Virus-like Particle Vaccine And Its Downstream Purification Process
Virus-like particle (VLP) vaccine is a vaccine technology that is based on the virus structure but does not contain the viral genetic materials. They stimulate a strong immune response by mimicking the morphology and surface antigens of natural viruses, and provide greater safety due to their inability to replicate. VLP vaccines are a better alternative to traditional vaccines (live attenuated and inactivated). VLP vaccine cannot replicate in the body, so it is suitable for everyone, including pregnant women or those with compromised immune systems. In addition, capsid proteins (including other structural proteins) from viruses with segmented genomes, such as influenza viruses, African horse sickness viruses, and bluetongue viruses, can also be used to develop VLP vaccines without worrying about genetic recombination issues like attenuated live vaccines. Second, VLPs are able to mimic the structure of real viruses, unlike inactivated vaccines, whose structural proteins may be modified during inactivation, resulting in immunogenic impairment.
FIG. 1. Formation mechanism of VLPs and chimeric VLPs
Features of VLP vaccine
1. Structure similar to natural viruses: VLPs are self-assembled by viral one or more structural proteins (such as capsid proteins) , and their size and shape are similar to real viruses.
2. Non-infectious: does not contain the viral genome, cannot replicate or cause disease.
3. High immunogenicity: granular structure can be recognized by the immune system efficiently and activate B cell and T cell responses. Can induce neutralizing antibodies and cellular immunity (e.g., HIV, HPV vaccines).
4. High safety: suitable for people with low immune function (such as hepatitis B VLP vaccine).
FIG. 2. Available VLP vaccines
VLP vaccine production system
VLPs can be produced through a variety of expression systems, common platforms include:
1. Insect cell-baculovirus system:
Advantages: High yield, low cost, suitable for complex protein assembly.
Application: HPV vaccine, Ebola vaccine.
2. Mammalian cells (e.g. HEK293 cells) :
Advantages: Post-translational modifications are closer to humans and suitable for enveloped VLPs (such as flu vaccines).
3. Yeast systems (e.g. Pichia pastoris) :
Advantages: Fast, low cost, has been used in hepatitis B vaccine.
4. Plant systems (e.g. tobacco chloroplasts) :
Environmentally friendly and scalable, under development (e.g., norovirus VLP vaccine).
Downstream purification process of VLP vaccine
The diversity of VLPs expression systems leads to the inuniversality of downstream purification processes.
FIG. 3 a general downstream process flow chart
Harvest and clarification:
In all kind of culture medium, propducing by cell culture, there are a large number of virus particles, as well as many cell tissue debris, metabolic products and other impurities, making the culture medium cloudy or semi-cloudy, if concentrating the culture medium directly, the ultrafiltration membrane will be blocked, result that the concentration can't be completed and the ultrafiltration membrane is scrap. Therefore, it must be clarified firstly before concentration, to remove the large particles in the culture medium, so that it becomes a translucent or transparent solution, and the concentration of the culture medium can make the concentration process normal and ensure the service life of the ultrafiltration membrane. Clarification before concentration of culture solution is a key technical link in the process of concentration.
An efficient clarification process requires a combination of high capacity to remove solid particles, high product yield, easy scale-up, and protection to downstream operating units.
Method
Centrifugation: Differential centrifugation removes large particles.
Deep filtration: Use a multistage filter (e.g. 1.2μm→0.45μm) to clarify the supernatant.
Tangential flow filtration (TFF) : Suitable for mass production, concentrates samples and removes small particles of impurities.
However, if the cultivation scale is too large, centrifugation takes a long time, a large number of deep filter membranes will be required, so the cost of consumables increase greatly. At the same time, the high density of cell culture will reduce the load of the deep filter membrane, resulting in increased costs and excessive product dilution.
There are two kind of TFF membrane components: flat cassette and hollow fiber. Tangential flow filtration (TFF) is driven by the transmembrane pressure difference. Substances and impurities which are smaller than the membrane pore pass through the membrane, while impurities such as cells with larger particles are trapped. The pore size of the membrane used for microfiltration is 0.45/0.22μm. Hollow fiber can process high solid content liquid directly, such as high-density cell culture medium, it can eliminate centrifugation and pre-filtration steps, fewer steps, simple operation, membrane can be used repeatedly through cleaning, reducing equipment investment and operating costs, in line with the requirements of modular automated production.
Ultrafiltration Concentration (TFF) :
Target: To reduce the amount of chromatographic treatment, improve the chromatographic efficiency and protect the chromatographic column.
In general, ultrafiltration (TFF) can concentrate more than 100 times of VLP, and the removal rate of heteroprotein can reach 99%. Among them, hollow fiber membrane filtration technology has the advantages of mild and low shear force, not easy to plug, flexible operation, long life, low cost and easy amplification, so it is recommended to choose hollow fiber for concentrated and purified VLP.
When ultrafiltration method is used for concentration and purification, it is very important to select the right membrane pore size, which determines the efficiency and quality of the concentration directly. On the one hand, it is necessary to select the membrane aperture to effectively trap the target molecules to ensure the yield, and on the other hand, the removal effect and processing speed of heteroproteins should be fully considered. Therefore, the best principle is to select the membrane with the largest pore size that can trap the target molecule, and try to select the filter membrane with uniform pore size distribution.
Primary purification:
1. Precipitation method
Polyethylene glycol (PEG) precipitation: Selective precipitation of VLPs requires optimized PEG concentration and salt concentration to balance yield and purity.
2. Chromatography
Affinity chromatography:
Heparin affinity: Using VLPs properties of surface negative charge (e.g., HPV VLP).
Antibody coupling chromatography: high specificity but expensive.
Ion exchange chromatography (IEX) : Choose anion exchange (e.g., Q column) or cation exchange (e.g., SP column) depending on the VLP surface charge characteristics, and optimize pH and salt gradient to avoid particle aggregation.
Hydrophobic interaction chromatography (HIC) : Based on VLPs surface hydrophobicity, suitable for some non-encapsulated VLPs.
Fine purification:
Molecular exclusion chromatography (SEC, gel filtration) :
Removal of residual host proteins, nucleic acids or aggregates, accompanied by buffer replacement. High resolution but low flux, often used as the final refinement step.
Multi-mode chromatography:
For example, Capto Core series resins, combined with ion exchange and molecular sieve effect, can remove a variety of impurities in one step.
Virus inactivation/nucleic acid removal (if required) :
Nuclease treatments: such as Benzonase degrade host DNA/RNA.
UF/DF: coordinating TFF system to remove small fragments of nucleic acids and enzymes.
Concentration and preparation:
Tangential Flow Filtration (TFF) :
Concentrate to target titers while displacing buffers (such as PBS or formulation buffers).
Sterile filtration:
0.22μm membrane filtration ensures sterility.
Typical case
The following are the downstream purification processes for typical VLP vaccines.
About Guidling
Guidling Technology is a production-oriented and high-tech enterprise focusing on the downstream clarification, separation and purification of biopharmaceuticals. The products are widely used in the filtration process of MAB, vaccine, diagnosis, blood products, serum, endotoxin and other biological products; Guidling Technology has "cassettes filter and tangential flow filtration device", "hollow fiber membrane", "virus filter", "deep membrane", "sterilizaation filter", "centrifugal filter devices" and other products, and has a large number of product lines, from small disposable laboratory filtration to production filtration system, meet the needs of testing and production. Guidling Technology is looking forward to cooperating with you!
