Purification of infective virus

Abstract

The invention relates to the field of ion exchange based purification processes and provides an improved method for the purification of lipid enveloped viruses. Use of the elution solution significantly enhances the efficiency of the recovery of the infective virus, in particular intact SARS-CoV-2 virions.

Description

[0001] Purification of infective virus

[0002] FIELD OF THE INVENTION

[0003] The invention relates to the field of ion exchange based purification processes and provides an improved method for the purification of lipid enveloped viruses. Use of the elution solution significantly enhances the efficiency of the recovery of the infective virus, in particular intact SARS-CoV-2 virions.

[0004] BACKGROUND OF THE INVENTION

[0005] The field of virus purification plays a critical role in medical research and public health, particularly highlighted by the COVID-19 pandemic. Efficient virus purification is essential for developing vaccines, diagnostic assays, and understanding viral pathogenesis. The scientific literature of virus purification is enormous. E.g. WO9706243 describes a method for virus purification comprising anion exchange chromatography followed by a cation exchange chroma- tograpy step, WO2023274989 describes a support material being functionalized with a ligand comprising a diamine functionality for purification of enveloped virus particles or exosomes, W02005080556 describes a method for virus purification comprising lysing cells with a detergent, EP4188414 describes a method using a wash solution with a pH between 3.0 and 6.5 to enhance the removal of host cell proteins.

[0006] Traditional purification methods, however, face challenges in yield and purity, especially with complex viruses like SARS-CoV-2. CN115305242A describes an anion exchange based purification method of inactivated SARS-CoV-2.

[0007] Current methods often rely on ultracentrifugation or filtration, which can be time-consuming and may compromise the integrity of the virus or other substance of interest.

[0008] The need for improved techniques is evident, considering the limitations of existing purification processes.

[0009] SUMMARY OF THE INVENTION

[0010] Non-ionic detergents are widely used in ion exchange chromatography, mainly to solubilize membranes by the extraction of integral membrane proteins from the lipid bilayer in which they are embedded. Non-ionic detergents are also used in ion exchange chromatography to inactivate lipid enveloped viruses by disrupting the lipid envelope. It has been surprisingly found that adding a non-ionic detergent to the elution solution used in the purification of an infective form of a virus with a lipid envelope in ion exchange chromatography enhances the efficacy of the purification, i.e. increases the yield of purified infective virus. The addition of the non-ionic detergent to the elution solution suprisingly did not inactivate (disrupted the lipid envelope of) the virus.

[0011] A method for the ion exchange chromatographic purification of a lipid enveloped virus is provided, comprising a step of eluting the lipid enveloped virus from a support used in the ion exchange chromatographic purification method using an elution solution comprising a non- ionic detergent.

[0012] A method for increasing the yield of lipid enveloped virus in an ion exchange chromatography purification method is provided, comprising a step of eluting the lipid enveloped virus from a support used in the ion exchange chromatographic purification method using an elution solution comprising a non-ionic detergent.

[0013] Use of a non-ionic detergent for the purification of a lipid enveloped virus in an ion exchange chromatograpic purification method is provided, comprising a step of eluting the lipid enveloped virus from a support used in the ion exchange chromatographic purification method using an elution solution comprising the non-ionic detergent.

[0014] Use of a non-ionic detergent for increasing the yield of a lipid enveloped virus in an ion exchange chromatograpic purification method is provided, comprising a step of eluting the lipid enveloped virus from a support used in the ion exchange chromatographic purification method using an elution solution comprising the non-ionic detergent.

[0015] Preferably the non-ionic detergent is polysorbate, highly preferably polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate; CAS Number: 9005-64-5). In another embodiment the non- ionic detergent is polyoxyethylenesorbitan monopalmitate (polysorbate 40, CAS No. 9005-66- 7; e.g. Tween 40), polyethylene glycol sorbitan monooleate, (polysorbate 80, CAS No. 9005- 65-6; e.g. Tween 80), polyethylene glycol sorbitan monostearate, (polysorbate 60, CAS No. 9005-67-8; e.g. Tween 60), polyoxyethylenesorbitan (CAS No. 9005-70-3; e.g. Tween 85), trioleatepolyethylene glycol dodecyl ether (CAS No. 9002-92-0; e.g. Brij L4), polyoxyethylene (20) oleyl ether (CAS No. 9004-98-2; e.g. Brij 020), polyoxyethylene (12) isooctylphenyl ether (CAS No. 9036-19-5; e.g. IGEPAL® CA-720), Triton non-ionic detergents (e.g. CAS No. 9036-19-5; CAS No 92046-34-9) or n-dodecyl P-D-glucopyranoside (CAS No. 59122-55-3). Highly preferably the non-ionic detergent is Tween-20 (e.g. https: / / www.bio-rad.com / en- hu / sku / 1706531 -tween-20- 100-nonionic-detergent?ID= 1706531 as of 2002 2024). Preferably the charged support comprises or is a membrane, resin applied on a column, highly preferably magnetic beads. Highly preferably the positively charged support is Dynabeads™ Intact Virus Enrichment, Catalog Numbers 10700D, 10701D as of 8 February, 2024.

