Manufacturing of glycoproteins

Abstract

A method of producing recombinant alkaline phosphatase comprising control of production parameters, particularly harvest clarified culture fluid (HCCF) and filtration pool (UFDF), to provide a defined total sialic acid content.

1 . A method of producing recombinant alkaline phosphatase comprising:
 (A) (a) inoculating Chinese Hamster Ovary (CHO) cells expressing recombinant alkaline phosphatase in culture medium;
 (b) culturing the CHO cells in the culture medium; 
 (c) isolating the recombinant alkaline phosphatase from the cell medium by at least one purification step to form harvest clarified culture fluid (HCCF) with a total sialic acid content (TSAC) of from about 2.1 mol/mol to about 4.3 mol/mol; 
 (d) performing at least one additional protein purification step to form a filtration pool (UFDF), wherein the UFDF is held at a temperature of from about 13° C. to about 27° C. for from about 1 hour to about 60 hours, and at a protein concentration of from about 1.7 g/L to about 5.3 g/L; and 
 (e) subjecting the UFDF to at least one chromatography step to obtain partially purified recombinant alkaline phosphatase, wherein the recombinant alkaline phosphatase has a TSAC of about 0.7 mol/mol to about 3.5 mol/mol; or 
   (B) (a) inoculating CHO cells expressing recombinant alkaline phosphatase in culture medium;
 (b) culturing the CHO cells in the culture medium; 
 (c) adding a nutrient supplement(s) to the cell medium; 
 (d) isolating the recombinant alkaline phosphatase from the cell medium by at least one purification step to form a filtration pool (UFDF); 
 and 
   (C) recovering the recombinant alkaline phosphatase from the UFDF, wherein the recombinant alkaline phosphatase in the UFDF has a total sialic acid content (TSAC) of from about 2.1 mol/mol to about 4.3 mol/mol.   
     
     
         2 . The method of  claim 1 , wherein sialidase is selectively removed from the cell culture, the HCCF, and/or the UFDF and/or an exogenous sialyltransferase is added to the cell culture, the HCCF, and/or the UFDF. 
     
     
         3 . The method of  claim 1 , wherein:
 (i) the culturing of the CHO cells in the culture medium is at a temperature of from about 36° C. to about 38° C.;   (ii) the nutrient supplement(s) is added to the cell culture at least one day after inoculation of CHO cells into the culture medium;   (iii) the nutrient supplement(s) is added at more than 2 different times;   (iv) the culture medium is selected from the group consisting of EX-CELL® 302 Serum-Free Medium; CD DG44 Medium; BD Select™ Medium; SFM4CHO Medium; and combinations thereof;   (v) the culturing of the CHO cells is in a 0.25 L to a 25,000 L bioreactor;   (vi) the temperature of the cell culture is decreased from about 80 hours to about 120 hours after the inoculation;   (vii) step (d) of (A) or (B) occurs about 10 to about 14 days after inoculation;   (viii) the TSAC of the HCCF is from about 2.2 mol/mol to about 3.6 mol/mol; and/or   (ix) the at least one additional purification step comprises at least one of harvest clarification, filtration, ultrafiltration, diafiltration, viral inactivation, affinity capture, and combinations thereof.   
     
     
         4 . The method of  claim 3 , wherein:
 (a) the culturing of the CHO cells in the culture medium is at a temperature of about 37° C.;   (b) the culturing of the CHO cells is in a 100 L to 25,000 L bioreactor;   (c) the TSAC of the HCCF is from about 2.2 mol/mol to about 3.4 mol/mol; and/or   (d) the at least one additional purification step comprises ultrafiltration and/or diafiltration.   
     
     
         5 . The method of  claim 4 , wherein the culturing of the CHO cells is in a 2000 L to 20,000 L bioreactor. 
     
     
         6 . The method of  claim 1 , wherein:
 (i) the UFDF is held at a temperature of about 14° C. to about 26° C.;   (ii) the UFDF is held for about 10 hours to about 50 hours;   (iii) the UFDF has a protein concentration from about 2.0 g/L to about 4.3 g/L;   (iv) the UFDF has an alkaline phosphatase concentration from about 3.0 g/L to about 4.5 g/L; and/or   (v) the at least one additional chromatography step and/or the at least one additional protein purification step is performed to obtain recombinant alkaline phosphatase with a TSAC of about 0.9 mol/mol to about 3.9 mol/mol.   
     
     
         7 . The method of  claim 6 , wherein:
 (a) the UFDF is held at a temperature of about 15° C. to about 26° C.;   (b) the UFDF is held for about 12 hours to about 48 hours;   (c) the UFDF has a protein concentration of about 3.1 g/L;   (d) the UFDF has an alkaline phosphatase concentration from about 3.3 g/L to about 4.1 g/L; and/or   (e) the at least one additional chromatography step and/or the at least one additional protein purification step is performed to obtain recombinant alkaline phosphatase with a TSAC of about 1.1 mol/mol to about 3.2 mol/mol.   
     
