Method for purifying albumin fusion proteins

Abstract

This invention provides a method for isolating albumin fusion proteins to reduce the oxidation level of sensitive amino acid residues. [Solution] A method for isolating an albumin fusion protein having less than 10% oxidized tryptophan residues relative to the total number of amino acid residues in the albumin fusion protein, comprising the steps of subjecting a composition containing the albumin fusion protein to the following purification process: (a) affinity matrix chromatography process, (b) anion exchange chromatography process, and (c) hydrophobic interaction matrix chromatography process, wherein an elution buffer containing octanoic acid is applied to the affinity matrix to separate the albumin fusion protein with oxidized tryptophan from the albumin fusion protein with unoxidized tryptophan.

Claims

1. A composition comprising a Tn3 scaffold containing a monomer subunit specific to CD40L, wherein the monomer subunit comprises SEQ ID NO: 146 A composition wherein multiple Tn3 scaffolds contain an unoxidized tryptophan residue at W46 of SEQ ID NO: 145, the Tn3 scaffolds are isolated using a hydrophobic interaction matrix, the oxidized and unoxidized scaffolds are eluted from the hydrophobic interaction matrix at different times, the monomeric subunit specific to CD40L binds to a heterologous moiety, the heterologous moiety is human serum albumin (HSA), the HSA is a mutant HSA compared to wild-type HSA, and the mutant HSA contains SEQ ID NO:

133.

2. The composition according to claim 1, wherein the Tn3 scaffold comprises the polypeptide of Sequence ID No.

145.

3. A composition comprising a Tn3 scaffold comprising a monomer subunit specific to CD40L, wherein the monomer subunit comprises seven β-strands named A, B, C, D, E, F, and G, and six loop regions named AB, BC, CD, DE, EF, and FG, the AB loop comprising SEQ ID NO: 4, the BC loop comprising SEQ ID NO: 86, the CD loop comprising SEQ ID NO: 6, the DE loop comprising SEQ ID NO: 96, the EF loop comprising SEQ ID NO: 8, and the FG loop comprising SEQ ID NO: 139, wherein a plurality of the Tn3 scaffolds comprises an unoxidized tryptophan residue in the DE loop sequence of the monomer subunit specific to CD40L, and the Tn3 scaffolds are isolated using a hydrophobic interaction matrix, wherein the oxidized scaffolds and the unoxidized scaffolds are eluted from the hydrophobic interaction matrix at different times.

4. The composition according to claim 3, wherein the unoxidized tryptophan residue is W46 of SEQ ID NO:

145.

5. The composition according to claim 3 or 4, wherein the Tn3 scaffold comprises a monomer subunit specific to the second CD40L.

6. The composition according to claim 5, wherein the unoxidized tryptophan residue is W46 and / or W151 of SEQ ID NO:

145.

7. The composition according to claim 3 or 4, wherein the monomer subunit specific to CD40L comprises the polypeptide sequence of SEQ ID NO:

167.

8. The composition according to claim 5, wherein the monomer subunit specific to CD40L and the second monomer subunit specific to CD40L are linked in tandem by a polypeptide linker.

9. The composition according to claim 8, wherein the polypeptide linker comprises a sequence selected from the group consisting of SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 142, SEQ ID NO: 143 and combinations thereof.

10. The composition according to claim 5, wherein one monomer subunit specific to CD40L is bound to an HSA, the HSA is a mutant HSA compared to a wild-type HSA, and the mutant HSA contains Sequence ID No.

133.

11. The composition according to claim 5, wherein the monomer subunit specific to CD40L and the second monomer subunit specific to CD40L have the same arrangement.

12. The composition according to claim 5, wherein the monomer subunit specific to CD40L and the second monomer subunit specific to CD40L have different arrangements.

13. The composition according to claim 3 or 4, wherein β-strand A is sequence number 11, β-strand B is sequence number 12, β-strand C is sequence number 14, β-strand D is sequence number 15, β-strand E is sequence number 16, β-strand F is sequence number 17, and β-strand G is sequence number 18.

