Purification Of Virus-like Particles Research Articles

Investigation of the Electrokinetic Properties of HIV-Based Virus-Like Particles.

The antigen density on the surface of HIV-based virus-like particles (VLPs) plays a crucial role in the improvement of HIV vaccine potency. HIV VLPs consist of a dense protein core, which is surrounded by a lipid bilayer and whose surface is usually decorated with antigenic glycoproteins. The successful downstream processing of these particles is challenging, and the high-resolution and cost-efficient purification of HIV-based VLPs has not yet been achieved. Chromatography, one of the major unit operations involved in HIV VLP purification strategies, is usually carried out by means of ion exchangers or ion-exchange membranes. Understanding the electrokinetic behavior of HIV-based VLPs may help to improve the adjustment and efficiency of the corresponding chromatographic processes. In this study, we investigated the electrokinetics and aggregation of both undecorated and decorated VLPs and interpreted the data from the perspective of the soft particle model developed by Ohshima (OSPM), which fails to fully predict the behavior of the studied VLPs. Post-Ohshima literature, and particularly the soft multilayer particle model developed by Langlet et al., provides an alternative theoretical framework to overcome the limits of the OSPM. We finally hypothesized that the electrophoretic mobility of HIV-based VLPs is controlled by an electrohydrodynamic interplay between envelope glycoproteins, lipid bilayer, and Gag envelope.

A simple and high efficiency purification of His-tagged turnip yellow mosaic virus-like particle (TYMV-VLP) by nickel ion affinity precipitation

Virus-like particles (VLPs) is one of the most favourable subjects of study, especially in the field of nanobiotechnology and vaccine development because they possess good immunogenicity and self-adjuvant properties. Conventionally, VLPs can be tagged and purified using affinity chromatography or density gradient ultracentrifugation which is costly and time-consuming. Turnip yellow mosaic virus (TYMV) is a plant virus, where expression of the viral coat protein (TYMVc) in Escherichia coli (E. coli) has been shown to form VLP. In this study, we report a non-chromatographic method for VLP purification using C-terminally His-tagged TYMVc (TYMVcHis6) as a protein model. Firstly, the TYMVcHis6 was cloned and expressed in E. coli. Upon clarification of cell lysate, nickel (II) chloride [NiCl2; 15 µM or equivalent to 0.0000194% (w/v)] was added to precipitate TYMVcHis6. Following centrifugation, the pellet was resuspended in buffer containing 1 mM EDTA to chelate Ni2+, which is then removed via dialysis. A total of 50% of TYMVcHis6 was successfully recovered with purity above 0.90. Later, the purified TYMVcHis6 was analysed with sucrose density ultracentrifugation, dynamic light scattering (DLS), and transmission electron microscopy (TEM) to confirm VLP formation, which is comparable to TYMVcHis6 purified using the standard immobilized metal affinity chromatography (IMAC) column. As the current method omitted the need for IMAC column and beads while significantly reducing the time needed for column washing, nickel affinity precipitation represents a novel method for the purification of VLPs displaying poly-histidine tags (His-tags).

Downstream process design for Gag HIV-1 based virus-like particles.

Virus-like particles-based vaccines have been gaining interest in recent years. The manufacturing of these particles includes their production by cell culture followed by their purification to meet the requirements of its final use. The presence of host cell extracellular vesicles represents a challenge for better virus-like particles purification, because both share similar characteristics which hinders their separation. The present study aims to compare some of the most used downstream processing technologies for capture and purification of virus-like particles. Four steps of the purification process were studied, including a clarification step by depth filtration and filtration, an intermediate step by tangential flow filtration or multimodal chromatography, a capture step by ion exchange, heparin affinity and hydrophobic interaction chromatography and finally, a polishing step by size exclusion chromatography. In each step, the yields were evaluated by percentage of recovery of the particles of interest, purity, and elimination of main contaminants. Finally, a complete purification train was implemented using the best results obtained in each step. A final concentration of 1.40 × 1010 virus-like particles (VLPs)/mL with a purity of 64% after the polishing step was achieved, with host cell DNA and protein levels complaining with regulatory standards, and an overall recovery of 38%. This work has resulted in the development of a purification process for HIV-1 Gag-eGFP virus-like particles suitable for scale-up.

