Packing material for liquid chromatography and process for separation and purification of biopolymer by means of the packing material

A technology for liquid chromatography and packing materials, which is applied in the field of packing materials for liquid chromatography, can solve the problems of reduction, the protein adsorption capacity is not improved, and it is impossible to increase the adsorption capacity, so as to improve the binding capacity, effectively separate and purify or concentrate and recycling

Active Publication Date: 2010-08-04
TOSOH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0020] In addition, also in hydrophobic interaction chromatography and reversed-phase partition type chromatography, as in the case of the above-mentioned packing material for ion exchange chromatography, it is possible to introduce a hydrophobic group on the graft polymer, but maintain the protein (including peptide Under the solvent conditions of ), the grafted polymer with the introduced hydrophobic groups undergoes coalescence and shrinkage, in which the protein adsorption capacity is substantially not improved, or can be reduced considerably
Therefore, it is impossible to increase the adsorption capacity by adsorption and desorption, or by chromatography, based on the use of hydrophobic bonds of this type of packing material (eg, Patent Document 5)

Method used

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  • Packing material for liquid chromatography and process for separation and purification of biopolymer by means of the packing material
  • Packing material for liquid chromatography and process for separation and purification of biopolymer by means of the packing material
  • Packing material for liquid chromatography and process for separation and purification of biopolymer by means of the packing material

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Embodiment

[0116] Now, the present invention will be described in further detail based on examples, but it should be construed that the present invention is by no means limited thereto. In addition, various substrates used in the following Examples and Comparative Examples are supports (hydrophilic substrates) having alcoholic hydroxyl groups on their surfaces. The performance of the matrix with respect to the porous structure under aqueous conditions was evaluated by exclusion limiting molecular weight and porosity. The measurement method is as follows.

[0117] Measurement of Exclusion Limit Molecular Weight and Porosity

[0118] By using an aqueous gel slurry solution of a hydrophilic matrix, the matrix was put into a stainless steel column with an inner diameter of 10.7 mm and a length of 150 mm at the highest packing density, and then, the packed column was installed on a RI-8020 detector (by TOSOH CORPORATION) HPLC system (manufactured by TOSOH CORPORATION).

[0119] Then, by us...

preparation example 1

[0125] A polymethacrylate porous filling material having alcoholic hydroxyl groups on the surface [by TOYOPEARL HW-60C (manufactured by TOSOH CORPORATION)] was repeatedly suspended with a dioxane solvent and filtered on a glass filter to remove moisture, and was filtered by suction The dispersion solvent in such filler material slurry is removed to prepare a drained gel mass.

[0126] 50 g of gel mass and 100 ml of dioxane were added to a 300 ml separatory bottle and stirred. 60 mmol of CDI was dissolved in 30 g of dioxane, and the CDI solution was added dropwise to the separatory bottle at a constant temperature of 30°C. After the dropwise addition, stirring was continued for 1 hour. Then, the slurry was filtered with a glass filter, and the gel was washed with dioxane solvent to remove unreacted CDI or by-products, thereby synthesizing a CDI-activated drained gel piece.

[0127] The entire amount of the resulting gel mass was added again to a 300 ml separating bottle and 1...

preparation example 2

[0132] The drained gel block activated by CDI was synthesized in the same manner as in Preparation Example 1. The entire amount of the resulting gel mass was added again to a 300 ml separating bottle, and 100 ml of DMF was added, followed by stirring. 24 mmol DL-phenylalanine and 6 mmol ethanolamine were dissolved in 25 ml of 1 mol / L sodium hydroxide aqueous solution, 50 ml of DMF was added and mixed. This amino acid solution was put into a separatory bottle at one time and stirred to perform a reaction at room temperature for 16 hours.

[0133] After the reaction, the obtained gel was washed again in this order with DMF, 50% acetone, 0.1 mol / L sodium hydroxide and pure water on the glass filter. The gel obtained from this reaction is referred to as filling material 2. Its ion exchange capacity was measured in the same manner as in Preparation Example 1, and was found to be 80 meq / liter. The introduction amount of the phenylalanine ligand of the filling material 2 correspon...

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Abstract

To provide a novel packing material for liquid chromatography capable of separating and purifying, or collecting and recovering, a biopolymer such as a protein or a peptide by adsorption and desorption by a pH change without being influenced by the isoelectric point of the protein or by the salt concentration in a solvent in which the biopolymer such as the protein is dissolved, and to provide a process for concentrating and recovering a desired biopolymer such as a protein or a peptide from a large amount of dilute cell culture solution by means of such a packing material. Separation and purification, or collection and recovery, of a biopolymer, is carried out by liquid chromatography by means of a packing material for liquid chromatography comprising a base matrix and a ligand immobilized to the base matrix, wherein the base matrix is a hydrophilic base matrix having alcoholic hydroxy groups on its surface, the ligand is at least one ligand selected from the group consisting of an +--amino acid represented by the following formula (1): RCH(NH 2 )COOH (1) wherein R is an aromatic group or a C 5-7 non-ionic aliphatic group, and an aminomethyl benzoic acid, and the ligand is immobilized to the base matrix by an amide bond or an urethane bond via the amino group contained in the compound represented by the formula (1).

Description

technical field [0001] The present invention relates to a packing material for liquid chromatography suitable for separating and purifying or collecting and recovering ionic substances dissolved in an aqueous solution, particularly biopolymers such as proteins or peptides, and to methods for adsorbing and desorbing proteins through the packing material . [0002] More specifically, the present invention relates to a packing material for liquid chromatography for separating and purifying biopolymers such as proteins or peptides by utilizing the relationship between the hydrophobic groups of the packing material and the hydrophobic groups on the surface of the biopolymer. The interaction between adsorbs the solute polymer in the acidic aqueous solution, and then makes the pH of the eluent become neutral or slightly alkaline to make the packing material hydrophilic, so that the adsorbed biopolymers such as proteins or peptides are desorbed or eluted for recovery and involve meth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01D15/08B01J20/286B01J20/30C07K1/22
Inventor 小宫克夫中村孝司
Owner TOSOH CORP
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