Methods and materials for expression of a recombinant protein

Abstract

Recombinant expression vectors are provided comprising a 3′ UTR of a light chain and an Epstein-Barr virus origin of replication. Also provided are host cells comprising such vectors and methods of producing recombinant protein with such vectors. Additional methods of producing a recombinant protein involve contacting cells with a first and second vector, each of which encode a different polypeptide chain, and wherein the second vector is present in an amount which is about 1.5 to 2.5 times as much as that of the first vector. Cells also can be transfected with a recombinant transient expression vector encoding a protein and are cultured in a medium in a membrane-enhanced culturing vessel to produce recombinant protein.

1 . A recombinant expression vector comprising a 3′ UTR of a light chain gene and an Epstein-Barr virus origin of replication (oriP).  
     
     
         2 . The recombinant expression vector of  claim 1 , wherein the light chain gene is a murine light chain gene.  
     
     
         3 . The recombinant expression vector of  claim 2 , wherein the 3′ UTR comprises a nucleotide sequence of nucleotides 1062-2560 of SEQ ID NO: 1.  
     
     
         4 . The recombinant expression vector of  claim 1 , wherein the recombinant expression vector is a recombinant transient expression vector.  
     
     
         5 . The recombinant expression vector of  claim 1  further comprising a pUC19 origin of replication, a viral promoter, a 5′ UTR intron, or a combination of any of the foregoing.  
     
     
         6 . The recombinant expression vector of  claim 5  further comprising a pUC19 origin of replication, a viral promoter, and a 5′ UTR intron.  
     
     
         7 . The recombinant expression vector of  claim 5 , wherein the viral promoter is a CMV promoter.  
     
     
         8 . The recombinant expression vector of  claim 5 , wherein the 5′ UTR intron comprises nucleotides 888-974 of SEQ ID NO: 1.  
     
     
         9 . The recombinant expression vector of  claim 5 , wherein the pUC19 origin of replication comprises the nucleotide sequence of nucleotides 4551-5220 of SEQ ID NO: 1.  
     
     
         10 . The recombinant expression vector of  claim 1  further comprising an antibody signal sequence.  
     
     
         11 . The recombinant expression vector of  claim 1 , wherein the recombinant expression vector does not comprise an antibody signal sequence.  
     
     
         12 . The recombinant expression vector of  claim 1  further comprising a nucleotide sequence encoding a protein or a functional fragment thereof.  
     
     
         13 . The recombinant expression vector of  claim 12 , wherein the nucleotide sequence encodes an immunoglobulin chain.  
     
     
         14 . The recombinant expression vector of  claim 13 , wherein the immunoglobulin chain is selected from the group consisting of a γ 1  heavy chain, a γ 2  heavy chain, a γ 4  heavy chain, a κ light chain, and a λ light chain.  
     
     
         15 . A method of producing a recombinant protein, comprising contacting a cell with the recombinant expression vector of  claim 1 , whereupon a recombinant protein is produced.  
     
     
         16 . A host cell comprising the recombinant expression vector of  claim 1 .  
     
     
         17 . The host cell of  claim 16 , wherein the host cell is a mammalian cell.  
     
     
         18 . The host cell of  claim 17 , wherein the mammalian cell is a human cell.  
     
     
         19 . The host cell of  claim 18 , wherein the human cell is a human embryonic kidney cell.  
     
     
         20 . The host cell of  claim 19 , wherein the human embryonic kidney cell expresses an Epstein Barr virus nuclear antigen-1 (EBNA-1) protein.  
     
     
         21 . The host cell of  claim 20 , wherein the host cell is a 293E cell.  
     
     
         22 . A method of producing a recombinant protein, comprising culturing the host cell of  claim 16 , whereupon a recombinant protein is produced.  
     
     
         23 . A method of producing a recombinant protein, the method comprising contacting cells in a medium with a first vector and a second vector, wherein the first vector encodes a first polypeptide chain and the second vector encodes a second polypeptide chain, wherein the first polypeptide chain is different from the second polypeptide chain, and wherein the second vector is present in the medium in an amount which is about 1.5 to 2.5 times as much as the amount of the first vector, whereupon a recombinant heterodimeric or heteromultimeric protein is produced.  
     
     
         24 . The method of  claim 23 , wherein the second vector is present in the medium in an amount which is about 1.75 to 2.25 times as much as the amount of the first vector.  
     
     
         25 . The method of  claim 24 , wherein the second vector is present in the medium in an amount which is twice as much as the amount of the first vector.  
     
