The method of producing sorbents

 

(57) Abstract:

Usage: in the field of chromatography for the separation of biologically active substances (BAS). The inventive method of producing sorbents containing fotogruppe includes processing hydroxyl-containing matrices, pre-treated with epichlorohydrin in protectii alkaline (pH greater than 12). Phosphorylation conduct POC13or Na2HPO4at a temperature not exceeding 50oC. the Obtained sorbents combine high hydrodynamic characteristics with more effective performance. 2 C. p. F.-ly, 3 tables.

The invention relates to chromatography, and more specifically to methods for sorbents containing fotogruppe for separation of biologically active substances (BAS).

Postactivity widely and effectively used for separation and purification of various biologically active substances [1] the Lack of them are usually low hydrodynamic characteristics.

The prototype of the invention is a method of producing phosphocellulose [2] which consists in dispersing a dry powder of cellulose in dry pyridine followed by treatment of the POCl3at a temperature of 115aboutC.

is trillionare in boiling pyridine, which leads to the destruction of the matrix and change in its composition due to the enrichment with more low molecular weight fractions;

thus treated, the matrix contains fotogruppe different ionic strength and different chemical species, because not only formed mono-, and di - and triphosphopyridine group, which reduces chromatographic quality of the sorbent.

The use of sorbent in the form of a powder or particles of irregular shape complicates the process chromatography, because it is a clogged filter columns and shrinkage of the layer of sorbent, which requires their frequent perebivki also virtually eliminates the possibility of scaling processes chromatography.

Object of the invention is the simplification of phosphorylation and the simultaneous achievement of sorbents in the form of spherical granules with the aim of improving their mechanical and hydrodynamic characteristics.

The problem is solved by the introduction of pre-processing matrix, obtained in the form of spherical granules, with epichlorohydrin in the presence of alkali with a pH > 12 with subsequent processing fosforiliruyusciye agents at a temperature not exceeding 50aboutC.

The technical level of the claimed invention conditioned which is the stabilization of the structure of the matrix and activation of its reactivity. This allows the phosphorylation reaction under mild conditions, to obtain pellets of the sorbent in its metric (microspheric) sizes while maintaining its performance.

The combination of granulation processes with cross-linking with epichlorohydrin and epoxidation and subsequent phosphorylation of such polymers are not described in literature.

As a result, when using a pre-granular matrix (3, 4) the proposed method of obtaining spherical phosphoserine for chromatography of biological products allows you to create chromatographic material of high quality sorbents not only have the form of spherical granules, but:

only contain chemically equivalent phosphomonoesterase group that excludes additional nonspecific interaction matrices sorbents with biopolymers, reducing the effectiveness chromatography;

have large pores, which allows simultaneous gelfiltration biopolymers;

have elevated hydrodynamic characteristics;

allow the cleaning processes of biological products with high efficiency.

P R I m e R 1 (prototype). 100 g of dry pulp, suspender filytrovali, and the precipitate washed three times with pyridine. The residue hydrolyzed crushed ice, filtered, washed with water 4 times with 1% NaOH, washed with water residues of alkali, 4 times washed with 5% HCl followed by treatment with water to neutral pH, dried. The percentage of mono-, di - and triphosphates 70, 20 and 10, respectively.

P R I m m e R 2.

2.1. 100 ml spherical granulated cellulose obtained by the method [3] was placed in a flask with a stirrer, was added a mixture of 65 ml of 0.5 M NaOH solution with 3 ml of epichlorohydrin and was stirred for 1 h at t 60oC. the temperature was Then raised to 95aboutC and stirred for 1 h was Cooled to room temperature and washed granules until neutral with water on the filter. The exclusion limit of the sorbent for dextrans (GPC method) 4107D. Density D-glucose links from 0.2 to 1.0 mgecw/ml, the granule size of 80-120 microns.

2.2. 100 ml of the cellulose granules (2.1) was placed in a container with a filter, washed successively with two volumes of acetone (or ethanol), then chloroform or toluene) and introduced to 9.5 ml of POCl3then added 10 ml of dry pyridine or triethylamine. The mixture was left for 1 h at a temperature of 25-30about. The matrix was washed successively with chloroform, dioxane, then 50%-n is Krom-H), obtained by the method [4] and prepared according to example 2.1, suspended in 0.5 l of water at t 45-50oC, under stirring introduced 0,1-0,15 l of epichlorohydrin and 0.1 l 10-15 M aqueous NaOH solution for 2-3 h, filtered, the matrix was washed with water to neutral pH.

