JPH02668B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a fully porous spacer molecule-attached carrier. More specifically, the present invention relates to a completely porous and hard spacer molecule-attached carrier suitable primarily as a precursor of a filler for high-speed affinity chromatography. [Prior Art] In the field of biochemistry, separating proteins, enzymes, and other biological substances from mixtures containing them is one of the important issues, and a great deal of effort has been made in the past. Many methods are currently used to separate biological materials. For example, (i) a method that uses differences in solubility, (ii) a method that uses differences in charge, (iii) a method that uses differences in molecular size or shape,
(iv) Examples include methods that utilize differences in chemical or physical affinity. Furthermore, methods for separating, purifying, and removing substances using biologically specific affinity are highly selective and widely used. In particular, affinity chromatography, in which one substance exhibiting biological affinity (hereinafter referred to as coordination molecule) is immobilized on an insoluble carrier and the other is selectively separated, is widely used from the viewpoint of operability ( (See “Affinity Chromatography,” co-authored by Ichiro Chibata, Tetsuya Tosa, and Yushi Matsuo, Kodansha). Conventionally, for affinity chromatography, a natural insoluble carrier such as agarose or cellulose is activated with Promcyan or epichlorohydrin, and then 1,6-diaminohexane or the like is immobilized as a spacer molecule. I've been exposed to it. In particular, agarose has been widely used (for example, the product name Cepharose, Pharmacia Co., Ltd.,
Sweden). However, agarose has the following drawbacks. That is, agarose can hold an extremely large amount of water, and its strength when wet is insufficient, resulting in many operational limitations. For example, they have drawbacks such as being destroyed during operations such as activation and immobilization, and being unable to flow a liquid containing the target substance at a high flow rate when packed in a column. [Problems to be solved by the invention] By the way, immobilizing a ligand molecule on a carrier is
Covalent methods are preferred. The ligand molecule may be directly covalently bonded to the active group on the support, or a linear molecule called a spacer molecule may be covalently bonded to the active group, and then the ligand molecule may be attached to the end of the spacer molecule. may be covalently bonded. In order to perform Affinity chromatography effectively,
Preferably, a spacer molecule is interposed between the carrier and the ligand molecule. For this purpose, a so-called spacer molecule-attached carrier is required, on which a spacer molecule having a functional group at its end that can be covalently bonded to a ligand molecule is immobilized. A carrier with a spacer molecule has (i) a spacer molecule with an appropriate molecular length immobilized so that the ligand molecule can be immobilized at a position where the distance from the carrier is sufficient to interact with the target substance; (ii) the ligand molecule can be covalently attached to the end of the spacer molecule immobilized on the support without losing its biological affinity; (iii) the spacer density can be adjusted depending on the purpose. (iv) When performing chromatography with ligand molecules bound, non-specific adsorption of the carrier is small so that only the target substance is specifically adsorbed; (v) (vi) Porousness of the carrier during the operation of immobilizing the ligand molecules; (vii) Changes in the solvent, denaturing agent, and pH used when performing chromatography; Properties such as resistance to temperature and (viii) non-rotation during storage are desired. Furthermore, affinity chromatography is often performed by filling a column with a carrier on which ligand molecules are immobilized. In that case, sufficient mechanical strength is required to allow liquid to flow at a high flow rate. Further, in some cases, it may be necessary to perform freeze-drying and ethylene oxide sterilization, heat sterilization, or radiation sterilization, so it is desirable that the chemical structure of the carrier is not destroyed by these sterilizations. Here, high-speed affinity chromatography, which applies the principles of affinity chromatography to high-performance liquid chromatography, utilizes interactions specific to target components, so it can be used, for example, in clinical tests that focus on specific enzymes. It is expected to be effective for equipment, etc. [Means for solving the problem] Therefore, as a result of intensive studies to develop a carrier that is particularly effective when used as a precursor for a packing material for high-speed affinity chromatography (hereinafter referred to as packing material), , alcoholic hydroxyl group 1.0-14.0
mmol/g, and the amount of water that can be retained is 0.5 to 4.0