Bacterial expression systems based on Tymovirus-like particles for the presentation of vaccine antigens.

Virus-like particles (VLPs) are virus-derived artificial nanostructures that resemble a native virus-stimulating immune system through highly repetitive surface structures. Improved safety profiles, flexibility in vaccine construction, and the ease of VLP production and purification have highlighted VLPs as attractive candidates for universal vaccine platform generation, although exploration of different types of expression systems for their development is needed. Here, we demonstrate the construction of several simple Escherichia coli expression systems for the generation of eggplant mosaic virus (EMV) VLP-derived vaccines. We used different principles of antigen incorporation, including direct fusion of EMV coat protein (CP) with major cat allergen Feld1, coexpression of antigen containing and unmodified (mosaic) EMV CPs, and two coexpression variants of EMV VLPs and antigen using synthetic zipper pair 18/17 (SYNZIP 18/17), and coiled-coil forming peptides E and K (Ecoil/Kcoil). Recombinant Fel d 1 chemically coupled to EMV VLPs was included as control experiments. All EMV-Feld1 variants were expressed in E. coli, formed Tymovirus-like VLPs, and were used for immunological evaluation in healthy mice. The immunogenicity of these newly developed vaccine candidates demonstrated high titers of Feld1-specific Ab production; however, a comparably high immune response against carrier EMV was also observed. Antibody avidity tests revealed very specific Ab production (more than 50% specificity) for four out of the five vaccine candidates. Native Feld1 recognition and subclass-specific antibody tests suggested that the EMV-SZ18/17-Feld1 complex and chemically coupled EMV-Feld1 vaccines may possess characteristics for further development.

Yeast-Based Virus-like Particles as an Emerging Platform for Vaccine Development and Delivery.

Virus-like particles (VLPs) are empty, nanoscale structures morphologically resembling viruses. Internal cavity, noninfectious, and particulate nature with a high density of repeating epitopes, make them an ideal platform for vaccine development and drug delivery. Commercial use of Gardasil-9 and Cervarix showed the usefulness of VLPs in vaccine formulation. Further, chimeric VLPs allow the raising of an immune response against different immunogens and thereby can help reduce the generation of medical or clinical waste. The economically viable production of VLPs significantly impacts their usage, application, and availability. To this end, several hosts have been used and tested. The present review will discuss VLPs produced using different yeasts as fermentation hosts. We also compile a list of studies highlighting the expression and purification of VLPs using a yeast-based platform. We also discuss the advantages of using yeast to generate VLPs over other available systems. Further, the issues or limitations of yeasts for producing VLPs are also summarized. The review also compiles a list of yeast-derived VLP-based vaccines that are presently in public use or in different phases of clinical trials.

Production and Purification of Virus-Like Particles by Transient Expression in Plants.

Transient expression in plants has become a useful production system for virus-like particle (VLP) expression. High yields and flexible approaches to assembling complex VLPs, combine with ease of scale-up and inexpensive reagents to provide an attractive method for recombinant protein expression in general. Plants have demonstrated excellent capacity for the assembly and production of protein cages for use in vaccine design and nanotechnology. Furthermore, numerous virus structures have now been determined using plant-expressed VLPs, showing the utility of this approach in structural virology. Transient protein expression in plants uses common microbiology techniques, leading to a straightforward transformation procedure that does not result in stable transgenesis. In this chapter, we aim to provide a generic protocol for transient expression of VLPs in Nicotiana benthamiana using soil-free plant cultivation and a simple vacuum infiltration procedure, along with methodology for purifying VLPs from plant leaves.

Assembly and Annotation of Viral Metagenomes from Short-Read Sequencing Data.

Viral metagenomics enables the detection, characterization, and quantification of viral sequences present in shotgun-sequenced datasets of purified virus-like particles and whole metagenomes. Next generation sequencing (Illumina) derived short single or paired-end read runs are a principal platform for metagenomics, and assembly of short reads allows for the identification of distinguishing viral signatures and complex genomic features for taxonomy and functional annotation. Here we describe the identification and characterization of viral genome sequences, bacteriophages, and eukaryotic viruses, from a cohort of human stool samples, using multiple methods. Following the purification of virus-like particles, sequencing, quality refinement, and genome assembly, we begin the protocol with raw short reads deposited in an open-source nucleotide archive. We highlight the use of VIBRANT, an automated computational tool for the characterization of microbial viruses and their viral community function. Finally, we also describe an alternative assembly-free option of mapping reads to established databases of reference genomes and previously characterized metagenome-assembled viral genomes.

