Methods of producing long acting CTP-modified growth hormone polypeptides

A purified pharmaceutical composition comprising a highly glycosylated polypeptide and a pharmaceutically acceptable carrier, the polypeptide comprising an amino terminal chorionic gonadotropin carboxy terminal peptide (CTP), a human growth hormone (hGH) polypeptide, and two carboxy terminal chorionic gonadotropin CTPs arranged in tandem (CTP-modified hGH polypeptide), wherein the CTP-modified hGH polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

• 7, wherein at least 60% of the CTP-modified hGH polypeptides present in the purified pharmaceutical composition are highly glycosylated and comprise 13 to 18 O-linked glycans per molecule, and wherein the purified pharmaceutical composition is manufactured by a method comprising the steps of:
• (a) stably transfecting a predetermined number of cells with an expression vector comprising a coding portion encoding the CTP-modified hGH polypeptide, wherein the transfected cells express and secrete the CTP-modified hGH polypeptide;
• (b) obtaining cell clones that overexpress the CTP-modified hGH polypeptide;
• (c) expanding the clones in solution to a predetermined scale by culturing at a dissolved oxygen (DO) content of 20-30%, wherein the pH of the solution is shifted from 7.2 to 6.9 during the expansion;
• (d) harvesting the solution containing the clones;
• (e) filtering the solution containing the clones to obtain a clarified harvest solution;
• and
• (f) purifying the clarified harvest solution to obtain a desired concentration of the CTP-modified hGH polypeptide.