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 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, wherein the composition containing the albumin fusion protein is purified using the following process: (a) Affinity matrix chromatography process; (b) Anion exchange chromatography process; and (c) Hydrophobic interaction matrix chromatography process A method comprising the step of applying an elution buffer containing octanoic acid to the affinity matrix to separate an albumin fusion protein with oxidized tryptophan from an albumin fusion protein with unoxidized tryptophan.

2. A method for purifying albumin fusion proteins, (a) the step of applying the composition containing the albumin fusion protein to an affinity matrix; (b) The step of eluting the albumin fusion protein from the affinity matrix of (a) to obtain a first eluate; (c) The step of applying the first eluate to an anion exchange matrix; (d) The step of eluting the albumin fusion protein from the anion exchange matrix to obtain a second eluate; (e) the step of applying the second eluate to the anion exchange membrane; (f) The step of passing the albumin fusion protein through an anion exchange membrane to obtain flow-through; (g) the step of applying the flow-through to a hydrophobic interaction matrix; (h) The step of eluting the albumin fusion protein from the hydrophobic interaction matrix to obtain a third eluate. A method comprising the third eluate comprising a purified albumin fusion protein.

3. The method according to claim 1 or 2, wherein the albumin in the albumin fusion protein is human serum albumin (HSA).

4. The method according to claim 3, wherein the HSA is a mutant HSA.

5. The method according to claim 4, wherein the amino acid sequence of the mutant HSA is sequence number 133.

6. The method according to any one of claims 1 to 5, wherein the albumin fusion protein comprises a scaffold portion containing a third fibronectin type III (FnIII) ​​domain.

7. The method according to claim 6, wherein the FnIII domain is derived from human tenascin C (Tn3 scaffold).

8. The method according to any one of claims 1 to 7, wherein the albumin fusion protein comprises a scaffold.

9. The method according to claim 8, wherein the scaffold comprises a tryptophan residue.

10. The method according to claim 9, wherein oxidation of the tryptophan residue reduces the activity of the albumin fusion protein.

11. The method according to any one of claims 8 to 10, wherein the scaffold specifically binds to CD40L.

12. The method according to claim 11, wherein the scaffold comprises a monomer subunit specific to a single CD40L.

13. The method according to claim 11, wherein the scaffold comprises two tandem-connected monomer subunits specific to CD40L.

14. The method according to claim 13, wherein the monomer subunits specific to the two CD40L are directly linked.

15. The method according to claim 13, wherein the two monomer subunits specific to the CD40L are linked by a linker.

16. The method according to claim 15, wherein the linker comprises a peptide linker.

17. The aforementioned peptide linker is (G m X) n Includes an array, where, (a) X is serine (S), alanine (A), glycine (G), Leu (L), isoleucine (I), or valine (V); (b) m and n are integers; (c) m is 1, 2, 3 or 4; and (d) The method according to claim 16, wherein n is 1, 2, 3, 4, 5, 6, or 7.

18. The method according to claim 17, wherein the peptide linker includes SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 142, or SEQ ID NO:

143.

19. The method according to any one of claims 11 to 15, wherein the scaffold comprises a β-strand, and the β-strand of at least one monomer subunit specific to CD40L has at least 90% sequence identity with respect to the β-strand of SEQ ID NO:

3.

20. The monomeric subunit specific to the aforementioned CD40L has the following amino acid sequence: 【Chemistry 1】 Including, here, (a) X AB , X BC , X CD , X DE , X EF , and X FG These represent amino acid residues present in the sequences of the AB, BC, CD, DE, EF, and FG loops, respectively; (b) X 1 represents an amino acid residue A or T; and (c) The method according to any one of claims 11 to 15, wherein the length n of the loop is an integer from 2 to 26.

21. The method according to claim 20, wherein the sequence of the AB loop includes sequence number 4 or sequence number 136, the sequence of the CD loop includes sequence number 6, and the sequence of the EF loop includes sequence number 8 or sequence number 137.

22. (a) The sequence of the BC loop includes sequence number 83, the sequence of the DE loop includes sequence number 94, and the sequence of the FG loop includes sequence number 9 or 139; (b) The sequence of the BC loop includes sequence number 83, the sequence of the DE loop includes sequence number 94, and the sequence of the FG loop includes sequence number 99; (c) The sequence of the BC loop includes sequence number 84, the sequence of the DE loop includes sequence number 95, and the sequence of the FG loop includes sequence number 9 or 139; (d) The sequence of the BC loop includes sequence number 85, the sequence of the DE loop includes sequence number 94, and the sequence of the FG loop includes sequence number 9 or 139; (e) The sequence of the BC loop includes sequence number 86, the sequence of the DE loop includes sequence number 96, and the sequence of the FG loop includes sequence number 9 or 139; (f) The sequence of the BC loop includes sequence number 87, the sequence of the DE loop includes sequence number 97, and the sequence of the FG loop includes sequence number 9 or 139; (g) The sequence of the BC loop includes sequence number 88, the sequence of the DE loop includes sequence number 95, and the sequence of the FG loop includes sequence number 9 or 139; (h) The sequence of the BC loop includes sequence number 89, the sequence of the DE loop includes sequence number 94, and the sequence of the FG loop includes sequence number 9 or 139; (i) The sequence of the BC loop includes sequence number 90, the sequence of the DE loop includes sequence number 94, and the sequence of the FG loop includes sequence number 9 or 139; (j) The sequence of the BC loop includes sequence number 91, the sequence of the DE loop includes sequence number 95, and the sequence of the FG loop includes sequence number 9 or 139; (k) The sequence of the BC loop includes sequence number 92, the sequence of the DE loop includes sequence number 98, and the sequence of the FG loop includes sequence number 9 or 139; or (l) The method according to claim 21, wherein the sequence of the BC loop includes sequence number 93, the sequence of the DE loop includes sequence number 94, and the sequence of the FG loop includes sequence number 9 or 139.

