SE459095B - Fixed biologically active materials - Google Patents

Abstract

An immobilised biologically active substance is described. The active substance is immobilised on a silica carrier. The biologically active material is carried on silicon gel or glass particles of 2-200 microns esp. 5-10 microns. The pore size of the silica material can be up to 10000 Angstroms esp. 1000-4000 Angstroms. The carrier is used as a packing material is a alumin. The active material has an affinity for immunoglobulins e.g. protein A, protein G, antibodies and antigens. Used for immunological purposes e.g. analytical and preparative purposes e.g. for diagnosis and monitoring during the course of an illness by isolation of the immunoglobulin fraction. The substance is used as a column packing. (Provisional Basic previously advised in WeekK18).

Description

The objects are achieved according to the invention with the substance and the column and the method, respectively, which are defined in the following claims and which will be described in more detail in the following.

Description of the invention According to the invention there is provided an immunoadsorbent for HPLAC consisting of an inorganic, porous support to which covalently attached adsorption-specific ligands, characterized in that the support consists of silica with a particle size of 2-200 μm and a pore size of 1000-4000 Å or titanium oxide with a particle size of 2-200 μm and a pore size of 500-1000 Å, and the ligands are Protein A, Protein G, Conglutinin, C1q, Fc receptors and C3b receptors.

Preferred examples of silica materials according to the invention are particles of silica gel or glass, preferably with sizes of 5-10 microns.

The titanium oxide preferably has sizes of 20-50 μm.

The invention also relates to the use as a filler in an HPLAC column of an immunoadsorbent according to claim 1.

The invention can be used in a process for the separation of immunoglobulins in a liquid, in particular a blood liquid, wherein the liquid is passed through a column with an immunoadsorbent according to claim 1 with affinity for said immunoglobulins.

In the following, the invention will be elucidated by means of concrete exemplary embodiments using protein A (biologically active substance) immobilized on preferred examples of carriers according to the invention for separation of immunoglobulins.

Preparation of epoxy silica gel: 11.2 g of silica gel (Li Chrospher SI 4000, 10 pm, E. Merck, Darmstadt, West Germany) dried at 160 ° C, suspended in 375 ml of Na-dried toluene and 275 μl of triethylamine. 8 ml of V -glycidoxypropyltrimethoxysilane were added and the mixture was refluxed under N for b h.

The epoxysilanized silica was filtered through a glass filter and washed with acetone and ether (500 ml each). The silica gel was dried 3 ~ 459 095 overnight at 110 ° C.

The concentration of epoxy groups was determined in a known manner according to the method described by R. Axén et al, Acta Chem. Scand., B 29, 471-474 (1975), in which 200 mg of silica gel sample was suspended in 2 ml of 3M sodium thiosulfate and 4 ml of H 2 O. The hydroxy ions formed were titrated with 1 mM HCl. The amount of epoxy groups was then estimated to be 13 pmol / g silica gel (dry).

Preparation of aldehyde silica gel: 8.5 g of epoxy silica gel were hydrolyzed with HCl (pH 2.0, 90-9500, 2 hours, 75 ml) to give dihydroxy groups (diol). The gel was filtered off on a glass filter and washed with H 2 O. The diol silica gel was suspended in 20 ml of periodate reagent (2.8 g of potassium periodate in 100 ml of H 2 O and 400 ml of glacial acetic acid). The oxidation was allowed to proceed for 1 hour at 22 ° C.

Then the aldehyde gel was washed on glass filters with H 2 O (250 mL) and dried overnight at QOOC.

Preparation of protein A covalently immobilized on deactivated silica gel: 44 g of dry aldehyde silica gel was suspended in 9.0 ml of 0.1M NaHCO 3 buffer (pH 8.3) containing 15.0 mg of protein A. 5 mg of Na (CN) BH 3 was added and the mixture was shaken on a tilting table for 48 hours (22 ° C).

The reaction was terminated by the addition of 2 x 3 mg NaBH 4 (20 min. Intervals). The protein A gel was filtered off and washed with 3 x 1 port. 1mM HCl, 0.1M NaHCO 3, pH = 8.3, and 0.5M NaCl, H 2 O.

The amount of immobilized protein A on silica gel was determined by acid hydrolysis of the bound protein with subsequent colorimetric analysis with ninhydrin of the amino acid content of the hydrolyzate. 50 mg of dry protein A silica gel was suspended in 1 ml of 6M HCl. The mixture was allowed to react at 90-9500 for 4 hours. After completion of hydrolysis, the mixture was neutralized to pH 5 with 6M NaOH. To the supernatant were added 0.5 ml of 0.2M citrate, pH 5.0 and 0.5 ml of 2% (w / v) ninhydrin (EtOH). The solution was incubated at 90-95 ° C for 1 hour. The resulting color development was measured spectrophotometrically at 570 nm.

Using this method, the protein A content of the gel was determined to be 2.9-3.0 mg protein A / g silica gel (dry). A reference column without protein A was prepared in an analogous manner as above.

