US2009203076A1PendingUtilityA1

Compositions and methods for increasing protein production

57
Individually held — no corporate assignee on recordPriority: Jul 20, 2004Filed: Sep 9, 2008Published: Aug 13, 2009
Est. expiryJul 20, 2024(expired)· nominal 20-yr term from priority
Inventors:Alexander V. KabanovValery Alakhov
C12P 21/02
57
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Claims

Abstract

Compositions and methods for increasing protein production are provided.

Claims

exact text as granted — not AI-modified
1 . A method for producing a protein comprising:
 a) providing cells comprising a heterologous nucleic acid encoding a recombinant protein; and   b) incubating the cells in media containing at least one amphiphilic block copolymer.   
   
   
       2 . The method of  claim 1 , comprising the further step of:
 c) replacing said media containing an amphiphilic block copolymer with media lacking said amphiphilic block copolymer.   
   
   
       3 . The method of  claim 1 , comprising the further step of isolating the expressed recombinant protein. 
   
   
       4 . The method of  claim 2 , comprising the further step of isolating the expressed recombinant protein. 
   
   
       5 . The method of  claim 1 , wherein said cells are mammalian cells. 
   
   
       6 . The method of  claim 1 , wherein said amphiphilic block copolymer comprises at least one block of poly(oxyethylene) and at least one block of poly(oxypropylene). 
   
   
       7 . The method of  claim 6 , wherein said amphiphilic block copolymer is a Pluronic® copolymer. 
   
   
       8 . The method of  claim 6 , wherein said amphiphilic block copolymer is a Tetronic® copolymer. 
   
   
       9 . The method of  claim 6 , wherein said amphiphilic block copolymer has a hydrophilic-lipophilic balance (HLB) of between 1 and 20. 
   
   
       10 . The method of  claim 9 , wherein said amphiphilic block copolymer has an HLB of between 8 and 16. 
   
   
       11 . The method of  claim 7 , wherein said Pluronic® copolymer is selected from the group consisting of Pluronic® P123, Pluronic® P103, Pluronic® P85, and Pluronic® L64. 
   
   
       12 . The method of  claim 1 , wherein said at least one amphiphilic block copolymer is a mixture of different amphiphilic block copolymers. 
   
   
       13 . The method of  claim 1 , wherein said amphiphilic block copolymer is a mixture of a Pluronic® copolymer and a polycation conjugated Pluronic® copolymer. 
   
   
       14 . The method of  claim 13 , wherein said mixture comprises Pluronic® P123 and Pluronic® P123 conjugated to polyethyleneimine. 
   
   
       15 . The method of  claim 14 , wherein the ratio of Pluronic® P123 to Pluronic(D P123 conjugated to polyethyleneimine in said mixture is 9:1 by weight. 
   
   
       16 . The method of  claim 1 , wherein said incubation of the cells in media comprising at least one amphiphilic block copolymer is for at least three hours. 
   
   
       17 . The method of  claim 16 , wherein said incubation is for at least nine hours. 
   
   
       18 . The method of  claim 6 , wherein said amphiphilic block copolymer is present in the media at a concentration ranging from about 0.0001% to about 5%. 
   
   
       19 . The method of  claim 18 , wherein said concentration ranges from about 0.1% to about 2%. 
   
   
       20 . The method of  claim 1 , wherein said heterologous nucleic acid is stably incorporated into said cells. 
   
   
       21 . The method of  claim 1 , wherein said heterologous nucleic acid encoding a recombinant protein is controlled by the cytomegalovirus promoter. 
   
   
       22 . The method of  claim 1 , wherein said recombinant protein is selected from the group consisting of cytokines, enzymes, clotting factors, vaccines, antibodies, growth factors and hormones, insulin, hemoglobin, alpha-1-antitrypsin (AAT), lactoferrin, cystic fibrosis transmembrane conductase (CFTR), human protein C, anti-viral agents, and interleukins. 
   
   
       23 . The method of  claim 22 , wherein said recombinant protein is Factor VIII and said amphiphilic block copolymer has a hydrophilic-lipophilic balance (HLB) of between 1 and 20. 
   
   
       24 . A method for producing a protein in a host comprising:
 a) providing a cell comprising a heterologous nucleic acid encoding a recombinant protein;   b) incubating the cells in media containing at least one amphiphilic block copolymer; and   c) introducing the cells into a host.   
   
   
       25 . The method of  claim 24 , wherein the cells in step a) are obtained from the host and the heterologous nucleic acid was subsequently incorporated into the cells in vitro. 
   
   
       26 . A method for enhancing production an RNA comprising:
 a) providing cells comprising a heterologous DNA encoding an RNA; and   b) incubating the cells in media containing at least one amphiphilic block copolymer.   
   
   
       27 . The method of  claim 26 , wherein said encoded for RNA is an siRNA. 
   
   
       28 . The method of  claim 26 , wherein said heterologous nucleic acid encoding a recombinant protein is controlled by the cytomegalovirus promoter or a polymerase III promoter. 
   
   
       29 . A composition comprising stably transformed cells, at least one amphiphilic block copolymer, and nucleic acid free media. 
   
   
       30 . A kit for practicing the method of  claim 1  comprising an amphiphilic block polymer, reagents to transform a cell, and at least one selection agent to isolate stably transformed cells. 
   
   
       31 . The kit of  claim 30  further comprising at least one of frozen stocks of host cells and instruction material.

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