RU2389022C2 - Sorbent for removal of immunoglobulins - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to immunology, in particular to the sorbent for the removal of G-immunoglobulin from biological fluids and solutions containing immunoglobulins including plasma and human blood. Sorbent is a polymer matrix, covalently linked through a spacer with the ligand, substances as 6-aminocaproic acid, glutaraldehyde, remainder of 6-aminokaproila condensed with glutaraldehyde are used as a spacer, and synthetic di-and tripeptides containing two residues of aromatic amino acids are used as a ligand. The sorbent is a polymer matrix covalently linked to ligand represented by synthetic di-and tripeptide containing two residues of aromatic amino acids.

EFFECT: proposed sorbent has a simplified synthesis scheme with minimal use of toxic substances and organic solvents, provides reduction of sorption of serum albumin and other impurity proteins, increase of sorption capacity and specificity of the sorbent, in addition, the proposed sorbent may be subjected to sterilisation.

2 cl, 4 tbl, 7 ex

Description

The invention relates to biotechnology and medicine, in particular to immunology, and is intended for binding of immunoglobulins of various subclasses. The invention can be used both in biotechnology for the isolation of immunoglobulins from various biological fluids or solutions containing immunoglobulins, and in medicine for the removal of immunoglobulins from plasma / blood of patients with severe forms of autoimmune pathologies, such as systemic lupus erythematosus, rheumatoid arthritis, idiomatic thrombocytoma purpura [1], Guillain-Barré syndrome, myasthenia gravis, dilated cardiomyopathy [2, 3], when drug therapy is not effective or cannot be applied [4-7].

Isolation of immunoglobulins from various biological fluids is one of the urgent tasks of modern biotechnology and preparative biochemistry. Among the sorbents used to isolate immunoglobulins, the leading place is occupied by sorbents with immobilized protein A from the cell wall of Staphylococcus aureus. The main disadvantage of these ligands is the high cost, the lack of stability under sterilization conditions and the limited ability to bind individual subclasses of immunoglobulins G.

In clinical practice, sorbents with immobilized polyclonal antibodies to human immunoglobulins are most widely used for Ig apheresis (removal of immunoglobulins) (Ig Adsopak ^® , NPO POCARD, Russia; Ig Therasorb ^® , "Miltenyi Biotec" ^® , Germany), [5, 8], columns with immobilized protein a ( «Immunosorba» ^® and ^{«Prosorba» ®, «Fresenius HemoCare} », Germany) [9].

The main disadvantages of these sorbents: 1) the use of products of animal or bacterial origin in the production process (for all of the listed sorbents), 2) the inability to sterilize a product containing protein in the last stages of production, which requires the use of aseptic conditions throughout almost the entire process of column production also for all of the listed sorbents), 3) for some of the above sorbents - only a single use [9], 4) leakage of the biologically active ligand, which m It can lead to side effects (primarily for sorbents with immobilized protein A).

A column with immobilized peptides of 11–13 amino acid residues capable of binding immunoglobulins G Globaffin (Fresenius, Germany) containing 67 ml of an agarose gel with immobilized PGAM146 peptide was described [10]. Globaffin columns remove a wide range of human immunoglobulins and are designed for repeated use. The sorption capacity of the sorbent averages 17 mg of IgG per ml of gel [10]. Despite the fact that the columns were developed in 2003, new data on the use of columns in clinical practice over the past 5 years have not appeared.

A close analogue of the invention is an sorbent with immobilized tryptophan used in Asahi Medicals TR-350 columns [11]. The columns contain 350 ml of gel, which is tryptophan, immobilized on a polyvinyl alcohol-based polymer matrix. The main disadvantage of this sorbent is limited specificity, for example, a high level of sorption of fibrinogen.

A prototype of the present invention is a sorbent with a Sepharose-immobilized L-tyrosine derivative (N-benzyloxycarbonyl-L-tyrosine) [12]. The concentration of ligand on the matrix is 35 μmol / ml (15 mg / ml gel). The sorbent is capable of selectively binding immunoglobulins G from human blood plasma in a series of several chromatographic cycles. The maximum sorption capacity of the sorbent was 17 mg IgG per ml of gel. The ratios of immunoglobulins and albumin in the eluate from the column are 33.7 and 64.5%, respectively, and the number of immunoglobulins G sorbed from 10 ml of blood plasma is 9.5 mg per 2 ml of gel [12].

The main disadvantages of the prototype: 1) the complexity of the synthesis of the sorbent (for example, the synthesis scheme proposed in the prototype includes 4 stages, each of which uses organic solvents); 2) a high level of sorption of serum albumin - a transport protein; 3) in the event of a ligand leak, the toxicity of N-carbobenzoxithyrosine and its metabolites is unknown; 4) there have been no studies on the thermal stability of the sorbent (the possibility of sterilization by autoclaving).

