Purification of virus like particles
Methods of purifying virus-like particles (VLPs) that are substantially free of process contaminants and infectious agents. The methods incorporate, for example, low-pH treatment during harvest and/or inactivation by a solvent and/or detergent during VLP capture.
1. A method for purifying norovirus or sapovirus virus like particles (VLPs), comprising:
generating or obtaining a cell lysate, culture supernatant, or filtrate containing said norovirus or sapovirus VLPs, wherein said VLPs comprise at least a first subpopulation of the VLPs comprising VLPs with a full length VP1 subunit and a second subpopulation of the VLPs comprising VLPs with a truncated VP1 subunit;
adjusting the pH of the lysate, supernatant, or filtrate to a pH value less than about 5 to generate a pH-adjusted solution; and
removing non-VLP particulates/aggregates from the pH-adjusted solution to generate a purified solution comprising the VLPs,
wherein said removing the non-VLP particulates/aggregates comprises selectively removing the second subpopulation of the VLPs from the pH-adjusted solution.
2. The method of claim 1 , wherein the purified solution contains less than about 10% truncated VP1 subunit.
3. The method of claim 1 , wherein the purified solution substantially retains the first subpopulation of VLPs and is substantially free of the second subpopulation of the VLPs.
4. The method of claim 3 , further comprising removing, after removing the second subpopulation of VLPs from the pH-adjusted solution to generate the purified solution, the first subpopulation of VLPs from the purified solution.
5. The method of claim 1 , wherein the VLPs of the cell lysate or culture supernatant/filtrate are norovirus VLPs.
6. The method of claim 1 , wherein said adjusting the pH is carried out for a duration between about 30 minutes and about 48 hours.
7. The method of claim 1 , wherein the VLPs comprise acid-stable VLPs.
8. The method of claim 1 , wherein said removing is done via one or more of the following types of processes: centrifugation, precipitation, flocculation, settling, and filtration.
9. The method of claim 8 , wherein said removing includes clarifying the pH-adjusted solution by depth filtration followed by additional filtration to generate the purified solution.
10. The method of claim 1 , wherein at least about 98% of residual contaminating nucleic acid is removed from the lysate, supernatant, or filtrate as a result of said purifying.
11. The method of claim 1 further comprising:
purifying the VLPs using a multistep chromatographic process, comprising:
contacting the purified solution comprising the VLPs with a chromatographic material, wherein the VLPs bind to said chromatographic material;
treating the chromatographic material with a solvent and/or detergent; and
eluting, after said treating, the VLPs from the chromatographic material.
12. The method of claim 11 , wherein the solvent and/or detergent comprises one or more of the following: TnBP, octylphenol, ethyleneoxide condensate, polyoxyethylene sorbitan monooleate, and sodium cholate, Triton X-100, and tributyl phosphate.
13. The method of claim 5 , wherein the norovirus VLPs are one or more of: norovirus genogroup I VLPs, norovirus genogroup II VLPs, norovirus genogroup IV VLPs, and chimeric norovirus VLPs.
14. The method of claim 11 , wherein the ratio of the VLPs to residual contaminating protein is increased at least about ten-fold as compared to the lysate, supernatant or filtrate.
15. The method of claim 11 , wherein at least about 50% of residual contaminating protein is removed from the lysate, supernatant or filtrate as a result of said purifying.
16. The method of claim 11 , wherein no more than about 50% of the VLPs are lost during said purification.
17. The method of claim 11 , further comprising separating the first subpopulation of VLPs from the purified solution using the one or more chromatographic processes, wherein each chromatographic process is independently selected from: hydroxyapatite chromatography, hydrophobic interaction chromatography, size exclusion chromatography, ion exchange chromatography, mixed mode chromatography, membrane-based chromatography, and affinity chromatography.
18. The method of claim 1 , wherein the VLPs are produced in bacterial cells, insect cells, yeast cells, plant, or mammalian cells.
19. The method of claim 1 , wherein the removing of the second subpopulation of VLPs from the pH-adjusted solution comprises using a filtration process to generate a filtered, purified solution, wherein the filtered, purified solution substantially retains the first subpopulation of VLPs.
20. The method of claim 1 , wherein the removing non-VLP particulates/aggregates from the pH-adjusted solution comprises clarifying the pH-adjusted solution by a depth filtration process to generate a purified, depth-filtered solution.
21. The method of claim 20 , wherein at least about 75% of the VLPs are recovered from the pH-adjusted solution as compared to the lysate, supernatant or filtrate.