Generation of Multiple Arbovirus-like Particles Using a Rapid Recombinant Vaccinia Virus Expression Platform.

As demonstrated by the 2015 Zika virus outbreak in the Americas, emerging and re-emerging arboviruses are public health threats that warrant research investment for the development of effective prophylactics and therapeutics. Many arboviral diseases are underreported, neglected, or of low prevalence, yet they all have the potential to cause outbreaks of local and international concern. Here, we show the production of virus-like particles (VLPs) using a rapid and efficient recombinant vaccinia virus (VACV) expression system for five tick- and mosquito-borne arboviruses: Powassan virus (POWV), Heartland virus (HRTV), severe fever with thrombocytopenia syndrome virus (SFTSV), Bourbon virus (BRBV) and Mayaro virus (MAYV). We detected the expression of arbovirus genes of interest by Western blot and observed the expression of VLPs that resemble native virions under transmission electron microscopy. We were also able to improve the secretion of POWV VLPs by modifying the signal sequence within the capsid gene. This study describes the use of a rapid VACV platform for the production and purification of arbovirus VLPs that can be used as subunit or vectored vaccines, and provides insights into the selection of arbovirus genes for VLP formation and genetic modifications to improve VLP secretion and yield.

A Hydrodynamic Approach to the Study of HIV Virus-Like Particle (VLP) Tangential Flow Filtration.

Emerging as a promising pathway to HIV vaccines, Virus-Like Particles (VLPs) have drawn considerable attention in recent years. A challenge of working with HIV VLPs in biopharmaceutical processes is their low rigidity, and factors such as shear stress, osmotic pressure and pH variation have to be reduced during their production. In this context, the purification and concentration of VLPs are often achieved by means of Tangential Flow Filtration (TFF) involving ultrafiltration hollow fiber modules. Despite the urgent need for robust upscaling strategies and further process cost reduction, very little attention has been dedicated to the identification of the mechanisms limiting the performance of HIV VLP TFF processes. In this work, for the first time, a hydrodynamic approach based on particle friction was successfully developed as a methodology for both the optimization and the upscaling of HIV VLP TFF. Friction forces acting on near-membrane HIV VLPs are estimated, and the plausibility of the derived static coefficients of friction is discussed. The particle friction-based model seems to be very suitable for the fitting of experimental data related to HIV VLP TFF as well as for upscaling projections. According to our predictions, there is still considerable room for improvement of HIV VLP TFF, and operating this process at slightly higher flow velocities may dramatically enhance the efficiency of VLP purification and concentration. This work offers substantial guidance to membrane scientists during the design of upscaling strategies for HIV VLP TFF.

Baculovirus-Free SARS-CoV-2 Virus-like Particle Production in Insect Cells for Rapid Neutralization Assessment.

Virus-like particles (VLPs) resemble authentic virus while not containing any genomic information. Here, we present a fast and powerful method for the production of SARS-CoV-2 VLP in insect cells and the application of these VLPs to evaluate the inhibition capacity of monoclonal antibodies and sera of vaccinated donors. Our method avoids the baculovirus-based approaches commonly used in insect cells by employing direct plasmid transfection to co-express SARS-CoV-2 envelope, membrane, and spike protein that self-assemble into VLPs. After optimization of the expression plasmids and vector ratios, VLPs with an ~145 nm diameter and the typical “Corona” aura were obtained, as confirmed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Fusion of the membrane protein to GFP allowed direct quantification of binding inhibition to angiotensin II-converting enzyme 2 (ACE2) on cells by therapeutic antibody candidates or sera from vaccinated individuals. Neither VLP purification nor fluorescent labeling by secondary antibodies are required to perform these flow cytometric assays.

A Four-Step Purification Process for Gag VLPs: From Culture Supernatant to High-Purity Lyophilized Particles.

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.

VLP-factory™ and ADDomer© : Self-assembling Virus-Like Particle (VLP) Technologies for Multiple Protein and Peptide Epitope Display.