The objective of the invention is to simplify the synthesis of the sorbent, minimizing the adsorption of albumin by the sorbent, increasing the security and capacity of the sorbent.

The problem is solved using covalently immobilized on an insoluble polymer matrix synthetic low molecular weight peptides from the series:

tyrosyl tryptophan (I),

tryptophil tyrosine (II),

tyrosyl-glycyl-tryptophan (III),

tryptophil-threonyl-tyrosine (IV).

Peptides are composed of amino acids of protein origin and have an affinity for human and animal immunoglobulins. Each peptide incorporates the remains of two aromatic amino acids. In the event of a ligand leak, peptides break down to amino acids of protein origin, which are widely represented in the human body and are components of solutions for parenteral nutrition [Russian Drug Register].

Peptides I-IV were synthesized by classical methods of peptide chemistry in solution. To block the α-amino group, benzyloxycarbonyl or tert-butyloxycarbonyl protection was used; tert-butyl ether was used to protect the lateral function of tyrosine. Condensation was carried out by the method of activated esters or mixed anhydrides. After the release of the final products with trifluoroacetic acid, the peptides were purified by HPLC to 96-98% purity and characterized by NMR spectroscopy. Covalent immobilization of peptides on a sorbent was carried out in two main ways: 1) by attachment of an amino group-containing ligand to a polysaccharide matrix previously activated with cyanogen bromide; 2) by attachment of an amino group ligand to an aminated polysaccharide matrix by condensation with glutaraldehyde, with preliminary amination of the polysaccharide matrix through stages of polymer oxidation with sodium periodate and subsequent addition of diamine derivatives.

A comparison of the inventive sorbent with the literature data of analogues and prototype are given in table 1, it also contains its own data comparing the sorption capacity of the proposed sorbent with commercially available analogues and prototype.

List of abbreviations

Boc is tert-butyloxycarbonyl;

Bu ¹ is tert-butyl;

Bzl is benzyl;

DMF — N, N′-dimethylformamide;

NMM is N-methylmorpholine;

ONSu N-oxysuccinimide ester;

ONp p-nitrophenyl ether;

Z is benzyloxycarbonyl;

Tos is n-toluenesulfonropyl;

DCHA - dicyclohexylamine;

TLC - thin layer chromatography;

HPLC - High Performance Liquid Chromatography

experimental part

In the synthesis, derivatives of L-amino acids from Bachem (Switzerland) were used. DMF was distilled over ninhydrin and barium oxide. For the extraction and crystallization of peptides, “h” and “hch” solvents were used.

The identity of the intermediate and final compounds was confirmed by TLC on Kiselgel 60 chromatographic plates (Merck, Germany) in the following solvent systems:

A - chloroform: methanol: 32% AcOH 15: 4: 1 B - chloroform: methanol: AcOH 9: 1: 0.5

Chromatogram substances were detected with chlorobenzidine reagent and ninhydrin.

Peptide hydrogenation was carried out in the presence of 5% palladium on charcoal (Pd / C, Merck, Germany).

Peptides were purified by HPLC on a column (25 × 250 mm) with C16T diasorb. The elution was carried out with a concentration gradient (0.5% per minute) of CH ₃ CN in 0.1% trifluoroacetic acid, a flow rate of 9 ml per minute, detection at 226 nm. The fractions corresponding to the target products were combined, evaporated. The residue was dissolved in water and lyophilized.

For immobilization on an insoluble matrix, 6-aminocaproyl derivatives of peptides were used. 6-aminocaproic acid is added to the structure as an auxiliary compound (spacer) that binds the ligand to the matrix.

For analytical HPLC, a Ultrasphere ODS column, 5 μm (4.6 × 200 mm) Beckman (USA) was used, buffer A — 0.1% trifluoroacetic acid and buffer B — 80% acetonitrile in buffer A were used as eluents. Flow rate 1 ml / min, detection at 220 nm. Gradient from 10% to 70% B in A in 30 minutes.

Example 1. 6-aminocaproyl-tryptophyl-tyrosine (I)

1.65 g (5 mmol) of Z-Trp-OH was dissolved in 25 ml of ethyl acetate, 0.55 ml (5 mmol) of NMM was added, cooled to -30 ° C, 0.65 ml (5 mmol) of isobutyl chloroformate was added. The reaction mixture was kept for 10 minutes at −30 ° C., 1.65 g (5 mmol) of HCl-Tyr · (Bu ^t ) OBu ^t and 0.55 ml (5 mmol) of NMM were added. The reaction mixture was kept for 30 minutes at room temperature (the completeness of the reaction was monitored by TLC; R _{f of the} target product 0.85 (A), 0.55 (B)). The reaction mixture was washed in a separatory funnel with 5% NaHCO ₃ , water, 5% citric acid solution, water, and was evaporated. The residue was dissolved in 50 ml of ethanol and hydrogenated. The catalyst was filtered off, washed on the filter with ethanol, the filtrate was evaporated, the residue was dissolved in 10 ml of ethyl acetate, cooled to -30 ° C (solution 1).

