In vivo active erythropoietin produced in insect cells
AssigneeProtein Sciences Corporation
InventorsGale E. Smith, John Knell, Andrei I. Voznesensky
Life of this patent
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Abstract
Disclosed and claimed is a human erythropoietin (EPO) expressed and produced in Spodoptera frugiperda Sf900+ cell line (ATCC: CRL 12579) transfected with a baculovirus construct containing the EPO gene. The EPO has an in vivo activity of 200,000 U/mg to 500,000 U/mg.
Claims14
A method for producing a substantially pure, recombinant, glycosylated erythropoietin (EPO) that has in vivo activity, including stimulating erythropoiesis, comprising:
- a) infecting Spodoptera frugiperda insect cells that grow in serum-free media with a baculovirus expression system comprising a recombinant baculovirus that comprises DNA coding for EPO such that the recombinant EPO is expressed;
- b) culturing the infected insect Spodoptera frugiperda cells in serum-free media;
- and
- c) purifying the recombinant EPO 10 95% or greater purity, whereby the substantially pure, recombinant, glycosylated EPO that has in vivo activity of between 200,000 U/mg protein and 500,000 U/ mg protein, including stimulating erythropoiesis, is produced.
A method for producing substantially pure, recombinant, glycosylated eryrhropoietin (EPO) that has in vivo activity, including stimulating erythropoiesis, comprising:
- a) infecting Spodoptera frugiperda SF900+ insect cells that grow in serum-free media with a baculovirus expression system comprising a recombinant baculovirus that comprises DNA coding for EPO such that the recombinant EPO is expressed;
- b) culturing the infected Spodoptera frugiperda SF900+ insect cells in serum-free media;
- and
- c) purifying the recombinant EPO to 95% or greater purity,
- whereby the substantially pure recombinant, glycosylated EPO that has in vivo activity, including stimulating erythropoiesis, is produced.
A method for producing substantially pure, recombinant, glycosylated eryrhropoietin (EPO) that has in vivo activity, including stimulating erythropoiesis, comprising:
- a) infecting Spodoptera frugiperda insect cells that grow in serum-free media with a baculovirus expression system comprising a recombinant baculovirus that comprises DNA coding for EPO such that the recombinant EPO is expressed;
- b) culturing the infected Spodoptera frugiperda insect cells in serum-free media;
- and
- c) purifying the recombinant EPO to 95% or greater purity, whereby the substantially pure recombinant, glycosylated EPO that has in vivo activity, including stimulating erythropoiesis, is produced, wherein the infecting of the insect cells with the recombinant baculovirus, the culturing of the insect cells, or both is in an apparatus for growing cells, wherein the apparatus comprises:
- (i) at least one bioreactor for cell culture;
- (ii) at least one vessel for culture media;
- whereby the bioreactor and vessel are in fluid communication, and
- wherein the bioreactor, vessel, or both are optionally stirred;
- (iii) a dialysis means for circulating culture media, cell culture, or both,
- whereby there is a first cell culture loop between the bioreactor and the dialysis means and a second media replenishment loop between the vessel and the dialysis means;
- (iv) in-operation dialysis between the culture media and the cell culture;
- (v) at least one means for delivery of oxygen comprising a hollow fiber filter oxygenator,
- whereby the oxygen is delivered directly to cells in a circulating loop of cells before cell entry into the hollow fiber filter.
Dependencies are inferred from the claim text.