US 9,808,534 B2
Method of increasing the hydrodynamic volume of polypeptides by attaching to gonadotrophin carboxy terminal peptides
Oren Hershkovitz, Rishon Lezion (IL); and Ahuva Bar-Ilan, Rehovot (IL)
Assigned to OPKO Biologics Ltd., Kiryat Gat (IL)
Filed by Opko Biologics Ltd., Kiryat Gat (IL)
Filed on Nov. 20, 2013, as Appl. No. 14/85,221.
Claims priority of provisional application 61/728,662, filed on Nov. 20, 2012.
Prior Publication US 2014/0316112 A1, Oct. 23, 2014
Int. Cl. A61K 38/24 (2006.01); C07K 14/59 (2006.01); A61K 47/48 (2006.01); A61K 38/17 (2006.01); A61K 38/18 (2006.01); A61K 38/26 (2006.01); A61K 38/27 (2006.01); A61K 38/48 (2006.01)
CPC A61K 47/48246 (2013.01) [A61K 38/1709 (2013.01); A61K 38/1816 (2013.01); A61K 38/26 (2013.01); A61K 38/27 (2013.01); A61K 38/4846 (2013.01); C07K 2319/00 (2013.01)] 8 Claims
 
1. A method of incrementally increasing the hydrodynamic size of a recombinant polypeptide of interest wherein said recombinant polypeptide of interest is selected from the group consisting of a coagulation factor IX (FIX) and a coagulation factor VIIa (FVIIa),
said method comprising a step of recombinantly fusing
(a) three chorionic gonadotrophin carboxy terminal peptide CTP units to the carboxy terminus of said coagulation factor IX (FIX) and no CTPs attached to the N-terminus; or
(b) three CTP units to the carboxy terminus of said coagulation factor VIIa (FVIIa) and no CTPs attached to the N-terminus;
followed by a step of expressing the recombinant CTP-modified polypeptides in a Chinese hamster ovary (CHO) host cell, wherein expression comprises glycosylating said CTP-units and the glycosylation comprises O-glycosylation;
wherein said glycosylated CTP units incrementally increase the hydrodynamic size of the FIX by an increment of about 48-53 kDA per each of said glycosylated CTP units, and,
wherein said glycoslyated CTP units incrementally increase the hydrodynamic size of the FVIIa by an increment of about 43-50 kDA per each of said glycosylated CTP units,
thereby incrementally increasing the hydrodynamic size of said recombinant polypeptide of interest.