     
         26 . The method of  claim 23 , wherein the recombinant protein is a heterotetrameric protein.  
     
     
         27 . The method of  claim 26 , wherein the heterotetrameric protein is an immunoglobulin.  
     
     
         28 . The method of  claim 27 , wherein the first vector encodes a heavy chain of an immunoglobulin, or a functional fragment thereof, and the second vector encodes a light chain of an immunoglobulin, or a functional fragment thereof.  
     
     
         29 . The method of  claim 28 , wherein the heavy chain is a human γ 1  heavy chain, human γ 2  heavy chain, or a human γ 4  heavy chain.  
     
     
         30 . The method of  claim 28 , wherein the light chain is a human κ light chain or a λ light chain.  
     
     
         31 . The method of  claim 23 , wherein each of the first vector and the second vector is a recombinant transient expression vector.  
     
     
         32 . The method of  claim 23 , wherein each of the first vector and the second vector comprises a 3′ untranslated region (UTR) of a light chain gene and an oriP.  
     
     
         33 . The method of  claim 23 , wherein each of the first vector and the second vector comprises a viral promoter, a pUC19 origin of replication, a 5′ UTR intron, or a combination of any of the foregoing.  
     
     
         34 . The method of  claim 33 , wherein the viral promoter is a CMV promoter.  
     
     
         35 . The method of  claim 33 , wherein the 5′ UTR intron comprises a nucleotides 888-974 of SEQ ID NO: 1.  
     
     
         36 . The method of  claim 23 , wherein each of the first vector and second vector comprises an antibody signal sequence.  
     
     
         37 . The method of  claim 23 , wherein the cells are contacted with the first vector and second vector simultaneously.  
     
     
         38 . The method of  claim 23 , wherein the cells are contacted with the first vector and second vector in the presence of a cationic polymer.  
     
     
         39 . The method of  claim 38 , wherein the cationic polymer is polyethyleneimine (PEI).  
     
     
         40 . The method of  claim 39 , wherein the PEI is a linear PEI.  
     
     
         41 . The method of  claim 40 , wherein the linear PEI is present in an amount that is about 1.5 to 4.5 times the amount of the first vector and second vector.  
     
     
         42 . The method of  claim 41 , wherein the linear PEI is present in an amount that is about 2.5 to 3.5 times the amount of the first vector and second vector.  
     
     
         43 . The method of  claim 42 , wherein the linear PEI is present in an amount that is twice the amount of the first vector and second vector.  
     
     
         44 . The method of  claim 23 , wherein the cells are mammalian cells.  
     
     
         45 . The method of  claim 44 , wherein the mammalian cells are human cells.  
     
     
         46 . The method of  claim 45 , wherein the cells are human embryonic kidney cells.  
     
     
         47 . The method of  claim 46 , wherein the human embryonic kidney cells express Epstein-Barr virus nuclear antigen-1 protein (EBNA-1).  
     
     
         48 . The method of  claim 47 , wherein the cells are 293E cells.  
     
     
         49 . The method of  claim 23  further comprising isolating the cells from the medium and culturing the cells in a second medium in a membrane-enhanced culturing vessel, wherein the second medium is different from the medium.  
     
     
         50 . The method of  claim 49 , wherein the second medium is a serum-free cell culture medium.  
     
     
         51 . The method of  claim 50 , wherein the second medium is IS293™ medium.  
     
     
         52 . The method of  claim 49 , wherein the membrane-enhanced culturing vessel is an Integra CL1000.  
     
     
         53 . The method of the  claim 49  further comprising purifying the recombinant protein from the second medium.  
     
     
         54 . The method of  claim 53 , wherein the purifying comprises centrifuging the second medium through a column comprising Protein A.  
     
     
         55 . The method of  claim 53 , wherein the purifying occurs after 3 days of culturing the cells in the second medium.  
     
     
         56 . The method of  claim 53 , wherein the purifying occurs after 7 days of culturing the cells in the second medium.  
     
     
         57 . The method of  claim 55 , wherein at least 300 μg/ml recombinant protein is produced in the second medium.  
     
     
         58 . The method of  claim 57 , wherein at least 500 μg/ml recombinant protein is produced in the second medium.  
     
     
         59 . The method of  claim 58 , wherein at least 700 μg/ml recombinant protein is produced in the second medium.  
     
     
         60 . The method of  claim 49 , wherein the culturing comprises seeding cells in the second medium at a cell density between about 1.0×10 6  and 2.0×10 7  cells/ml.  
     