14. The composition according to claim 3 or 4, wherein the Tn3 scaffold comprises the polypeptide of Sequence ID No.

145.

15. Use of a Tn3 scaffold in the manufacture of a pharmaceutical for the treatment of a CD40-mediated immune response, wherein the treatment comprises the administration of a composition comprising a Tn3 scaffold comprising a monomer subunit specific to CD40L, the monomer subunit comprising seven β-strands named A, B, C, D, E, F, and G, and six loop regions named AB, BC, CD, DE, EF, and FG, the AB loop comprising SEQ ID NO: 4, the BC loop comprising SEQ ID NO: 86, the CD loop comprising SEQ ID NO: 6, and the DE loop comprising SEQ ID NO: The Tn3 scaffold contains 96, the EF loop contains SEQ ID NO: 8, the FG loop contains SEQ ID NO: 139, and multiple Tn3 scaffolds contain unoxidized tryptophan residues in the DE loop sequence of the monomer subunit specific to CD40L, and the administration is effective in reducing the CD40-mediated immune response, and the Tn3 scaffold is isolated using a hydrophobic interaction matrix, and the oxidized and unoxidized scaffolds are eluted from the hydrophobic interaction matrix at different times.

16. The use according to claim 15, wherein the unoxidized tryptophan residue is W151 of SEQ ID NO:

145.

17. The use according to claim 15 or 16, wherein the Tn3 scaffold comprises a monomer subunit specific to the second CD40L.

18. The use according to claim 17, wherein the unoxidized tryptophan residue is W46 and / or W151 of SEQ ID NO:

145.

19. The use according to claim 15 or 16, wherein the monomer subunit specific to CD40L comprises the polypeptide sequence of SEQ ID NO:

167.

20. The use according to claim 17, wherein the monomer subunit specific to CD40L and the second monomer subunit specific to CD40L are linked in tandem by a polypeptide linker.

21. The use according to claim 20, wherein the polypeptide linker comprises a sequence selected from the group consisting of SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 142, SEQ ID NO: 143 and combinations thereof.

22. The use according to claim 17, wherein one monomer subunit specific to CD40L is bound to an HSA, the HSA is a mutant HSA compared to a wild-type HSA, and the mutant HSA includes Sequence ID No.

133.

23. The use according to claim 17, wherein the monomer subunit specific to CD40L and the monomer subunit specific to the second CD40L have the same arrangement.

24. The use according to claim 17, wherein the monomer subunit specific to CD40L and the monomer subunit specific to the second CD40L have different arrangements.

25. The use according to claim 15 or 16, wherein β-strand A is sequence number 11, β-strand B is sequence number 12, β-strand C is sequence number 14, β-strand D is sequence number 15, β-strand E is sequence number 16, β-strand F is sequence number 17, and β-strand G is sequence number 18.

26. The use according to claim 15 or 16, wherein the Tn3 scaffold comprises the polypeptide of Sequence ID No.

145.

27. ​​The following: (a) A Tn3 scaffold comprising a monomer subunit specific to CD40L, wherein the monomer subunit comprises seven β-strands named A, B, C, D, E, F, and G, and six loop regions named AB, BC, CD, DE, EF, and FG, where the AB loop comprises SEQ ID NO: 4, the BC loop comprises SEQ ID NO: 86, the CD loop comprises SEQ ID NO: 6, the DE loop comprises SEQ ID NO: 96, the EF loop comprises SEQ ID NO: 8, and the FG loop comprises SEQ ID NO: 139, and a plurality of the Tn3 scaffolds comprising an unoxidized tryptophan residue contained in the DE loop sequence of the monomer subunit specific to CD40L; and (b) pharmaceutically acceptable carrier A composition comprising, The Tn3 scaffold is isolated using a hydrophobic interaction matrix, and the oxidized and unoxidized scaffolds are eluted from the hydrophobic interaction matrix at different times, in a composition.

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