# US 7,608,583 B2 · Process for the extraction and isolation of insulin from recombinant sources

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- Number: US 7,608,583 B2
- Document ID: US7608583B2
- Status: Granted
- Kind: B2
- Application number: 10/523,999
- Filed: 2003-11-14
- Published: 2006-07-27
- Granted: 2009-10-27
- Earliest claimed: 2002-12-05
- Assignees: Wockhardt Limited
- Inventors: Maharaj K Sahib; Edupuganti B Raju; Umesh S Shaligram
- Web page: https://patentgrep.com/patent/US7608583B2

## CPC

- C07K 14/62: Insulins
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The present invention relates to the extraction and isolation of insulins from recombinant sources, particularly those expressed in and secreted by yeasts. Organic solvents have been used to extract host surface bound forms of insulin polypeptide. In addition, procedures for combining the steps medium clarification, solvent extraction and chromatography, in order to effect the simultaneous isolation and purification of soluble and membrane bound forms of insulin, is disclosed.

## Claims

### Claim 1

A process for enhanced recovery of recombinant insulin comprising:
(a) expressing the insulin in yeast, wherein the expressed insulin is secreted into the culture medium;
(b) adding a water miscible organic solvent to the fermentation culture broth, wherein the fermentation culture broth contains the secreted insulin and the yeast, wherein the water miscible organic solvent is selected from the group consisting of ethanol, isopropanol, t-butanol, acetic acid, dimethylformamide, dimethylsulfoxide, acetonitrile, dioxan, ethylene glycol, and propylene glycol, and wherein the water miscible organic solvent optionally contains additives;
(c) clarifying the fermentation culture broth containing the added water miscible organic solvent by centrifugation and decanting the supernatant from the pellet;
(d) isolating the insulin from the supernatant.

### Claim 2 (depends on 1)

The process of claim 1, wherein the water miscible organic solvent is isopropanol.

### Claim 3 (depends on 1)

The process of claim 1, wherein the water miscible organic solvent is used at a concentration range from about 10% (v/v) to about 40% (v/v).

### Claim 4 (depends on 1)

The process of claim 1, wherein the additives comprise one or more of sodium chloride, calcium chloride, arginine, aspartic acid, urea, guanidinum hydrochloride, or polyethylene glycol.

### Claim 5 (depends on 1)

The process of claim 1, wherein the process is carried out at a pH range from about 2 to about 5.

### Claim 6 (depends on 1)

The process of claim 1, wherein the insulin is isolated using a cation exchange chromatography in expanded bed mode.

### Claim 7 (depends on 1)

The process of claim 1, wherein the yeast comprises genera Hansenula, Saccharomyces, Pichia, or Kluyveromyces.

### Claim 8 (depends on 1)

The process of claim 1 further comprising
(e) resuspending the pellet of step (c) with a miscible organic solvent selected from the group consisting of ethanol, isopropanol, t-butanol, acetic acid, dimethylformamide, dimethylsulfoxide, acetonitrile, dioxan, ethylene glycol, and propylene glycol, and wherein the water miscible organic solvent optionally contains additives;
(f) centrifuging the resuspended pellet and decanting the supernatant;
(g) isolating the insulin from the supernatant of step (f).