Methods of manufacturing therapeutic proteins

Abstract

Disclosed herein are methods of manufacturing therapeutic proteins.

1 .- 42 . (canceled) 
     
     
         43 . A method of manufacturing a therapeutic protein, comprising: transfecting a cholesterol-auxotrophic cell with (i) a nucleic acid encoding a protein that restores cholesterol biosynthesis in the cell; (ii) an aminoglycosidic antibiotic resistance gene; and (iii) a nucleic acid encoding the therapeutic protein. 
     
     
         44 . The method of  claim 43 , wherein the protein that restores cholesterol biosynthesis in the cell is a 3-ketosteroid reductase (3-KSR). 
     
     
         45 . The method of  claim 43 , wherein the aminoglycosidic antibiotic resistance gene is selected from the group consisting of: a neomycin resistance gene, a blasticidin resistance gene, a hygromycin resistance gene, a puromycin resistance gene, a zeocin resistance gene, and a mycophenolic acid resistance gene. 
     
     
         46 . The method of  claim 43 , comprising transfecting the cell with a first vector and a second vector, wherein the first vector comprises the nucleic acid encoding the protein that restores cholesterol biosynthesis in the cell and the nucleic acid encoding the therapeutic protein; and the second vector comprises the aminoglycosidic antibiotic resistance gene. 
     
     
         47 . The method of  claim 43 , comprising transfecting the cell with a first vector and a second vector, wherein the first vector comprises the nucleic acid encoding the protein that restores cholesterol biosynthesis in the cell; and the second vector comprises the antibiotic resistance gene and the nucleic acid encoding the therapeutic protein. 
     
     
         48 . The method of  claim 43 , comprising transfecting the cell with a first vector and a second vector, wherein the first vector comprises the nucleic acid encoding the protein that restores cholesterol biosynthesis in the cell and the nucleic acid encoding the therapeutic protein; and the second vector comprises the antibiotic resistance gene and the nucleic acid encoding the therapeutic protein 
     
     
         49 . The method of  claim 46 , wherein the method comprises transfecting the cell with the first vector and the second vector, and wherein the transfecting the cell with the first vector is carried out prior to the transfecting with the second vector, or the transfecting the cell with the first vector is carried out after the transfecting with the second vector. 
     
     
         50 . The method of  claim 47 , wherein the method comprises transfecting the cell with the first vector and the second vector, and wherein the transfecting the cell with the first vector is carried out prior to the transfecting with the second vector, or the transfecting the cell with the first vector is carried out after the transfecting with the second vector. 
     
     
         51 . The method of  claim 48 , wherein the method comprises transfecting the cell with the first vector and the second vector, and wherein the transfecting the cell with the first vector is carried out prior to the transfecting with the second vector, or the transfecting the cell with the first vector is carried out after the transfecting with the second vector. 
     
     
         52 . The method of  claim 46 , wherein the method comprises transfecting the cell with the first vector and the second vector, and wherein the transfecting the cell with the first vector is carried out simultaneously with the transfecting the cell with the second vector. 
     
     
         53 . The method of  claim 47 , wherein the method comprises transfecting the cell with the first vector and the second vector, and wherein the transfecting the cell with the first vector is carried out simultaneously with the transfecting the cell with the second vector. 
     
     
         54 . The method of  claim 48 , wherein the method comprises transfecting the cell with the first vector and the second vector, and wherein the transfecting the cell with the first vector is carried out simultaneously with the transfecting the cell with the second vector. 
     
     
         55 . The method of  claim 43 , further comprising culturing the cell in the presence of a 3-KSR inhibitor after transfecting the cell with (i). 
     
     
         56 . The method of  claim 43 , further comprising culturing the cell in the absence of at exogenously introduced cholesterol after transfecting with (i). 
     
     
         57 . The method of  claim 43 , further comprising culturing the cell n the presence of an aminoglycosidic antibiotic after transfecting the cell with (ii). 
     
     
         58 . The method of  claim 52 , comprising culturing the cell in the absence of an exogenously introduced cholesterol and in the presence of an aminoglycosidic antibiotic after simultaneously transfecting the cell with the first vector and the second vector. 
     
     
         59 . The method of  claim 53 , comprising culturing the cell in the absence of an exogenously introduced cholesterol and in the presence of an aminoglycosidic antibiotic after simultaneously transfecting the cell with the first vector and the second vector. 
     
     
         60 . The method of  claim 54 , comprising culturing the cell in the absence of an exogenously introduced cholesterol and in the presence of an aminoglycosidic antibiotic after simultaneously transfecting the cell with the first vector and the second vector. 
     
     
         61 . The method of  claim 43 , wherein the nucleic acid encoding the therapeutic protein comprises a nucleic acid encoding a heavy chain and a light chain of an antibody. 
     
     
         62 . The method of  claim 61 , wherein the antibody is selected from the group consisting of: adalimumab, infiliximab, palivizumab, cetuximab, natalizumab, eculizumab, ustekinumab, golimumab, ofatumab, canakinumab, belimumab, alirocumab, mepolizumab, necitumumab, nivolumab, dinutuximab, secukinumab, evolocumab, blinatumomab, pembrolizumab, ramucirumab, vedolizumab, siltuximab, obinutuzumab, trastuzumab, raxibacumab, pertuzumab, brentuximab, ipilimumab, denosumab, tocilizumab, ofatumumab, canakinumab, certolizumab, catumaxomab, ranibizumab, panitumumab, bevacizumab, cetuximab, efalizumab, omalizumab, tositumomab, ibritumomab, alemtuzumab, gemtuzumab, basiliximab, daclizumab, rituximab, abciximab, alefacept, entanercept, abatacept, belatacept, aflibercept, ziv-aflibercept, rilonacept, romiplostim, apocept, trebananib, blisibimod, and dulaglutide. 
     
     
         63 . The method of  claim 43 , wherein the therapeutic protein is selected from the group consisting of: an antibody, a fusion protein, an anticoagulant, a blood factor, a bone morphogenic protein, an engineered protein scaffold, an enzyme, a growth factor, a hormone, a hormone releasing factor, an interleukin, or a thrombolytic protein and the method comprises transfecting the cell with a nucleic acid encoding an antibody, a fusion protein, an anticoagulant, a blood factor, a bone morphogenic protein, an engineered protein scaffold, an enzyme, a growth factor, a hormone, a hormone releasing factor, an interferon, an interleukin, and a thrombolytic protein. 
     
     
         64 . The method of  claim 43 , wherein the cell is an NS0 cell. 
     
     
         65 . A transformed mammalian host cell, transfected with an aminoglycosidic antibiotic resistance gene, and a nucleic acid encoding a therapeutic protein, wherein the transformed mammalian host cell is derived from a parental mammalian host cell that is cholesterol-auxotrophic. 
     
     
         66 . A therapeutic protein produced by a method comprising culturing the transformed mammalian host cell of  claim 65 , in the presence of an aminoglycosidic antibiotic.