23. (a) The sequence of the BC loop includes sequence number 100, the sequence of the DE loop includes sequence number 118, and the sequence of the FG loop includes sequence number 129; (b) The sequence of the BC loop includes sequence number 101, the sequence of the DE loop includes sequence number 119, and the sequence of the FG loop includes sequence number 129; (c) The sequence of the BC loop includes sequence number 102, the sequence of the DE loop includes sequence number 120, and the sequence of the FG loop includes sequence number 129; (d) The sequence of the BC loop includes sequence number 103, the sequence of the DE loop includes sequence number 121, and the sequence of the FG loop includes sequence number 129; (e) The sequence of the BC loop includes sequence number 104, the sequence of the DE loop includes sequence number 122, and the sequence of the FG loop includes sequence number 129; (f) The sequence of the BC loop includes sequence number 105, the sequence of the DE loop includes sequence number 121, and the sequence of the FG loop includes sequence number 129; (g) The sequence of the BC loop includes sequence number 106, the sequence of the DE loop includes sequence number 123, and the sequence of the FG loop includes sequence number 129; (h) The sequence of the BC loop includes sequence number 107, the sequence of the DE loop includes sequence number 123, and the sequence of the FG loop includes sequence number 129; (i) The sequence of the BC loop includes sequence number 108, the sequence of the DE loop includes sequence number 118, and the sequence of the FG loop includes sequence number 129; (j) The sequence of the BC loop includes sequence number 109, the sequence of the DE loop includes sequence number 123, and the sequence of the FG loop includes sequence number 129; (k) The sequence of the BC loop includes sequence number 110, the sequence of the DE loop includes sequence number 121, and the sequence of the FG loop includes sequence number 129; (l) The sequence of the BC loop includes sequence number 111, the sequence of the DE loop includes sequence number 123, and the sequence of the FG loop includes sequence number 130; (m) The sequence of the BC loop includes sequence number 108, the sequence of the DE loop includes sequence number 121, and the sequence of the FG loop includes sequence number 129; (n) The sequence of the BC loop includes sequence number 112, the sequence of the DE loop includes sequence number 124, and the sequence of the FG loop includes sequence number 129; (o) The sequence of the BC loop includes sequence number 113, the sequence of the DE loop includes sequence number 125, and the sequence of the FG loop includes sequence number 129; (p) The sequence of the BC loop includes sequence number 114, the sequence of the DE loop includes sequence number 118, and the sequence of the FG loop includes sequence number 129; (q) Whether the sequence of the BC loop includes sequence number 115, the sequence of the DE loop includes sequence number 126, and the sequence of the FG loop includes sequence number 129; (r) The sequence of the BC loop includes sequence number 116, the sequence of the DE loop includes sequence number 127, and the sequence of the FG loop includes sequence number 129; or (s) The method according to claim 21, wherein the sequence of the BC loop includes sequence number 117, the sequence of the DE loop includes sequence number 128, and the sequence of the FG loop includes sequence number 129.

24. The method according to claim 23, wherein the monomer subunit specific to CD40L includes a sequence selected from the group consisting of SEQ ID NOs: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, and 146.

25. The method according to claim 7, wherein the albumin fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 134, 135, 201, 202, 203, 204, 205, 206, 207, and 208.

26. An albumin fusion protein composition obtained by the method described in any one of claims 1 to 25.

27. A composition comprising the albumin fusion protein of SEQ ID NO: 134, 135, 201, 202, 203, 204, 205, 206, 207, or 208, wherein the host cell protein is less than 20 ng / mg and the tryptophan at position 46, position 151, or both is not oxidized.

28. (a) The composition according to claim 26 or 27; (b) buffer; (c) sugars; and (d) Emulsifier A pharmaceutically acceptable formulation containing [the specified substance].

29. The formulation according to claim 28, wherein the buffer is a sodium phosphate buffer, the sugar is sucrose, and the emulsifier is polysorbate 80.

30. The formulation according to claim 28 or 29, which is freeze-dried.

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