1 liter of prepared matrix was kept in 1 l of 0.5-1.0 N solution of Na2HPO4within 6-8 hours at a temperature of 40-50aboutC. the Results are given in table. 1.

P R I m e R 4. 100 ml of neutral gel (Biochrom-N 1000) obtained by the method [4] and prepared according to the method 2.1, was placed in a glass container with a porous filter, washed successively with two volumes of acetone (or ethanol), toluene (or chloroform) and introduced to 9.5 ml of POCl3then 10 ml of anhydrous pyridine or triethylamine. The mixture was left for 1 h at a temperature of 25-30aboutC. the matrix was washed successively with two volume, respectively, chloroform, dioxane, 50% aqueous solution of pyridine, 1% aqueous NaOH solution and water to neutral pH. Properties of the sorbent similar to those shown in table. 2.

P R I m e R 5. 100 ml spherical granulated cellulose obtained by the method [3] and prepared for modifications in example 2.1, suspended in 50 ml of water at those who e 2-3 hours The matrix was washed on the filter with water until neutral pH was kept in 100 ml of 0.5-1.0 n solution of Na2HPO4within 6-8 hours at a temperature of 40-50aboutWith, washed with water until neutral pH.

Properties experiments are shown in table. 2.

The size of the granules during the reaction has not changed. Thus obtained sorbents contain only monophosphatase group.

Physico-chemical characteristics of the sorbents in comparison with the prototype and analog (postkatrina R-11 company Whatman, England) were compared, passing through a column of 10 x 100 mm in 0.1 n NaCl solution at a pressure equal to 1 (in cm water column (see column heights).

The efficiency of the sorbents tested in the cleanup process lactoperoxidase (similar to R-11) and insulin.

P R I m e R 6. Column 10 x 100 mm was applied a solution of a mixture of proteins containing lactoperoxidase 2 ml of 0.03 N. Na-phosphate buffer (pH 7.5) at a flow rate of 20 ml/hour, the Column was washed with the same buffer, v, 15 ml, was suirable proteins in a linear gradient of 0-0,5 N. NaCl in the same buffer. Fractions 4.4 ml were collected and protein content was determined and the activity of the enzyme. The results are given in table. 3.

P R I m e R 7. Chromatographic purification of the reaction mixture after enzymatic the second mixture with a volume of 2.3 l, containing 3 l of phospho-Biochrom and balanced source buffer, 50 mm sodium acetate pH 4.0 and 3.5 M urea. After applying the column was washed 7,2 l source buffer (2 h) with a flow rate of 3.6 l/h While C-peptide, without resting on the column, came out first, followed suiryudan impurities, which in the starting conditions are sorbed on the column. Next aluinum protein in linear gradient from 0 to 100% buffer 50 mm sodium acetate pH 4.0, 5 M urea and 0.5 M NaCl against the source buffer for 7 hours (total volume of eluent 25 l). At the end of the elution column equilibrated with 9 l of the source buffer for the next chromatographic cycle. The fractions containing C-peptide without impurities collected in a volume of 2.5-12, the Fractions containing insulin without impurities are collected in a volume of 7.5 l 14, Purity obtained in this way C-peptide is 85-95% Purity thus insulin is 95%

Sorbent SP-Toyopearl 550 With firm Toson Corp, Japan, allow the separation of insulin with a purity of at least 95% of the protein in pure form in one chromatographic stage could not be obtained, because together with the protein With out any impurities, resulting fractions containing protein that is highly contaminated.

As shown by the Kie features with more effective performance.

1. The METHOD of producing SORBENTS containing fotogruppe, including the processing of hydroxyl-containing matrices fosforiliruyusciye agent, characterized in that the matrix is pre-treated with epichlorohydrin in the presence of alkali (pH > 12), and the process of postlarvae carried out at a temperature of not more than 50oC.

2. The method according to p. 1, characterized in that the phosphorylation is performed by processing the matrix POCl3.

3. The method according to p. 1, characterized in that the phosphorylation is performed by processing the matrix Na2HPO4.

 

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