Abstract
Gag-based virus-like particles (VLPs) have high potential as scaffolds for the development of chimeric vaccines and delivery strategies. The production of purified preparations that can be preserved independently from cold chains is highly desirable to facilitate distribution and access worldwide. In this work, a nimble purification has been developed, facilitating the production of Gag VLPs. Suspension-adapted HEK 293 cells cultured in chemically defined cell culture media were used to produce the VLPs. A four-step downstream process (DSP) consisting of membrane filtration, ion-exchange chromatography, polishing, and lyophilization was developed. The purification of VLPs from other contaminants such as host cell proteins (HCP), double-stranded DNA, or extracellular vesicles (EVs) was confirmed after their DSP. A concentration of 2.2 ± 0.8 × 109 VLPs/mL in the lyophilized samples was obtained after its storage at room temperature for two months. Morphology and structural integrity of purified VLPs was assessed by cryo-TEM and NTA. Likewise, the purification methodologies proposed here could be easily scaled up and applied to purify similar enveloped viruses and vesicles.
Highlights
Published: 9 October 2021Virus-like particles (VLPs) are recognized as a promising strategy in recombinant vaccine development due to their ability to mimic native viruses with the lack of a viral genome
The thermostability and aggregation of Gag virus-like particles (VLPs) were firstly studied to assess their resistance to different storage conditions and prevent further purification loss during the different unit operations
Spectrofluorometry enables the quantification of Gag-enhanced green fluorescence protein (eGFP) polyprotein that can be indirectly related to VLP concentrations [17], while nanoparticle tracking analysis (NTA) tracks individual GFP fluorescent nanoparticles [30]; the specific quantification of assembled VLP from free Gag monomer and the particle size distribution (PSD) of the VLP population could be assessed, simultaneously
Summary
Virus-like particles (VLPs) are recognized as a promising strategy in recombinant vaccine development due to their ability to mimic native viruses with the lack of a viral genome. Their highly organized and repetitive antigen-presenting structure has been shown to stimulate both cellular and humoral immune responses [1,2]. Their easy and flexible production, based on recombinant protein expression, makes them an excellent nanocarrier for the delivery of immunogens, proteins, enzymes, and DNA in vitro and in vivo [1,3]. The final nanoparticles are enveloped by a host cell lipid membrane [5], with an expected size between 100 and 200 nm
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