Method of purifying albumin-fusion proteins

Patent metadata

Field	Value
Publication number	US-11548933-B2
Application number	US-202016901812-A
Country	US
Kind code	B2
Filing date	Jun 15, 2020
Priority date	Mar 12, 2015
Publication date	Jan 10, 2023
Grant date	Jan 10, 2023

Abstract

The present invention relates to a method of purifying albumin-fusion proteins to reduce the level of oxidation of susceptible amino acid residues. The method comprises an affinity matrix chromatography step and an anion exchange chromatography step. The purified albumin-fusion proteins have low levels of oxidation and retain their enhanced half-life in vivo and its bioactivity. In some embodiments, the albumin-fusion protein comprises a scaffold, such as human Tenascin C scaffold. Compositions comprising the albumin-fusion protein are further disclosed.

First claim

What is claimed is:

1. A method of isolating an albumin-fusion protein essentially free from oxidation of tryptophan residues, the process comprising subjecting a composition comprising an albumin-fusion protein to the following purification processes:
(a) an affinity matrix chromatography process;
(b) an anion exchange chromatography process; and
(c) a hydrophobic interaction matrix chromatography process,

wherein an elution buffer comprising octanoate is applied to the affinity matrix, and wherein oxidized tryptophan albumin-fusion protein and non-oxidized tryptophan albumin-fusion protein are eluted from the hydrophobic interaction matrix at different times, thereby separating the oxidized tryptophan albumin-fusion protein from the non-oxidized tryptophan albumin-fusion protein.

2. The method of claim 1 , wherein the albumin in the albumin-fusion protein is a human serum albumin (HSA).

3. The method of claim 2 , wherein the HSA is a variant HSA.

4. The method of claim 3 , wherein the amino acid sequence of the variant HSA is SEQ ID NO: 133.

5. The method of claim 1 , wherein the albumin-fusion protein comprises a scaffold moiety comprising a third fibronectin type III (FnIII) domain.

6. The method of claim 5 , wherein the FnIII domain is derived from human Tenascin C (Tn3 scaffold).

7. The method of claim 1 , wherein the albumin-fusion protein comprises a scaffold.

8. The method of claim 7 , wherein the scaffold comprises a tryptophan residue.

9. The method of claim 8 , wherein oxidation of the tryptophan residue reduces the activity of the albumin-fusion protein.

10. The method of claim 7 , wherein the scaffold specifically binds to CD40L.

11. The method of claim 10 , wherein the scaffold comprises a single CD40L-specific monomer subunit.

12. The method of claim 10 , wherein the scaffold comprises two CD40L-specific monomer subunits connected in tandem.

13. The method of claim 12 , wherein the two CD40L-specific monomer subunits are directly connected.

14. The method of claim 12 , wherein the two CD40L-specific monomer subunits are connected by a linker.

15. The method of claim 14 , wherein the linker comprises a peptide linker.

16. The method of claim 15 , wherein the peptide linker comprises a (G m X) n sequence wherein:
(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) n is 1, 2, 3, 4, 5, 6, or 7.

17. The method of claim 16 , wherein the peptide linker comprises SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 142 or SEQ ID NO: 143.

18. The method of claim 6 , wherein the Tn3 scaffold comprises a beta strand, and wherein the beta strand comprises at least one CD40L-specific monomer subunit having at least 90% sequence identity to the beta strand of SEQ ID NO: 3.

19. An albumin-fusion protein composition obtained by the method of claim 1 .

Assignees

• Medimmune Llc
• Medimmune Llc

Inventors

• Pabst Timothy
• Fonseca Mariko
• Thompson Christopher
• Hunter Alan
• Wang Xiangyang
• Tie Liu
• Li Yiming

Classifications

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• C07K14/78 Primary

  Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG] · CPC title

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• C07K1/14 Primary

  Extraction; Separation; Purification · CPC title

• A61K47/643

  Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL] · CPC title