1.16 g (5 mmol) of Boc-ε-aminocaproic acid was dissolved in 25 ml of ethyl acetate, 0.55 ml (5 mmol) of NMM was added, cooled to -30 ° C, 0.65 ml (5 mmol) of isobutyl chloroformate was added. The reaction mixture was kept for 10 minutes at -30 ° C and added to solution 1 obtained previously. The reaction mixture was kept at room temperature for 30 minutes, 10 ml of n-butanol was added, washed in a separatory funnel with water, evaporated, the residue was crystallized from ethyl acetate, the precipitate was filtered off, the filter was washed with ethyl acetate, hexane, and dried. Received 2.14 g (yield 58%) of the product. The resulting product was dissolved in a mixture of 25 ml of trifluoroacetic acid and 0.25 ml of mercaptoethanol, kept for 1 hour at room temperature, evaporated, the residue was treated with ether, the precipitate was filtered off, washed on the filter with ether, and dried. The resulting product was purified by HPLC as described above. Received 1.28 g (yield 43% based on Z-Trp-OH) of compound (I) 98% purity according to analytical HPLC. R _t 18.15; R _f 0.24 (A).

¹ H-NMR spectrum (DMSO-d ₆ , δ, ppm):

6-aminocaproic acid - 2.00 (α-CH ₂ , 2H); 1.35 (β-CH ₂ , 2H); 1.11 (γ-CH ₂ , 2H); 1.43 (δ-CH ₂ , 2H); 2.68 (ε-CH ₂ , 2H); 7.60 (NH, 1H)

Trp 7.87 (NH, 1H); 4.57 (α-CH, 1H); 3.10 / 2.85 (β-CH ₂ , 2H)

Tyr 8.1 (NH, 1H); 4.36 (α-CH, 1H); 2.95 / 2.81 (β-CH ₂ , 2H); 9.13 (OH)

Example 2. 6-aminocaproyl-tyrosyl-tryptophan 7 (II)

2.04 g (10 mmol) of tryptophan was dissolved in a mixture of 30 ml of DMF and 5 ml of 40% Triton B (10 mmol) in methanol. The methanol was evaporated, the residue was cooled to -5 ° C (solution 2).

3.72 g (10 mmol) was dissolved in 25 ml of DMF, 1.11 ml (10 mmol) of NMM was added, cooled to -30 ° C, 1.3 ml (10 mmol) of isobutyl chloroformate was added. The reaction mixture was kept for 10 minutes at -10 ° C, solution 2 was added. The reaction mixture was kept for 30 minutes at room temperature (the completeness of the reaction was monitored by TLC; R _{f of the} target product 0.7 (B)), was evaporated, the residue was dissolved in water, acidified with 15% citric acid to pH 3.0, the product was extracted with 200 ml of ethyl acetate, washed in a separatory funnel with water, and evaporated. The residue was crystallized in hexane, filtered off, washed with hexane on the filter, and dried. Received 5.4 g (yield 97%). The resulting product was dissolved in 100 ml of ethanol and hydrogenated in the presence of 5% Pd / C. After completion of the hydrogenation (TLC: R _f 0.25 (A)), 5 ml of a 40% solution of Triton B in methanol was added to the reaction mixture. The catalyst was filtered off, washed on the filter with ethanol, evaporated, the residue was dissolved in 50 ml of DMF, and evaporated by half. To the resulting solution was added 2.6 g (8 mmol) of Boc-6-aminocaproic acid N-hydroxysuccinimide ester. The reaction mixture was kept at room temperature for 10 hours, evaporated, the residue was dissolved in 100 ml of water, extracted with ether, the aqueous layer was acidified with 15% citric acid to pH 3.0, extracted with ethyl acetate, washed with water until neutral, evaporated, the residue was treated with hexane . The precipitate was filtered off, washed on the filter with hexane, and dried. The resulting product was dissolved in a mixture of 25 ml of trifluoroacetic acid and 0.25 ml of mercaptoethanol, kept for 1 hour at room temperature, evaporated, the residue was treated with ether, the precipitate was filtered off, washed on the filter with ether, and dried. The resulting product was purified by HPLC as described above. Received 3.65 g (60% based on the first stage) of compound (II) 97% purity according to analytical HPLC. R _t 18.52; R _f 0.21 (A).