[0016] Preferably the lipid enveloped virus is SARS-CoV-2. Highly preferably the lipid enveloped virus is intact virion. Highly preferably the lipid enveloped virus is infective.

[0017] Preferably the elution solution comprises the non-ionic detergent in a concentration of 0.005 - 0.1% (v / v), preferably 0.01-0.1% (v / v), highly preferably about 0.1 % (v / v), highly preferably about 0.01 % (v / v).

[0018] Preferably the elution solution comprises polysorbate 20 in a concentration of 0.005 - 0.1% (v / v), preferably 0.01-0.1% (v / v), highly preferably about 0.1 % (v / v), highly preferably about 0.01 % (v / v). Preferably the elution solution comprises the non-ionic detergent (preferably polysorbate 20) in an amount that is the same as 0.005 - 0.1%, preferably 0.01-0.1% (v / v), highly preferably about 0.1 % (v / v), highly preferably about 0.01 % (v / v) Tween 20.

[0019] Preferably the concentration of the non-ionic detergent in the elution solution is such that it does not impair the physical activity and / or structure of the enveloped virus, e.g. such that the enveloped virus retains its infectivity. Preferably the concentration of the non-ionic detergent in the elution solution is such that it does not damage the lipid envelope of the lipid enveloped virus. Preferably the concentration of the non-ionic detergent in the elution solution is such that it does not kill the virus to be purified or leaves the virus functional.

[0020] Highly preferably the elution solution comprises or consists of 0.25 M KI in 20 mM triethanolamine and 0.01% (v / v) Tween 20; pH=6.

[0021] Preferably the method comprises the provision of a viral supernatant. Preferably the viral supernatant is provided by clarification of viral cultures.

[0022] Preferably the method comprises inactivation of the virus, preferably inactivation by UV-C light.

[0023] Preferably the method comprises filtering the viral supernatant, preferably through a 0.22 pm filter.

[0024] Preferably the positively charged surface comprises or is magnetic beads, highly preferably Dynabeads™ Intact Virus Enrichment, Catalog Numbers 10700D, 10701D as of 8 February, 2024, highly preferably 10 pl.

[0025] Preferably the elution solution is 0.25 M KI in 20 mM triethanolamine and 0.01-0.1% (v / v) Tween 20, preferably about 0.1 % (v / v), preferably about 0.01 % (v / v); pH=6.

[0026] Preferably the concentration of Tween 20 is 0.01-5 (v / v), preferably about 0.1 % (v / v), preferably about 0.01 % (v / v) and the volume of the beads is 10 pl. BRIEF DESCRIPTION OF THE FIGURES

[0027] Figure 1 Recovery efficiency of SARS-CoV-2 RNA in 1-ml virus isolates, with elution volumes ranging from 50 to 500 pL. We compare two purification methods, RoLink and Thermo Fisher, relative to the RNA content in the original 1 ml virus stock solution. The graph showcases RNA-based recovery efficiency as a percentage of the stock solution RNA content (calculated as elution RNA content / RNA content of 1 ml stock 100). qPCR.

[0028] Figure 2 Comparison of BCA based total protein content from two purification methods, Rolink and Thermo. The analysis covers protein content before and after desalting for both methods. Rolink was assessed at two elution volumes (50 pL and 250 pL), pre- and post-desalting, while Thermo was evaluated at a fixed 50 pL elution volume. This assessment illustrates the purification methods' effectiveness in terms of protein yield and purity, relative to the initial stock solution (708 pg / ml). The total protein content of the samples was determined by fitting curves to the internal standard curve. BCA assay.

[0029] Figure 3 displays the remaining SARS-CoV-2 RNA in 1-ml virus isolates post-magnetic bead pulldown from the supernatant, four bead-to-virus ratios ranging from 5 pl to 20 pl were evaluated and the residual RNA content of the 1 ml stock after the pulldown process was analyzed. The graph illustrates the quantity of virus remaining in the solution, with data expressed in TCIDso / ml, employing qPCR for analysis.

