Cell culture process for making a glycoprotein

Abstract

The instant application provides a method for screening batches of soy hydrolysate for a desired amount of a component thereof, such as ornithine or putrescine, and selecting only those batches of soy hydrolysate that have a desired amount of such component. The present disclosure also sets forth methods for culturing cells in media supplemented with selected batches of soy to produce more consistent, high quality lots of a protein of interest. Further, the present disclosure provides a plurality of protein preparations that have each been produced by culturing cells in media supplemented with separate batches of soy hydrolysate containing a desired amount of ornithine or putrescine, whereby each batch of protein produced exhibits improved quality of the protein of interest or amount of quality protein produced.

What is claimed is: 
     
         1 . A method comprising:
 culturing a population of cells expressing a recombinant heterologous glycoprotein in cell culture media comprising soy hydrolysate to produce the recombinant heterologous glycoprotein, and wherein the soy hydrolysate comprises ≤0.067% (w/w) ornithine or putrescine.   
     
     
         2 . The method of  claim 1 , wherein the population of cells is obtained by clonal expansion of a cell expressing a recombinant heterologous glycoprotein. 
     
     
         3 . The method of  claim 1 , wherein the soy hydrolysate comprises 0.003%-0.027% (w/w) ornithine or putrescine. 
     
     
         4 . The method of  claim 1 , wherein the culture media comprises ≤5 mg/L ornithine or putrescine. 
     
     
         5 . The method of  claim 1 , wherein the culture media comprises 0.6-3 mg/L ornithine or putrescine. 
     
     
         6 . The method of  claim 1 , wherein the glycoprotein is a trap molecule. 
     
     
         7 . The method of  claim 6 , wherein the trap molecule is selected from the group consisting of etanercept, rilonacept, and aflibercept. 
     
     
         8 . The method of  claim 6 , wherein the glycoprotein comprises an A1 N-glycan and at least one other N-glycan species, wherein the relative amount of A1 N-glycan is ≥10% (w/w) of the total amount of all N-glycan species of the glycoprotein. 
     
     
         9 . A method comprising:
 a. culturing a cell expressing a glycosylated protein in a cell culture media to produce the glycoprotein;   b. purifying the glycosylated protein;   c. subjecting the purified glycosylated protein to oligosaccharide fingerprint analysis;   d. determining the relative amount of an A1 N-glycan compared to total amount of N-glycan species of the glycoprotein; and   e. selecting a soy hydrolysate that provides for at least 10% (w/w) A1 N-glycan compared to total amount of N-glycan species of the glycoprotein.   
     
     
         10 . The method of  claim 9 , wherein the selected soy hydrolysate comprises ≤0.067% (w/w) ornithine or putrescine. 
     
     
         11 . The method of  claim 9 , wherein the selected soy hydrolysate comprises 0.003%-0.027% (w/w) ornithine or putrescine. 
     
     
         12 . The method of  claim 9 , wherein the culture media comprises 0.6-3 mg/L ornithine or putrescine. 
     
     
         13 . The method of  claim 9 , wherein the glycoprotein is a trap molecule. 
     
     
         14 . The method of  claim 13 , wherein the trap molecule is selected from the group consisting of etanercept, rilonacept, and aflibercept. 
     
     
         15 . The method of  claim 9 , wherein the glycoprotein comprises 8-12 moles of sialic acid per mole of glycoprotein, or 35-65 moles of sialic acid per mole of glycoprotein. 
     
     
         16 . A method of selecting a soy hydrolysate for use in manufacturing a glycoprotein, the method comprising:
 a. measuring the amount of ornithine or putrescine in a soy hydrolysate;   b. selecting a soy hydrolysate with ≤0.067% (w/w) ornithine or putrescine; and   c. combining the selected soy hydrolysate with an additional ingredient to form a cell culture media with ≤5 mg/L ornithine or putrescine.   
     
     
         17 . The method of  claim 16 , wherein the selected soy hydrolysate comprises 0.003%-0.027% (w/w) ornithine or putrescine. 
     
     
         18 . The method of  claim 16 , wherein the culture media comprises 0.6-3 mg/L ornithine or putrescine. 
     
     
         19 . The method of  claim 16 , wherein the glycoprotein is a trap molecule. 
     
     
         20 . The method of  claim 19 , wherein the trap molecule is selected from the group consisting of etanercept, rilonacept, and aflibercept. 
     
     
         21 . The method of  claim 16 , wherein the glycoprotein comprises an A1 N-glycan and at least one other N-glycan species, wherein the relative amount of A1 N-glycan is ≥10% (w/w) of the total amount of all N-glycan species of the glycoprotein. 
     
     
         22 . A glycoprotein comprising an A1 N-glycan and at least one other N-glycan species, wherein the relative amount of the A1 N-glycan is at least 10% (w/w) of the total amount of N-glycans of said glycoprotein. 
     
     
         23 . The glycoprotein of  claim 22 , wherein said glycoprotein is a trap molecule. 
     
     
         24 . The glycoprotein of  claim 23 , wherein said trap molecule is selected from the group consisting of etanercept, rilonacept, and aflibercept. 
     
     
         25 . The glycoprotein of  claim 22 , wherein said glycoprotein further comprises an A2 N-glycan, an A2F N-glycan, an A1F N-glycan, an NGA2F N-glycan, an NA2G1F N-glycan, an NA2 N-glycan, and an NA2F N-glycan. 
     
     
         26 . The glycoprotein of  claim 22 , wherein the relative amount of the A1 N-glycan is determined by comparing the area under the peak of the A1 N-glycan to the total areas under the peak for all N-glycans of an oligosaccharide fingerprint obtained by capillary electrophoresis. 
     
     
         27 . The glycoprotein  claim 22 , wherein the relative amount of the A1 N-glycan is 10%-17% (w/w). 
     
     
         28 . The glycoprotein of  claim 24 , wherein the glycoprotein is rilonacept with 35-65 moles of sialic acid per mole of glycoprotein. 
     
     
         29 . The glycoprotein of  claim 28 , wherein the rilonacept comprises an A1 N-glycan at any one or more of residues N37, N98, N418, and N511 of SEQ ID NO: 1. 
     
     
         30 . The glycoprotein of  claim 24 , wherein the glycoprotein is aflibercept with 8-12 moles of sialic acid per mole of glycoprotein. 
     
     
         31 . The glycoprotein of  claim 30 , wherein the aflibercept comprises an A1 N-glycan at any one or more of residues N123 and N196 of SEQ ID NO: 2. 
     
     
         32 . A method comprising:
 a. enzymatically digesting soy extract in a residue-free reaction vessel to manufacture a soy hydrolysate;   b. measuring the amount of ornithine or putrescine in the soy hydrolysate; and   c. selecting soy hydrolysate with ≤0.067% (w/w) ornithine or putrescine for use in a cell culture media.