¹ H-NMR spectrum (DMSO-d ₆ , δ, ppm):

6-aminocaproic acid - 1.99 (α-CH ₂ , 2H); 1.36 (β-CH ₂ , 2H); 1.12 (γ-CH ₂ , 2H); 1.44 (δ-CH ₂ , 2H); 2.70 (ε-CH ₂ , 2H); 7.61 (NH, 1H)

Tyr 7.86 (NH, 1H); 4.47 (α-CH, 1H); 2.89 / 2.58 (β-CH ₂ , 2H); 9.13 (OH)

Trp 8.80 (NH, 1H); 4.47 (α-CH, 1H); 3.18 / 3.06 (β-CH ₂ , 2H)

Example 3. 6-aminocaproyl-tyrosyl-glycyl-tryptophan (III)

0.55 g (1 mmol) of Z-Tyr (Bu ^t ) -OH · DCHA was suspended in 50 ml of ethyl acetate, the organic layer was washed in a separatory funnel with 2% sulfuric acid, water, and evaporated to dryness. The residue was dissolved in 5 ml of DMF, 0.11 ml (1 mmol) of NMM was added, cooled to -30 ° C, 0.13 ml (1 mmol) of isobutyl chloroformate was added. The reaction mixture was kept for 3 minutes at -10 ° C, 0.38 g (1 mmol) of Tos · H-Gly-OBzl and NMM were added. The reaction mixture was kept at room temperature for 1 hour, evaporated, the residue was dissolved in 25 ml of ethyl acetate, washed in a separatory funnel with 5% NaHCO ₃ , water, 2% sulfuric acid solution, water, and evaporated. The residue was dissolved in 20 ml of ethanol and hydrogenated. The catalyst was filtered off, washed on the filter with ethanol, evaporated, the residue was dissolved in 10 ml of DMF, 0.11 ml (1 mmol) of NMM and 0.35 g (1 mmol) of Boc-ε-aminocaproic acid N-hydroxysuccinimide ester were added, and they were kept at room temperature for 18 hours.

Then the reaction mixture was evaporated, the residue was dissolved in ethyl acetate, the organic layer was acidified with 2% sulfuric acid, washed with water until neutral, and evaporated to dryness. The residue was dissolved in 25 ml of ethyl acetate, the resulting solution was applied to a column of 1.6 cm ² × 20 cm with silica gel 60, the product was eluted with ethyl acetate. After evaporation of the fractions corresponding to the target product, and crystallization of the residue in hexane, 0.45 g (86% yield) of the product are obtained.

The resulting product was dissolved in 10 ml of DMF, 0.11 ml (1 mmol) of NMM was added, cooled to -20 ° C, 0.13 ml (1 mmol) of isobutyl chloroformate was added. The reaction mixture was kept for 3 minutes at -5 ° C. A solution of tryptophan triton salt prepared from 0.21 g of tryptophan, as described above, was added cooled to -5 ° C. The reaction mixture was kept at room temperature for 1 hour, evaporated, the residue was dissolved in 25 ml of ethyl acetate, washed in a separatory funnel, 15% citric acid solution, water until neutral, and evaporated to dryness. 0.57 g of product was obtained as an amorphous powder. The resulting product was dissolved in 25 ml of trifluoroacetic acid and 0.25 ml of mercaptoethanol, kept for 1 hour at room temperature, evaporated, the residue was treated with ether, the precipitate was filtered off, washed on the filter with ether, and dried. The resulting product was purified by HPLC as described above. Obtained 0.36 g (yield 53.8%) of compound (III) 97% purity according to analytical HPLC. R _t 17.74; R _f 0.18 (A).

¹ H-NMR spectrum (DMSO-d ₆ , δ, ppm):

6-aminocaproic acid - 2.03 (α-CH ₂ , 2H); 1.38 (β-CH ₂ , 2H); 1.13 (γ-CH ₂ , 2H); 1.44 (δ-CH ₂ , 2H); 2.70 (ε-CH ₂ , 2H); 7.60 (NH, 1H)

Tyr 7.96 (NH, 1H); 4.41 (α-CH, 1H); 2.88 / 2.61 (β-CH ₂ , 2H); 9.14 (OH)

Gly 8.18 (NH, 1H); 3.78 / 3.63 (α-CH ₂ , 2H);

Trp - 8.09 (NH, 1H); 4.48 (α-CH, 1H); 3.18 / 3.04 (β-CH ₂ , 2H)

Example 4. 6-aminocaproyl-tryptophil-threonyl-tyrosine (IV)

