Method of increasing the hydrodynamic volume of polypeptides by attaching to gonadotrophin carboxy terminal peptides
This invention is directed to the use of a chorionic gonadotrophin carboxy terminal peptide (CTP) or fragments thereof for modifying a polypeptide or a fragment thereof in order to increase the hydrodynamic volume of the polypeptide or fragment thereof.
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.
2. The method of claim 1 , wherein said O-glycosylation is a GaINAc attachment to serine (Ser) or threonine (Thr) in the polypeptide chain by an α-glycosidic linkage or a core 1 glycosylation, O-fucosylation, O-mannosylation, or O-glycosylation.
3. The method of claim 2 , wherein said O-glycosylation is followed by the addition of one to sixty galactose molecules or by the addition of one to 120 sialic acid molecules.
4. The method of claim 1 , wherein increasing said hydrodynamic size increases the bioavailability of said polypeptide.
5. The method of claim 1 , wherein the amino acid sequence of at least one of said CTP units is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.
6. The method of claim 1 , wherein at least one of said CTP units is truncated.
7. The method of claim 1 , wherein at least one of said CTP units is attached to said polypeptide via a linker.
8. The method of claim 7 , wherein said linker is a peptide bond.