Binding molecules for human factor VIII and factor VIII-like proteins
View Patent ↗It is an object of the present invention to provide novel binding molecules for factor VIII and factor VIII-like proteins. Preferred binding molecules of the present invention exhibit not only distinct characteristics for binding of the target factor VIII polypeptides but also specific and desirable characteristics for release (elution) of the target polypeptides. Especially preferred binding molecules according to the invention are short polypeptide sequences, characterized by a stable loop structure.
1. A method for purifying human factor VIII, or a fragment thereof that retains the procoagulant properties of factor VIII, from a solution, the method comprising:
(a) immobilizing a polypeptide that binds human factor VIII or the fragment thereof, wherein the polypeptide is attached to a solid support;
(b) contacting a solution containing human factor VIII or the fragment thereof with the polypeptide under conditions such that human factor VIII or the fragment thereof binds to the polypeptide; and
(c) separating bound factor VIII or the fragment thereof from the solution, thereby purifying human factor VIII or the fragment thereof from the solution;
wherein the polypeptide comprises an amino acid sequence Ser-Trp-X 1 -X 2 -Pro-Cys (SEQ ID NO: 45), wherein X 1 is Val, Ile, Leu or Phe, and wherein X 2 can be any amino acid.
2. The method of claim 1 , wherein said polypeptide comprises the amino acid sequence X 1 -X 2 -Cys-Ser-Trp-X 3 -X 4 -Pro-Cys-X 5 -X 6 (SEQ ID NO: 55), wherein:
X 1 is Arg or Phe;
X 2 is Leu or Gly;
X 3 is Val, Ile, Leu or Phe;
X 4 is Ser or Phe;
X 5 is Ser or Pro; and
X 6 is Ala or Phe.
3. The method of claim 1 , wherein said amino acid sequence is selected from the group consisting of
Ser-Trp-Val-Ser-Pro-Cys (SEQ ID NO:46),
Ser-Trp-Leu-Phe-Pro-Cys (SEQ ID NO:47),
Ser-Trp-Ile-Ser-Pro-Cys (SEQ ID NO:48),
Ser-Trp-Leu-Arg-Pro-Cys (SEQ ID NO:49),
Ser-Trp-Phe-Arg-Pro-Cys (SEQ ID NO:50),
Ser-Trp-Leu-Phe-Pro-Cys (SEQ ID NO:51),
Ser-Trp-Phe-Asn-Pro-Cys (SEQ ID NO:52),
Ser-Trp-Leu-His-Pro-Cys (SEQ ID NO:53), and
Ser-Trp-Phe-Arg-Pro-Cys (SEQ ID NO:54).
4. The method of claim 3 , wherein said amino acid sequence is Ser-Trp-Leu-His-Pro-Cys (SEQ ID NO:53).
5. The method of claim 1 , wherein said polypeptide has a terminal amino acid residue that comprises a chemical modification.
6. The method of claim 3 , wherein the carboxyl terminus of said polypeptide comprises a chemical modification.
7. The method of claim 6 , wherein the chemical modification comprises a hydrazide functional group.
8. The method of claim 5 , wherein the amino terminus of said polypeptide comprises a chemical modification.
9. The method of claim 8 , wherein the chemical modification is an acetylation.
10. The method of claim 1 , wherein the polypeptide is attached to the solid support via a covalent or non-covalent linkage.
11. The method of claim 10 , wherein the solid support is selected from the group consisting of sepharose, agarose and cellulose.
12. The method of claim 1 , wherein the method further comprises eluting bound factor VIII from the polypeptide.