0.12 g (1 mmol) of threonine was dissolved in 1 ml of 1 M NaOH, cooled to 0 ° C, a solution of 0.46 g of Z-Trp-ONp in 5 ml of DMF cooled to 0 ° C was added to the resulting solution. The reaction mixture was kept at room temperature for 18 hours, evaporated, the residue was dissolved in 50 ml of water, extracted with ethyl acetate-hexane 1: 1 (2 × 25 ml), the aqueous layer was acidified with 15% citric acid to pH 3.5, extracted with ethyl acetate , the organic layer was washed with water until neutral, evaporated to dryness, the residue was dissolved in 10 ml of DMF, 0.11 ml (1 mmol) of NMM was added, cooled to -20 ° C, 0.13 ml (1 mmol) of isobutyl chloroformate was added. The reaction mixture was kept for 5 minutes at −10 ° C., 0.33 g (1 mmol) of HCl Tyr (Bu ^t ) OBu ^t and NMM were added. The reaction mixture was kept at room temperature for 1 hour, evaporated, the residue was dissolved in 25 ml of ethyl acetate, washed in a separatory funnel with 5% NaHCO ₃ , water, 5% citric acid solution, water, and evaporated. The residue was dissolved in 20 ml of ethanol and hydrogenated in the presence of 5% Pd / C (R _f 0.45 (A)). The catalyst was filtered off, washed on the filter with ethanol, evaporated, the residue was dissolved in 10 ml of DMF, 0.3 g (0.86 mmol) of Boc-ε-aminocaproic acid N-hydroxysuccinimide ester was added to it and kept at room temperature for 18 hours.

Then the reaction mixture was evaporated, the residue was dissolved in ethyl acetate, the organic layer was washed in a separatory funnel 5% NaHCO ₃ , water, 15% citric acid, evaporated, the residue was dissolved in a mixture of 10 ml trifluoroacetic acid and 0.1 ml mercaptoethanol, kept for 1 hour at room temperature, evaporated, the residue was treated with ether, the precipitate was filtered off, washed on the filter with ether, and dried. The resulting product was purified by HPLC as described above. Received 0.3 g (yield 42%) of the target product 97% purity according to analytical HPLC. R _t 17.66; R _f - 0.17 (A).

¹ H-NMR spectrum (DMSO-d ₆ , δ, ppm):

6-aminocaproic acid - 2.04 (α-CH ₂ , 2H); 1.44 (β-CH ₂ , 2H); 1.14 (γ-CH ₂ , 2H); 1.38 (δ-CH ₂ , 2H); 2.69 (ε-CH ₂ , 2H); 7.60 (NH, 1H)

Tyr 7.87 (NH, 1H); 4.40 (α-CH, 1H); 2.93 / 2.84 (β-CH ₂ , 2H); 9.20 (OH)

Thr 7.77 (NH, 1H); 4.24 (α-CH, 1H); 3.96 (β-CH, 1H); 1.01 (γ-CH ₃ , 3H)

Trp 8.01 (NH, 1H); 4.60 (α-CH, 1H); 3.13 / 2.93 (β-CH ₂ , 2H)

Example 5. Synthesis of sorbents with immobilized peptides. Synthesis of sorbent on a matrix activated by bromine cyan.

As an agarose matrix, commercial Sepharose 4 Fast Flow matrices and a matrix suitable for perfusion of Sepharose 6MB cells were used. In 15 ml of a 30% suspension of the agarose matrix in a solution containing 4 M KOH and 1.6 M KH ₂ PO ₄ , 1 ml of a solution of 0.6 g / ml BrCN in dioxane was added at a temperature of 5 °. The mixture was incubated for 10 min at the same temperature, stirring constantly. Then, the gel was washed with a ten-fold volume of distilled water on a glass filter using a Bunsen flask with a water-jet pump, and then a gel suspension was prepared in a buffer mixture of 0.2 M H ₃ BO ₃ -NaOH pH 8.0. A ligand solution was added to the resulting suspension at the rate of 20-30 μmol per ml of settled gel. Immobilization was carried out with continuous stirring at a temperature of 20 ° C. After 2 hours, the amount of bound ligand was 65-70% of the amount taken in immobilization. Then 1 ml of 1 M ethanolamine pH 8.0 was added and after 30 minutes the gel was washed with a ten-fold volume of water and then with a five-fold volume of a 0.1 M KH ₂ PO ₄ -NaOH buffer mixture with a pH of 7.0. The amount of immobilized ligand was estimated by the difference between the added amount and the amount in the first washings from the obtained sorbent.

Example 6. Synthesis of sorbents with immobilized peptides. Synthesis of a sorbent on a matrix activated with sodium periodate followed by amination and ligand condensation with glutaraldehyde.

