IP Library Granted Patent US 7,420,035
Granted Patent B2
US 7,420,035 · App. 10/258,191 · Granted Sep 2, 2008

Purification of polypeptides

Assignee: Atheris Laboratories, Dr. Reto Stocklin et Sylvie Stocklin Associes
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Quick Facts
Patent No.
US 7,420,035
App. No.
10/258,191
Granted
Sep 2, 2008
Kind
B2
Abstract

This invention relates to a process for purifying a polypeptide, a capture tag useful for purifying a polypeptide and a periodate-cleavable amino acid derivative useful for purifying a polypeptide. The polypeptide to be purified comprises a vicinal-amino-thiol, vicinal-amino-hydoxyl, vicinal-diol or vicinal-diamino group. The purification process comprises attaching the polypeptide to a purification matrix by contacting the polypeptide with a purification matrix comprising aldehyde or ketone groups under conditions which favor formation of a heterocyclic ring system, washing the purification matrix, and releasing the polypeptide from the purification matrix.

Claims (87)

1. A process for the purification of a polypeptide comprising a vicinal-amino-thiol, vicinal-amino-hydroxyl, vicinal-diol or vicinal-diamino group, the process comprising:

(i) attaching the polypeptide to a purification matrix by contacting the polypeptide with a purification matrix comprising aldehyde or ketone groups under conditions which favour formation of a heterocyclic ring system,

(ii) washing the purification matrix, and

(iii) releasing the polypeptide from the purification matrix.

2. A process according to claim 1 , for the purification of a polypeptide comprising an N-terminal cysteinyl (Cys), threonyl (Thr) or seryl (Ser) amino acid, the process comprising:

(i) attaching the polypeptide to a purification matrix by contacting the polypeptide with a purification matrix comprising aldehyde or ketone groups under conditions favouring formation of a thiazolidine or oxazolidine ring,

(ii) washing the purification matrix to remove unbound material, and

(iii) releasing the polypeptide from the matrix by exposure to conditions which reverse thiazolidine or oxazolidine formation.

3. A process according to claim 2 , comprising the further step of cleavage of the N-terminal Cys, Thr, or Ser amino acid and optionally further N-terminal amino acids.

4. A process for the purification of a polypeptide comprising a group selected from

—CR(OH)—CR′(R″)—NH—CHR 1 CO—

—CR(NH 2 )—CR′(R″)—NH—CHR 1 CO—

wherein R 1 is the side chain of an amino acid,

R, R′ and R″ are selected from hydrogen, alkyl, aralkyl, aryl and heterocyclic groups, the process comprising the steps of

(i) attaching the polypeptide to a purification matrix by contacting the polypeptide with a purification matrix comprising aldehyde or ketone groups under conditions favouring formation of a heterocyclic ring,

(ii) washing the purification matrix to remove unbound material, and

(iii) releasing the polypeptide from the matrix by exposure to conditions which reverse heterocyclic ring formation.

5. A process according to claim 4 , comprising the further step of cleaving the group —R(OH)— or CR(NH 2 ) from the polypeptide with periodic acid or a salt thereof.

6. A process according to claim 4 , wherein the polypeptide comprises the group

HOCH 2 —CH 2 —NH—CHR 1 CO—.

7. A process for the purification of a polypeptide comprising a group selected from

T-L-CO—CR′(R″)—NH—CHR 1 —CO—

T-L-CR(OH)—CR′(R″)—NH—CHR 1 —CO— and

T-L-CR(NH 2 )—CR′(R″)—NH—CHR 1 —CO—,

wherein T is a capture tag group capable of binding, with or without the participation of the —CR—(OH)—,—CR(NH 2 )— or —CO— group, with a purification matrix,

L is a divalent linker moiety or may be absent,

R 1 is a side chain of an amino acid,

R, R′ and R″ are independently selected from hydrogen, alkyl, aralkyl, aryl and heterocyclic groups the process comprising the steps of

(i) attaching the polypeptide to a purification matrix by contacting the polypeptide with a purification matrix comprising a structure or chemical functionality capable of binding through the capture tag group,

(ii) washing the purification matrix to remove unbound material, and

(iii) releasing the bound polypeptide.

8. A process according to claim 7 , comprising the step of cleaving the group comprising

T-L-CO—, T-L-CR(OH)— or T-L-CR(NH 2 )—

from the polypeptide with periodic acid or a salt thereof.

9. A process according to claim 8 , wherein cleaving the group comprising

T-L-CO—, T-L-CR(OH)— or T-L-CR(NH 2 )—

from the polypeptide serves to release the polypeptide from the purification matrix.

10. A process according to claim 8 , wherein cleaving the group comprising

T-L-CO—, T-L-CR(OH)— or T-L-CR(NH 2 )—

from the polypeptide is performed subsequent to releasing the bound polypeptide from the purification matrix.

11. A polypeptide comprising a group selected from

T-L-CR(OH)—CR′(R″)—NH—CHR 1 —CO—

and

T-L-CO—CR′—(R″)—NH—CHR 1 —CO—,

wherein

T is a capture tag group capable of binding, with or without the participation of the

—CR—(OH)—, or —CO— group,

with a purification matrix,

L is a divalent linker moiety or may be absent,

R 1 is the side chain of an amino acid of the polypeptide,

R, R′ and R″ are independently selected from hydrogen, alkyl, aralkyl, aryl and heterocyclic groups,

and protected derivatives thereof.

