Vaccines are the most economical and effective means of preventing and controlling infectious diseases. Vaccination is a conventional method for preventing and controlling human and animal diseases by inducing protective immune responses. Vaccine technology has evolved from Pasteur's principle of pathogen "isolation, inactivation, and injection" to modern vaccine technologies based on the integration of genetic engineering, immunology, structural biology, reverse vaccinology, and systems biology, expanding into areas such as cancer, autoimmune diseases, and other chronic conditions.

Traditional vaccine approaches are inadequate for addressing today's major, complex, emerging, and highly variable pathogens. Furthermore, vaccines for several significant infectious diseases, including HIV, tuberculosis, malaria, and other widely spread pathogens, have yet to be successfully developed. Novel vaccine technologies represent powerful tools for addressing future global health challenges.

Figure 1. Production systems and cell substrates used in vaccine manufacturing.

1. Recombinant Protein Production Process

Recombinant protein vaccine technology is a novel vaccine preparation method that utilizes genetic engineering to clone specific pathogen protein genes into expression vectors, producing high-purity protein vaccines through large-scale cell culture and purification techniques. Compared to traditional inactivated and live vaccines, recombinant protein vaccines offer advantages including high safety, good stability, and ease of production.

Production Process:

1. Gene Cloning: Clone specific pathogen protein genes into expression vectors to construct recombinant expression vectors
2. Cell Culture: Transfect recombinant expression vectors into host cells and produce recombinant proteins through large-scale cell culture
3. Protein Purification: Purify recombinant proteins from cell culture medium using centrifugation, chromatography, and electrophoresis techniques to obtain high-purity protein vaccines
4. Vaccine Preparation: Mix purified recombinant proteins with appropriate adjuvants to formulate vaccines

The recombinant protein vaccine preparation process is relatively complex, requiring high-level technical and equipment support. However, these vaccines offer superior safety and stability compared to traditional inactivated and live vaccines, effectively preventing various infectious diseases, including hepatitis B, influenza, and AIDS.

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2. Virus-Like Particle (VLP) Vaccines

VLP vaccine production is a novel vaccine manufacturing technology that uses genetic engineering to express viral capsid proteins, forming virus-like particles. These particles possess the same appearance and structure as actual viruses but contain no viral nucleic acids, making them incapable of causing disease. VLP vaccines exhibit high safety and immunogenicity and are widely used for preventing various infectious diseases, including hepatitis B and human papillomavirus infection.

Production Process:

1. Gene Cloning: Clone viral capsid protein genes into expression vectors using PCR amplification
2. Cell Culture: Transfect expression vectors into host cells (yeast, insect cells, or mammalian cells) to produce VLP particles
3. Purification and Extraction: Separate VLP particles from cell debris and protein impurities using centrifugation, filtration, ultracentrifugation, column chromatography, and electrophoresis
4. Vaccine Preparation: Process purified VLP particles through inactivation and formulation into vaccines using freeze-drying or suspension methods

Currently, yeast expression systems are commonly used for VLP vaccine production. After fermentation and cell collection, yeast cells are disrupted, cell debris is removed through clarification filtration, and target proteins are captured using membrane chromatography. Following further purification, ultrafiltration buffer exchange, and formulation filling complete the process.

3. Polysaccharide and Polysaccharide Conjugate Vaccines

To enhance immune response, traditional bacterial polysaccharide vaccines are chemically conjugated with protein components to create polysaccharide conjugate vaccines, which increase immune response in infants and young children.

Production Process:

Upstream bacterial fermentation is followed by cell collection, detoxification with inactivating agents, and biocompatible deep filtration. Ultrafiltration systems concentrate the harvest volume and remove small molecular proteins. After clarification filtration to remove large molecules and particulate impurities, repeated ultrafiltration and clarification filtration complete the polysaccharide preparation.

For conjugate vaccines, protein conjugation uses chemical methods to bind proteins with polysaccharides. This process produces free polysaccharides and proteins alongside the target conjugated product, which are removed using ultrafiltration and chromatographic methods. Single-use liquid preparation and filling systems improve batch changeover efficiency and prevent cross-contamination between different serotype preparations.

4. Viral Vector Vaccine Production

Viral vector vaccines utilize attenuated vaccine strains or non-replicating viruses as vectors to effectively deliver antigen gene coding to host cell nuclei and trigger immune responses. Various viruses, including adenovirus, lentivirus, vesicular stomatitis virus, herpes virus, measles virus, and recombinant vaccinia virus Ankara strain, serve as vectors for vaccine development.

Viral genomes can express any specific antigen and stably accept large gene fragment insertions. Antigens can be accurately synthesized, modified, and targeted to specific cells in hosts, making viral vector technology applicable for various vaccine development.

