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 of producing aflibercept, comprising culturing a population of cells expressing aflibercept in a cell culture medium comprising soy hydrolysate, wherein the soy hydrolysate comprises ≤0.067% (w/w) ornithine or putrescine, thereby producing aflibercept. 
     
     
         2 . The method of  claim 1 , wherein the population of cells is obtained by clonal expansion of a cell comprising a recombinant polynucleotide encoding aflibercept. 
     
     
         3 . The method of  claim 1 , wherein the cell culture medium is chemically defined. 
     
     
         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 cell culture medium comprises the twenty basic amino acids and cysteine. The method of  claim 1 , wherein the cell culture medium comprises lipids or free fatty acids. 
     
     
         8 . The method of  claim 1 , wherein the cells are a primary cells, BSC cells, HeLa cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, CHO cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells or chicken embryo cells. 
     
     
         9 . The method of  claim 1 , wherein the cells are CHO cells, 293 cells, or BHK cells. 
     
     
         10 . The method of  claim 9 , wherein the CHO cells are CHO-K1 cells. 
     
     
         11 . The method of  claim 1 , further comprising supplementing the cell culture medium with one or more point-of-use additions. 
     
     
         12 . The method of  claim 11 , wherein the one or more point-of-use additions comprise a growth factor, buffer, energy source, salt, amino acid, metal, chelator or combination thereof. 
     
     
         13 . The method of  claim 11 , wherein the one or more point-of-use additions comprises insulin. 
     
     
         14 . The method of  claim 13 , wherein the amount of insulin in the cell culture medium after addition is from about 0.1 μM to 10 μM. 
     
     
         15 . The method of  claim 12 , wherein the buffer comprises NaHCO 3  or HEPES. 
     
     
         16 . The method of  claim 12 , wherein the energy source comprises glucose. 
     
     
         17 . The method of  claim 16 , wherein the concentration of glucose in the cell culture medium after addition is about 1 mM to about 20 mM. 
     
     
         18 . The method of  claim 12 , wherein the metal comprises a metal salt. 
     
     
         19 . The method of  claim 18 , wherein the metal salt comprises copper sulfate, zinc sulfate, ferric chloride, nickel sulfate or a combination thereof 
     
     
         20 . The method of  claim 12 , wherein the chelator comprises Tetrasodium EDTA dehydrate or citrate. 
     
     
         21 . The method of  claim 1 , wherein the aflibercept comprises N-linked glycans (N-glycans). 
     
     
         22 . The method of  claim 21 , wherein culturing the cells in a cell culture medium comprising a soy hydrolysate comprising ≤0.067% (w/w) ornithine or putrescine increases the percentage of aflibercept A1 N-glycans. 
     
     
         23 . The method of  claim 21 , wherein ≥10% (w/w) of the aflibercept N-glycans are A1 N-glycans. 
     
     
         24 . The method of  claim 1 , wherein the aflibercept comprises about 5-15 moles sialic acid per 1 mole of aflibercept (mol/mol), about 8-12 mol/mol, 4 mol/mol, 5 mol/mol, 6 mol/mol, 7 mol/mol, 8 mol/mol, 9 mol/mol, 10 mol/mol, 11 mol/mol, 12 mol/mol, 13 mol/mol, 14 mol/mol, 15 mol/mol, 16 mol/mol, 17 mol/mol, 18 mol/mol, 19 mol/mol, or 20 mol/mol. 
     
     
         25 . The method of  claim 1 , further comprising purifying the aflibercept.