SU762917A1 - Method for preparing sorbent for hydrophobic chromatography - Google Patents

Description

The invention relates to methods for separating biopolymers and other molecules with hydrophobic groups and can be used in medicine, pharmaceutical and microbiological industries and in other industries where enzyme preparations are produced or used.

Hydrophobic chromatography is a new type of separation and purification of chemical compounds, especially biopolymers, and is based on their different ability to form hydrophobic bonds with the carrier. Hydrophobic chromatography is superior to other types of chromatographic separation (gel filtration, ion exchange chromatography, etc.) during purification and the separation of proteins, lipids and their 20 mixtures.

Current sorbents for hydrophobic chromate? The decanter is a synthetic or natural compound (agar-25, rosa, Sephadex, cellulose derivatives, polyacrylamide, porous glass, etc.) modified by attaching side groups of a hydrophobic character to them.

2

A method of obtaining a sorbent for hydrophobic chromatography by covalent bonding of a hydrophobic ligand to an activated carrier through an insert is known. Agarase spheres are used as a sorbent, bromine cyanide as an insert, and phenylalanine as the hydrophobic ligand on the least hydrophobic column and n-hexylamine on the most hydrophobic. In this method, it is proposed to use a series of columns filled with a carrier with hydrophobic ligands covalently attached to it to separate a mixture of proteins. The degree of hydrophobicity of the carrier, determined by the type of ligand, increases with the growth of the column number. As a result, the most hydrophobic proteins are adsorbed on the first column, less hydrophobic on the next, etc. Protein elution is carried out. 5% solution of ethylene glycol by water [ί "] ·

The disadvantages of this method are the complexity. Activation processes of the carriers and attachments of inserts and ligands to them, instability of the connection between the Ligand and the carrier,

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activated by cyanogen bromide, low degree of ligand hydrophobicity, multistage purification of biopolymers on these sorbents and the impossibility of using them for biospecific chromatography.

The purpose of the invention is to obtain a stable sorbent for hydrophobic and biospecific chromatography, increasing its holding capacity and simplifying the processes of activating the carrier and attaching an insert and a ligand to it.

This goal is achieved by using powdered polyamide-kapron as a carrier, glutaraldehyde as an insert, and phosphatidylethanolamine as a hydrophobic ligand.

The proposed method for obtaining a sorbent for hydrophobic chromatography consists in activating solid 20 carrier nylon — 4 N, hydrochloric acid solution at 45 ° C for 1-2 h, attaching to the carrier. Insert — glutaraldehyde — in 0.1 M borate buffer a solution with a pH of 8.59.5 at 4-20 ° C for 1-2 hours and subsequent binding with an insert of a hydrophobic ligand-phosphatidyl-ethanolamine in the same buffer solution for 10 to 48 hours.

The use of capron as a carrier allows one to simplify the processes of its activation and the insertion of an insert and a ligand to it, and the use of phosphatidyl ethanolamine as a ligand can increase the degree of sorbent hydrophobicity and use it for biospecific adsorption chromatography of phospholipases and lipases.

Activate the media. A portion of the polyamide carrier (nylon fabric, granules, yarn, powder, etc.) is suspended in 5-fold amount of 4 M hydrochloric acid solution and incubated for 1 h at 45 ° C, washed with water and 0.1 M borate buffer

solution (pH 8.5} and the suspension is separated by decantation.

Attachment, inserts. To a wet suspension of polyamide, add a 5-fold amount of a 12.5% solution of glutaraldehyde in the same buffer solution and leave it under stirring for 1-2 hours. At the same time, a part of glutaraldehyde reacts with side amino groups ногθ carrier with the formation of the Schiff base. Unreacted glutaraldehyde is washed with a 10-fold amount of the same buffer solution. The ligand attachment. To the suspension of the modified polyamide in 0.1 M '5 borate buffer solution (pH 9.6) add a 5-fold volume of the suspension of ligand-phosphatidylethanolamine with a concentration of 40 mg-ml in the same buffer solution (the ratio of phospholipid and carrier 1 : 5 per dry weight.). The mixture is kept under constant stirring for 2 days. As a result, a covalent bond is formed between the 25 amino group of the hydrophobic ligand and the free aldehyde groups of the modified carrier. Unbound phospholipids are removed by washing the sorbent with a 10-fold volume of distil ether.

Blocking free aldehyde groups of the sorbent. Ethanolamine at the rate of 0.35 ml per 1 g dry weight of the sorbent is added to the obtained sorbent, equilibrated in a 0.1 M borate buffer solution (pH 9.6), and kept under stirring for 24 hours at 4-20 C. Excess, ethanolamine is removed by washing the sorbent with a 5-fold amount of the same 40 buffer solution.

