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Chromatography medium

Inactive Publication Date: 2011-02-24
GE HEALTHCARE BIOPROCESS R&D
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0018]The size exclusion property of the medium is determined by the porosity of one or both of its porous constituents. The certain size referred to above is below the size that excludes the target molecules / organism / particles such as cells, cell particles, virus, virus like particles, plasmids, any type of antibodies, lipids, proteins, peptides and nucleic acids. This medium will efficiently separate low size molecules from larger molecules of the type mentioned above.
[0024]Preferably, the core entity has a pore size preventing molecules over a certain size from entering the pores. Alternatively, or in addition thereto, a polymeric hydrogel e.g. dextran is provided in the lid to fill the pores and thereby further decrease and adjust the pore size to prevent high molecular weight molecules from entering the pores.
[0027]The invention also relates to a separation medium which comprises the lid bead medium described above in mixture with conventional chromatography media, such as HIC, IE or affinity media. Preferably the lid bead medium comprises up to 10% of the total media volume. In a preferred embodiment of this mixed media, the lid bead medium comprises octyl ligands and the chromatography media comprises a cation exchange media. Benefits of this mixed media are increased resolution between large and small molecules.

Problems solved by technology

One of the most challenges faced in the purification of mAbs is their separation from host cell proteins (HCPs) in the cell culture media.

Method used

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  • Chromatography medium

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of Octyl Media Based on SEPHAROSE™ 20 Fast Flow (A), SEPHAROSE™ 6 Fast Flow (B), and Spinning Disc Media (C)

[0053]Volumes of matrix refer to settled bed volume and weights of matrix given in gram refer to suction dry weight. For large scale reaction stirring is referring to a suspended, motor-driven stirrer since the use of magnet bar stirrer is prompt to damage the beads. Small-scale reactions (up to 20 mL of gel) were performed in closed vials and stirring refers to the use of a shaking table.

[0054]Conventional methods were used for the analysis of the functionality and the determination of the degree of allylation, epoxidation, or the degree of substitution of ion exchanger groups on the beads.

A: Preparation of Lid-OH core-octyl SEPHAROSE™ 20 Fast Flow

A1: Preparation of SEPHAROSE™ 20 Fast Flow

[0055]Agarose (50 g) was dissolved in water (250 g) by heating at 95° C. for approximately 10 hours. The solution was added to toluene (375 mL) and ethyl cellulose (35 g) in an e...

example 2

Chromatographic Evaluation of the Three Prototypes Based on Octyl Ligands in the Core of the Beads

[0074]The three different octyl media to be investigated (Prototypes: lid-OH core-octyl SEPHAROSE™ 20 Fast Flow, lid-OH core-octyl spinning disc and lid-dextran octyl core SEPHAROSE™ Fast Flow), with respect to breakthrough capacity, were packed in HR 5 / 5 columns and the sample solution was pumped at a flow rate of 0.3 or 1.0 mL / min through the column after equilibration with buffer solution. The breakthrough capacity was evaluated at 10% of the maximum UV detector signal (280 nm). The maximum UV signal was estimated by pumping the test solution directly into the detector. The breakthrough capacity at 10% of absorbance maximum (Qb10%) was calculated according to the formula:

Qb10%=(TR10%−TRD)×C / Vc

where TR10% is the retention time (min) at 10% of absorbance maximum, TRD the void volume time in the system (min), C the concentration of the sample (4 mg protein / mL) and VC the column volume ...

example 3

Purification of Influenza Virus

[0125]When producing influenza virus at large scale aiming at influenza vaccines it is important to reduce the levels of protein and DNA in the final preparation.

[0126]The particles of the present invention are well suited for the purification of viruses since viral particles are significantly larger in size than most of the contaminants.

[0127]This is illustrated in the following example.

Analysis Methods

Virus Concentration

[0128]The DotBlot HA assay was used according to a standard protocol.

Dna Concentration

[0129]The PICOGREEN® DNA assay was used according to the manufacturers instruction (available from Invitrogen).

Protein Concentration

[0130]The Bradford protein assay was used according to the manufacturers instruction. (Available from Bio-Rad)

Agarose Gel Electrophoresis for Analysis of Molecular Weight Distribution in DNA Sample.

[0131]An E-GEL® Agarose Gel 0.8% (Invitrogen) precast gel was used according to the manufacturers instructions

[0132]The DNA ...

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Abstract

The present invention is within the field of chromatography. More precisely, it relates to a novel chromatography medium, namely a hydrophobic medium provided with different lids excluding molecules over a certain size due to the porosity of the hydrophobic medium and / or the porosity of the lid. The invention also relates to use of the separation medium for purification of large molecules, which do not enter the separation medium, as well as small molecules, which enter the separation medium and are eluted from there.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a filing under 35 U.S.C. §371 and claims priority to international patent application number PCT / SE2009 / 050406 filed Apr. 21, 2009, published on Oct. 29, 2009 as WO 2009 / 131526, which claims priority to application number 0800923-5 filed in Sweden on Apr. 22, 2008.FIELD OF THE INVENTION[0002]The present invention is within the field of chromatography. More precisely, it relates to a novel chromatography medium, namely a hydrophobic medium provided with different lids excluding molecules over a certain size due to the porosity of the hydrophobic medium and / or the porosity of the lid.[0003]Within biotechnology, the chromatographic methods suggested up to date are based on different modes of interaction with a target. Thus, for example, in ion-exchange chromatography, the functional groups are permanently bonded ionic groups with their counter ions of opposite charge, while in hydrophobic interaction chromatography (HIC),...

Claims

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Application Information

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IPC IPC(8): C12N7/02H01F1/00C12N1/20C12N15/63B01J20/00B01J20/26C07K16/00C07K1/14C07H21/00C11B3/00
CPCB01D15/327B01J20/28009B01J20/285B01J20/3212B01J20/3293B01J39/26B01J20/287C07K1/20C07K16/00C12N7/00C12N2760/16151G01N1/405
Inventor BERGSTROM, JANGLAD, GUNNARJOHANSSON, BO-LENNARTMALOISEL, JEAN-LUCNORRMAN, NILSSODERMAN, TOBIAS E.
Owner GE HEALTHCARE BIOPROCESS R&D
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