The prepared 30% suspension of the washed agarose matrix in a 1% aqueous NaIO ₄ solution was incubated with stirring for 2 hours at 20 ° C. The gel was washed with a 20-fold volume of water on a glass filter. An equal volume of a 2 M aqueous solution of diaminohexane / diaminoethane was added to the squeezed gel. Incubated for 2 hours at 20 ° C. After 2 hours, the unbound ligand was removed on a glass filter and the sorbent was washed with a 20-fold volume of distilled water. A 2-fold volume of a 1.2% freshly prepared aqueous NaBH ₄ solution was added to the squeezed gel twice and incubated twice for 10 minutes. Then the gel was washed with a 10-fold volume of water, a 2-fold volume of a buffer mixture of 0.1 M Na ₂ CO ₃ -NaHCO ₃ pH 9.0, and an equal volume of the peptide solution in the same buffer and 25% glutaraldehyde solution were added at a rate of 30 μl per 1 ml of gel. The suspension was incubated with stirring for 2 hours at 20 ° C. Then, a twofold volume of a 1.2% freshly prepared aqueous NaBH ₄ solution was added. After an hour, it was washed with a 20-fold solution of distilled water. The amount of immobilized ligand was also controlled as described in claim 5.

It should be emphasized that in the above example 6, the synthesis of the sorbent is carried out mainly in aqueous solutions, and in the process of synthesis there are practically no toxic substances and organic solvents.

Example 7. Immobilization of the peptide on Toyopearl-AF65.

Toyopearl-AF65 epoxidized gel (Tosoh Bioscience, Japan) was washed with water (100 ml per 5 ml gel). To 1 g of the settled gel was added 1 ml of a 2 M solution of 1,6-diaminohexane and the mixture was stirred for 2 hours at a temperature of 50 ° C. Then, the resulting sorbent (Toyopearl-AF65-NH-C ₆ H ₁₂ -NH ₂ ) was washed with 50 ml of water. A solution of 6-aminocaproyl-Trp-Thr-Tyr peptide was prepared at a concentration of 17 mg / ml in a buffer mixture of 0.1 M NaHCO ₃ -NaOH with a pH of 9.6. To attach the peptide to 1 ml of the settled Toyopearl-AF65-NH-C ₆ H ₁₂ -NH ₂ gel, 1 ml of the peptide solution, 1 ml of dimethyl sulfoxide and 0.1 ml of a 2.6 M glutaraldehyde solution were added. The mixture was stirred for 2 hours at a temperature of 20 ° C. After 2 hours, 1 ml of freshly prepared 1% NaBH ₄ solution was added to the mixture, stirred for 30 minutes, then the gel was washed with 5 ml of water and then 1 ml of 0.1 M KH ₂ PO ₄ -NaOH pH 7.0. The amount of immobilized ligand was 30.2 mg per ml of gel.

Sorption capacity of the sorbent based on Sepharose with immobilized peptide.

When a solution containing immunoglobulins G is applied to a column with a sorbent with tryptophyl tyrosine (Trp-Tyr) peptide immobilized at a concentration of 30 μmol / ml gel (14.6 mg / ml gel), the amount of bound IgG is 19 ± 1 mg IgG per ml gel , which exceeds the same indicator for the prototype by 12%.

When applying human blood plasma to a sorbent with immobilized peptides under conditions similar to that described for the prototype [12], the number of bound immunoglobulins G is from 18.3 to 27.7 mg / ml of gel, in contrast to the prototype - 4.7 mg / ml gel (see table 2).

When passing 5 ml of human blood stabilized with sodium citrate at a final concentration of 0.6% through a chromatography column, 1 cm ² section containing 1 ml of Sepharose 6MB-6-aminocaproyl-TrpThrTyr gel, at a flow rate of 0.2 ml / min, the sorption capacity the sorbent was 11.5 ± 1.6 mg of IgG (table 2a).

Table 2. Sorption capacity of sorbents containing peptides as a ligand (bromine cyan immobilization variant). Ligand The concentration of the ligand on the sorbent (μmol / ml) Sorption capacity (mg IgG / ml gel) Sorption capacity (mg IgG / mol of ligand) one Tyr-Trp (I) fifteen 25.3 1,69 2 Trp-tyr (ii) 17 27.7 1,63 3 Tyr-Gly-Trp (III) fourteen 21.8 1,56 four Trp-Thr-Tyr (IV) 12 18.3 1,52

Table 2a. Removal of IgG on columns with Sepharose 6MB based sorbents in a hemosorption procedure (in vitro). Before chromatography After chromatography Sepharose 6MB-6-aminocaproyl-Trp-Thr-Tyr Sepharose 6MB-Trp-Thr-Tyr Sepharose 6MB Blood volume ml 5,0 6.0 6.0 6.0 Plasma volume, ml 3.0 4.0 4.0 4.0 IgG concentration, mg / ml 10.5 ± 04 5.0 ± 0.4 4.6 ± 0.3 7.2 ± 0.4 IgG total amount in the sample, mg 31.5 ± 1.2 20.0 ± 1.6 18.4 ± 1.2 28.8 ± 1.6 The amount of IgG bound to the column, mg per ml of sorbent gel - 11.5 ± 1.6 13.1 ± 1.2 3.0 ± 1.6

Data on hemocompatibility of the sorbent based on Sepharose 6MB in in vitro experiments with whole blood perfusion are shown in table 2b. A certain decrease in the concentration of formed elements (except for platelets) relative to the initial one occurs due to the effect of blood dilution with physiological saline during the chromatographic cycle.

