Process for purifying VLPs
Processes and compositions to produce, package, and purify virus like particles containing heterologous cargo molecules utilizing self assembling proteins and protease treatment coupled with simple precipitation and filtration methods are described.
1. A process for purifying a quantity of virus-like particles (VLPs) consisting of Enterobacteriophage MS2 capsid proteins (SEQ ID NO.: 3) enclosing at least one heterologous cargo molecule, the process comprising: (a) obtaining a cell lysate comprising a plurality of the VLPs; (b) contacting the cell lysate with a protease for a time and under conditions sufficient to hydrolyze cell lysis products other than the VLPs to form a hydrolysate; and (c) isolating the VLPs from the hydrolysate.
2. The process according to claim 1 , wherein the protease is a peptide bond hydrolase category E.C. 3.4.
3. The process according to claim 1 , wherein the protease is selected from Proteinase K, Protease from Streptomyces griseus , Protease from Bacillus licheniformis , pepsin and papain.
4. The process according to claim 1 , wherein step (b) is performed for about 30 minutes.
5. The process according to claim 1 , wherein step (b) is performed at about 37° C.
6. The process according to claim 1 , wherein the time and condition for hydrolysis are sufficient for at least 60, at least 70, at least 80, or at least 90 of every 100 individual polypeptides present in the whole cell lysate but not enclosed by the capsids to be cleaved, while at least 60, at least 70, at least 80, or at least 90 of every 100 capsids present in the whole cell lysate before such hydrolysis remain intact following the hydrolysis.
7. The process according to claim 1 , wherein step (b) further comprises, contacting the cell lysate with at least one of the group consisting of a nuclease, an amylase and a lipase.
8. The process according to claim 1 , wherein step (c) comprises centrifuging the hydrolysate to obtain a precipitate comprising at least 90% by weight of the VLPs.
9. The process according to claim 1 , wherein step (c) comprises (i) performing a first precipitation of the hydrolysate with ammonium sulfate followed by a first centrifugation to obtain a first precipitate and a first supernatant; and (ii) performing a second precipitation on the first supernatant with ammonium sulfate followed by a second centrifugation to obtain a second precipitate, wherein the second precipitate comprises at least 90% by weight of the VLPs.
10. The process according to claim 1 , wherein step (c) comprises (i) performing a first precipitation of the hydrolysate with ethanol followed by a first centrifugation to obtain a first precipitate and a first supernatant; and (ii) performing a second precipitation on the first supernatant with ammonium sulfate followed by a second centrifugation to obtain a second precipitate, wherein the second precipitate comprises at least 90% by weight of the VLPs.
11. The process according to claim 1 , wherein the heterologous cargo molecule comprises an oligonucleotide linker coupling the heterologous cargo molecule and the VLP.
12. The process according to claim 11 , wherein the oligonucleotide linker is an oligoribonucleotide.
13. The process according to claim 1 , wherein the heterologous cargo molecule comprises an oligoribonucleotide selected from siRNA, shRNA, sshRNA, lshRNA and miRNA.
14. The process according to claim 1 , wherein the heterologous cargo molecule comprises an oligoribonucleotide comprising a ribozyme.
15. The process according to claim 1 , wherein the heterologous cargo molecule comprises a peptide or a polypeptide.
16. The process according to claim 1 , wherein the VLPs isolated from the hydrolsate comprise less than 4 grams of one or more cell lysis products for every 100 grams of capsid present in the composition, wherein the cell lysis products are selected from proteins, polypeptides, peptides and any combination thereof.
17. The process of claim 1 , wherein the quantity of virus-like particles (VLPs) enclosing at least one heterologous cargo molecule is at least ten milligrams.
18. The process of claim 1 , wherein the quantity of virus-like particles (VLPs) enclosing at least one heterologous cargo molecule is at least one gram.
19. The process of claim 1 , wherein the quantity of virus-like particles (VLPs) enclosing at least one heterologous cargo molecule is at least one hundred grams.