     
         8 . The method of  claim 7 , wherein:
 (i) the UFDF is held at a temperature of about 15° C. to about 25° C.;   (ii) the UFDF is held for about 14 hours to about 42 hours;   (iii) the at least one additional chromatography step and/or the at least one additional protein purification step is performed to obtain recombinant alkaline phosphatase with a TSAC of about 1.4 mol/mol to about 2.6 mol/mol.   
     
     
         9 . The method of  claim 8 , wherein:
 (a) the UFDF is held at a temperature of about 19° C. to about 25° C.;   (b) the UFDF is held for about 17 hours to about 34 hours;   (c) the at least one additional chromatography step and/or the at least one additional protein purification step is performed to obtain recombinant alkaline phosphatase with a TSAC of about 1.2 mol/mol to about 3.0 mol/mol.   
     
     
         10 . The method of  claim 9 , wherein:
 (i) the UFDF is held for about 19 hours to about 33 hours; and/or   (ii) the TSAC of the recombinant alkaline phosphatase in the UFDF is from about 2.2 mol/mol to about 3.6 mol/mol.   
     
     
         11 . The method of  claim 10 , wherein:
 (a) the UFDF is held for about 25 hours to about 38 hours; and/or   (b) the TSAC of the recombinant alkaline phosphatase in the UFDF is from about 2.2 mol/mol to about 3.4 mol/mol.   
     
     
         12 . The method of  claim 11 , wherein the UFDF is held for about 29 hours to about 35 hours. 
     
     
         13 . The method  claim 1 , wherein:
 (i) the at least one chromatography step is protein affinity chromatography;   (ii) the at least one chromatography step is Protein A chromatography;   (iii) the at least one chromatography step comprises column chromatography;   (iv) the at least one chromatography step comprises hydrophobic interaction chromatography;   (v) the at least one additional protein purification step of (A) comprises an additional diafiltration;   (vi) the at least one chromatography step and/or the at least one additional protein purification step comprises hydrophobic interaction chromatography and/or at least one additional diafiltration step;   (vii) step (d) of (A) further comprises a viral inactivation step; and/or   (viii) step (e) of (A) or (B) further comprises at least one additional chromatography and/or purification step.   
     
     
         14 . The method of  claim 1 , wherein the recombinant alkaline phosphatase comprises the structure of W-sALP—X-Fc-Y-Dn-Z, wherein W is absent or is an amino acid sequence of at least one amino acid;
 X is absent or is an amino acid sequence of at least one amino acid; 
 Y is absent or is an amino acid sequence of at least one amino acid; 
 Z is absent or is an amino acid sequence of at least one amino acid; 
 F c is a fragment crystallizable region; 
 Dn is a poly-aspartate, poly-glutamate, or combination thereof, wherein n=10 or 16; and 
 sALP is a soluble alkaline phosphatase. 
 
     
     
         15 . The method of  claim 14 , wherein the recombinant alkaline phosphatase comprises asfotase alfa (SEQ ID NO: 1). 
     
     
         16 . The method of  claim 13 , wherein the recombinant alkaline phosphatase obtained from the protein affinity chromatography is stored at from about 2° C. to about 8° C. 
     
     
         17 . The method of  claim 1 , wherein (A) further comprises:
 (i) adding a nutrient supplement to the cell medium culture of (b) after inoculation; and/or   (ii) measuring recombinant alkaline phosphatase activity.   
     
     
         18 . A recombinant alkaline phosphatase produced in a mammalian cell culture, wherein the recombinant alkaline phosphatase in a harvest clarified culture fluid (HCCF) produced from the cell culture has a total sialic acid content (TSAC) always greater than or equal to about 1.2 mol/mol, and wherein the mammalian cell culture is about 100 L to about 25,000 L. 
     
     
         19 . A filtration pool (UFDF) comprising recombinant alkaline phosphatase, wherein the UFDF is produced from about 100 L to about 25,000 L cell culture, and wherein the UFDF is held at from about 19° C. to about 25° C., for from about 14 to about 42 hours, at a protein concentration of from about 2.0 to about 4.3 g/L. 
     
     
         20 . A composition comprising a recombinant alkaline phosphatase produced by the method of  claim 1 , wherein, optionally, the composition further comprises at least one pharmaceutically acceptable carrier, diluent, or excipient or a combination thereof. 
     
     
         21 . A method comprising administering the composition of  claim 20  to a subject to increase cleavage of inorganic pyrophosphate (PPi) or to treat a condition associated with an alkaline phosphatase deficiency in the subject. 
     
     
         22 . A method of controlling glycosidase activity or total sialic acid content (TSAC) in a sialic acid-containing recombinant protein, comprising culturing the recombinant protein in mammalian cell culture and conducting at least one purification step to provide a UFDF, wherein the temperature, protein concentration and/or hold time is controlled in the UFDF.