Methods and compositions for purifying human factor VIII, factor VIII-like proteins or fragments thereof
View Patent ↗Provided herein are methods and compositions for purifying human factor VIII, human factor VIII-like peptide or fragments thereof. The methods comprise immobilizing a binding molecule for human factor VIII, human factor VIII-like protein or fragments thereof to a solid support, contacting the immobilized binding molecule with a solution containing the human factor VIII, human factor VIII-like protein or fragments thereof, and purifying the factor by separating the solution from the solid support.
1. A method for purifying human factor VIII, a fragment of human factor VIII, or a factor VIII-like peptide wherein said fragment or factor VIII-like peptide retains the procoagulant properties of human factor VIII, the method comprising:
(a) immobilizing a polypeptide on a solid support, wherein the polypeptide comprises the amino acid sequence
X 1 —X 2 -Cys-X 3 —X 4 -Trp-X 5 —X 6 -Pro-Cys-X 7 —X 8 (SEQ ID NO:2), wherein: X 1 is Arg or His; X 2 is Gly, Ala, Arg, Leu or Pro; X 3 is Gly or Phe; X 4 is Ala or Ser; X 5 is Phe or Leu; X 6 is Arg, Asn or His; X 7 is Ala, Asp, His, Leu, Phe, Pro or Tyr; and X 8 is Ala, Arg, Asn, Asp or His, such that the polypeptide binds factor VIII and such that a terminal amino acid residue comprises a chemical modification;
(b) contacting a solution containing said human factor VIII, fragment of human factor VIII, or factor VIII-like peptide with the immobilized polypeptide on a solid support, and
(c) purifying said human factor VIII, fragment of human factor VIII, or factor VIII-like peptide by separating the solution from said solid support.
2. The method of claim 1 , wherein the carboxyl terminus has a chemical modification.
3. The method of claim 2 , wherein the chemical modification comprises a hydrazide functional group.
4. The method of claim 1 , wherein the amino terminus has a chemical modification.
5. The method of claim 4 , wherein the chemical modification is an acetylation.
6. The method of claim 1 , wherein the amino acid sequence is selected from the group consisting of:
(SEQ ID NO: 60)
His-Pro-Cys-Gly-Ala-Trp-Leu-Arg-Pro-Cys-Tyr-Asn;
(SEQ ID NO: 61)
His-Pro-Cys-Gly-Ser-Trp-Leu-Arg-Pro-Cys-Leu-His;
(SEQ ID NO: 62)
His-Pro-Cys-Gly-Ser-Trp-Phe-Arg-Pro-Cys-Phe-His;
(SEQ ID NO: 64)
His-Pro-Cys-Gly-Ser-Trp-Phe-Asn-Pro-Cys-Ala-His;
(SEQ ID NO: 65)
His-Leu-Cys-Phe-Ala-Trp-Phe-Arg-Pro-Cys-Asp-Ala;
(SEQ ID NO: 66)
His-Pro-Cys-Gly-Ser-Trp-Leu-His-Pro-Cys-Ala-Ala;
(SEQ ID NO: 67)
His-Ala-Cys-Gly-Ser-Trp-Phe-Arg-Pro-Cys-His-Ala;
(SEQ ID NO: 68)
His-Leu-Cys-Gly-Ala-Trp-Phe-Arg-Pro-Cys-Asp-Ala;
and
(SEQ ID NO: 69)
His-Arg-Cys-Gly-Ser-Trp-Leu-His-Pro-Cys-Leu-Ala.
7. The method of claim 1 , wherein the amino acid sequence is His-Arg-Cys-Gly-Ser-Trp-Leu-His-Pro-Cys-Leu-Ala (SEQ ID NO: 69).
8. The method of claim 1 , wherein factor VIII is purified.
9. The method of claim 1 , wherein a factor VIII-like peptide is purified.
10. A separation media comprising:
(a) a chromatographic matrix material, and, immobilized thereon,
(b) a polypeptide comprising the amino acid sequence:
X 1 —X 2 -Cys-X 3 —X 4 -Trp-X 5 —X 6 -Pro-Cys-X 7 —X 8 (SEQ ID NO:2), wherein: X 1 is Arg or His; X 2 is Gly, Ala, Arg, Leu or Pro; X 3 is Gly or Phe; X 4 is Ala or Ser; X 5 is Phe or Leu; X 6 is Arg, Asn or His; X 7 is Ala, Asp, His, Leu, Phe, Pro or Tyr; and X 8 is Ala, Arg, Asn, Asp or His, such that the polypeptide binds factor VIII and such that a terminal amino acid residue comprises a chemical modification.
11. The separation media of claim 10 , wherein the carboxyl terminus has a chemical modification.
12. The separation media of claim 11 , wherein the chemical modification comprises a hydrazide functional group.
13. The separation media of claim 10 , wherein the amino terminus has a chemical modification.
14. The separation media of claim 11 , wherein the chemical modification is an acetylation.
15. The separation media of claim 10 , wherein the amino acid sequence is selected from the group consisting of:
(SEQ ID NO: 60)
His-Pro-Cys-Gly-Ala-Trp-Leu-Arg-Pro-Cys-Tyr-Asn;
(SEQ ID NO: 61)
His-Pro-Cys-Gly-Ser-Trp-Leu-Arg-Pro-Cys-Leu-His;
(SEQ ID NO: 62)
His-Pro-Cys-Gly-Ser-Trp-Phe-Arg-Pro-Cys-Phe-His;
(SEQ ID NO: 64)
His-Pro-Cys-Gly-Ser-Trp-Phe-Asn-Pro-Cys-Ala-His;
(SEQ ID NO: 65)
His-Leu-Cys-Phe-Ala-Trp-Phe-Arg-Pro-Cys-Asp-Ala;
(SEQ ID NO: 66)
His-Pro-Cys-Gly-Ser-Trp-Leu-His-Pro-Cys-Ala-Ala;
(SEQ ID NO: 67)
His-Ala-Cys-Gly-Ser-Trp-Phe-Arg-Pro-Cys-His-Ala;
(SEQ ID NO: 68)
His-Leu-Cys-Gly-Ala-Trp-Phe-Arg-Pro-Cys-Asp-Ala;
and
(SEQ ID NO: 69)
His-Arg-Cys-Gly-Ser-Trp-Leu-His-Pro-Cys-Leu-Ala.
16. The separation media of claim 10 , wherein the amino acid sequence is His-Arg-Cys-Gly-Ser-Trp-Leu-His-Pro-Cys-Leu-Ala (SEQ ID NO: 69).
17. A method for separating human factor VIII, a fragment of human factor VIII, or a factor VIII-like peptide, wherein said fragment or factor VIII-like peptide retains the procoagulant properties of human factor VIII, from a solution, the method comprising:
(a) contacting said solution with separation media as defined in claim 8 under binding conditions, and
(b) removing unbound material, and
(c) eluting bound factor human factor VIII, fragment of human factor VIII, or factor VIII-like peptide from said separation media.
18. The method of claim 17 , wherein the amino acid sequence is His-Arg-Cys-Gly-Ser-Trp-Leu-His-Pro-Cys-Leu-Ala (SEQ ID NO: 69).
19. The method of claim 17 , wherein factor VIII is purified.
20. The method of claim 17 , wherein a factor VIII-like peptide is purified.