Sorption capacity of the sorbent based on Toyopearl-AF65.

The sorption capacity of the sorbent with the ligand 6-aminocaproyl-Trp-Thr-Tyr based on the Toyopearl-AF65 matrix was 8.3 ± 0.4 mg of human IgG per 1 ml of gel, which is almost 2 times higher than the same indicator for the prototype. The use of a gel based on crosslinked polyvinyl alcohol as a matrix confirms the possibility of using polymer matrices different in nature from the agarose matrix (Sepharose) for the synthesis of sorbents containing the claimed peptides as a ligand.

Binding of immunoglobulins of various species origin from protein solutions.

When applied to a sorbent with 6-aminocaproyl-Trp-Tyr-Tyr synthesized, as described in Example 6, IgG solutions of various species origin (IgG concentration of 5 mg / ml in physiological saline), the amount of bound immunoglobulins was 23.4 mg of goat IgG, 13.9 mg of sheep IgG and 10.5 mg of bovine IgG per ml gel. As a control of the binding specificity of immunoglobulins, sorption of bovine serum albumin (BSA) and human serum albumin (HSA) from solutions with a protein concentration of 5 mg / ml in physiological saline was also checked. No sorbent binding of BSL and HSA was observed. Therefore, the ability of sorbents based on the claimed peptides to selectively bind immunoglobulins of various species origin from protein solutions is shown.

Table 2b. The study of blood cells after the hemosorption procedure on chromatographic samples based on Sepharose 6MB. Blood counts Blood before passing through hemosorbent Blood after passing through a sorbent Sepharose 6MB-6-aminocaproyl-Trp-Thr-Tyr Sepharose 6MB-Trp-Thr-Tyr Sepharose 6M White blood cells, * 10 ⁹ cells / l 5.45 ± 0.25 4.05 ± 0.26 4.05 ± 0.25 3.90 ± 0.35 Red blood cells, 10 ¹² cells / l 4.7 ± 0.2 3.35 ± 0.37 3.42 ± 0.29 3.39 ± 0.24 Hematocrit% 40.3 ± 0.5 28.5 ± 3.2 29.3 ± 3.5 29.2 ± 1.9 The average volume of the red blood cell, fl 86 ± 11 86 ± 11 86 ± 10 86 ± 9 Red blood cell heterogeneity (distribution width),% 10.5 ± 1.3 10.5 ± 1.4 10.2 ± 1.3 9.7 ± 1.6 The average hemoglobin content in the red blood cell, PG / CL 28.8 ± 1.7 28.6 ± 2.0 28.4 ± 1.9 28.7 ± 2.2 The average concentration of hemoglobin in the red blood cell, g / dl 33.4 ± 2.6 33.3 ± 2.5 33.2 ± 2.8 32.7 ± 2.7 Hemoglobin concentration, g / dl 13.4 ± 1.0 9.5 ± 0.9 9.7 ± 1.1 9.5 ± 1.1 Platelets, 10 ⁹ cells / l 165 ± 20 70 ± 18 85 ± 16 95 ± 18 Lymphocytes,% 33 thirty 25 27 Monocytes,% four 8 5 3 Basophils. % one 0 0 0 Eosinophils,% 2 3 one one Band neutrophils,% one one one 2 Segmented neutrophils,% 59 58 68 67

The specificity of binding of sorbents to immobilized dipeptides.

The specificity of the interaction of peptides with immunoglobulins, i.e. the ability to specifically bind immunoglobulins G from a mixture of proteins (human blood plasma) was determined by the method of “sorption in volume” with human blood plasma.

The presence of human immunoglobulins G in eluates was confirmed by studying the composition of eluates by enzyme-linked immunosorbent assay and vertical electrophoresis in a gradient of polyacrylamide gel (gradient of polyacrylamide from 4 to 15%) under denaturing conditions. Analysis of the electrophoregram showed that the main component of the eluate with the sorbent is class G immunoglobulins. The eluate does not contain significant amounts of serum albumin and other impurity proteins, whereas in the prototype the amount of sorbed serum albumin is 64% of the total amount of protein bound to the sorbent [21].

Sorbent stability during autoclaving.

The stability of the sorbents under sterilization by autoclaving was confirmed by measuring the sorption capacity of the sorbents on model solutions of human G immunoglobulins with samples of sorbents before and after sterilization in an autoclave under standard conditions for sterilization of liquid media (121 ° C, 1.1 Bar, 30 min).

