mRNA Vaccine Manufacturing Strategy 1 --Plasmid DNA Production

mRNA Vaccine Manufacturing Strategy 1 --Plasmid DNA Production

As a branch field of gene therapy, mRNA vaccine production depends on the design and modification of specific gene sequences in the early stage of development. After in vitro transcription and purification of mRNA, packaging with LNP(lipid nanoparticles) vectors in microfluidic devices to obtain mRNA-LNP products. (Figure 1).

Figure 1-Production process of mRNA vaccine

The overall CMC production process of mRNA vaccines can be divided into three modules: pDNA production, mRNA production, and LNP encapsulation (Figure 2).

Figure 2-Overview of mRNA-CMC process

Plasmid DNA(pDNA) Manufacturing

The core goal of plasmid production is to obtain a certain volume of high-purity supercoiled plasmids. Plasmid amplification usually requires large-scale high-density fermentation of Escherichia coli. Due to the relatively high solid content of the bacterial culture, considering the efficiency and amplification issues, tangential flow filtration is often used to collect and wash the bacterial culture. Subsequently, the alkaline lysis method is utilized to release the plasmid in E. coli.

Under alkaline conditions, the proteins, RNA, DNA and plasmids of Escherichia coli will be denatured. After adding the neutralizing solution, a large number of denatured proteins and host DNA will combine together through hydrophobic interactions which will form a flocculent precipitate. While our target plasmid can be refolded and dissolved in the supernatant, it is not easy to collect and separate because this flocculent precipitate is loose and fragile and dispersed throughout the system.

Therefore, considering the cost, flocculation with CaCl is generally used to precipitate impurities. Or adding solid NHHCO to generate a large amount of gas which will drive the flocculent precipitate to float.These methods are more conducive to solid-liquid separation, and the harvested clear liquid will be clarified by filters.

Because the amount of feed liquid obtained after clarification is relatively large, concentration and diafiltration are required to reduce the column volume for subsequent purification. It will also remove impurities and realize liquid exchange at the same time.

Plasmid purification is usually processed by the classic three-step method. Size-exclusion chromatography (SEC) removes most of the RNA and reduces the content of endotoxin. Affinity chromatography(AC) is mainly used to remove open-circle DNA. Ion exchange chromatography(IEX) is used to remove small amounts of impurities HCP and endotoxin. Finally, with concentration, buffer exchange and sterile filtration, it will obtain high-purity supercoiled plasmid products (see Figure 3).

Figure 3 - Classic Process of Plasmid Production


There are two commonly used methods for harvesting bacterial cultures, centrifugation and tangential flow filtration. Centrifugal collection can be applied to small-volume solutions in the early research and development stage, and scale-up production requires tangential flow filtration.

Due to the relatively high solid content of the bacterial cultures, the application of microfiltration cassettes is easy to block, resulting in reduced flux and difficult cleaning. Hollow fiber products have open flow channels that can handle feed liquid with higher solid content. They are easy to scale up linearly and can exchange the resuspension culture medium at the same time.

For high concentration of E. coli fermentation, we recommends 750 kDa or 0.1-0.2μm membranes, 1mm inner diameter open flow channel hollow fiber modules. After harvesting bacteria, add 2-3 diavolumes for diafiltration, and exchanged with lysis buffer. Using hollow fiber products to concentrate or diafiltration can achieve a recovery rate of 99.99%.


In alkaline lysis process, use NaOH and optimize the lysis reaction time in 0.5-10min. If the lysis incubation time is too long, it will lead to irreversible damage to the plasmid DNA. In addition, a gentle and efficient mixing method should be used to prevent pH extremes.


For the clarification of Escherichia coli lysis feed, Cobetter offers product series including PP filter bags, PP filters and depth filters. There are many combinations for specific applications:

1. PP Filter Bag + PP(CHT) Filter.

2. PP filter bag + Claricap Depth Filter.

3. Multi-stage of CHT filters such as CHT90+CHT50+CHT05.

4. Single-stage large cavity depth filter (4070 or 0700).

All of them can achieve high filtration capacity.

Among them, PP filter bags are only suitable for small-scale primary clarification. For CHT filters (PP membrane material), the nano-membrane fibers are stacked in a gradually decreasing manner, which can greatly improve the dirt-holding capacity and retention efficiency, usually use with multi-stage application to achieve better clarification effect.

The depth filters compose of lignocellulose and inorganic filter aids as filter media. When feed liquid passes through the medium, the solid particles will be randomly adsorbed or retained. Its dirt-holding capacity and clarification effect are undoubtedly better than other options. Therefore, it can achieve a better clarification effect with a single-stage application, and it has better airtightness, flexibility, and compliance for scale-up production. In addition, Cobetter has developed a higher-capacity large-cavity depth filter that can be applied to kiloliter-scale clarification.


For the plasmid concentration step after clarification, it is necessary to consider the influence factors such as shear-sensitive, temperature, and nuclease contamination during the operation. Using ultrafiltration cassettes with low shear force or hollow fiber modules for plasmid concentration and exchange, under reasonable conditions can prevent DNA degradation. Based on the size of the plasmid, following the recommendation specifications in Table 1.

Sterile Filtration

The purified plasmid solution needs to have sterile filtration before final filling. Conventional 0.2um/0.22um precision sterile filters are widely used. Cobetter recommends high flow rate and low protein binding polyethersulfone (PES) or polyvinylidene fluoride (PVDF) material filter for sterile filtration. In order to make the operation more convenient and flexible, the sterile filter is often used in conjunction with single-use products.


Cobetter's product applications for the plasmid platform includes bacterial harvest, alkaline lysis, clarification, ultrafiltration concentration, ion exchange chromatography, sterile filtration etc. Considering factors such as process scale-up convenience, efficiency, and compliance, our recommendations have been successfully applied to overall solutions for different platforms in R&D, pilot testing, and commercial production.


Fang E, Liu X, Li M, Zhang Z, Song L, Zhu B, Wu X, Liu J, Zhao D, Li Y. Advances in COVID-19 mRNA vaccine development. Signal Transduct Target Therapy. 2022 Mar 23;7(1):94. DOI: 10.1038/s41392-022-00950-y. PMID: 35322018; PMCID: PMC8940982.