Method of purifying albumin-fusion proteins

Patent metadata

Field	Value
Publication number	US-10683340-B2
Application number	US-201615557358-A
Country	US
Kind code	B2
Filing date	Mar 11, 2016
Priority date	Mar 12, 2015
Publication date	Jun 16, 2020
Grant date	Jun 16, 2020

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 purifying an albumin-fusion protein, the method comprising subjecting a composition comprising an albumin-fusion protein to the following purification processes:
(a) an affinity matrix, wherein an elution buffer comprising octanoate is applied to the affinity matrix
and wherein the affinity matrix is washed with a wash buffer comprising: (1) about 2% to about 20% polyol, wherein the polyol is selected from the group consisting of 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,6 hexanediol, and 2-methyl-2,4-pentanediol; (2) 0.05 M to 2.0 salt, wherein the salt is selected from sodium chloride, potassium chloride, calcium chloride, lithium chloride, sodium bromide, potassium bromide and lithium bromide; (3) about 0.02 M to about 0.2 M sodium sulfate; (4) about 0.01% to about 1% nonionic surfactant; (5) about 0.05 M to about 1.0 M urea; or (6) about 0.02 M to about 0.5 M nicotinamide,
(b) an anion exchange matrix; and
(c) a hydrophobic interaction matrix,

wherein the resulting purified albumin-fusion protein is essentially free of oxidized tryptophan residues.

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 45, 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 CD40L-specific monomer subunit comprising the amino acid sequence:

IEV(X AB ) n ALITW(X BC ) n CELX 1 YGI(X CD ) n TTIDL(X DE ) n YSI

(X EF ) n YEVSLIC(X FG ) n KETFTT

wherein:
(a) X AB , X BC , X CD , X DE , X EF , and X FG represent the amino acid residues present in the sequences of the AB, BC, CD, DE, EF, and FG loops, respectively;
(b) X 1 represents amino acid residue A or T; and,
(c) length of the loop n is an integer between 2 and 26.

12. The method of claim 11 , wherein the sequence of the AB loop comprises SEQ ID NO: 4 or SEQ ID NO: 136, the sequence of the CD loop comprises SEQ ID NO: 6, and the sequence of the EF loop comprises SEQ ID NO: 8 or SEQ ID NO: 137.

13. The method of claim 12 , wherein:
(a) the sequence of the BC loop comprises SEQ ID NO: 83, the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139;
(b) the sequence of the BC loop comprises SEQ ID NO: 83, the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQ ID NO: 99;
(c) the sequence of the BC loop comprises SEQ ID NO: 84, the sequence of the DE loop comprises SEQ ID NO: 95, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139;
(d) the sequence of the BC loop comprises SEQ ID NO: 85, the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139;
(e) the sequence of the BC loop comprises SEQ ID NO: 86, the sequence of the DE loop comprises SEQ ID NO: 96, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139;
(f) the sequence of the BC loop comprises SEQ ID NO: 87, the sequence of the DE loop comprises SEQ ID NO: 97, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139;
(g) the sequence of the BC loop comprises SEQ ID NO: 88, the sequence of the DE loop comprises SEQ ID NO: 95, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139;
(h) the sequence of the BC loop comprises SEQ ID NO: 89, the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139;
(i) the sequence of the BC loop comprises SEQ ID NO: 90, the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139;
(j) the sequence of the BC loop comprises SEQ ID NO: 91, the sequence of the DE loop comprises SEQ ID NO: 95, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139;
(k) the sequence of the BC loop comprises SEQ ID NO: 92, the sequence of the DE loop comprises SEQ ID NO: 98, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139; or,
(l) the sequence of the BC loop comprises SEQ ID NO: 93, the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139.

14. The method of claim 12 , wherein:
(a) the sequence of the BC loop comprises SEQ ID NO: 100, the sequence of the DE loop comprises SEQ ID NO: 118, and the sequence of the FG loop comprises SEQ ID NO: 129;
(b) the sequence of the BC loop comprises SEQ ID NO: 101, the sequence of the DE loop comprises SEQ ID NO: 119, and the sequence of the FG loop comprises SEQ ID NO: 129;
(c) the sequence of the BC loop comprises SEQ ID NO: 102, the sequence of the DE loop comprises SEQ ID NO: 120, and the sequence of the FG loop comprises SEQ ID NO: 129;
(d) the sequence of the BC loop comprises SEQ ID NO: 103, the sequence of the DE loop comprises SEQ ID NO: 121, and the sequence of the FG loop comprises SEQ ID NO: 129;
(e) the sequence of the BC loop comprises SEQ ID NO: 104, the sequence of the DE loop comprises SEQ ID NO: 122, and the sequence of the FG loop comprises SEQ ID NO: 129;
(f) the sequence of the BC loop comprises SEQ ID NO: 105, the sequence of the DE loop comprises SEQ ID NO: 121, and the sequence of the FG loop comprises SEQ ID NO: 129;
(g) the sequence of the BC loop comprises SEQ ID NO: 106, the sequence of the DE loop comprises SEQ ID NO: 123, and the sequence of the FG loop comprises SEQ ID NO: 129;
(h) the sequence of the BC loop comprises SEQ ID NO: 107, the sequence of the DE loop comprises SEQ ID NO: 123, and the sequence of the FG loop comprises SEQ ID NO: 129;
(i) the sequence of the BC loop comprises SEQ ID NO: 108, the sequence of the DE loop comprises SEQ ID NO: 118, and the sequence of the FG loop comprises SEQ ID NO: 129;
(j) the sequence of the BC loop comprises SEQ ID NO: 109, the sequence of the DE loop comprises SEQ ID NO: 123, and the sequence of the FG loop comprises SEQ ID NO: 129;
(k) the sequence of the BC loop comprises SEQ ID NO: 110, the sequence of the DE loop comprises SEQ ID NO: 121, and the sequence of the FG loop comprises SEQ ID NO: 129;
(l) the sequence of the BC loop comprises SEQ ID NO: 111, the sequence of the DE loop comprises SEQ ID NO: 123, and the sequence of the FG loop comprises SEQ ID NO: 130;
(m) the sequence of the BC loop comprises SEQ ID NO: 108, the sequence of the DE loop comprises SEQ ID NO: 121, and the sequence of the FG loop comprises SEQ ID NO: 129;
(n) the sequence of the BC loop comprises SEQ ID NO: 112, the sequence of the DE loop comprise

Assignees

• Medimmune Llc

Inventors

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

Classifications

• C07K2319/31

  fusions, other than Fc, for prolonged plasma life, e.g. albumin · CPC title

• 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

• A61P43/00

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

• C07K14/765 Primary

  Serum albumin, e.g. HSA · CPC title

• A61K47/643

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