Virus-like particles (VLPs) play a prominent role in vaccination as safe and highly versatile alternatives to attenuated or inactivated viruses or subunit vaccines. We present here two innovations, VLP-factory™ and ADDomer© , for creating VLPs displaying entire proteins or peptide epitopes as antigens, respectively, to enable efficient vaccination. For producing these VLPs, we use MultiBac, a baculovirus expression vector system (BEVS) that we developed for producing complex protein biologics in insect cells transfected with an engineered baculovirus. VLPs are protein assemblies that share features with viruses but are devoid of genetic material, and thus considered safe. VLP-factory™ represents a customized MultiBac baculovirus tailored to produce enveloped VLPs based on the M1 capsid protein of influenza virus. We apply VLP-factory™ to create an array of influenza-derived VLPs presenting functional mutant influenza hemagglutinin (HA) glycoprotein variants. Moreover, we describe MultiBac-based production of ADDomer© , a synthetic self-assembling adenovirus-derived protein-based VLP platform designed to display multiple copies of pathogenic epitopes at the same time on one particle for highly efficient vaccination. © 2021 The Authors. Basic Protocol 1: VLP-factory™ baculoviral genome generation Basic Protocol 2: Influenza VLP array generation using VLP-factory™ Basic Protocol 3: Influenza VLP purification Basic Protocol 4: ADDomer© BioBrick design, expression, and purification Basic Protocol 5: ADDomer© candidate vaccines against infectious diseases.

Aqueous two‐phase systems and monolithic chromatography as alternative technological platforms for virus and virus‐like particle purification

AbstractThere is a rising interest in viral and non‐viral gene delivery vehicles for human gene therapy and vaccination applications, and these vehicles include nanoparticulate virus, virus‐like particles (VLP) and plasmids. This communication debates the importance of designing innovative platforms for virus and VLP concentration and purification, envisaging downstream process integration and intensification. Two alternative separation platforms are suggested and discussed: affinity‐aqueous two‐phase systems and monolithic chromatography. These can integrate clarification, concentration, and purification downstream unit operations, significantly decreasing industrial manufacturing costs. Additionally, high recovery yields are expected after process optimization,which is achieved through manipulation of the physicochemical parameters governing partition coefficients. Such cutting‐edge technologies encompass several advantages at industrial scale, particularly in continuous operations, for example ease of scalability, continuous operation potential, easy process integration, low toxicity, and high biocompatibility. Together with innovative processing conditions, they can effectively overcome the major bottlenecks of conventional virus and VLP downstream processing, contributing to more efficient, sustainable, and cost effective industrial systems. © 2020 Society of Chemical Industry (SCI)

Purification of virus-like particles using aqueous biphasic systems composed of natural deep eutectic solvents

Virus-like particles (VLPs) mimic the structure of viral particles, presenting antigens in a repetitive and ordered structure, which makes them excellent vaccine immunogens. However, a major bottleneck in the VLPs industrial production process is the lack of robust purification methods, which can account for up to 80% of total manufacturing cost.Aqueous biphasic systems (ABS) are portrayed as viable and efficient platform in purification of wide variety of biomolecules, since they can be designed to be versatile, biocompatible and easy to scale-up, depending on the phase forming components. In the last years, natural deep eutectic solvents (NADES) have emerged and gained a lot of attention in several fields and different applications, including the ABS implementation, where they have been proposed as versatile, cost-effective and biocompatible phase forming compounds.In this work, a study of the liquid-liquid equilibria (LLE) of ABS composed of carbohydrates and carbohydrates-based NADES is reported and the mechanisms associated with phase equilibria discussed. The carbohydrates used in the NADES preparation are osmolytes, which can be regarded both as extractants and excipients that stabilize the final formulation of biopharmaceuticals, avoiding the tricky back extraction of VLPs. It is here shown that the use of NADES leads to an increase of two-phase region, when compared to the use of only one of the NADES components, thus offering liquid-liquid demixing at low concentrations of phase forming compounds. Furthermore, the cytotoxicity studies of these NADES and their individual components using two different human cell lines, HEK 293 and Huh-7, was evaluated, in order to conclude about the biocompatibility and suitability of proposed systems in vaccine development. Finally, the potential of the proposed ABS in purification of VLPs from bovine serum albumin (BSA) is demonstrated, while their structural properties are also assessed.

