Process Development and Optimization of Nucleic Acid Vaccines

In 1989, the concept of mRNA vaccine was first mentioned. Thanks to the efforts of global nucleic acid vaccine companies represented by BioNTech and Moderna, mRNA vaccine technology has made breakthrough progress.

mRNA vaccines have the advantages of short research and development production cycle, high immunogenicity, high safety, and low risk of biochemical pollution. Currently, there are multiple companies developing vaccines for influenza, African swine fever, epidemic diarrhea, foot-and-mouth disease, and feline vaccination, among other categories.

I. Basic Principles of Nucleic Acid Vaccines

Nucleic acid vaccines introduce exogenous genes (DNA or RNA) encoding a certain antigen protein directly into animal somatic cells, where the antigen protein is synthesized through the expression system of the host cells. This process induces an immune response in the host against the antigen protein, ultimately achieving the goal of preventing and treating diseases. Based on the different main components, nucleic acid vaccines can be mainly classified into DNA vaccines and mRNA vaccines.

DNA vaccines consist of recombinant eukaryotic expression vectors that encode vaccine antigens. mRNA vaccines work on the same principle as DNA vaccines, but bypass the first step of nuclear translocation and transcription of DNA vectors into mRNA. Self-replicating RNA vaccines may induce protective immunity with lower doses because each cell expresses more vaccine antigens. Unlike traditional vaccines, mRNA vaccines do not require the use of the pathogen itself or its proteins, but instead directly utilize genetic information to direct cells to synthesize antigens. Below, we will primarily introduce the process scheme focusing on mRNA vaccines.

II. Production Process of mRNA

The production process of mRNA vaccines primarily consists of three parts: preparation of pDNA templates, production of mRNA drug substance (DS), and loading of delivery vectors followed by formulation and filling. The production process of mRNA DS encompasses upstream processes such as plasmid linearization, in vitro transcription (IVT), and downstream processes like mRNA purification and bulk filling.

1. Plasmid Production Process

Figure 1. BioLink Plasmid Production Solution

In the upstream process, Escherichia coli expression bacteria are used, and through bacterial harvest, downstream lysis, clarification filtration, concentration, and chromatography purification, high-purity supercoiled DNA (scDNA) can be obtained.

Figure 2. Plasmid Purification Solution

The three-step method using Chromstar® 6FF + MaXtar® PlasmidCap HR + MaXtar® Q HR chromatography for plasmid purification is the most classic and commonly used. Depending on different plasmid products and user needs, BioLink also offers various plasmid product purification strategies. For example, BARONHAP® Type II can be used to remove HCD and ocDNA, or after precipitating the sample with CaCl2 or ammonium sulfate, MaXtar® Q + MaXtar® PlasmidCap HR/ BARONHAP® Type II can be used for two-step chromatography purification of supercoiled plasmids.

Case Sharing:

Figure 3. Chromatogram of the three-step plasmid purification method

(From left to right: Chromstar® 6FF, MaXtar® PlasmidCap HR, MaXtar® Q HR)

Conclusion: Through the classic three-step method, the supercoiled purity can reach over 90%, and the overall yield is also at a high level.

Figure 4. Chromatogram of the two-step method

Using MaXtar® COLL 700 to remove impurities such as RNA and MaXtar® PlasmidCap HR resin to enhance the purity of supercoiled plasmids. Both the yield and purity meet regulatory requirements.

2. mRNA Process Flow

BioLink provides a range of solutions for the mRNA process, covering key equipment and consumables from linearization to formulation. For example, the CytoLinX® WB is used in the IVT process, catering to the characteristics of low-shear and small-volume in vitro transcription; innovative RNase-free single-use consumables address the potential issue of introducing RNase/DNase during production; laboratory and industrial-grade ultrafiltration equipment and consumables are also available. In addition, BioLink offers high-quality chromatography purification solutions, including chromatography systems, columns, and related resin products.

Figure 5. mRNA Production Process Flow

The purification of mRNA is performed using affinity Maxgo® Oligo dT+ hydrophobic MaXtar® Phenyl HR (or MaXtar® Butyl HR). After completion, further concentration and buffer exchange are typically required, which can be achieved through ultrafiltration. The final mRNA product is then filled and stored frozen to ensure its stability.

3. Delivery System

Lipid Nanoparticles (LNPs) are the critical carriers for mRNA vaccines, effectively protecting the mRNA and facilitating its delivery and expression within cells.

Figure 6. LNP Production Process Flow

The production of LNPs involves large-volume fluid transfer and ultrafiltration buffer exchange processes, for which BioLink can provide complete ultrafiltration and fluid handling solutions. Additionally, for filling processes, BioLink offers efficient and flexible customization, utilizing industry-leading particle control (Class B+A) during production. A diverse range of options and robust testing and validation help to manage risks in LNP production.

The development and process solutions for veterinary mRNA vaccines encompass all aspects from basic research to production. With continuous advancements in technology, mRNA vaccines will play an increasingly important role in the veterinary field, and BioLink will provide more efficient and safe solutions for animal nucleic acid vaccines.