[0030] Figure 4 Recovery efficiency of SARS-CoV-2 RNA in 1-ml virus isolates, with tween concentrations ranging from 0 to 0.1 %, and elution volumes ranging from 250 to 500 pL. Eight purification methods were compared, relative to the RNA content in the original 1 ml virus stock solution. The graph showcases RNA-based recovery efficiency as Cq change compared to 1 ml virus stock (ACq is calculated as mean Cq of lx virus stock - mean Cq of sample; all measurements were done in technical triplicates). qPCR.

[0031] Figure 5 Recovery efficiency of infective SARS-CoV-2 in 1-ml virus isolates, with tween concentration and elution volume of 0.01 % and 50 pL respectively. The purified 50 pL virus was added to 950 uL PBS. TCID50 results of the infective SARS-CoV-2 before and after purification (1. Plate and 2. Plate respectively) were compared. A 10-fold serial dilution using a purified and original SARS-CoV-2 stock solution was conducted. Subsequently, 100 pL of each sample dilution was pipetted into separate wells, beginning with the lOx dilution. Each row on the plate represented one dilution, with the 12th row serving as a negative control. In this control, cell culture media devoid of the virus was added. The rows and columns depicted in the figure correspond to those of a typical 96-well plate and were labeled accordingly. The figure showcases TCID50-based recovery efficiency as TCID50% change compared to 1 ml virus stock (TCID50% is calculated as TCID50 of purified virus / TCID50 of lx virus stock before purification * 100). TCID50 assay results. The recovery results based on infectivity for active SARS- CoV-2 (56%) closely align with RNA-based recovery findings for UV-inactivated SARS-CoV- 2 (57.64%).

[0032] Figure 6 Interaction of PBMC with UV-Inactivated SARS-CoV-2. (a) Native PBMC (Peripheral Blood Mononuclear Cells) after overnight incubation display a healthy monocyte population with minimal cellular debris, indicative of normal PBMC function. 106 PBMC, overnight, (b) PBMC exposed to unpurified UV-inactivated SARS-CoV-2 exhibit a striking absence of monocytes and a significant presence of cellular debris, highlighting extensive cellular damage resulting from interaction with the unpurified virus. 106PBMC, overnight. Figure 6 provides a visual comparison of PBMC responses to native and UV-inactivated SARS-CoV-2, shedding light on the impact of virus purification on cell health and viability.

[0033] Figure 7 SARS-CoV-2 rapid antigen tests. Samples: original (1), original 64-fold diluted in PBS (7), filtered (8), RoLink purified (13), Thermo purified desalted (18), and RoLink purified, desalted (23). Data demonstrate the superiority of the RoLink protocol.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] The invention provides a method for e.g virus purification that maintains virus integrity while significantly enhancing recovery efficiency. An added benefit of the method is, that it does not require specific laboratory equipment (e. g. ultracentrifuge) and is perfectly compatible with benchtop labwork.

[0036] The method is particularly useful for UV-inactivated SARS-CoV-2 (delta variant)

[0037] The elution solution described herein significantly enhances viral recovery efficiency. As shown in the examples the use of the elution solution provides a substantial improvement in both the purity and recovery rate of viruses compared to standard procedures. The purification method examplified by a methof for the purification of SARS-CoV-2, however, it is adaptable to e.g. various viruses, proteins and extracellular vesicles, promising broad applications in virology research and public health.

[0038] The elution solution may be used with magnetic bead technology, eg. employing Dynabeads™ Intact Virus Enrichment beads, which bind viruses through electrostatic interactions

[0039] A pivotal aspect of the invention is the novel elution solution, distinctively incorporating a nonionic detergent (e.g. Tween-20), at a specific concentration. This aspect of the invention breaks from traditional practices where the use of non-ionic detergents in anion exchange purification is typically avoided. The detergent's concentration may be optimized to detach the virus efficiently from the beads without compromising the virus's structural integrity.

[0040] Application for Various Viruses: While primarily optimized for SARS-CoV-2, the method can be adapted for other viruses, indicating broad applicability in virology.

[0041] The terms polysorbate 20 and Tween 20 are used interchangeably herein. The skilled person understands that an elution solution (or elution buffer) is a solution that is useful in ion exchange chromatography, i.e. in the release step of ion exchange chromatography, e.g. it is suitable to release a substance of interest from a surface (e.g. an ion chromatogrpahy column).

[0042] The term “RoLink protocol” or “RoLink” refers to an ion exchange based purification method, wherein a low concentration non-ionic detergent is used to enhance the elution or purification of a substance of interest. In the RoLink protocol the substance of interest may be a negatively charged substance that is bound to a positively charged support in a step of anion exchange chromatography .

[0043] The terms “Thermo protocol”, “ThermoFisher protocol”, “Thermo” refer to the manufacturer’s protocol provided with Dynabeads™ Intact Virus Enrichment magnetic beads (Dynabeads™ Intact Virus Enrichment (optimized for SARS-CoV-2), 100 reactions, obtained 02. 06. 2021, ThermoFisher Scientific, Catalog numbers 10700D, 10701D, Pub. No. MAN0019858 Rev.