Integrated Process for Capture and Purification of Virus-Like Particles: Enhancing Process Performance by Cross-Flow Filtration.

Virus-like particles (VLPs) are emerging nanoscale protein assemblies applied as prophylactic vaccines and in development as therapeutic vaccines or cargo delivery systems. Downstream processing (DSP) of VLPs comes both with challenges and opportunities, depending on the complexity and size of the structures. Filtration, precipitation/re-dissolution and size-exclusion chromatography (SEC) are potent technologies exploiting the size difference between product and impurities. In this study, we therefore investigated the integration of these technologies within a single unit operation, resulting in three different processes, one of which integrates all three technologies. VLPs, contained in clarified lysate from Escherichia coli, were precipitated by ammonium sulfate, washed, and re-dissolved in a commercial cross-flow filtration (CFF) unit. Processes were analyzed for yield, purity, as well as productivity and were found to be largely superior to a reference centrifugation process. Productivity was increased 2.6-fold by transfer of the wash and re-dissolution process to the CFF unit. Installation of a multimodal SEC column in the permeate line increased purity to 96% while maintaining a high productivity and high yield of 86%. In addition to these advantages, CFF-based capture and purification allows for scalable and disposable DSP. In summary, the developed set-up resulted in high yields and purities, bearing the potential to be applied as an integrated process step for capture and purification of in vivo-assembled VLPs and other protein nanoparticles.

Production of Human Papillomavirus Type-16 L1 VLP in Pichia pastoris

Human papillomavirus-16 (HPV-16) is primary etiological agent of invasive cervical cancer development in the world population. There is no treatment for HPV infection. As a possible tool for prophylactic vaccination, the development of virus-like particles (VLPs) comprising the HPV-16 L1 capsid protein is highly preferred. The expression of HPV-16 L1 gene was carried out in Pichia pastoris. It was done as a promising vaccine candidate against HVP-16 infection for developing countries. The codon optimization for HPV-16 L1 gene was done and synthesized in pPICZA plasmid. The expression of HPV-16 L1 was evaluated in P. pastoris after induction with methanol. The purification of HPV-16 VLPs was accomplished by the ultra-centrifugation using the sucrose density gradient. The sera of 101 subjects including 16 patients positive for HPV-16 and 85 individuals negative for HPV-16 were tested by ELISA assay using anti-HPV-16 antibodies. The results of ELISA test have depicted no false positive and no false negative as compared with commercial ELISA kit. The results of HPV-16 L1 expression in P. pastoris showed a band of about 56 kDa by SDS-PAGE, and were confirmed by Western-blot assay. The formation of VLP and the self-assembly of HPV-16 L1 major capsid protein in VLP was observed by transmission electron and atomic force microscopies.

Plant-produced chimeric virus-like particles - a new generation vaccine against African horse sickness

BackgroundAfrican horse sickness (AHS) is a severe arthropod-borne viral disease of equids, with a mortality rate of up to 95% in susceptible naïve horses. Due to safety concerns with the current live, attenuated AHS vaccine, alternate safe and effective vaccination strategies such as virus-like particles (VLPs) are being investigated. Transient plant-based expression systems are a rapid and highly scalable means of producing such African horse sickness virus (AHSV) VLPs for vaccine purposes.ResultsIn this study, we demonstrated that transient co-expression of the four AHSV capsid proteins in agroinfiltrated Nicotiana benthamiana dXT/FT plants not only allowed for the assembly of homogenous AHSV-1 VLPs but also single, double and triple chimeric VLPs, where one capsid protein originated from one AHS serotype and at least one other capsid protein originated from another AHS serotype. Following optimisation of a large scale VLP purification procedure, the safety and immunogenicity of the plant-produced, triple chimeric AHSV-6 VLPs was confirmed in horses, the target species.ConclusionsWe have successfully shown assembly of single and double chimeric AHSV-7 VLPs, as well as triple chimeric AHSV-6 VLPs, in Nicotiana benthamiana dXT/FT plants. Plant produced chimeric AHSV-6 VLPs were found to be safe for administration into 6 month old foals as well as capable of eliciting a weak neutralizing humoral immune response in these target animals against homologous AHSV virus.

