PROCESS FOR PURIFYING VLPS

• US 20140302593A1
• Filed: 05/16/2014
• Published: 10/09/2014
• Est. Priority Date: 12/21/2011
• Status: Active Grant

• Current Assignee: RNAiSSANCE AG LLC
• Original Assignee: APSE, LLC (RNAiSSANCE AG LLC)
• Inventors: Arhancet, Juan Pedro Humberto, Arhancet, Juan P., Delaney, Kimberly, Hall, Kathleen B., Summers, Neena
• Granted Patent: US 9,181,531 B2
• Time in Patent Office: Days
• US Class Current: 435/272
• CPC Class Codes: A01N 25/26 in coated particulate form
• CPC Class Codes: A01N 25/28 Microcapsules or nanocapsules
• CPC Class Codes: A01N 43/70 Diamino—1,3,5—triazines with only one oxygen, sulfur or halogen atom or only one cyano, thiocyano (—SCN), cyanato (—OCN) or azido (—N3) group directly attached to a ring carbon atom
• CPC Class Codes: A01N 47/40 the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
• CPC Class Codes: A01N 57/12 containing acyclic or cycloaliphatic radicals
• CPC Class Codes: A01N 57/14 containing aromatic radicals
• CPC Class Codes: A01N 57/30 containing aromatic radicals
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/01 DNA viruses
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/121 Hammerhead
• CPC Class Codes: C12N 2310/123 Hepatitis delta
• CPC Class Codes: C12N 2310/128 processing or releasing ribozyme
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/16 Aptamers
• CPC Class Codes: C12N 2310/531 Stem-loop; Hairpin
• CPC Class Codes: C12N 2330/51 Specially adapted vectors
• CPC Class Codes: C12N 2795/18122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2795/18123 Virus like particles [VLP]
• CPC Class Codes: C12N 2795/18142 : virus or viral particle as vehicle, e.g. encapsulating small organic molecule
• CPC Class Codes: C12N 7/00 : Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: C12N 7/04 : Inactivation or attenuation; Producing viral sub-units

Abstract

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.

Claims

1. A process for purifying VLPs 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 VLPs comprise a capsid protein having at least 40% sequence identity with the amino acid sequence of Enterobacteria phage MS2 capsid protein (SEQ ID NO:

3).

3. The process according to claim 1, wherein the protease is a peptide bond hydrolase category E.C. 3.4.

4. The process according to claim 1, wherein the protease is selected from Proteinase K, Protease from Streptomyces griseus, Protease from Bacillus lichenformis, pepsin and papain.

5. The process according to claim 1, wherein step (b) is performed for about 30 minutes.

6. The process according to claim 1, wherein step (b) is performed at about 37°

C.

7. 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.

8. 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.

9. The process according to claim 1, wherein step (c) comprises centrifuging the hydrolysate to obtain a precipitate comprising at least about 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 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 about 90% by weight of the VLPs.

11. 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 about 90% by weight of the VLPs.

12. The process according to claim 1, wherein the heterologous cargo molecule comprises an oligonucleotide linker coupling the heterologous cargo molecule and the VLP.

13. The process according to claim 12, wherein the oligonucleotide linker is an oligoribonucleotide.

14. The process according to claim 1, wherein the heterologous cargo molecule comprises an oligoribonucleotide selected from siRNA, shRNA, sshRNA, lshRNA and miRNA.

15. The process according to claim 1, wherein the heterologous cargo molecule comprises an oligoribonucleotide comprising a ribozyme.

16. The process according to claim 1, wherein the heterologous cargo molecule comprises a peptide or a polypeptide.

17. 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.