Method of increasing the hydrodynamic volume of polypeptides by attaching to gonadotrophin carboxy terminal peptides
First Claim
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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.
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Abstract
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.
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8 Claims
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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