Bioprocess optimization for purification of chimeric VLP displaying BVDV E2 antigens produced in yeast Hansenula polymorpha

Chimeric virus-like particles (VLP) are known as promising tools in the development of safe and effective subunit vaccines. Recently, a technology platform to produce VLP based on the small surface protein (dS) of the duck hepatitis B virus was established. In this study, chimeric VLP were investigated displaying the 195 N-terminal amino acids derived from the glycoprotein E2 of the bovine viral diarrhea virus (BVDV) on their surface. Isolation of the VLP from methylotrophic yeast Hansenula polymorpha was allowed upon co-expression of wild-type dS and a fusion protein composed of the BVDV-derived antigen N-terminally fused to the dS. It was shown the VLP could be purified by a process adapted from the production of a recombinant hepatitis B VLP vaccine. However, the process essentially depended on costly ultracentrifugation which is critical for low cost production. In novel process variants, this step was avoided after modification of the initial batch capture step, the introduction of a precipitation step and adjusting the ion exchange chromatography. The product yield could be improved by almost factor 8 to 93 ± 12 mg VLP protein per 100 g dry cell weight while keeping similar product purity and antigenicity. This allows scalable and cost efficient VLP production.

Formalin treatment increases the stability and immunogenicity of coxsackievirus B1 VLP vaccine

Type B Coxsackieviruses (CVBs) are a common cause of acute and chronic myocarditis, dilated cardiomyopathy and aseptic meningitis. However, no CVB-vaccines are available for human use. We have previously produced virus-like particles (VLPs) for CVB3 with a baculovirus-insect cell production system. Here we have explored the potential of a VLP-based vaccine targeting CVB1 and describe the production of CVB1-VLPs with a scalable VLP purification method. The developed purification method consisting of tangential flow filtration and ion exchange chromatography is compatible with industrial scale production. CVB1-VLP vaccine was treated with UV-C or formalin to study whether stability and immunogenicity was affected. Untreated, UV treated and formalin treated VLPs remained morphologically intact for 12 months at 4 °C. Formalin treatment increased, whereas UV treatment decreased the thermostability of the VLP-vaccine. High neutralising and total IgG antibody levels, the latter predominantly of a Th2 type (IgG1) phenotype, were detected in female BALB/c mice immunised with non-adjuvanted, untreated CVB1-VLP vaccine. The immunogenicity of the differently treated CVB1-VLPs (non-adjuvanted) were compared in C57BL/6 J mice and animals vaccinated with formalin treated CVB1-VLPs mounted the strongest neutralising and, CVB1-specific IgG and IgG1 antibody responses. This study demonstrates that formalin treatment increases the stability and immunogenicity of CVB1-VLP vaccine and may offer a universal tool for the stabilisation of VLPs in the production of more efficient vaccines.

High‐throughput screening of aqueous biphasic systems with ionic liquids as additives for extraction and purification of enveloped virus‐like particles

Due to their unique properties, virus‐like particles (VLPs) have been portrayed as a promising high‐value biopharmaceutical in VLP‐based vaccination and cancer therapy. Nevertheless, due to limited physical and economical capabilities of the current downstream processing of VLPs, their production is still difficult and seen as a major problem that needs to be tackled. In this work, high‐throughput screening on a liquid handling station (LHS) has been implemented for efficient selection of adequate polymer‐salt aqueous biphasic systems (ABS) for extraction and purification of enveloped Hepatitis C virus pseudoparticles (HCVpp). The effect of polyethylene glycol (PEG) molecular weight and salt type (citrate, sulfate, and phosphate) was first evaluated. Furthermore, to optimize extraction parameters, the effect of pH and tie‐line lengths (TLL) was also addressed. For the most promising ABS and extraction conditions, the addition of ten ionic liquids (ILs) as adjuvants was investigated. Insights on the chemical features of ILs that impact HCV‐VLPs partitioning are highlighted. Finally, the potential of studied ABS containing ILs as adjuvants in the extraction and purification of VLPs from cell culture supernatants was addressed. The 100% extraction efficiency of VLPs in the PEG‐rich phase was achieved, and VLP purity was increased compared to the system without IL. In conclusion, these promising results show that ILs can be very effective in modulating the phase properties of polymer‐salt ABS, achieving high HCV‐VLP purification.