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Separation method and separation matrix

A technology for separating matrices and matrices, which is used in the ion exchange separation of proteins, separation matrices with high alkali stability, and the preparation of such matrices, which can solve problems such as changing pH.

Active Publication Date: 2016-07-06
CYTIVA BIOPROCESS R&D AB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most commonly eluted by increasing salt concentration, but changing pH is also possible

Method used

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  • Separation method and separation matrix

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 11

[0080] The grafting of embodiment 1.1-vinyl-2-pyrrolidone (VP) and vinylsulfonic acid (VSA)

[0081] 1a) Allylation of Sepharose Bead Support

[0082] 50 g of highly rigid, drained agarose beads (prepared according to the method described in US6602990) were added to a 500 ml round bottom flask and mixed by stirring in 101.4 g of 50% aqueous NaOH and 77.04 g of distilled water. The temperature was raised to 50°C for 30 minutes. Thereafter, allyl glycidyl ether (AGE) was added in an amount to produce the AGE / reactant volume ratio according to Table 2, and the reaction was continued overnight at 50° C. for 17 hours. The beads were then filtered off and washed with ethanol (5x300ml) followed by distilled water (5x300ml).

[0083] Agarose beads of 4 different pore sizes were used, corresponding to Kd values ​​of 0.50, 0.67, 0.77 and 0.80 for dextran Mw110 kDa. Using an AKTA equipped with an A-900 autosampler TM The detector is evaluated for Kd. Connect Shimadzu RI-Detector (RI...

Embodiment 2

[0093] Example 2. Evaluation of prototypes grafted with VP and VSA

[0094] Column Packing and Testing

[0095] Prototype media was settled and compressed in Tricorn 5 / 50 columns. After filling, the column was tested in 0.4M NaCl at 0.065 ml / min by injecting 10 μl of 2M NaCl with 3% (v / v) acetone. Registration A 280 、A 260 and conductance peaks and integrated.

[0096] An acceptable criterion for a successful filling is an asymmetry value between 0.8 and 1.5.

[0097] Mab sample preparation

[0098] The protein A purified Mab was buffer-exchanged to 50 mM sodium acetate + 10 mM NaCl (pH 5.25) using a HiPrep Desalting 26 / 10 column. Protein concentrations were then determined spectrophotometrically.

[0099] by A 280 concentration determination

[0100] Protein concentration was determined spectrophotometrically at 280 nm using Lambert Beer's law (Equation 1).

[0101] Equation 1: C=A / (l*ε)

[0102] where C is the concentration (mg / ml), l is the path length ...

Embodiment 3

[0138] Example 3. Grafting of VP and 3-allyloxy, 2-hydroxyl-1-propanesulfonic acid (APS)

[0139] Place the agarose beads on a glass filter and wash several times with 40% APS solution (2x gel volume). The suspension was sucked dry, after which 75 g were added to a 250 ml three necked round bottom flask. 1.5 g ADBA was added to the flask along with 30 g APS solution and 25.5 ml VP. Polymerization was started at 65°C and stirred for about 17h. Wash the gel thoroughly with distilled water.

[0140] Measurement of Static Binding Capacity in 96-well Filter Plates

[0141] Using a pre-programmed Gilson automaton, 1% gel slurries were prepared and filled into 96-well filter plates corresponding to 2 microliters of settled gel / well.

[0142] Equilibration of the gel filled in the plate was performed by adding 200 microliters of equilibration buffer from the plate prepared in the Tecan automaton, followed by agitation at 1100 rpm for 1 minute, after which the buffer was removed by v...

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Abstract

The invention discloses a method of separating a biomolecule from at least one other component in a liquid, comprising a step of contacting said liquid with a separation matrix comprising a solid support and polymer chains bound to said solid support. The polymer chains comprise units derived from a first monomer of structure CH2═CH-L-X, where L is a covalent bond or an alkyl ether or hydroxysubstituted alkyl ether chain comprising 2-6 carbon atoms, and X is a sulfonate or phosphonate group.

Description

[0001] Technical Field of the Invention [0002] The present invention relates to the separation of biomolecules, and more particularly to the ion exchange separation of proteins. The invention also relates to separation matrices suitable for the separation of biomolecules and to methods for preparing such matrices. Background of the invention [0003] There are many situations where it is necessary to separate a compound, such as a contaminant or a desired molecule, from a liquid. Charge-charge based interactions are used in many fields to capture and thus separate charged or chargeable compounds. [0004] In the fields of chemistry and biotechnology, target compounds such as drugs or drug candidates often need to be separated from contaminating substances originating from the production process. For example, a protein drug or drug candidate produced by expression of a recombinant host cell will need to be isolated, for example, from the host cell and possible cellular debr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01D15/36B01J20/26B01J20/289B01J39/26G01N30/96
CPCB01J39/26B01D15/1864B01D15/34B01D15/361B01D15/362B01D15/3809B01J20/24B01J20/261B01J20/265B01J20/267B01J20/28014B01J20/28033B01J20/286B01J20/289B01J20/3085B01J20/3212B01J20/3278B01J20/3282B01J2220/80C07H1/08C07K1/22G01N2030/8827Y02P20/582
Inventor J.汉斯森G.罗德里戈T.E.塞德曼
Owner CYTIVA BIOPROCESS R&D AB
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