[0044] A.0.

[0045] It has been suprisingly found that adding a non-ionic detergent to the elution solution (release buffer) used in an ion (preferably anion) exchange chromatography process to remove a substance of interest bound to a charged surface from the charged surface enhances the efficiency of the recovery of the substance of interest.

[0046] Aspects of the invention are listed below.

[0047] 1. An elution solution for ion exchange chromatography, wherein the elution solution comprises a non-ionic detergent.

[0048] 2. Use of a non-ionic detergent in an elution solution useful in ion exchange chromatography.

[0049] 3. Use of a non-ionic detergent for the purification of a charged substance.

[0050] 4. Use of a non-ionic detergent for the recovery of a charged substance having a lipid surface.

[0051] 5. Use of a non-ionic detergent for removing / eluting a charged substance from a charged support.

[0052] 6. Use of a non-ionic detergent for increasing the yield of a charged substance in a process of removing / eluting the charged substance from a charged support.

[0053] 7. Use of a non-ionic detergent for increasing the purity of a charged substance in a process of removing / eluting the charged substance from a charged support. 8. Use of a non-ionic detergent for the enrichment of a charged substance in an elution solution, wherein the elution solution comprises a non-ionic detergent.

[0054] 9. A method for the purification of a charged substance, comprising removing / eluting the charged substance from a charged support.

[0055] 10. A kit comprising a charged support and an elution solution useful in ion exchange chromatography, wherein the elution solution comprises a non-ionic detergent.

[0056] Preferably the ion exchange chromatography is anion exchange chromatography.

[0057] Preferably the charged support is positively charged.

[0058] Preferably the charged substance is negatively charged.

[0059] Preferably the non-ionic detergent is polysorbate, highly preferably polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate; CAS Number: 9005-64-5). In another embodiment the non- ionic detergent is polyoxyethylenesorbitan monopalmitate (polysorbate 40, CAS No. 9005-66- 7; e.g. Tween 40), polyethylene glycol sorbitan monooleate, (polysorbate 80, CAS No. 9005- 65-6; e.g. Tween 80), polyethylene glycol sorbitan monostearate, (polysorbate 60, CAS No. 9005-67-8; e.g. Tween 60), polyoxyethylenesorbitan (CAS No. 9005-70-3; e.g. Tween 85), trioleatepolyethylene glycol dodecyl ether (CAS No. 9002-92-0; e.g. Brij L4), polyoxyethylene (20) oleyl ether (CAS No. 9004-98-2; e.g. Brij 020), polyoxyethylene (12) isooctylphenyl ether (CAS No. 9036-19-5; e.g. IGEPAL® CA-720), Triton non-ionic detergents (e.g. CAS No. 9036-19-5; CAS No 92046-34-9) or n-dodecyl P-D-glucopyranoside (CAS No. 59122-55-3). Highly preferably the non-ionic detergent is Tween-20 (e.g. https: / / www.bio-rad.com / en- hu / sku / 1706531 -tween-20- 100-nonionic-detergent?ID= 1706531, as of 20 02 2024).

[0060] Preferably the purification or recovery or removing / eluting of the charged substance is performed by an ion (preferably anion) exchange chromatography method.

[0061] Preferably the purification or recovery or removing / eluting of the charged substance is performed in a step of ion (preferably anion) exchange chromatography.

[0062] Preferably the charged support comprises or is a membrane, resin applied on a column, highly preferably magnetic beads. Highly preferably the positively charged support is Dynabeads™ Intact Virus Enrichment, Catalog Numbers 10700D, 10701D as of 8 February, 2024.

[0063] Preferably the charged substance comprises a lipid surface.

[0064] Preferably the charged substance is a virus, preferably a virus having a lipid envelope, highly preferably SARS-CoV-2. Highly preferably the charged substance is an intact virion.

[0065] Preferably the charged substance is a virus like particle.

[0066] Preferably the charged substance is an extracellular vesicle.

[0067] Preferably the charged substance is a protein, polypeptide or nucleic acid. Preferably the elution solution comprises the non-ionic detergent in a concentration of 0.005 - 0.1% (v / v), preferably 0.01-0.1% (v / v), highly preferably about 0.1 % (v / v).

[0068] Preferably the elution solution comprises polysorbate 20 in a concentration of 0.005 - 0.1% (v / v), preferably 0.01-0.1% (v / v), highly preferably about 0.1 % (v / v). Preferably the elution solution comprises the non-ionic detergent (preferably polysorbate 20) in an amount that is the same as 0.005 - 0.1%, preferably 0.01-0.1% (v / v), highly preferably about 0.1 % (v / v) Tween 20.

