Insight

Virus-like particles (VLPs) bear high structural similarity to native viruses but lack viral genetic material, rendering them non-infectious. They can be expressed via multiple expression systems, thereby attracting extensive research interest. VLPs self-assemble from one or more structural subunit proteins and are categorized into enveloped and non-enveloped types. For instance, human immunodeficiency virus (HIV)-like particles acquire an envelope derived from the host cell membrane during budding, while non-enveloped VLPs consist solely of protein subunits with a symmetric icosahedral morphology. Multilayered VLPs are formed by the simultaneous intracellular expression and assembly of distinct structural proteins, as exemplified by rotavirus-like particles.

VLPs can effectively mimic the epitopes of native viruses and elicit robust immune responses, which enables their development into a variety of VLP-based vaccines. Additionally, VLPs hold promising potential in drug delivery, capable of encapsulating therapeutic drugs and targeting specific cell types. Studies have demonstrated that VLPs can cross the blood-brain barrier, exert synergistic effects with chemotherapeutic agents, and significantly inhibit brain tumor growth. VLPs also represent a novel modality for gene therapy with high structural modifiability. Although no commercial VLP-based gene therapy products are currently available on the market, their application prospects remain broad.

A standard downstream processing workflow for VLP purification has been established to date. Scalable downstream purification procedures are indispensable for isolating VLPs from complex impurities. The downstream process initiates with the harvesting of VLP-expressing cells, followed by pretreatment to lyse cells and remove cell debris, host cell proteins (HCPs), and protein aggregates. Subsequent chromatographic purification is performed, ending with final quality assurance of VLPs. Nevertheless, major challenges persist in achieving high VLP purity, eliminating nucleic acid and viral contamination, as well as realizing cost control and scalable purification.

Purification of VLPs from diverse expression systems poses a considerable challenge due to high levels of residual host cell proteins. Although numerous studies have attempted to establish purification workflows, most rely on non-scalable strategies such as protein affinity tags or sucrose density gradient ultracentrifugation, with limited efficiency in host protein removal. Moreover, even with scalable processes, the yield and purity of VLPs fail to reach maximum levels. Ribonucleases are occasionally adopted for DNA digestion during purification, yet their high cost increases overall production expenses.

Given the structural complexity of VLPs, their purification workflow differs substantially from that of conventional recombinant proteins. VLPs exhibit unique behaviors in standard chromatographic media and are more sensitive to salt concentration, pressure, and pH variations. In the downstream processing of modern biotechnological products, the removal of large aggregates and cell debris is critical to prevent column clogging and fouling in subsequent purification steps. Proper sample pretreatment can enhance overall purification efficiency and ensure economic feasibility. Common pretreatment techniques include centrifugation, precipitation and filtration, among which filtration is the preferred option.

Precipitants such as polyethylene glycol (PEG) and ammonium sulfate serve as cost-effective alternatives, capable of reducing subsequent chromatographic steps without compromising the integrity of recombinant proteins, viruses or VLPs. Ammonium sulfate is a traditional reagent for protein precipitation; increasing ammonium sulfate concentration and centrifugal force facilitates more protein sedimentation, yet it cannot achieve efficient separation of target components from impurities. Polyethylenimine (PEI) also exhibits favorable protein precipitation performance, particularly for negatively charged DNA. However, PEI is cytotoxic in vivo and must be completely removed in subsequent purification steps.

Ion exchange chromatography (IEX) is a conventional protein purification technique applicable to the isolation and purification of VLPs. Firstly, crude VLP extracts are obtained through preliminary treatments including cell lysis and centrifugation. Ion exchange columns filled with charged resins are employed for further purification, enabling electrostatic interactions with charged molecules in the sample. An appropriate anion or cation exchange resin is selected according to the surface charge properties of target VLPs.

After sample loading onto the column, buffer pH and ionic strength are adjusted to promote binding between VLPs and the resin. Gradient elution is subsequently performed by gradually increasing the salt concentration of the buffer, which disrupts electrostatic interactions and elutes bound VLPs from the column. Featuring high efficiency and excellent selectivity, ion exchange chromatography markedly improves VLP purity and provides qualified samples for subsequent structural analysis and vaccine development.

Hydrophobic interaction chromatography (HIC) is also applicable to VLP purification. Selection of suitable HIC columns and optimization of buffer conditions are critical throughout the process. Tuning buffer salt concentration and pH can minimize non-specific binding, thereby improving the purity and recovery rate of target VLPs.

Size exclusion chromatography (SEC) is commonly used for final polishing and concentration in purification workflows, yet it is unsuitable for crude samples due to the high risk of column clogging and fouling. Hydroxyapatite chromatography is a typical multimodal chromatography (MMC) technique that offers unique advantages for protein purification. Ceramic hydroxyapatite (CHT) has shown great potential in the purification of viruses and VLPs.

CHT consists of spherical particles with pore sizes of 20 μm, 40 μm and 80 μm, containing positively charged calcium ion sites (C-sites) and negatively charged oxygen atoms associated with hydroxyl groups and three phosphate groups (P-sites). Owing to this structural feature, acidic proteins tend to bind to C-sites, while alkaline proteins interact with P-sites. Calcium ion-modified CHT columns support scalable purification of recombinant adeno-associated viruses, achieving a high binding capacity of 65 mg total protein per milliliter of medium and a vector recovery rate of approximately 70%.