     
         61 . The method of  claim 60 , wherein the cell density is about 3.0×10 6  to about 1.0×10 7  cells/ml.  
     
     
         62 . A method of producing a recombinant protein comprising culturing cells, which have been contacted with a recombinant transient expression vector encoding a recombinant protein, in a medium in a membrane-enhanced culturing vessel or in a Fembach flask, whereupon the recombinant protein is produced.  
     
     
         63 . The method of  claim 62 , wherein the medium is a serum-free cell culture medium.  
     
     
         64 . The method of  claim 63 , wherein the serum-free medium is IS293™ medium.  
     
     
         65 . The method of  claim 62 , wherein the membrane-enhanced culturing vessel is an Integra CL1000.  
     
     
         66 . The method of  claim 62 , wherein the method further comprises purifying the recombinant protein from the medium.  
     
     
         67 . The method of  claim 66 , wherein the purifying comprises centrifuging the medium through a column comprising Protein A.  
     
     
         68 . The method of  claim 66 , wherein the purifying occurs after 3 days of culturing the cells in the medium.  
     
     
         69 . The method of  claim 66 , wherein the purifying occurs after 7 days of culturing the cells in the medium.  
     
     
         70 . The method of  claim 68 , wherein at least 300 μg/ml recombinant protein is produced in the medium.  
     
     
         71 . The method of  claim 70 , wherein at least 500 μg/ml recombinant protein is produced in the medium.  
     
     
         72 . The method of  claim 71 , wherein at least 700 μg/ml recombinant protein is produced in the medium.  
     
     
         73 . The method of  claim 62 , wherein the culturing comprises seeding cells in the medium at a cell density between about 1.0×10 6  and 2.0×10 7  cells/ml.  
     
     
         74 . The method of  claim 73 , wherein the cell density is 3.0×10 6  to about 1.0×10 7  cells/ml.  
     
     
         75 . The method of  claim 62 , wherein the recombinant transient expression vector comprises a 3′ untranslated region (UTR) of a light chain gene and an oriP.  
     
     
         76 . The method of  claim 62 , wherein the recombinant transient expression vector comprises a pUC19 origin of replication, a viral promoter, a 5′ UTR intron, or a combination of any of the foregoing.  
     
     
         77 . The method of  claim 76 , wherein the viral promoter is a CMV promoter.  
     
     
         78 . The method of  claim 76 , wherein the 5′ UTR intron comprises nucleotides 888-974 of SEQ ID NO: 1.  
     
     
         79 . The method of  claim 62 , wherein the cells have been contacted with a recombinant transient expression vector in the presence of a cationic polymer.  
     
     
         80 . The method of  claim 79 , wherein the cationic polymer is a polyethyleneimine (PEI).  
     
     
         81 . The method of  claim 80 , wherein the PEI is a linear PEI.  
     
     
         82 . The method of  claim 81 , wherein the PEI is present in an amount that is about 1.5 to 4.5 times the amount of the recombinant transient expression vector.  
     
     
         83 . The method of  claim 82 , wherein the PEI is present in an amount that is about 2.5 to 3.5 times the amount of the recombinant transient expression vector.  
     
     
         84 . The method of  claim 83 , wherein the PEI is present in an amount that is twice the amount of the recombinant transient expression vector.  
     
     
         85 . The method of  claim 62 , wherein the cells are mammalian cells.  
     
     
         86 . The method of  claim 85 , wherein the mammalian cells are human cells.  
     
     
         87 . The method of  claim 86 , wherein the human cells are human embryonic kidney cells.  
     
     
         88 . The method of  claim 87 , wherein the human embryonic kidney cells express Epstein-Barr virus nuclear antigen-1 protein (EBNA-1).  
     
     
         89 . The method of  claim 88 , wherein the cells are 293E cells.  
     
     
         90 . The method of  claim 62 , wherein the protein is an immunoglobulin chain or a functional fragment thereof.  
     
     
         91 . The method of  claim 90 , wherein the immunoglobulin chain is a heavy chain or a light chain.  
     
     
         92 . The method of  claim 91 , wherein the immunoglobulin chain is a heavy chain, and the heavy chain is a human γ 1  heavy chain, human γ 2  heavy chain, or a human γ 4  heavy chain.  
     
     
         93 . The method of  claim 91 , wherein the immunoglobulin chain is a light chain, and the light chain is a human κ light chain or a λ light chain.