[0069] Preferably the concentration of the non-ionic detergent in the elution solution is such that it does not impair the physical activity and / or structure of the charged substance, e.g. such that the charged substance retains its physiological function, such as enzyme activity. Preferably the concentration of the non-ionic detergent in the elution solution is such that it does not damage the lipid surface of the charged substance. Preferably the concentration of the non-ionic detergent in the elution solution is such that it does not kill the virus to be purified or leaves the virus functional. Preferably the concentration of the non-ionic detergent in the elution solution is such that it does not disrupt or lyse the extracellular vesicle.

[0070] Highly preferably the elution solution comprises or consists of 0.25 M KI in 20 mM triethanolamine and 0.01% (v / v) Tween 20; pH=6.

[0071] 11. A method for the purification of SARS-CoV-2 from a sample, comprising contacting the sample comprising SARS-CoV-2 with a positively charged surface eluting SARS-CoV-2 from the positively charged surface using an elution solution comprising a non-ionic detergent.

[0072] Preferably the method comprises the provision of a viral supernatant. Preferably the viral supernatant is provided by clarification of viral cultures.

[0073] Preferably the method comprises inactivation of the virus, preferably inactivation by UV-C light.

[0074] Preferably the method comprises filtering the viral supernatant, preferably through a 0.22 pm filter.

[0075] Preferably the positively charged surface comprises or is magnetic beads, highly preferably Dynabeads™ Intact Virus Enrichment, Catalog Numbers 10700D, 10701D as of 8 February, 2024, highly preferably 10 pl.

[0076] Preferably the elution solution is 0.25 M KI in 20 mM triethanolamine and 0.01-0.1% (v / v) Tween 20, preferably about 0.1 % (v / v); pH=6.

[0077] Preferably the concentration of Tween 20 is 0.01-5 (v / v), preferably about 0.1 % (v / v) and the volume of the beads is 10 pl. EXAMPLES

[0078] The invention is described by way of experiments carried out using SARS-CoV-2.

[0079] Propagation in BSL-4 Facility

[0080] Active SARS-CoV-2 delta variant was propagated using Vero E6 cells within a Biosafety Level 4 (BSL-4) facility. This environment ensured strict containment and safety protocols for handling the highly infectious virus.

[0081] Clarification of Viral Supernatant

[0082] Following propagation, the viral cultures were clarified to obtain supernatants, which are essential for subsequent purification steps.

[0083] UV-C Irradiation for Inactivation

[0084] The clarified viral supernatants were treated with 254 nm UV-C light in a stainless- steel irradiation chamber. This method was chosen for its effectiveness in inactivating the virus while preserving structural integrity, crucial for further analysis. To confirm complete inactivation of the virus, a series of assays were conducted, including TCID50 assay, plaque assay, and RT-PCR. These tests are critical for ensuring the safety of subsequent handling in lower biosafety level laboratories and for validating the integrity of the viral particles for research purposes.

[0085] Virus Purification

[0086] For the purification of UV-inactivated SARS-CoV-2, the study employed the Dyna-beads™ Intact Virus Enrichment kit (Thermo Fisher, 10700D) for virus binding. A novel elution buffer, containing 0.01% Tween-20, was developed and used for the effective release of the virus from the beads. This step was pivotal in ensuring the integrity and recovery of the virus for subsequent analyses.

[0087] The UV-inactivated viral supernatants (SARS-CoV-2 delta variant) were filtered through a 0.22 pm filter. Subsequent anion exchange chromatography was employed as per manufacturer’s instructions with slight modifications. Briefly, after gentle agitation 10 pl magnetic beads were placed into a 1.5 ml Eppendorf tube. Subsequently the beads were washed with 500 pl Binding & Washing Buffer (B&W Buffer: 10 mM NaCL in 20 mM tri-ethanolamine, pH 6), and the B&W Buffer was removed from the beads on a magnetic rack. 1000 pl of filtered, UV-inactivated SARS-CoV-2 (delta variant) was added to the beads, and the bead-virus mixture was agitated for 10 minutes on a MixMate (Eppendorf, 5353000537) at 1200 rpm. After the agitation, the tube was placed on the magnetic rack, and the virus solution was removed from the beads. The beads were then washed with 500 pl B&W Buffer. After removing the B&W Buffer, the varying volumes of Release Buffer (R Buffer: 0.25 M KI in 20 mM triethanolamine, pH 6) or Release Buffer + 0.01% tween (R+T Buffer) were added to the beads. The bead-R or R+T Buffer mixtures were agitated for 10 minutes on a MixMate (Eppendorf, 5353000537) at 1200 rpm. After 10 minutes the beads were separated from the virus containing R or R+T buffers on a magnetic rack. Optional desalting was carried out using disposable PD-10 desalting columns (Merck, GE17-0851-01) according to manufacturer’s protocol. Due to the innate volume requirements of our desalting columns, the 50 pl elutions were diluted in 950 pl PBS (1000 pl total volume). The total protein content in the samples was quantified using a BCA assay to assess purity, and viral recovery was evaluated using RT-PCR and SARS-CoV-2 rapid antigen tests.

