ROBUST ANTIBODY PURIFICATION

• US 20170073394A1
• Filed: 02/18/2015
• Published: 03/16/2017
• Est. Priority Date: 03/04/2014
• Status: Active Grant

• Current Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Original Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Inventors: EDELMANN, Bianca, BRAUN, Simon, KOZLOV, Mikhail, GALIPEAU, Kevin, SKUDAS, Romas, JOEHNCK, Matthias, STONE, Matthew T.
• Granted Patent: US 10,457,720 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: B01D 15/327 with hydrophobic interaction
• CPC Class Codes: B01D 15/361 Ion-exchange
• CPC Class Codes: B01D 15/3809 of the antigen-antibody type, e.g. protein A, G, L chromatography
• CPC Class Codes: C07K 1/20 Partition-, reverse-phase or hydrophobic interaction chromatography
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002

Abstract

The present invention refers to a method for the separation of host cell proteins (HCPs), antibody fragments and low molecular weight substances from solutions containing antibodies.

Claims

1. Method for the separation of host cell proteins (HCPs), antibody fragments and low molecular weight substances from solutions containing antibodies, characterized in that a solution containing antibodies is contacted with a hydrophobic chromatography material for a period of time whereby the antibodies stay in solution and HCPs, antibody fragments and low molecular weight substances are adsorbed by the hydrophobic chromatography material.

2. Method of claim 1, characterized in that the hydrophobic chromatography material is particulate made of cross-linked vinylbenzene, crosslinked ethylvinylbenzene, polystyrene-divinylbenzene, polystyrene-divinylbenzene, polyethylstyrene-divinylbenzene, or of polyethylstyrene-divinylbenzene ethyleneglycol-dimethylacrylate resin.

3. Method according to claim 1, characterized in that a chromatography material is applied which is composed of cross-linked polymer composed of monovinylaromatic monomer and divinylaromatic monomer in a ratio 98:

2 up to 10;

90% by weight.

4. Method according to claim 1, characterized in that a chromatography material is applied which is composed of cross-linked polymer composed of monovinylaromatic monomer and mixture of divinylaromatic and di(meth)acrylester monomer in a ratio 98:

2 up to 10;

90% by weight.

5. Method according to claim 1, characterized in that particulate chromatography material is applied, consisting of polystyrene or poly(ethyl)styrene, which is cross-linked with copolymer of divinylbenzene and ethylenglycol methacrylate in a ratio of 98:

2 up to 10;

90% by weight.

6. Method according to claim 1, characterized in that an aqueous solution is applied having a pH value in the range of 2-11, preferably in a range of 5-9 and a conductivity in the range of 1-150 mS/cm, preferably in the range of 2-50 mS/cm.

7. Method according to claim 1, characterized in that the aqueous solution is passed through at a flow rate in the range of 150-1000 cm/min, preferably in the range of 300-900 cm/min.

8. Method according to claim 1, characterized in that the separation is processed after the Protein A affinity binding step.

9. Method according to claim 1, characterized in that the sequence of purification treatments includes a treatment with ion exchange resin.

10. Method according to claim 1, characterized in that the separation treatment is processed using particulate, hydrophobic chromatographic separation materials having 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 preferred in the range of 20 μ

m to 63 μ

m.

11. Method according to claim 1, characterized in that the separation is processed using hydrophobic chromatographic separation materials 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.

12. Method according to claim 1, characterized in that at least one separation treatment with an ion exchange resin, specific for the separation of Protein A, is combined with a treatment with porous hydrophobic polymer beads resulting in a depletion of up to <

10 ng HCP and removal of leached Protein A.

13. Method according to claim 12, characterized that treatment(s) with ion exchange resin(s), specific for the separation of Protein A, are (is) combined with a treatment with hydrophobic polymer beads consisting of cross-linked vinylbenzene, poly(ethyl)styrene-divinylbenzene, or of polystyrene-divinylbenzene ethyleneglycol-dimethylacrylate resin.

16. Method according to claim 10, characterized in that the separation is processed using hydrophobic chromatographic separation materials 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.

14. Use of hydrophobic chromatographic separation materials having pore sizes in the range of 4 nm to 500 nm, preferably in the range of 10 nm-30 nm, most preferred in the range of 13 nm to 25 nm for the separation of host cell proteins (HCPs), antibody fragments and low molecular weight substances from solutions containing antibodies.

15. Use of hydrophobic, rigid polymer beads, having 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 preferred in the range of 20 μ

m to 63 μ

m, and pore sizes in the range of 4 nm to 500 nm, preferably in the range of 10 nm-30 nm, most preferred in the range of 13 nm to 25 nm, for the separation of host cell proteins (HCPs), antibody fragments and low molecular weight substances from solutions containing antibodies.