CELL-BASED BIOPROCESSING

• US 20140004565A1
• Filed: 07/06/2010
• Published: 01/02/2014
• Est. Priority Date: 07/06/2009
• Status: Abandoned Application

• Current Assignee: Alnylam Pharmaceuticals Incorporated
• Original Assignee: Alnylam Pharmaceuticals Incorporated
• Inventors: Rossomando, Anthony, Manoharan, Muthiah, Hinkle, Gregory, Bettencourt, Brian, Pollard, Stuart, Maraganora, John M., Kocisko, David, Hogan, Shannon
• Application Number:
  US13/380,182
• Publication Number:
  US 20140004565A1
• Time in Patent Office: Days
• US Class Current: 435/70.3
• CPC Class Codes: C07K 16/2887 against CD20
• CPC Class Codes: C12N 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12Y 101/01027 L-Lactate dehydrogenase (1.1.1.27)

Abstract

The invention provides compositions and methods for producing an immunogenic agent from a host cell. In various embodiments, the immunogenic agent is a polypeptide, an antigen, a virus particle, or a vaccine In one aspect, the invention provides for a method for producing an immunogenic agent from a host cell. The method generally comprises contacting the cell with a RNA effector molecule, a portion of which is complementary to a target gene, maintaining the cell in a large-scale bioreactor for a time sufficient to modulate expression of the target gene, wherein the modulation enhances production of the immunogenic agent from the cell, and isolating the immunogenic agent from the cell.

Claims

1. A method for producing an immunogenic agent in a large scale host cell culture, comprising:

(a) contacting a host cell in a large scale host cell culture with at least a first RNA effector molecule, a portion of which is complementary to at least one target gene of a host cell,

(b) maintaining the host cell culture for a time sufficient to modulate expression of the at least one first target gene, wherein the modulation of expression improves production of an immunogenic agent in the host cell;

(c) isolating the immunogenic agent from the host cell;

wherein the large scale host cell culture is at least 1 Liter in size, and wherein the host cell is contacted with at least a first RNA effector molecule by addition of the RNA effector molecule to a culture medium of the large scale host cell culture such that the target gene expression is transiently inhibited.

3. The method of claim 1, wherein the host cell in the large scale host cell culture is contacted with a plurality of RNA effector molecules, wherein the plurality of RNA effector molecules modulate expression of at least one target gene, at least two target genes, or a plurality of target genes.

6. The method of claim 1, wherein the RNA effector molecule, comprises a double-stranded ribonucleic acid (dsRNA), wherein said dsRNA comprises at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is substantially complementary to at least part of a target gene, and wherein said region of complementarity is 10-30 nucleotides in length.

7. The method of claim 1, wherein the contacting step is performed by continuous infusion of the RNA effector molecule into the culture medium used for maintaining the host cell culture to produce the immunogenic agent.

8. The method of claim 1 wherein the modulation of expression is inhibition of expression, and wherein the inhibition is a partial inhibition.

9. The method of claim 8, wherein the partial inhibition is no greater than a percent inhibition selected from the group consisting of:

10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and 85%.

10. The method of claim 1 wherein the contacting step is repeated at least once.

11. The method of claim 1 wherein the contacting step is repeated multiple times at a frequency selected from the group consisting of:

6 hr, 12 hr, 24 hr, 36 hr, 48 hr, 72 hr, 84 hr, 96 hr, and 108 hr.

12. The method of claim 1 wherein the modulation of expression is inhibition of expression and wherein the contacting step is repeated multiple times, or continuously infused, to maintain an average percent inhibition of at least 50% for the target gene(s) throughout the production of the immunogenic agent.

13. The method of claim 12, wherein the average percent inhibition is selected from the group consisting of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%.

14. The method of claim 1 wherein the RNA effector molecule is contacted at a concentration of less than 100 nM.

15. The method of claim 1 wherein the RNA effector molecule is contacted at a concentration of less than 20 nM.

16. The method of claim 1 wherein said contacting a host cell in a large scale host cell culture with a RNA effector molecule is done at least 6 hr, at least 12 hr, at least 18 hr, at least 36 hr, at least 48 hr, at least 60 hr, at least 72 hr, at least 96 hr, or at least 120 hr, or at least 1 week, before isolation of the immunogenic agent or prior to harvesting the supernatant.

17. The method of claim 1 wherein the RNA effector molecule is composition formulated in a lipid formulation.

19. The method of claim 1 wherein the RNA effector molecule is not shRNA.

20. The method of claim 1 wherein the RNA effector molecule is siRNA.

21. The method of claim 1 wherein the RNA effector molecule is chemically modified.

2. (canceled)

4. A method for production of an immunogenic agent in a cell, the method comprising:

(a) contacting a host cell with a plurality of RNA effector molecules, wherein the two or more RNA effector molecules modulate expression of a plurality of target genes;

(b) maintaining the cell for a time sufficient to modulate expression of the plurality of target genes, wherein the modulation of expression improves production of the immunogenic agent in the cell; and

(c) isolating the immunogenic agent from the cell,

wherein the plurality of target genes comprises at least Bax, Bak, and LDH.

5. The method of claim 4, wherein the host cell is contacted with the plurality of RNA effector molecules by addition of the RNA effector molecule to a culture medium of the large scale host cell culture such that the target gene expression is transiently inhibited.

18. (canceled)

22-143. -143. (canceled)