Methods for expression and purification of recombinant human growth hormone mutants

• US 7,959,926 B2
• Filed: 10/17/2007
• Issued: 06/14/2011
• Est. Priority Date: 12/22/2004
• Status: Expired due to Fees

• Current Assignee: Ambrx, Inc. (Johnson & Johnson)
• Original Assignee: Ambrx, Inc. (Johnson & Johnson)
• Inventors: Cho, Ho Sung, Bussell, Stuart, Ong, Michael, Lieu, Ricky, Knudsen, Nick, Buechler, Ying
• Primary Examiner(s): STOICA, ELLY GERALD
• Application Number:
  US11/874,068
• Publication Number:
  US 20080085538A1
• Time in Patent Office: 1,336 Days
• Field of Search: None
• US Class Current: 424/200.1
• CPC Class Codes: A61P 5/06 of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
• CPC Class Codes: C07K 14/61 Growth hormone [GH], i.e. somatotropin

Abstract

The present invention relates generally to the production, purification, and isolation of human growth hormone (hGH). More particularly, the invention relates to the production, purification, and isolation of substantially purified hGH comprising non-naturally encoded amino acids and hGH from recombinant host cells or culture medium including, for example, yeast, insect, mammalian and bacterial host cells. The process of the present invention is also useful for purification of hGH linked to polymers or other molecules.

Claims

1. A process comprising:

a) culturing Escherichia coli host cells comprising an O-tRNA/O-RS pair and capable of producing human growth hormone (hGH) comprising a non-naturally encoded amino acid in culture medium;

b) producing the hGH comprising the non-naturally encoded amino acid wherein the non-naturally encoded amino acid is at a position corresponding to 35, 92, 143, or 145 of SEQ ID NO;

2, or the corresponding amino acid positions in SEQ ID NO;

1 or 3;

c) coupling the non-naturally encoded amino acid to a water soluble polymer; and

d) purifying the coupled hGH comprising the non-naturally encoded amino acid with one or more steps, wherein at least about 100 mg substantially purified hGH is produced per liter of recombinant host cell culture medium.

2. The method of claim 1, wherein the coupling of hGH to a water soluble polymer produces a substantially purified hGH polypeptide.

3. The method of claim 1, wherein step d) comprises contacting the coupled hGH with an anion exchange chromatography matrix under conditions that allow binding of the coupled hGH to the matrix followed by elution and collection of the coupled hGH.

4. The method of claim 3 further comprising contacting the eluted coupled hGH with a hydroxyapatite chromatography matrix under conditions that allow binding of the coupled hGH to the matrix.