Moveable chromatography column separator

Abstract

The current invention is directed to a chromatography column separator which separates the chromatography column in an upper chromatography column chamber and a lower chromatography column chamber, and which has a variable position within the chromatography column, and which is embedded by the chromatography material. The separator allows the replacement of the chromatography material in the upper chromatography column chamber without the need to replace the chromatography column material in the lower chromatography column chamber and it allows also the combination of two different chromatography materials with different chromatographical functional groups in one chromatography column.

Claims

1. A chromatography column comprising a separator comprising an O-shaped guide ring having a vertical cross-section comprising two separate axially symmetric cross-section areas, wherein each of the axially symmetric cross-section areas has i) a tapering structure, wherein the tapering is from the outside to the inside of the guide ring, and ii) a notch with an opening directed to the inside of the guide ring for mounting a frit, wherein the notch is a rectangular notch, wherein further each of the cross-section areas has a triangular shape and the longest side has a length of at least 1.5 times the diameter of the notch, and a frit mounted into the guide ring; wherein the separator is slidably disposed inside a chromatography column such that the separator is freely movable within the chromatography column by sliding along the inner wall of the chromatography column; wherein an outside of the guide ring is in contact with the inner wall of the chromatography column.

2. The chromatography column of claim 1, wherein the frit is selected from the group consisting of a) a single frit, or b) two frits with an upper frit and a lower frit.

3. The chromatography column of claim 2, wherein a) the single frit has a pore size of from 1 m to 20 m or b) each of the two frits has a pore size of from 1 m to 20 m independently of each other whereby the pore size of the upper frit is smaller than the pore size of the lower frit.

4. The chromatography column of claim 1, wherein the frit is made of metal, silicone, polypropylene, polyethylene, polytetrafluoroethylene, sintered materials or combinations thereof.

5. The chromatography column of claim 4, wherein the separator comprises distance holders all attached to one side of the separator; wherein the separator has a shape.

6. A chromatography column comprising more than one chromatography column separators according to claim 1, comprising more than one separator.

7. The chromatography column of claim 6 wherein at least one of the chromatography column separators is in contact with a first chromatography material and in contact with a second chromatography material, whereby the first and the second chromatography material are a) chromatography materials with the same chromatographical functional group and of the same or different particle size, or b) chromatography materials with different chromatographical functional groups.

8. The chromatography column of claim 1, wherein the separator separates the chromatography column into an upper chromatography column chamber and a lower chromatography column chamber.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 Exemplary chromatography column separators according to the invention: a) separator with a single frit comprising a frit (1) and a fitting (2); b) separator with an upper frit (1) and a lower frit (3) and an upper fitting (2) and a lower fitting (4); c) vertical cross-section of the guide ring of the separator comprising two axially symmetric cross-section areas (5 and 6) each having i) a tapering structure, wherein the tapering is from the outside to the inside of the guide ring, and i) a notch (8) with an opening directed to the inside of the guide ring for mounting a frit.

(2) FIG. 2 Cross-section areas of different fittings: a) cross-section of a triangular fitting, b) cross-section area of a trapezoid fitting, c) cross-section area of a rectangular fitting, d) cross-section area of an upper fitting and a lower fitting.

(3) FIG. 3 Perspective views of a fitting comprising three holes with screw thread are shown.

(4) FIG. 4 Cross-section of a chromatography column separator with a fitting comprising three holes with a screw thread attached via three connectors to a chromatography column separator application device.

(5) FIG. 5 Chromatography columns comprising one (a), two (b), and three (c) chromatography column separators.

(6) FIG. 6 UV-absorption elution diagram of a chromatography of erythropoietin with a Vydac C4 chromatography material, whereby the chromatogram a) is obtained with a chromatography column comprising no chromatography column separator according as reported herein and chromatogram b) is obtained with a chromatography column comprising one chromatography column separator as reported herein.

(7) FIG. 7 Increase of the backpressure of a chromatography column comprising a chromatography column separator as reported herein in successive regeneration cycles of a multi-use chromatography column.

(8) FIG. 8 Determination of the plate number of a Vydac C4 chromatography column.

(9) FIG. 9 UV-absorption Q-sepharose elution diagram of a chromatogram of an IL13 receptor alpha antibody: a) SP-sepharose cation exchange chromatography; b) Q-sepharose anion exchange chromatography.

(10) FIG. 10 Analytical SEC elution diagram: a) diagram of the analysis of the product peak of FIG. 9; b) diagram of the product peak of a Hybrid-Chromatography with the SP-sepharose in the upper chamber of the Hybrid-Chromatography column and the Q-Sepharose in the lower chamber of the Hybrid-Chromatography column; c) diagram of the product peak of a Hybrid-Chromatography with the Q-sepharose in the upper chamber of the Hybrid-Chromatography column and the SP-Sepharose in the lower chamber of the Hybrid-Chromatography column.

(11) FIG. 11 Diagram showing resulting column (back) pressure vs. set mobile phase flow for an anion exchange chromatography material (DEAE Sepharose). Diamonds: chromatography column without a separator as reported herein; triangles: chromatography column with separator as reported herein. It can be seen that a reduction of back-pressure for higher bed heights can be achieved with a separator as reported herein.

EXAMPLE 1

Fermentation and Purification of Erythropoietin

(12) The Fermentation and purification of Erythropoietin was carried out as reported in European patent No. 1 064 951 B1. The data presented herein were obtained in the reversed phase HPLC on a Vydac C4 chromatography material as reported in Example 1d) of EP 1 064 951.

(13) The RP-HPLC material Vydac C4 (Vydac) consists of silica gel particles, the surfaces of which carry C4-alkyl chains. The separation of Erythropoietin from the proteinaceous impurities is based on differences in the strength of hydrophobic interactions. Elution is performed with an acetonitrile gradient in diluted trifluoroacetic acid. Preparative HPLC is performed using a stainless steel column (filled with 2.8 to 3.2 liter of Vydac C4 silica gel). The Hydroxyapatite Ultrogel eluate is acidified by adding trifluoro-acetic acid and loaded onto the Vydac C4 column. For washing and elution an acetonitrile gradient in diluted trifluoroacetic acid is used. Fractions are collected and immediately neutralized with phosphate buffer.

EXAMPLE 2

Production of an Anti-(IL-13R1) Antibody

(14) An anti-IL13 receptor alpha antibody was produced according to the data and methods reported in WO 2006/072564, especially in accordance with Examples 10 to 12.

(15) Sequential-Chromatography

(16) For the ion exchange chromatography the protein A eluate comprising the anti-IL13R alpha antibody is adjusted to pH 6.5 and applied to a SP-Sepharose cation exchange chromatography column that has been equilibrated with 10 mM potassium phosphate buffer. After a wash step with 10 mM potassium phosphate buffer the antibody is eluted with a 50 mM potassium phosphate buffer pH 6.5. The pH value of the SP-Sepharose eluate is adjusted to pH 7.1 and applied to a Q-Sepharose anion exchange chromatography column that has been equilibrated with 35 mM potassium phosphate buffer. The purified antibody is obtained from the flow-though of the anion exchange chromatography column.

(17) Hybrid-Chromatography

(18) For the Hybrid-ion exchange chromatography with a chromatography column containing a chromatography column separator according to the current invention the protein A eluate comprising the anti-IL13R alpha antibody is adjusted to pH 7.1 and applied to the Hybrid-Chromatography column that has been equilibrated with 10 mM potassium phosphate buffer. After a wash step with 10 mM potassium phosphate buffer the antibody is eluted with a 20 mM potassium phosphate buffer pH 7.1.