Virus like particle purification
View Patent ↗Methods for purifying human Calciviruses are disclosed, including Noroviruses and Sapoviruses.
1. A method of purifying Calicivirus virus-like particles (VLPs) using a multistep chromatographic process, wherein said VLPs are produced in insect cells, said method comprising
(a) contacting a cell lysate or culture supernatant containing said VLPs with an ion exchange chromatographic material, wherein said VLPs bind to said ion exchange chromatographic material;
(b) contacting the VLPs eluted from said ion exchange chromatographic material with a hydrophobic interaction chromatographic material, wherein said VLPs bind to said hydrophobic interaction chromatographic material; and
(c) eluting said VLPs from said hydrophobic interaction chromatographic material, wherein said VLPs are purified to greater than about 70%,
wherein the chromatographic materials used in the method do not comprise hydroxyapatite.
2. The method of claim 1 , wherein the hydrophobic interaction chromatographic material is a methyl HIC resin.
3. The method of claim 1 , wherein the ionic strength of the VLP-containing solution in step (b) is adjusted to cause the retention of VLPs with contaminating materials passing through the hydrophobic interaction chromatographic material.
4. The method of claim 3 , wherein the ionic strength is adjusted by the addition of ammonium sulfate.
5. The method of claim 1 , wherein the eluate from step (c) is ultrafiltered and/or diafiltered.
6. The method of claim 1 further comprising (d) contacting the eluate from step (c) with a third chromatographic material, wherein contaminating materials are retained and said VLPs pass through the third chromatographic material; and (e) collecting said solution containing said VLPs.
7. The method of claim 6 , wherein the third chromatographic material comprises an anion-exchanger.
8. The method of claim 6 , wherein the solution containing said VLPs from step (e) is ultrafiltered and/or diafiltered.
9. The method of claim 6 , wherein said VLPs are purified to greater than about 90%.
10. The method of claim 1 , further comprising contacting the VLPs eluted from said ion exchange chromatographic material with an adsorption material prior to contact with the hydrophobic interaction chromatographic material, wherein contaminating materials are retained and said VLPs pass through the adsorption material.
11. The method of claim 1 , wherein the Calicivirus VLPs are Norovirus VLPs.
12. The method of claim 11 , wherein the Norovirus VLPs are Norovirus genogroup I VLPs.
13. The method of claim 11 , wherein the Norovirus VLPs are Norovirus genogroup II VLPs.
14. The method of claim 1 , wherein the ion exchange chromatographic material comprises a cation-exchanger.
15. The method of claim 1 , wherein the cell lysate or the culture supernatant is produced using recombinant methodologies.
16. The method of claim 15 , wherein the contaminant level of host cell DNA content is less than 1%.
17. The method of claim 15 , wherein the contaminant level of host cell protein content is less than 5%.
18. The method of claim 1 , wherein the ion exchange chromatographic material comprises an anion-exchanger.
19. The method of claim 1 , wherein the cell lysate or the culture supernatant is filtered, centrifuged, or clarified prior to contact with the ion exchange chromatographic material.
20. The method of claim 19 , wherein the cell lysate or the culture supernatant is clarified by precipitation or by size exclusion chromatography.
21. A method of purifying Calicivirus virus-like particles (VLPs) wherein the VLPs are produced in insect cells, said method comprising contacting a solution containing said VLPs with two or more chromatographic materials, wherein the chromatographic materials do not comprise hydroxyapatite, and wherein at least one chromatographic material retains said VLPs.
22. The method of claim 21 , wherein the two or more chromatographic materials are selected from ion-exchange, affinity, hydrophobic interaction, mixed mode, reverse phase, size exclusion or adsorption material.
23. The method of claim 22 , wherein the two or more chromatographic materials are ion-exchange and hydrophobic interaction.
24. The method of claim 23 , wherein the ion-exchange material comprises a cation-exchanger.
25. The method of claim 21 , wherein said VLPs are purified to greater than about 70%.
26. The method of claim 21 , wherein the Calicivirus VLPs are Norovirus VLPs.
27. A method of purifying Calicivirus virus-like particles (VLPs) using a multistep chromatographic process wherein said VLPs are produced in insect cells, said method comprising
(a) contacting a cell lysate or culture supernatant containing said VLPs with an ion exchange chromatographic material, wherein said VLPs bind to said ion exchange chromatographic material, and wherein said ion exchange chromatographic material comprises an anion exchanger;
(b) contacting the VLPs eluted from said ion exchange chromatographic material with a hydrophobic interaction chromatographic material, wherein said VLPs bind to said hydrophobic interaction chromatographic material; and
(c) eluting said VLPs from said hydrophobic interaction chromatographic material, wherein said VLPs are purified to greater than about 70%,
wherein the chromatographic materials used in the method do not comprise hydroxyapatite.
28. A method of purifying Calicivirus virus-like particles (VLPs) using a multistep chromatographic process wherein said VLPs are produced in insect cells, said method comprising
(a) contacting a cell lysate or culture supernatant containing said VLPs with an ion exchange chromatographic material, wherein said VLPs bind to said ion exchange chromatographic material, and wherein said cell lysate or culture supernatant is filtered, centrifuged, or clarified prior to contact with the ion exchange chromatographic material;
(b) contacting the VLPs eluted from said ion exchange chromatographic material with a hydrophobic interaction chromatographic material, wherein said VLPs bind to said hydrophobic interaction chromatographic material; and
(c) eluting said VLPs from said hydrophobic interaction chromatographic material, wherein said VLPs are purified to greater than about 70%,
wherein the chromatographic materials used in the method do not comprise hydroxyapatite.
29. A method of purifying Calicivirus virus-like particles (VLPs) using a multistep chromatographic process wherein said VLPs are produced in insect cells, said method comprising
(a) contacting a cell lysate or culture supernatant containing said VLPs with an ion exchange chromatographic material, wherein said VLPs bind to said ion exchange chromatographic material, and wherein said cell lysate or culture supernatant is clarified by precipitation or by size exclusion chromatography;
(b) contacting the VLPs eluted from said ion exchange chromatographic material with a hydrophobic interaction chromatographic material, wherein said VLPs bind to said hydrophobic interaction chromatographic material; and
(c) eluting said VLPs from said hydrophobic interaction chromatographic material, wherein said VLPs are purified to greater than about 70%,
wherein the chromatographic materials used in the method do not comprise hydroxyapatite.