Given the particle size range of viruses and VLPs (30–300 nm), their interactions with conventional chromatographic media are limited. Such large particle sizes prevent their diffusion into porous packed beds. Monolithic columns fabricated from modified polymethacrylate with controllable large pore sizes provide a promising alternative; their enlarged pores enhance resolution, increase binding capacity and accelerate flow rates.

Immobilized metal affinity chromatography (IMAC) is a specialized technique for the adsorption and purification of proteins and peptides with metal ion affinity. In IMAC, metal ions are coordinated to carriers functionalized with metal chelating ligands, followed by specific binding of target proteins or peptides to the immobilized metal ions.

Advancements in magnetic nanoparticle technology have driven significant progress in IMAC, enabling magnetic separation and elution of proteins under mild conditions without denaturation. Multiple fabrication methods are available for magnetic nanoparticles, including grinding, thermal decomposition, microemulsification, chemical vapor deposition and co-precipitation. Nickel ions (Ni²⁺) are the most widely used metal ions in IMAC due to their high binding affinity for histidine residues.

Purification of enveloped VLPs can be achieved via one-step or two-step workflows. For intracellularly expressed VLPs, centrifugation or filtration is first applied to remove debris and solid substances from cell lysates. Following pretreatment, one-step purification is conducted using ion exchange chromatography, CHT chromatography, membrane separation or tangential flow filtration (TFF). SDS-PAGE analysis verifies that chromatographic methods deliver a recovery rate of 50–70% and superior purity.

CHT-based one-step purification achieves a VLP recovery rate of 60–64% and a dsDNA removal rate of 96.6%, demonstrating excellent scalability, reproducibility and stability. The lipid envelope of enveloped VLPs imposes restrictions on buffer and pH conditions during purification. Accordingly, molecular sieving is adopted as the initial step to allow large VLP particles to flow through while small molecular impurities bind to ligands. Capto Core 700 is widely used in primary polishing workflows, featuring a non-functionalized outer layer and a ligand-functionalized inner core. The outer bead layer possesses a molecular weight cutoff of approximately 700 kDa, excluding macromolecules such as viruses and VLPs while retaining small molecules via core ligand binding. Due to sample dilution after primary elution, SEC is subsequently employed in the second step for VLP concentration.

Non-enveloped VLPs exhibit higher tolerance to physicochemical conditions including pH, temperature and ionic strength compared with enveloped VLPs. Rigorous pretreatment prior to chromatography efficiently eliminates host cell-derived impurities. Precipitation, heat treatment, filtration and centrifugation are all effective strategies for sample pretreatment.

Hepatitis B virus core antigen (HBc) VLPs are found in the soluble fraction of disrupted Escherichia coli cells. HBc VLPs are thermostable: heat treatment at 60 °C for 30 minutes removes 78% of host cell proteins, while treatment at 70 °C for 30 minutes increases the removal rate to 94%. HIC-based polishing further elevates the purity of HBc VLPs to 99%. Two-stage heat treatment does not alter the particle size, morphology or antigenicity of HBc VLPs, achieving an overall recovery rate of 78% and purity of 99%. Additionally, heat treatment eliminates the majority of non-specific nucleic acids.

Chromatographic workflows are scalable for VLPs with high tolerance to buffer and elution pH. Ion exchange purification (IEX) is the preferred primary purification step due to its efficient removal of host cell proteins and DNA. For example, in the purification of polyomavirus VLPs expressed in yeast, ammonium sulfate precipitation is followed by primary IEX chromatography. A small fraction of VLPs flows through the column, while most contaminating proteins are eluted via linear sodium chloride gradient, yielding a purity of 65–80%.

Given the molecular weight of these VLPs far exceeding 700 kDa, they cannot be retained by the medium and flow through during secondary multimodal chromatography, which removes residual contaminating proteins. The final product achieves a protein recovery rate of 42% and a purity of 90–95%. Multi-step purification strategies are also widely applied in VLP production.

Non-enveloped VLPs are generally more stable than enveloped counterparts, with an optimal pH range of approximately 7–8. Enveloped viral particles require storage and processing temperatures maintained between 0 °C and 30 °C. Ionic strength and salt concentration significantly affect VLP stability, resulting in a narrower stable condition range for enveloped VLPs relative to non-enveloped ones. PEG and ammonium sulfate can be used for the precipitation and purification of non-enveloped VLPs.

Assembly-derived VLPs may carry residual host DNA and proteins after purification, compromising structural integrity. High purity is imperative for medical-grade VLPs. Conventional characterization and quality control methods include sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), and transmission electron microscopy (TEM). An ideal universal purification workflow applicable to all recombinant proteins, viruses and VLPs remains challenging to develop due to the unique physicochemical properties of individual particles. To meet pharmaceutical-grade purity requirements, high-performance liquid chromatography (HPLC), capillary electrophoresis and mass spectrometry are increasingly recommended for VLP purity analysis and detection.

A variety of expression systems have been successfully applied to produce pharmaceutical-grade virus-like particles. Despite remaining challenges in post-translational modification for Escherichia coli and yeast expression systems, advances in fermentation technology have facilitated large-scale VLP production. Insect cell expression systems hold particularly promising prospects, enabling large-scale cultivation in serum-free media using bioreactors. Furthermore, silkworm larvae have recently emerged as alternative host organisms, featuring mammalian-like post-translational modification and easy cultivation, though their supporting purification technology is still in the initial developmental stage.

Downstream purification typically involves two to three procedural steps dominated by ion exchange chromatography and affinity chromatography. The development of innovative chromatographic matrix materials has driven major advances in VLP purification and vaccine research and development.