# US 7,744,862 B2 · Production process of recombinant placental growth factor

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- Number: US 7,744,862 B2
- Document ID: US7744862B2
- Status: Granted
- Kind: B2
- Application number: 10/503,911
- Filed: 2002-02-05
- Published: 2005-03-31
- Granted: 2010-06-29
- Earliest claimed: 2002-02-05
- Assignees: Geymonat S.p.A.
- Inventors: Domenico Maglione; Mauro Battisti; Ettore Conti; Giuseppe Salvia; Marina Tucci
- Web page: https://patentgrep.com/patent/US7744862B2

## CPC

- C07K 14/515: Angiogenesic factors; Angiogenin
- 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

Process for extracting and purifying the recombinant Placental Growth Factor (PLGF) expressed in inducible prokaryotic expression systems comprising the following steps: I) fermentation of the bacterial cells, II) extraction and purification of the inclusion bodies, III) renaturation of the expressed protein, IV) ion-exchange chromatography, V) reverse-phase chromatography.

## Claims

### Claim 1

A process for extracting and purifying recombinant Placental Growth Factor (PLGF) protein in dimeric and multimeric active form containing no more than 1.5% of monomeric form, by expression of the PLGF protein in E. Coli cells modified with an inducible expression system comprising an insert coding for the PLGF protein, the process comprising in the following order:
I) inoculating in a culture medium the E. Coli cells and fermenting the E. Coli cells until obtaining optical density of the culture medium from 14 to 50 at 600 nm (OD600 14-50);
II) inducing the expression of PLGF protein via the inducible expression system;
III) extracting the inclusion bodies by lysing the cultured cells, rupturing DNA of the cultured cells, isolating and purifying the inclusion bodies;
IV) solubilizing the inclusion bodies into a denaturing buffer to form a solution and renaturating the expressed protein PLGF by adding to the solution oxidizing/reducing agents to bring the expressed protein to a dimeric form, at least partially;
V) passing the solubilized protein on a high flow resin for anion exchange chromatography separating and isolating the dimeric and multimeric forms of the expressed protein from the monomeric form; and
VI) further separating and finally isolating the dimeric and multimeric forms of the expressed protein from the monomeric form by passing them through a reverse-phase chromatography resin with two subsequent elution stages with increasing gradient of an organic solvent separated by an elution stage under isochratic conditions, wherein the organic solvent comprises ethanol, methanol, or acetonitrile, and wherein the monomeric form comprises no more than 1.5% of the total PLGF protein.

### Claim 2 (depends on 1)

The process according to claim 1, wherein the Placental Growth Factor (PLGF) is human PLGF-1 or of non-human origin.

### Claim 3 (depends on 1)

The process according to claim 1, comprising performing the fermentation in a medium comprising at least one selection agents, yeast extract, glycerol and ammonium salts.

### Claim 4 (depends on 1)

The process according to claim 1, wherein the inducible expression system is a T7 RNA polymerase IPTG inducible system.

### Claim 5 (depends on 4)

The process according to claim 4, comprising inducing the expression by adding a compound selected from the group consisting of lactose, isopropyl-beta.-D-thiogalactopyranoside (IPTG) and functionally equivalent analogues.

### Claim 6 (depends on 1)

The process according to claim 1, wherein the bacterial strain is E. Coli {B121(DE3)pLysS}.

### Claim 7 (depends on 1)

The process according to claim 1, comprising inducing expression when the optical density of the culture is 14 to 30 OD at 600 nm (14-30 OD600).

### Claim 8 (depends on 1)

The process according to claim 1, comprising performing the cell lysis by one of the group consisting of freezing/thawing and French press.

### Claim 9 (depends on 1)

The process according to claim 1, comprising performing the DNA rupture by one of the group consisting of DNAse of extraction origin, DNAase of recombinant origin, and chemical-mechanical action.

### Claim 10 (depends on 1)

The process according to claim 1, comprising performing the DNA rupture by mixing with a mixer.

### Claim 11 (depends on 1)

The process according to claim 1, comprising isolating the inclusion bodies by centrifuging for at least two cycles and washing into a suitable buffer.

### Claim 12 (depends on 1)

The process according to claim 1, wherein the extracting step further comprises homogenizing or sonicating the solution of inclusion bodies, and wherein the denaturing buffer comprising a compound selected from the group consisting of urea, guanidine isothiocyanate, and guanidine-hydrochloride.

### Claim 13 (depends on 1)

The process according to claim 1, wherein in the renaturing step IV, the solution of the denatured inclusion bodies is incubated with the oxidising/reducing agents for 10 to 30 hours at a temperature of 10° C. to 30° C.

### Claim 14 (depends on 13)

The process according to claim 13, further comprising diluting the solution of denatured inclusion bodies until obtaining an optical density at 280 nm (OD280) from 0.01 to 2 and adding reduced/oxidised glutathione to form the dimeric form of PLGF protein.

### Claim 15 (depends on 1)

The process according to claim 1, wherein, in step V, said passing is done with a ratio Volume loaded/Volume column from 1:1 to 10:1.

### Claim 16 (depends on 15)

The process according to claim 15 wherein the ratio Volume loaded/Volume column is 10:1.

### Claim 17 (depends on 15)

The process according to claim 15, comprising eluting the protein with an ethanolamine-HCl, NaCl elution buffer, wherein the protein in monomeric form is mainly comprised in the a percolate not bound to the resin of the anionic-exchange column, and wherein the protein in essentially dimeric form is eluted with elution buffers comprising NaCl at a concentration from 150 to 250 mM.

### Claim 18 (depends on 17)

The process according to claim 17, comprising diluting the eluted fraction in essentially dimeric form and loading the eluted fraction of the protein in essentially dimeric form onto a reverse-phase chromatography column in an amount corresponding to an optical density from 4.5 to 5.5 OD at 280 nm per ml of resin.

### Claim 19 (depends on 18)

The process according to claim 18, additionally comprising performing with first and second elution buffers a first elution stage under conditions of increasing gradient of organic solvent until the elution peak of the monomeric form appears, prosecuting the elution under isochratic conditions until exhaustion of the elution peak of the monomeric form, and performing a second elution stage under conditions of increasing gradient of organic solvent until complete elution of the protein in mainly dimeric form.

### Claim 20 (depends on 19)

The process according to claim 19, wherein the first and the second elution buffers comprise a solvent selected from the group consisting of ethanol, methanol, and acetonitrile.

### Claim 21 (depends on 19)

The process according to claim 19, wherein the first and the second elution buffers are a hydroalcoholic solution comprising increasing percentages of ethanol.

### Claim 22 (depends on 18)

The process according to claim 18 comprising performing the reverse-phase chromatography on resin having particles with an average diameter of 30-micron.

### Claim 23 (depends on 1)

The process according to claim 1, comprising performing after step IV an additional ultrafiltration step followed by lyophilisation.

### Claim 24 (depends on 1)

A product obtained by the process of claim 1, the product comprising not less than 98.5% of Placental Growth Factor (PLGF) in active dimeric and multimeric form, the monomeric form in a percentage not higher than 1.5%, the product being free from bacterial contaminants.