Virus and antigen purification and conjugation
Disclosed herein are methods and exemplary compositions associated with virus purification, antigen purification, and conjugation of virus and proteins (e.g., antigen) to form vaccines for delivery of immunological and other therapeutic agents, exemplary aspects of which may include harvesting viral and antigenic substances from source organisms; a purification platform comprising chemical separation and size-difference separation for the removal of contaminants, debris and impurities from the viral and protein (e.g. antigenic, including influenza hemagglutinin antigens) substances, as well as their concentration and collection; and a conjugation platform providing activation of the virus at a pH that increases binding rate and binding propensity between the virus and the protein, wherein embodiments related to the conjugation platform include controlling the ratio of virus to protein.
1. A method for conjugating a protein and a virus, comprising: (a) extracting a virus from a source organism and purifying the extracted virus; (b) then activating the purified virus by exposing the purified virus to an environment at a pH of about 5.5 or less; and (c) after the activating step, mixing the purified virus and the protein in a conjugation reaction to form a conjugate mixture.
2. The method of claim 1 , wherein the source organism is a plant and the virus activating step further comprises chemically modifying surface-exposed lysine residues on the virus to facilitate amine-targeted conjugation of the protein.
3. The method of claim 1 , wherein the virus activating step is conducted in a buffer for a period of time between about 18 hours and about 72 hours.
4. The method of claim 2 , wherein the protein is an antigen.
5. The method of claim 4 , further comprising preparing the antigen by sub-steps of purifying the antigen and exposing the antigen to a pH of about 6.5 or less prior to the mixing step.
6. The method of claim 5 , further comprising harvesting the antigen from a source organism, wherein purifying the antigen comprises removing cellular debris from the antigen, concentrating the antigen, separating host cell contaminants from the antigen, subjecting the antigen to affinity chromatography to elute the antigen, and subjecting the antigen to multi-modal chromatography to separate residual impurities from the antigen based on size differences between the impurities as retentate and the antigen as permeate.
7. The method of claim 6 , wherein separating host cell contaminants from the antigen comprises subjecting the concentrated antigen to ion-exchange chromatography.
8. The method of claim 1 , wherein the protein is an antigen comprising an influenza hemagglutinin antigen (HA) and the virus is a tobacco mosaic virus (TMV).
9. The method of claim 8 , wherein the TMV and HA are mixed at a ratio of at least about 4:1.
10. The method of claim 8 , wherein the TMV and HA are mixed at a ratio between about 1:1 and 4:1.
11. The method of claim 8 , further comprising adding chemical linkers to the HA and TMV mixture.
12. The method of claim 1 , further comprising quenching the conjugation reaction.
13. The method of claim 1 , wherein the protein is an antigen and the virus is chosen from the group consisting of red clover mosaic virus; commelina yellow mottle virus, cauliflower mosaic virus, Soybean chlorotic mottle virus, Cassava vein mosaicvirus, rice tungro bacilliformvirus, petunia vein clearing virus, maize streak virus, beet curly top virus, bean golden mosaic virus, alfalfa mosaic virus, tobacco streak virus, brome mosaic virus, cucumber mosaic virus, beet yellows virus, lettuce infectious yellows virus, cowpea mosaic virus, broad bean wilt virus 1 , tobacco ringspot virus, potato virus Y, ryegrass mosaic virus, barley yellow mosaic virus, parsnip yellow fleck virus, rice tungro spherical virus, carnation mottle virus, carnation ringspot virus, maize chlorotic mottle virus, tobacco necrosis virus, tomato bushy stunt virus, apple stem grooving virus, carnation latent virus, pea enation mosaic virus, soil-borne wheat mosaic virus, barley stripe mosaic virus, raspberry bushy dwarf virus, barley yellow dwarf virus, maize rayado fino virus, potato virus X, clover mosaic viruses, Southern bean mosaic virus, rice stripe virus, tobacco mosaic virus, tobacco rattle virus, apple chlorotic leaf spot virus, turnip yellow mosaic virus, carrot mottle virus.
14. The method of claim 13 , further comprising, following the quenching step, separating residual impurities from the conjugate mixture based on size differences between the impurities as retentate and the conjugate mixture as permeate.
15. The method of claim 1 , wherein the conjugation reaction achieves at least about 50% reduction in non-conjugated, native molecular weight, virus coat protein.
16. The method of claim 2 , wherein the virus is TMV and the TMV has a radius, and the step of mixing the virus and protein is performed after the TMV radius has increased by at least a factor of 2.75 during the activation step.
17. The method of claim 2 , wherein the virus is TMV and the TMV has a radius, and the protein is an antigen, and the step of mixing the virus and antigen is performed after the TMV radius has increased by at least a factor of 2.75 during the activation step.
18. The method of claim 2 , wherein the virus is TMV and the TMV has a radius, and the step of mixing the virus and protein is performed after the TMV radius has increased to at least about 195 nm during the activation step.
19. The method of claim 2 , wherein the virus is TMV and the TMV has a radius, and the protein is an antigen, and the step of mixing the virus and antigen is performed after the TMV radius has increased to at least about 195 nm during the activation step.
20. The method of claim 2 , wherein the virus activating step is conducted in a buffer for a period of time between about 18 hours and about 72 hours.
21. The method of claim 2 , wherein the protein is an antigen comprising an influenza hemagglutinin antigen (HA) and the virus is a tobacco mosaic virus (TMV).
22. The method of claim 21 , wherein the TMV and HA are mixed at a ratio of at least about 4:1.
23. The method of claim 21 , wherein the TMV and HA are mixed at a ratio between about 1:1 and 4:1.
24. The method of claim 21 , further comprising adding chemical linkers to the HA and TMV mixture.
25. The method of claim 2 , wherein the conjugation reaction achieves at least about 50% reduction in non-conjugated, native molecular weight, virus coat protein.