US 12,459,982 B2
Methods of producing long acting CTP-modified growth hormone polypeptides
Oren Hershkovitz, Shikmim (IL); and Laura Moschcovich, Givat Shmuel (IL)
Assigned to OPKO BIOLOGISTICS LTD., Kiryat Gat (IL)
Appl. No. 15/533,910
Filed by OPKO BIOLOGICS LTD., Kiryat Gat (IL)
PCT Filed Dec. 10, 2015, PCT No. PCT/IL2015/051196
§ 371(c)(1), (2) Date Jun. 7, 2017,
PCT Pub. No. WO2016/092549, PCT Pub. Date Jun. 16, 2016.
Claims priority of provisional application 62/090,104, filed on Dec. 10, 2014.
Claims priority of provisional application 62/090,116, filed on Dec. 10, 2014.
Claims priority of provisional application 62/090,124, filed on Dec. 10, 2014.
Prior Publication US 2018/0111974 A1, Apr. 26, 2018
Int. Cl. A61K 38/27 (2006.01); B01D 15/00 (2006.01); C07K 14/505 (2006.01); C07K 14/555 (2006.01); C07K 14/575 (2006.01); C07K 14/59 (2006.01); C07K 14/61 (2006.01); C07K 19/00 (2006.01); C12N 5/00 (2006.01); C12N 9/64 (2006.01); C12N 15/09 (2006.01); C12N 15/85 (2006.01); G06Q 10/0633 (2023.01); G06Q 10/0637 (2023.01); G06Q 10/0639 (2023.01); G06Q 30/0201 (2023.01); A61K 38/00 (2006.01); C12M 1/00 (2006.01)
CPC C07K 14/59 (2013.01) [C07K 14/505 (2013.01); C07K 14/555 (2013.01); C07K 14/575 (2013.01); C07K 14/61 (2013.01); C12N 9/6437 (2013.01); C12N 9/644 (2013.01); C12N 15/85 (2013.01); G06Q 10/0633 (2013.01); G06Q 10/0637 (2013.01); G06Q 10/06393 (2013.01); G06Q 30/0201 (2013.01); A61K 38/00 (2013.01); C07K 2319/00 (2013.01); C07K 2319/31 (2013.01); C12M 21/00 (2013.01); C12Y 304/21021 (2013.01); C12Y 304/21022 (2013.01)] 31 Claims
 
1. 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.