[0088] Efficacy of Different Purification Protocols and Elution Volumes on SARS-CoV-2 Recovery: A Comparative Analysis (Experiment: UV-062)

[0089] This study aims to evaluate the efficacy of two different purification protocols - RoLink and Thermo - on SARS-CoV-2 virus recovery, using Dynabeads™ Intact Virus Enrichment magnetic beads (Dynabeads™ Intact Virus Enrichment (optimized for SARS-CoV-2), 100 reactions, obtained 02. 06. 2021, ThermoFisher Scientific, Catalog numbers 10700D, 10701D, Pub. No. MAN0019858 Rev. A.0). The study further investigates the impact of varying elution volumes on virus recovery. The efficacy is assessed by comparing quantitative PCR (qPCR) cycle threshold (Cq) values adjusted for dilution factors.

[0090] Materials and Methods: The experiment involved purifying UV-inactivated SARS-CoV-2 virus from VeroE6 cell supernatant. Two purification protocols (RoLink and Thermo, i.e. manufacturer’s protocol provided with the beads) and four different elution volumes (50 pL, 125 pL, 250 pL, and 500 pL) were evaluated.

[0091] Nucleic Acid Extraction: Cellular supernatant was processed using the Zybio Nucleic Acid Extraction Kit (B200-32, Zybio, Catalog No. 5112043) alongside the Zybio Robot, ensuring efficient and consistent extraction of viral RNA.

[0092] Quantitative PCR (qPCR): The extracted RNA was quantified via qPCR using the SARS- CoV-2 (COVID19) RdRP 530 kit (Roche, 9155376001). This involved specific primers and a probe targeting the SARS-CoV-2 RdRp gene. The sequences used were:

[0093] Forward Primer: GTGARATGGTCATGTGTGGCGG; Reverse Primer:

[0094] CARATGTTAAASACACTATTAGCATA; Probe: FAM-CAGGTGGAACCTCATCAGGA- GATGC-BBQ

[0095] The Cq values from qPCR were adjusted based on the relative gross dilution factor for a standardized comparison.

[0096] Droplet Digital PCR (ddPCR) Following qPCR, ddPCR analysis was conducted on the QX200 Droplet Digital PCR System. This step provided a highly accurate quantification of viral RNA copies, offering a more sensitive measure of viral load in the samples.

[0097] Rapid Antigen Tests

[0098] We employed multiple commercially available rapid antigen test kits, including BOSON and CLUNGENE, to evaluate their performance under varied conditions. Samples were prepared according to each manufacturer's instructions. Special attention was paid to the handling of reagents and the preparation of test surfaces to ensure consistency across tests. The concentration of Tween 20 detergent was systematically varied to deter-mine its optimal concentration for antigen release and detection. This was performed by preparing a range of dilutions and assessing their efficacy in antigen release. We explored various release step conditions, including temperature and incubation times, to establish the most effective method for antigen extraction. These conditions were tested in a con-trolled environment to ensure replicability. Each test was evaluated based on sensitivity, specificity, and the time required to obtain a result. The performance was compared against a standard PCR test to benchmark the results.

[0099] BCA Protein Assay

[0100] The BCA Protein Assay Kit (Merck, 71285-3) was used to quantify total protein content in the purified virus samples. This assay, following User Protocol TB38O Rev. D Oi l UN, allows for the accurate determination of protein concentration, which is crucial for assessing the purity of the viral samples. The process involves a colorimetric reaction, where the intensity of the color formed is proportional to the protein concentration. This method provided a reliable measure of sample purity post-purification, an essential factor in evaluating the efficacy of the virus purification process.

[0101] Immunological Interaction Studies

[0102] Human blood samples from healthy donors were used for isolating PBMCs via density gradient centrifugation. These PBMCs were then co-incubated with both purified and unpurified UV- inactivated SARS-CoV-2 suspensions. The interactions were analyzed using FACS Canto II flow cytometry and further visualized via light microscopy post-incubation.

