Purification of nerve growth factor (.beta. subunit) by subunit exchange chromatography

• US 5,057,223 A
• Filed: 06/28/1990
• Issued: 10/15/1991
• Est. Priority Date: 07/20/1989
• Status: Expired due to Term

• Current Assignee: Fidia S.P.A.
• Original Assignee: Fidia S.P.A.
• Inventors: Callegaro, Lanfranco, Della Valle, Francesco
• Primary Examiner(s): Therkorn, Ernest G.
• Application Number:
  US07/544,531
• Time in Patent Office: 474 Days
• Field of Search: 210/635, 210/656, 210/659, 210/198.2, 210/502.1, 530/399, 530/413, 530/417
• US Class Current: 210/635
• CPC Class Codes: C07K 14/48 Nerve growth factor [NGF]

Abstract

A process is disclosed for the purification of Nerve Growth Factor (β subunit) by subunit exchange chromatography.

Claims

1. A method for the purification of the β

subunit of a Nerve Growth Factor which comprises;

a) covalent binding a first β

subunit of Nerve Growth Factor (NGF β

subunit) to a solid matrix having a coupling density sufficient to achieve subunit exchange chromatography;

b) combining the matrix having said first NGF (β

subunit) immobilized thereon with a preparation containing a second β

subunit of Nerve Growth Factor (NGF β

subunit), whereby said second NGF (β

subunit) is bound to said matrix;

c) eluting said matrix with a releasing agent to release said second NGF (β

subunit) from said matrix; and

d) recovering said released second NGF (β

subunit).

2. A method according to claim 1, wherein said first NGF (β

subunit) is a murine NGF and said second NGF (β

subunit) is a murine NGF, a bovine NGF, a human NGF prepared from human placental tissue or a human NGF prepared from recombinant DNA technology.

3. A method according to claim 1, wherein said first NGF (β

subunit) is a bovine NGF, and said second NGF (β

subunit) is a murine NGF, a bovine NGF, a human NGF prepared from human placental tissue or a human NGF prepared from recombinant DNA technology.

4. A method according to claim 1, wherein said first NGF (β

subunit) is a human NGF prepared from human placental tissue, and said second NGF (β

subunit) is a murine NGF, a bovine NGF, a human NGF prepared from human placental tissue or a human NGF prepared from recombinant DNA technology.

5. A method according to claim 1, wherein said first NGF (β

subunit) is a human NGF prepared from recombinant DNA technology, and said second NGF (β

subunit) is a murine NGF, a bovine NGF, a human NGF prepared from human placental tissue or a human NGF prepared from recombinant DNA technology.

6. A method according to any one of claims 1-5, wherein said releasing agent is guanidine hydrochloride, urea, thiocyanate or magnesium chloride.

7. A method according to any one of claims 1-5, wherein prior to said elution step said matrix having said second NGF (β

subunit) bound thereto is washed to remove unreacted biological material.

8. A method according to claim 7, wherein said washing comprises stepwise washing with phosphate buffer, sodium chloride and a releasing agent selected from guanidine hydrochloride, urea, thiocyanate and magnesium chloride.