Chromatography with protein Arsilica: About 2.3 g of protein A-silica (hereinafter referred to as pA-Si) "slurry" - packed in a column ('316' stainless steel, 200 x 5 mm) at 100-200 bar pressure. The packing medium used was 50 mM Na-phosphate, pH 7.5 (250 ml) The chromatography equipment consisted of a pump (Water Associates, 6000A), an injector (Waters, UGK) and a detector (UV, Waters 440A). 20-2200) with a normal pressure of 600 psi (1 ml / min.) And injection volumes 03; 0.01 -1ml.

To ascertain the function of pA- of dog IgG on pA-Si. A solution of canine IgG (0.5 mg / ml, 0.1M phosphorus pH 7.5) was applied continuously at 1.0 ml / min. The amount of IgG adsorbed is a good measure of the operating conditions. The non-specific adsorption- IgG on a reference column without pA is negligible (<1%) under corresponding conditions. Using this frontal analysis technique, the operative capacity of pA-Si (2.4 g, was estimated to be 29 mg IgG / g Si (dry). at step pH 2.5) amounted to> 95%.

Separation of immunoglobulins: A normal plasma (0.5 ml) was applied to the pA-Si column with 0.1 M phosphate, pH 7.5 (1 ml / min.) As a mobile phase.

IgG3 and most of IgM and IgA (> 90%) went undefeated in the front. de fraction on pA- dient (The adsorber- Si was eluted using a linear pH-gra- 0.1M citrate) from pH 5.0 to pH 2.5. A characteristic fraction pattern of immunoglobulins (in combination with any antigens) was obtained. Analysis with specific antisera revealed that IgG2 eluted at pH 4.6, IgG1 at pH 4.3 and a number of smaller immunoglobulin fractions in the pH range 3.8-2.6. What these represent is not yet clear, possibly they are aggregated immunoglobulins or immune complexes. IgG4 has not yet been identified. The resolution is satisfactory at the fast analysis time here, about 40 minutes. The analysis time can be reduced to about 10-15 minutes without "performance" significantly deteriorating.

For alternative coupling methods for the immobilization of the biologically active substance, for example protein A, to deactivated silicon or glass, see K. Mosbach, Methods in ENZYMOLOGY, vol. XLIV, 1976. Examples of such methods may be 1) Direct reaction, in which protein A binds to the epoxy groups of the matrix via the amino groups of the protein. 2) In a first step, the epoxy glass is hydrolyzed to the corresponding diol, which in turn is activated by bromocyanide. The activated gel then binds the amino groups of protein A. 3) The diol gel (prepared according to 2, above) can be tosylated either directly or after periodate oxidation. The tosyl activated gel can then react with the amino groups of protein Az. 4) The diol gel (according to 3, above) can be activated with carbonyldiimidazole, which then binds to amino groups. 5) The diol gel (according to 3, above) can, after activation with cyanuric chloride or derivatives thereof, bind the amino groups of protein A. 6) Epoxy, diol or aldehyde (periodate oxidation of diol gel) -based matrix can be modified with spacers of different functionalities, e.g. amino, carboxyl, thio. These mats with spacers can then be used for immobilization, where a number of established coupling methods are available.

Preparation of protein A immobilized on titanium oxide particles: 10 g of porous titanium oxide (1000 Å, 20-50 μm) was shaken in 100 ml of 1% SnCl 2 for 2 hours at 40 ° C. After filtration on a glass filter, the support was washed with 3 x 50 ml H 2 O. 5 g of the above-activated titanium oxide was suspended in 0.1M Na-phosphate, pH 7.5, containing 25 mg of protein A. The mixture was shaken on a tilting table at 35 ° C for 24 hours. The protein A-titanium oxide particles were filtered and washed. with 3 x 1 port. 0.5M NaCl resp. H20. 459 095 6 Industrial usability The invention is useful for HBLAC technology in immunological fields. This includes both analytical and, for example, methods for diagnosing the course of disease and eating, in particular a blood condition. The utility of the invention as preparative applications, nostics and monitoring and / or treatment of immunoglobulins in a liquid, for example plasma.

Claims (2)

51 459 095 attentkrav Immunosorbent for HPLAC consisting of an inorganic. porous support to which covalently bound adsorption-specific ligands are attached. characterized in that the support consists of silica with a particle size of 2-200 μm and a pore size of 1000-4000 Å or titanium oxide with a particle size of 2-200 μm and a pore size of 500-1000 Å. and the ligands consist of Protein A Protein G. Conglutinin. C lq. Fc receptors and C3 receptors. b Use as a filler in an HPLAC column of an immunosorbent according to claim 1 consisting of a silica or titanium oxide having a particle size of about 10 μm and a pore size of about 500-4000 Å. With covalently bound ligands of Protein A. Protein B, Conglutinin, C. Fc receptors and C3b receptors.