12. A polypeptide according to claim 11 , wherein the capture tag group T is capable of binding with a purification matrix with the participation of the

—CR—(OH)—, or —CO— group.

13. A polypeptide according to claim 12 , comprising the group

HS—CH 2 —CH 2 —CH(OH)CH 2 NH——CHR 1 CO—.

14. A polypeptide according to claim 12 , comprising the group

H 2 N—CH 2 —CH 2 —CH(OH)CH 2 NH—CHR 1 CO—.

15. A polypeptide according to claim 12 , comprising the group

H 2 N—CH 2 —CH(OH)CH 2 NH—CHR 1 CO—.

16. A polypeptide according to claim 11 , wherein the capture tag group T is capable of binding with a purification matrix without the participation of the

—CR(OH), or —CO— group.

17. A polypeptide according to claim 16 , comprising the group

H-Cys-NH-CH 2 —CH 2 —CH(OH)—CH 2 NH—CHR 1 CO—.

18. A polypeptide according to claim 16 , comprising the group

H-Thr-NH—CH 2 —CH 2 13 CH(OH)—CH 2 NH—CHR 1 CO—.

19. A polypeptide according to claim 16 , comprising the group

H 2 N—O—CH 2 —CO—NH—CH 2 —CH 2 —CH(OH)—CH 2 NH—CH—R 1 CO—.

20. A polypeptide according to claim 11 , wherein the polypeptide is capable of binding with a purification matrix comprising aldehyde or ketone groups.

21. A process for removing the capture tag group T and, when present, the linker L from a polypeptide comprising a group selected from

T-L-CR(OH)—CR′(R″)—NH—CHR 1 —CO

T-L-CR(NH 2 )—CR′(R″)—NH—CH—R 1 —CO and

T-L-CO—CR′(R″)—NH—CHR 1 —CO,

the process comprising the step of treating the polypeptide with periodic acid or a salt thereof.

22. A process according to claim 21 , wherein the polypeptide is treated in solution.

23. A process according to claim 21 , wherein the polypeptide is treated while bound to a matrix and treatment serves to release the polypeptide from the matrix.

24. A process according to claim 4 , wherein a thiazolidine ring is formed upon attaching the polypeptide to a purification matrix.

25. A polypeptide comprising the group:

T-L-CR(NH 2 )—CR′(R″)—NH—CH R 1 —CO—

wherein

T is a capture tag group capable of binding, with the participation of the

—CR(NH 2 )— group

with a purification matrix,

L is a divalent linker moiety or may be absent,

R 1 is the side chain of an amino acid of the polypeptide,

R, R′ and R″ are independently selected from hydrogen, alkyl, aralkyl, aryl and heterocyclic groups,

and protected derivatives thereof.

Assignments (7)
CHANGE OF NAME Recorded Feb 22, 2013
From: UNIVERSITE DE GENEVE
To: ATHERIS LABORATORIES, DR RETO STOCKLIN
Reel/Frame 029859/0249 →
CORRECTIVE ASSIGNMENT TO CORRECT THE NAME OF THE RECEIVING PARTY PREVIOUSLY RECORDED ON REEL 017039 FRAME 0306. ASSIGNOR(S) HEREBY CONFIRMS THE CORRECTION OF ATHERIS LABORATORIES, DR. RETO STOCKLIN ET SYLVIE STOCKLIN ASSOCIES.. Recorded May 12, 2008
From: UNIVERSITE DE GENEVE
To: ATHERIS LABORATORIES, DR. RETO STOCKLIN ET SYLVIE STOCKLIN ASSOCIES
Reel/Frame 020932/0383 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 17, 2005
From: UNIVERSITE DE GENEVE
To: ATHERIA LABORATORIES, DR. RETO STOCKLIN ET SYLVIA STOCKLIN ASSOCIES
Reel/Frame 017039/0306 →
CORRECTIVE ASSIGNMENT ORIGINALLY RECORDED ON JUNE 27, 2005 TO CORRECT NATURE OF CONVEYANCE. Recorded Jul 18, 2005
From: GENEVE, UNIVERSITE DE
To: ATHERIS LABORATORIES, DR. RETO STOCKLIN ET SYLVIE STOCKLIN ASSOCIES
Reel/Frame 016811/0175 →
SECURITY AGREEMENT Recorded Jun 27, 2005
From: UNIVERSITE DE GENEVE
To: ATHERIS LABORATORIES DR. RETO STOCKLIN ET SYLVI. STCKLIN ASSOCIOS
Reel/Frame 016421/0604 →
CORRECTIVE ASSIGNMENT TO CORRECT THE NAME OF THE THIRD CONVEYING PARTY, PREVIOUSLY RECORDED AT REEL/FRAME: 013941/0940. Recorded Oct 17, 2003
From: ROSE, KEITH; VILLAIN, MATTEO; VIZZAVONA, JEAN
To: UNIVERSITE DE GENEVE
Reel/Frame 014600/0280 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 18, 2002
From: ROSE, KEITH; VILLAIN, MATTEO; VIZZAVONA, JEAN
To: UNIVERSITE DE GENEVE
Reel/Frame 013941/0940 →
Priority Claims (1)
GB 009918.4 · Apr 20, 2000 · national
Continuity (1)
Related Publication 20030135031A1 · Jul 17, 2003