[0103] Data Analysis

[0104] Viral RNA was quantified using RT-PCR techniques post-purification to assess re-covery rates. For PCR data analysis, the MyGo Pro ESR software (v3.6.4) and MyGo Mini software (v3.6.3) were utilized accordingly, corresponding to the MyGo Mini or MyGo Pro equipment used. These software tools enabled precise quantification and assessment of viral RNA concentration in the samples. The integration of this software facilitated a streamlined analysis, contributing to the reliability and reproducibility of the PCR data across different experimental setups. Protein purity was determined through BCA assays, utilizing the specified kit protocol. Immunological interactions were analyzed using flow cytometry (FACS Canto II) with FACSDiva software, focusing on PBMC responses to the UV-inactivated SARS-CoV-2. This comprehensive approach validated the purification method and assessed the immunogenicity of the virus. Results were systematically rec-orded in a detailed Excel database, ensuring traceability and aiding in the robust analysis of purification efficacy and immune response potential

[0105] Results: The comparative analysis between RoLink and Thermo protocols revealed significant differences in recovery efficiencies across various elution volumes. The RoLink protocol consistently outperformed the Thermo protocol in terms of recovery efficiency. The RoLink protocol achieved 99.7% total protein purity (BCA assay) and viral RNA recovery between 36- 75%. (Fig. 1-3)

[0106] Rapid Antigen Test and Cell Viability: Strong rapid antigen test signals were observed, with healthy leukocytes maintained post-purification (Fig. 6; Fig. 7)

[0107] Optimal Elution Volume for RoLink Protocol: The highest recovery efficiency for the RoLink protocol was observed at a 250 pL elution volume, reaching up to 88.92%. This indicates the protocol's effectiveness in virus recovery, especially with Tween-20 as the eluent.

[0108] Comparative Analysis with Thermo Protocol: The Thermo protocol, in contrast, achieved its highest recovery efficiency of 53.93% at a 50 pL elution volume. This comparison underscores the superior performance of the RoLink protocol in virus recovery when using Tween-20 as the eluent.

[0109] Impact of Elution Volume on Recovery Efficiency: Both protocols exhibited a decrease in recovery efficiency with increasing elution volumes. This pattern suggests that lower elution volumes, particularly with Tween-20, are more favorable for virus recovery in this experimental setup.

[0110] Implications for Virus Purification Process Optimization: Enhanced Yields with RoLink Protocol: The results strongly indicate that the RoLink protocol, especially with a 250 pL elution volume using Tween-20, can significantly improve virus purification processes, yielding higher recovery rates.

[0111] Concentration Factor: A noteworthy enhancement in virus concentration was achieved using the RoLink protocol with a 50 pL elution volume containing 0.01% Tween-20, leading to an 11.5-fold increase in virus concentration. This result was substantially higher compared to other methods, emphasizing the efficiency of Tween-20 in the purification process. Table 1. Comparative Analysis of SARS-CoV-2 Recovery Efficiencies and TCID50 Calculation Across Different Protocols.

[0112] This table presents a detailed comparison of recovery efficiencies and TCID50 values for different protocols applied to SARS-CoV-2 virus purification. TCID50, or the tissue culture infectious dose required to infect 50% of cells, was calculated based on quantitative PCR (qPCR) Cq values following PCR analysis of a serially diluted active SARS-CoV-2 stock. The data encompass various elution volumes and their corresponding recovery efficiencies, providing critical insights into the performance of each protocol in virus recovery and infectivity assessment. The RoLink protocol shows higher recovery efficiencies across all elution volumes compared to the Thermo protocol. The highest recovery efficiency for RoLink is observed at the 250 pL elution volume, reaching up to 88.92%. Thermo protocol efficiencies are generally lower, with the highest observed at 53.93% for the 50 pL elution volume.

[0113] Across the tested elution volumes, the RoLink protocol outperforms the Thermo protocol in terms of recovery efficiency, as indicated by the higher percentages and calculated TCID50 values. Recovery efficiencies decrease with increasing elution volumes for both protocols, which suggests that lower elution volumes may be more effective for virus recovery in this experimental setup. These results can guide optimization efforts for virus purification processes to achieve higher yields, particularly when using the RoLink protocol.

[0114] We also evaluated four bead-to-virus ratios ranging from 5 pl to 20 pl and analyzed the residual RNA content of the 1 ml stock after the pulldown process. Table 2 and Fig 3 show the results of this analysis.

[0115] Table 2

[0116] Cq represents the quantitative PCR cycle quantification values.

[0117] Recovery efficiency of SARS-CoV-2 RNA in 1-ml virus isolates, with tween concentrations ranging from 0 to 0.1 %, and elution volumes ranging from 250 to 500 pL is shown on Fig 4. We compared eight purification methods, relative to the RNA content in the original 1 ml virus stock solution. The graph showcases RNA-based recovery efficiency as Cq change compared to 1 ml virus stock (ACq is calculated as mean Cq of lx virus stock - mean Cq of sample; all measurements were done in technical triplicates).

