TARGET MOLECULE CAPTURE FROM CRUDE SOLUTIONS

• US 20170340989A1
• Filed: 11/18/2015
• Published: 11/30/2017
• Est. Priority Date: 12/15/2014
• Status: Active Grant

• Current Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Original Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Inventors: SKUDAS, Romas, ADRIAN, Klaus, EDELMANN, Bianca, JOEHNCK, Matthias
• Granted Patent: US 10,617,975 B2
• Time in Patent Office: Days
• CPC Class Codes: B01D 15/265 Adsorption chromatography
• CPC Class Codes: B01D 15/327 with hydrophobic interaction
• CPC Class Codes: C04B 2103/0052 Hydrophobic polymers
• CPC Class Codes: C07K 1/20 Partition-, reverse-phase or hydrophobic interaction chromatography

Abstract

The present invention refers to a method for the separation of peptide aggregates and fragments from solutions containing target peptide.

Claims

1. A method for separating peptide aggregates and fragments from solutions containing the target peptide, comprising the steps of

(a) providing the sample containing the target peptide;

(b) contacting the sample with a hydrophobic chromatography material for a suitable period of time and adsorbing the peptides,

(c) recovering the target peptide by use of different solvent compositions and

thereby separating aggregated peptides and peptide fragments from the target peptide.

2. A method of claim 1, wherein the hydrophobic chromatography material, is particulate, and which is made of cross-linked polymer, selected from the group vinylbenzene, styrene, ethylstyrene, poly(ethyl)styrene-divinylbenzene, and poly(ethyl)styrene-divinylbenzene ethyleneglycol-dimethylacrylate resin.

3. A method of claim 1, wherein the particulate hydrophobic chromatography material is a resin, which is composed of a cross-linked polymer consisting of styrene and divinylbenzene in a ratio 98:

2 up to 10;

90% by weight or of polystyrene, which is cross-linked with copolymer of divinylbenzene and ethylenglycoldi-methacrylate in a ratio of 98;

2 up to 10;

90% by weight.

4. A method according to claim 1, wherein the particulate hydrophobic chromatographic separation material has mean particle diameters in the range of 10 μ

m to 600 μ

m, preferably in the range of 20 μ

m to 150 μ

m, most preferably in the range of 20 μ

m to 63 μ

m.

5. A method according to claim 1, wherein the particulate hydrophobic chromatographic separation material consists of hydrophobic porous polymer beads having pore sizes in the range of 4-500 nm, preferably in the range of 10-30 nm, most preferred in the range of 13 nm to 25 nm.

6. A method according to claim 1, wherein in step b) an aqueous solution containing the target peptide, having a pH value in the range of 2-11, preferably in a range of 3-8 and a conductivity in the range of 1-150 mS/cm, preferably in the range of 2-50 mS/cm, is contacted with a hydrophobic chromatography material.

7. A method according to claim 1, wherein in step b) the hydrophobic chromatographic material is exposed to 30-100 mg of target peptide per ml of packed bed, preferably to 50-80 mg of target peptide per ml of packed bed at a flow rate in the range of 150-1000 cm/min, preferably in the range of 300-900 cm/min.

8. A method according to claim 1, wherein in step c) the hydrophobic chromatographic material is exposed to an aqueous solution comprising organic solvent in direct or gradient manner, whereby depending on the concentration of the contained organic solvent a partial separation between the aggregated and target peptide is achieved.

9. A method according to claim 1, wherein in step c) a selective desorption and separation of the bound components is achieved using various ratios of organic solvents contained in an aqueous mixture.

10. A method according to claim 1, wherein the separation of peptide aggregates is processed after a target molecule refolding step.

11. A method according to claim 1, wherein after contacting the sample with a hydrophobic chromatography material for a suitable period of time and adsorbing the peptides, the loaded chromatography material is subjected to post peptide refolding solutions for selectively reducing the level of aggregated substances for a suitable period of time.

12. A method according to claim 1, wherein the separation and purification sequence includes a treatment with an ion exchange resin.

13. A method according to claim 1, wherein the separation and purification is carried out in a bind and elute or flow-through mode, whereby the flow velocity is adjusted in the range of 150 cm/min-1000 cm/min, and especially between 300-900 cm/min.

14. A method according to claim 1, wherein in step c) organic solvents selected from the group ethanol, 1-propanol and dipropylenglycol are used to selectively desorb the adsorbed substance from the hydrophobic chromatography material.

15. A method according to claim 1, wherein in step b) the sample is contacted with a hydrophobic chromatography material in form of polymer beads in a liquid chromatography column having a diameter ranging from 1 to 100 cm, preferably in the range of 5 to 50 cm, and where the column is operated at pressures up to 100 bar, and preferably at pressure ranging from 0.2 to 80 bar.

16. A method according to claim 1, wherein the sample is contacted with a hydrophobic chromatography material in form of polymer beads in a liquid chromatography column having a diameter in the range of 10 to 50 cm and where the column is operated at pressures in the range of 0.2 to 80 bar.

17. A method according to claim 1, wherein in step a) a crude insulin solution originating from E. coli expression system is provided.