Methods for removal, purification, and concentration of viruses and methods of therapy based thereupon

• US 7,160,464 B2
• Filed: 12/21/2004
• Issued: 01/09/2007
• Est. Priority Date: 12/02/1999
• Status: Expired due to Term

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Yarmush, Martin L., Lee, William, Morgan, Jeffrey R.
• Primary Examiner(s): FORTUNA, ANA M
• Application Number:
  US11/018,939
• Publication Number:
  US 20050247630A1
• Time in Patent Office: 749 Days
• Field of Search: 210/645, 210/651, 210/195.1, 210/650, 210/653, 210/500.36, 210/500.35, 210/321.8, 210/175, 210/184, 210/500.37, 264/48, 435/325, 435/235.1, 435/320.1, 435/69.2, 435/252.3, 424/484
• US Class Current: 210/651
• CPC Class Codes: A61L 2/0017 Filtration
• CPC Class Codes: A61L 2/0082 using chemical substances
• CPC Class Codes: C12N 2740/10051 Methods of production or purification of viral material
• 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

Abstract

The invention is based on the discovery that certain membranes, which include side chains or molecular “brushes” having, for example, tertiary amino functional groups, can be used as highly effective filters to capture viruses/virus particles from liquids without removal of proteins. New methods based on this discovery include removing viruses from liquids such as blood or plasma, removing viruses from pharmaceuticals, concentrating and/or purifying viruses, e.g., for use in gene therapy, and producing recombinant viruses in new bioreactors. The invention also includes new methods of therapy or adjunct therapy for viral infections, in which a patient'"'"'s blood or plasma is filtered through the membranes to remove viruses to reduce the viral load. The invention also includes new bioreactors and viral filters containing the membranes.

Claims

1. A method of removing viruses from the blood of an individual having a viral infection, the method comprising:

obtaining a membrane engrafted with polymeric side chains, each side chain having one or more positively charged functional groups that interact with viruses, the polymeric side chains each having one functional group per repeat unit, each functional group consisting of a single secondary, tertiary, or quaternary amine, and having a single positive charge at physiological pH wherein the membrane has a nominal pore size between 20 nm and 1000 nm;

sequestering blood cells from the individual'"'"'s blood using a plasma separator to produce sequestered blood cells and plasma;

passing the plasma through the membrane to produce filtered plasma;

combining the filtered plasma with the sequestered blood cells to produce filtered blood; and

returning the filtered blood to the individual;

thereby removing viruses from the blood.

2. The method of claim 1, wherein the patient is infected with a hepatitis virus or human immunodeficiency virus.

3. A device for removing viruses from a liquid sample, the device comprising a membrane engrafted with polymeric side chains, each side chain having one or more positively charged functional groups that interact with viruses, the polymeric side chains each having one functional group per repeat unit, each functional group consisting of a single secondary, tertiary, or quaternary amine, and having a single positive charge at physiological pH, wherein the membrane has a nominal pore size between 20 nm and 1000 nm.

4. The device of claim 3, wherein the membrane has a nominal pore size between 100 nm and 500 nm.

5. The device of claim 3, wherein the membrane comprises polyethylene and the polymeric side chains comprise diethylaminated poly(glycidyl methacrylate) (“

DEA-PGMA”

).

6. The device of claim 3, wherein the polymeric side chains have an average length between 50 nm and 2000 nm.

7. The device of claim 3, wherein the polymeric side chains have an average length between 500 nm and 1000 nm.

8. The device of claim 3, wherein between 1.0e16 and 1.0e20 of the PGMA side chains are present per square meter of the membrane'"'"'s surface area.

9. The device of claim 3, wherein the membrane has a degree of grafting (“

DG”

) between 50% and 500%.

10. The device of claim 3, wherein the sample flows through the membrane at a rate of 1 to 1000 ml/min per centimeter length of said membrane.

11. A method of isolating virus particles, the method comprising

culturing a virus infected cell line in a culture medium under conditions suitable for the production of viruses;

flowing culture medium containing virus particles through a filter comprising a membrane engrafted with polymeric side chains, each side chain having one or more positively charged functional groups that interact with viruses, the polymeric side chains each having one functional group per repeat unit, each functional group consisting of a single secondary, tertiary, or quaternary amine, and having a single positive charge at physiological pH, wherein the membrane has a nominal pore size between 20 nm and 1000 nm; and

eluting virus particles from the membrane.

12. The method of claim 11, further comprising storing the virus particles under conditions suitable for long-term storage.

13. The method of claim 12, wherein long-term storage is at 4°

C.

14. A device for isolating virus particles comprising:

a bioreactor comprising a vessel suitable for containing a culture medium for the generation of viruses; and

a filter comprising at least one membrane engrafted with polymeric side chains, each side chain having one or more positively charged functional groups that interact with viruses, the polymeric side chains each having one functional group per repeat unit, each functional group consisting of a single secondary, tertiary, or quaternary amine, and having a single positive charge at physiological pH, wherein the membrane has a nominal pore size between 20 nm and 1000 nm,

wherein the filter is functionally connected to the bioreactor so as to allow virus particles produced in the bioreactor to contact the membrane.

15. The device of claim 14, wherein the bioreactor further comprises a heater to heat the vessel.

16. The device of claim 14, wherein the bioreactor further comprises a refrigerator to cool the filter.

17. The device of claim 14, wherein the one or more membranes comprise hollow fibers.