[0118] Table 3 Recovery efficiency of SARS-CoV-2 RNA in 1-ml virus isolates, with different tween concentrations

[0119] Comparison of BCA based total protein content from two purification methods, Rolink and Thermo is shown on Fig 2. Our analysis covers protein content before and after desalting for both methods. Rolink was assessed at two elution volumes (50 pL and 250 pL), pre- and postdesalting, while Thermo was evaluated at a fixed 50 pL elution volume. This assessment illustrates the purification methods' effectiveness in terms of protein yield and purity, relative to the initial stock solution (708 pg / ml). The total protein content of the samples was determined by fitting curves to the internal standard curve.

[0120] Statistical Analysis

[0121] RoLink protocol showed standard deviations ranging from 0.16 to 0.23 across different elution volumes, indicating moderate variability. The Thermo protocol exhibited slightly higher variability, especially at larger elution volumes (250 pL and 500 pL).

[0122] A one-way ANOVA test comparing the adjusted Cq values between the RoLink and Thermo protocols yielded a p-value of 0.00074 and an F-statistic of 15.83, indicating significant differences in virus recovery efficacy between the two protocols.

[0123] Efficacy of Purification Protocols:

[0124] The RoLink protocol consistently showed lower mean adjusted PCR Cq values across all elution volumes compared to the Thermo protocol, suggesting a higher efficiency in virus recovery.

[0125] Impact of Elution Volume: Smaller elution volumes (50 pL and 125 pL) generally resulted in higher viral concentrations (lower Cq values) compared to larger volumes (250 pL and 500 pL) for both protocols. The findings demonstrate that the choice of purification protocol significantly impacts the efficiency of SARS-CoV-2 virus recovery. The RoLink protocol was more effective compared to the Thermo protocol across all tested elution volumes. Additionally, the study highlights the importance of optimizing elution volume in the purification process, with smaller volumes favoring higher virus recovery.

[0126] Conclusion:

[0127] In summary, our investigation reveals that the RoLink protocol, when employing Tween-20 as the eluent, to be markedly superior in recovering viruses across a range of elution volumes when compared to the Thermo protocol. This superiority is particularly pronounced at a 250 pL elution volume, where the RoLink protocol achieves its peak efficiency. Such insights are invaluable for refining virus purification methodologies, especially those pertinent to the fields of UV inactivation and vaccine development. This study underscores the RoLink protocol's role in enhancing SARS-CoV-2 virus recovery, with a notable emphasis on the effectiveness of smaller elution volumes.

Claims

CLAIMS1. A method for increasing the yield of recovered lipid enveloped virus in an ion exchange chromatography purification method, comprising a step of eluting the lipid enveloped virus from a support used in the ion exchange chromatographic purification method using an elution solution comprising a non-ionic detergent.

2. Use of a non-ionic detergent for increasing the yield of recovered lipid enveloped virus in an ion exchange chromatograpic purification method, comprising a step of eluting the lipid enveloped virus from a support used in the ion exchange chromatographic purification method using an elution solution comprising the non-ionic detergent.

3. The method according to claim 1 or the use according to claim 2, wherein the non-ionic detergent is polyoxyethylene (20) sorbitan monolaurate (CAS No. 9005-64-5).

4. The method according to claim 1 or 3 or the use according to claim 2 or 3, wherein the recovered lipid enveloped virus has an intact lipid envelope.

5. The method according to any one claims 1 and 3-4 or the use according to any one of claims 2-4, wherein the recovered lipid enveloped virus retained its infectivity.

6. The method according to any one claims 1 and 3-5 or the use according to any one of claims 2-5, wherein the lipid enveloped virus is SARS-CoV-2.

7. The method according to any one claims 1 and 3-6 or the use according to any one of claims 2-6, wherein the elution solution comprises the non-ionic detergent in a concentration of 0.001 - 0.5% (v / v).

8. The method according to any one claims 1 and 3-7 or the use according to any one of claims 2-7, wherein the support is magnetic beads.

9. The method according to claim 8, wherein the magnetic beads are essentially identical to Dynabeads™ Intact Virus Enrichment, Catalog Numbers 10700D, 10701D as of 8 February, 2024.

10. The method according to any one claims 1 and 3-10 or the use according to any one of claims 2-10, wherein the elution solution comprises 0.25 M KI in 20 mM triethanolamine and 0.01-0.5% (v / v) polyoxyethylene (20) sorbitan monolaurate; pH=6.

11. The method according to claim 10, wherein the concentration of the non-ionic detergent is about 0.01 % (v / v).

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