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5. Inactivated Vaccine Production

Inactivated vaccines are produced by culturing viruses or bacteria and then inactivating them through physical or chemical methods to obtain vaccines without infectious activity. Compared to attenuated vaccines and genetic engineering vaccines, inactivated vaccines offer advantages including short development cycles, relatively mature preparation processes, no infectious toxicity, and high safety. However, they typically require multiple doses to produce protective immunity and generally induce humoral immune responses with relatively weak cellular immunity.

Production Process:

1. Pathogen Culture: Cultivate pathogens (viruses, bacteria, or other microorganisms) in appropriate culture media
2. Pathogen Inactivation: Inactivate pathogens using chemical, thermal, or radiation methods while preserving antigenicity
3. Pathogen Extraction and Purification: Extract and purify inactivated pathogens through centrifugation, filtration, precipitation, and washing to remove impurities
4. Vaccine Formulation and Packaging: Mix pathogens with appropriate excipients, including preservatives, stabilizers, and buffers, then package in suitable containers
5. Quality Control: Conduct physicochemical testing, microbiological testing, and animal experiments to ensure safety and efficacy

Figure 2. Example of a typical Inactivated viral vaccine production process.

6. DNA Vaccine Production

DNA vaccines insert eukaryotic expression cassettes encoding antigen proteins into bacterial plasmids, with antigen protein expression driven by efficient eukaryotic promoters. DNA vaccines can induce both humoral and cellular immunity with long-lasting immune effects, simple preparation, and easy large-scale production. However, they have limitations, including weak immunogenicity, low plasmid DNA delivery efficiency, and low expression efficiency, as well as risks of foreign gene integration into host genomes, causing mutations and tumor formation.

Production Process:

1. Plasmid Library and Seed Bank Establishment: Construct quality plasmid libraries and seed banks for efficient production of specific plasmid DNA vaccines
2. Engineered Bacteria Fermentation: Optimize cultivation schemes to obtain high-quality bacterial populations
3. Cell Lysis: Use gentle and stable reaction systems with controlled lysis time (within 5 minutes), pH range of 12.0-12.5, and mild uniform stirring
4. Plasmid DNA Recovery: Perform clarification treatment using centrifugation and filtration to remove cell debris and large aggregates
5. Initial Purification: Remove impurities using ultrafiltration membranes with different molecular weight cutoffs
6. High-Degree Purification: Achieve optimal results through gel filtration chromatography combined with other chromatographic methods
7. Concentration, Formulation, Sterile Filtration, and Packaging: Complete concentration using membrane components, followed by sterile filtration and packaging according to requirements

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7. mRNA Vaccine Production

mRNA vaccine development involves selecting specific antigen proteins from target pathogens, sequencing and synthesizing protein genes, cloning them into DNA template plasmids, transcribing into mRNA in vitro, and then inoculating subjects. mRNA vaccines mainly include two types: traditional non-replicating mRNA and self-amplifying mRNA.

Compared to traditional vaccines, mRNA vaccines offer immune response mechanisms similar to live viruses, no risk of infection or integration into host genomes, greater stability with efficient antigen protein expression, and simple, rapid chemical synthesis, enabling easier, low-cost, large-scale production.

Production Process (Using SARS-CoV-2 mRNA vaccine as an example):

1. DNA Stock Preparation: Design and construct vectors containing S protein gene sequences, then amplify through E. coli culture to obtain large quantities of plasmids carrying S protein gene sequences
2. mRNA Stock Preparation: Linearize plasmids containing S protein gene sequences and perform in vitro transcription (IVT) with 5' capping. Subsequent purification removes enzymes, free nucleotides, residual DNA, exogenous RNA fragments, dsRNA, and immunogenic impurities from the reaction system
3. Lipid Nanoparticle Encapsulation: Prepare delivery systems such as polymer- or lipid-based nanoparticles (LNP), then encapsulate purified mRNA using manufacturer-specific methods. The resulting mRNA/delivery nanoparticle assemblies undergo concentration, buffer exchange, and sterile filtration before final evaluation and packaging as drug substance, typically frozen before final drug product filling

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As a key partner in vaccine research and development, Creative Biogene provides comprehensive one-stop services for recombinant protein vaccines, viral vector vaccines, and VLP vaccines through advanced genetic engineering technologies and integrated production platforms. We offer efficient gene cloning and expression systems for precise construction of high-quality expression vectors, enabling large-scale production via optimized mammalian and yeast cell culture technologies. With emphasis on process reproducibility and scalability, Creative Biogene helps clients accelerate development timelines and enhance market competitiveness. From gene cloning and protein expression to purification, assembly, and vaccine formulation, Creative Biogene delivers flexible, efficient technical support to facilitate seamless translation of innovative vaccines from laboratory to commercial production.