The shelf life of the obtained sorbent in a 0.1 M borate buffer solution with a pH of 8.5 for at least 1 month, and in the dried state is unlimited.

45 The structural fragment of the obtained sorbent for hydrophobic and biospecific chromatography is as follows:

1-Ν gsn- (SI _about \ -SI τΝ-CB ~ CH „

3 ¹ u 2 5

nastekh

where 8 8 ₂ - the remains of fatty acids,

containing 16-20 carbon atoms in the acyl part,

The resulting sorbent is used for hydrophobic chromatography as follows. Proteins sorption is carried out at a high concentration (1-3 m) '60 different neutral salts (e.g. MAC1, KC1, _and 50 at _4, ΝΗ ^ ϋΙ et al.) And the maximum possible temperature (30 ° C) desorbing the proteins osuschest- . are consistently conducted 65

ABOUT"

-o-pc

about

about

II

sn-o-s-k ’

I -3

SN „-O-С-Ко

2 .. II 2.

about

the following operations: a gradual decrease in temperature, a decrease in ionic strength, an increase in pH, and the use of a concentration gradient of solvents that weaken the hydrophobic interaction (for example, ethylene glycol) or surfactants (for example, dimethyl sulfoxide).

PRI me R 1. Separation and purification. human serum albumin. A 1.0 x 16 cm column is filled with a sorbent prepared as *

five

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described, and equilibrated with 1 M solution of KCl in a 0.05 M solution of Tris-HC 1 buffer, pH 7.5. 10 mg of human serum albumin (with a protein content of 2.75 mg) are passed through the column. The <sorption temperature is 25 ° C. Desorption is carried out by successively carrying out the following operations: reducing the temperature in the column to 15 ° C, reducing the ionic strength and washing the column with a gradient, dimethyl sulfoxide, 0-40%. In the results ^and Tate, human serum albumin is separated into fractions b differing degrees of hydrophobic interaction with the sorbent. The quantitative distribution of protein in fractions is 0.6, 0.2, 0.4, 0.3, 0.35, and 0.8 mg. The first three fractions are impurity proteins, and the last three are tetramer, dimer and monomer of serum albumin. Calculations 20

show that the column holding capacity is 0.99, i.e. 99.2% albumin binds to a hydrophobic carrier.

Example 2: Separation and purification of 25 hemoglobin. Sorption of hemoglobin is carried out as in example 1 (15 mg with a protein content of 7.5 mg).

Desorption is carried out by successively lowering the temperature of the column. to 15 ° C, reducing ionic strength, increasing the pH and washing the sorbent first with a concentration gradient of dimethyl sulfoxide (0-40%), then of ethylene glycol (0-10%). As a result, the hemoglobin preparation is divided into 5 fractions in the following quantitative distribution of protein: 1.2, 1.0, 0.9,

0.9 and 3.5 mg. The retention capacity of the sorbent is 0.98.

Example Separation and purification of phospholipase (acetone powder from cabbage leaves). The sorption of 50 mg of acetone powder of phospholipase D (with a protein content of 9.2 mg) is carried out analogously to example 1. Desorption is carried out as in example 2. The preparation of phospholipase D is divided into 6 fractions in the following proportion: 4, 3, 0.9, 0, 8, 0.8, 1.0 'and 1.4 mg. Holding capacity 0.95. , θ

The degree of purification of phospholipase D 50 times (the second fraction).

Thus, the proposed method for obtaining a sorbent for hydrophobic chromatography as compared with the known ones makes it possible to obtain a sorbent with a stable bond between the ligand and carrier, and the processes of activating the carrier and attaching the insert and ligand to it are simplified, the resulting sorbent can be used as a hydrophobic and for biospecific chromatography of phospholipases, the multistage purification of biopolymers is eliminated, the retention capacity of the sorbent with respect to proteins is increased and is 0.95-0.98 against 0.60 - 0.85 in the famous.

Claims (1)

Claim A method for producing a sorbent for hydrophobic chromatography by covalently attaching a hydrophobic ligand to an activated carrier through an insert, characterized in that, in order to obtain a sorbent for both hydrophobic and biospecific adsorption chromatography of biopolymers, increase the retention capacity of the sorbent and simplify the activation processes of the carrier and attach to inserts and ligand; powdered polyamide is used as a carrier; glutaraldehyde is used as an insert; hydrophobic fosfatidiletonolamin of ligand, the activation medium is carried out in a 4 N solution of hydrochloric acid at 40-5 (/ ⁵ C for 1-2 h, joining the insert to the carrier is carried out in reverse buffer solution at pH 8.5 - 9.5 at 420 ° C for 1-2 hours, and binding of the ligand to the insert is carried out in the same buffer solution for 10 to 48 hours.