasmid supercoiling and mRNA integrity.

Table 1. USP-Recommended Methods for Plasmid and mRNA Integrity Testing

The principle of capillary gel electrophoresis (CGE) involves a capillary filled with gel, which connects two containers filled with buffer solution, serving respectively as the inlet and outlet. A voltage is applied across the system, and a detection window connected to a detector collects and processes data; this constitutes the CGE apparatus. Under buffer-gel conditions, the phosphate groups in nucleic acid molecules dissociate and carry a negative charge. When a reverse voltage is applied, nucleic acids move from the negative electrode toward the positive electrode. Under the same electric field conditions, nucleic acid molecules of different sizes and conformations migrate at different speeds and reach the detector at different times, thus achieving separation and detection.

Plasmid Supercoiling
High-quality plasmid DNA is a critical starting material in the production of mRNA, AAV vectors, and cell therapies. Plasmid DNA exists in three primary conformations: Supercoiled (SC), Linear, and Open Circular (OC). Among these, the supercoiled form is the most desirable for biomanufacturing applications. Accurate quantification of the supercoiled plasmid proportion is essential for ensuring product quality and process consistency.

Overview of Supercoiled Plasmid Detection Methods

Agarose Gel Electrophoresis (AGE)

Principle: Separates plasmids based on conformational differences that affect migration rates in the gel; qualitative analysis is performed by assessing band positions.

Advantages: Fast operation and low cost; suitable for preliminary analysis.

Limitations: Supercoiled dimers may be confused with open circular monomers, affecting qualitative and quantitative accuracy.

Anion Exchange Chromatography (AEX-HPLC)

Principle: Separates based on differences in the number of negatively charged phosphate groups in plasmid DNA.

Advantages: High resolution and capable of quantitative analysis.

Limitations: High cost and low throughput; not ideal for rapid detection of large sample volumes.

Capillary Gel Electrophoresis (CGE)

Principle: Gel-filled capillaries act as a support medium, separating plasmid conformations by size through a molecular sieving effect.

Advantages: Requires small sample volumes, offers high sensitivity and short detection time, with excellent resolution of plasmid DNA conformations.

Limitations: Requires specialized equipment.

Hzymes Biotech
Capillary Gel Electrophoresis Plasmid DNA Detection Kit
This kit is designed for quantifying the proportions of supercoiled, linearized, and open circular plasmid DNA. It is compatible with various capillary electrophoresis instruments and offers high reproducibility and excellent resolution.

Figure 1. DNA Ladder Profile

Figure 2. Electrophoresis Profile of Plasmid DNA

mRNA Integrity
After large-scale synthesis via in vitro transcription (IVT), mRNA crude solutions are typically analyzed using capillary gel electrophoresis to assess integrity and detect potential truncated fragments. CGE enables precise quantification of mRNA integrity and allows for estimation of mRNA length using an RNA ladder.

Hzymes Biotech
Capillary Gel Electrophoresis (CGE) Plasmid DNA Detection Kit
Designed for assessing mRNA integrity, this tool enables rapid sample detection within 30 minutes. It offers high sensitivity, capable of detecting RNA at concentrations as low as 1 ng/μL, and is suitable for various RNA types including mRNA and total RNA.

Figure 3. Electrophoresis Profile of mRNA Samples

Figure 4. Standard Curve

Table 2. Integrity Analysis of mRNA Raw Material at Different Lengths

RNA ladder 6000

The RNA Ladder is a molecular weight mixture composed of single-stranded RNA (ssRNA) fragments of varying lengths. When analyzed by gel electrophoresis or capillary electrophoresis, it produces distinct bands that serve as size markers for identifying the length of single-stranded RNA molecules. Based on the principle that the migration rate through a gel matrix is inversely proportional to molecular weight, the RNA Ladder allows for the estimation of the size of unknown RNA fragments by comparison to the known reference bands.

Hzymes Biotech
RNA Ladder 6000
The RNA Ladder 6000 is a mixture of single-stranded RNA molecules of varying lengths, comprising eight reference bands at 200 nt, 500 nt, 1000 nt, 1500 nt, 2000 nt, 3000 nt, 4000 nt, and 6000 nt. With clear band resolution and precise molecular sizes, it is suitable for a variety of electrophoresis techniques, including non-denaturing gel electrophoresis, denaturing gel electrophoresis, and capillary gel electrophoresis.

Figure 5. RNA 6000 Ladder Capillary Gel Electrophoresis Profile

Figure 6. RNA 6000 Ladder Gel Electrophoresis Profile

Ordering