The number of immunoglobulins bound to non-autoclaved and autoclaved samples of sorbents with the immobilized tryptophyl-tyrosine peptide (Trp-Tyr) for the periodical immobilization method is 19.5 ± 0.7 mg / ml gel and 15.2 ± 1.9 mg / ml gel , respectively; the drop in sorption capacity during autoclaving did not exceed 22%.

The main advantages of the proposed sorbent relative to the prototype are:

1) simplification of the synthesis of sorbents,

2) minimizing the use of toxic substances and organic solvents in the synthesis of sorbents,

3) the use of biocompatible natural amino acids,

4) the absence of sorption of the albumin transport protein,

5) a higher sorption capacity compared to the prototype,

6) the possibility of sterilization of the sorbent.

List of sources used

1. McMillan R. // Autoantibodies and autoantigens in chronic immune thrombocytopenic purpura. / Semin. Hematol. - 2000. - V.37 (3). - P.239-248.

2. Schulze K., Becker B.F., Schauer R., Schultheiss H.P. // Antibodies to ADP-ATP carrier - an autoantigen in myocarditis and dilated cardiomyopathy - impair cardiac function. / Circulation. - 1990 - V.81. - P.959-969.

3. Magnusson Y., Wallukat G., Waagstein F., Hjalmarson A., Hoebeke J. // Autoimmunity in idiopathic dilated cardiomyopathy. Characterization of antibodies against the β1-adrenoceptor with positive chronotropic effect. / Circulation. - 1994 - V.89. - P.2760-2767.

4. Muller J., Wallukat G., Dandel M., Bieda H., Brandes K., Spiegelsberger S., Nissen E., Kunze R., Hetzer R. / Immunoglobulin adsorption in patients with idiopathic dilated cardiomyopathy // Circulation. - 2000 - V.101. - P. 385-391.

5. Konovalov G.A., Belenkov Yu.N., Zvezdkin P.V., Chebyshev AM, Semin S.N., Kuznetsova Yu.V., Adamova I.Yu., Kipor S.G., Pokrovsky S. N. / Apheresis of immunoglobulins - a new approach to the treatment of severe forms of dilated cardiomyopathy. // Cardiology. - 2002. - T.6. - S. 92-96.

6. Moreso F., Poveda R., Gil-Vernet S., Carreras L., Garcia-Osuna R., Grino J.M., Alsina J. // Therapeutic immunoadsorption in Goodpasture disease. / Med. Clin. (Barc) - 1995 Jun. 10 .-- V.105 (2) - P.59-61.

7. Robinson J.A. // Apheresis in thoracic organ transplantation. / Ther. Apher. - 1999 Feb. - V.3 (1) - P.34-39.

8. Koll R.A. // Ig-Therasorb immunoadsorption for selective removal of human immunoglobulins in diseases associated with pathogenic antibodies of all classes and IgG subclasses, immune complexes, and fragments of immunoglobulins. / Ther. Apher. - 1998 May. - V.2 (2). - P.147-152.

9. Euler H.H., Schwab U.M., Schroeder J.O., Hasford J. // The Lupus Plasmapheresis Study Group: rationale and updated interim report. / Artif Organs. - 1996 .-- V.20 (4). - P.356-359.

10. Ronspeck W., Brinekmann R., Egner R., et. al. // Peptide based adsorbers for therapeutic immunoadsorption. / Ther Apher Dial. - 2003. - V.7 (1). - P.91-97.

11. Hirata N., Kuriyama T., Yamawaki N. Immusorba TR and PH. Ther Apher Dial. 2003 Feb; 7 (1): 85-90.

12. Feng H., Jia L., Li H., Wang X. // Screening and chromatographic assessing of a novel IgG biomimetic ligand. / Biomed Chromatogr. 2006; 20 (10): 1109-15.

Claims (2)

1. Sorbent for removing class G immunoglobulins from biological fluids and solutions containing immunoglobulins, including from human plasma or blood, which is a polymer matrix covalently linked through a spacer to a ligand, characterized in that substances from the series are used as a spacer: 6-aminocaproic acid, glutaraldehyde, the remainder of 6-aminocaproyl condensed with glutaraldehyde, and synthetic peptides from the series: tyrosyl tryptophan, tryptophil tyrosine, tyrosis are used as a ligand l-glycyl-tryptophan, tryptophil-threonyl-tyrosine. 2. A sorbent for removing class G immunoglobulins from biological fluids and solutions containing immunoglobulins, including from human plasma or blood, which is a polymer matrix covalently linked to a ligand, characterized in that synthetic peptides from the series: tyrosyl are used as a ligand tryptophan, tryptophil-tyrosine, tyrosyl-glycyl-tryptophan, tryptophil-threonyl-tyrosine.