We have found that a spacer molecule-attached carrier made of a crosslinked copolymer having a structure in which spacer molecules are covalently bonded is suitable, and has a specific surface area of 5 to 1000 m ² /g, and has led to the present invention. . The spacer molecule-attached carrier of the present invention contains alcoholic hydroxyl groups in a range of 1.0 to 14.0 mmol/g. Among the alcoholic hydroxyl groups, hydroxyl groups derived from vinyl alcohol units are preferred. By containing hydroxyl groups in this range, the filler obtained from this spacer molecule carrier has hydrophilic properties and does not exhibit hydrophobic adsorption or partitioning of many water-soluble substances in water. The amount of hydroxyl group is practically 1.5 to 11.0 mmol/
It is better to be in the g range. The amount of hydroxyl groups can be determined by reacting the hydroxyl groups with acetic anhydride and measuring the amount of acetic anhydride consumed or the change in weight of the spacer molecule-attached carrier. At this time, if the functional group at the end of the immobilized spacer molecule also reacts, it can be determined by the above method after protecting the functional group.
1 g of dry carrier with spacer molecules is 1 mmol/
The amount of hydroxyl groups when reacting with g of acetic anhydride is 1 m
Let be mol/g. In the following physical property measurements, if the functional group at the end of the immobilized spacer molecule reacts under the measurement conditions, the physical properties can be determined by protecting the functional group in the same way as when measuring the amount of hydroxyl groups. You can know. The spacer molecules covalently bonded to the spacer molecule-attached carrier are present in an amount of 0.001 to 3000 μmol/g, preferably 0.001 to 250 μmol/g. This range of binding of spacer molecules makes it possible to create a packing material suitable for high-speed affinity chromatography purposes. The amount of immobilized spacer molecules can be determined by, for example, when the spacer molecules are amino acids or peptides, hydrolyze the spacer molecule-attached carrier with 6N hydrochloric acid, etc., and then measure the amount of amino acids in the supernatant using an amino acid analyzer. It can be found through analysis.
If a titratable functional group such as an amino group or a carboxyl group is present at the end of the spacer molecule, it can be determined by titration as a simple method. The spacer molecules immobilized on the carrier with spacer molecules preferably have two or more hydrophilic functional groups, are linear, and do not have strong ionic groups. The molecular length of the spacer molecule needs to be long enough to allow sufficient interaction between the target substance and the ligand molecule immobilized at the end of the spacer molecule. Usually, the spacer molecule has a methylene chain, amide bond, ether bond, etc. between the functional groups at both ends, and is composed of a molecule with a molecular length corresponding to 1 to 10 carbon atoms. Examples of typical spacer molecules include 6-aminohexanoic acid, β-alanine, 1,6-diaminohexane, bis(3-aminopropyl)amine, glycine, glycylglycine, glycylglycylglycine, and succinamide. Examples include ethylamine, succinamidobutylamine, aminoethanol, 2,2'-diaminodiethyl ether, and the like. A single type of spacer molecule may be bound to the spacer molecule-attached carrier, or two or more types of spacer molecules may be bound to the carrier. The spacer molecule-attached carrier of the present invention is made of a crosslinked copolymer. The crosslinked structure may be a structure formed by bonding epichlorohydrin or a bisepoxy compound with a hydroxyl group, but a structure crosslinked by a crosslinkable monomer unit having a triazine ring is preferable. Among these, a structure crosslinked by a crosslinkable monomer unit having a triazine ring such as triallyl isocyanurate or triallyl cyanurate is preferred. The crosslinkable monomer unit having a triazine ring is represented by the following formula:
It represents a structure formed by polymerization or copolymerization of the monomers shown in (A) and (B).

【formula】

[Formula] (where R ₁ , R ₂ and R ₃ are each independently −
CH ₂ −CH=CH ₂ , −CH ₂ −C≡CH or

〔Effect of the invention〕

The spacer molecule-attached carrier of the present invention is hard,
For example, when used as a packing material for high-speed affinity chromatography, the eluent can be flowed at a high flow rate, making rapid analysis possible. In addition, the carrier with spacer molecules of the present invention has a wide range of
It is stable within the pH range, and has the advantage that it can be used stably without deterioration even under alkaline conditions, which cannot be applied to carriers with a silica gel skeleton, for example. Furthermore, since the spacer molecule-attached carrier of the present invention has a large amount of hydroxyl groups, it has sufficient hydrophilicity and has the advantage of less hydrophobic adsorption to biological components and the like. By reacting the functional group at the end of the spacer molecule on the spacer molecule-attached carrier of the present invention with a ligand molecule, or by activating all or part of the functional group with an activating reagent or the like, the active group can be transferred to the spacer molecule. After forming a carrier with a spacer molecule at the end thereof, a filler on which a ligand molecule is immobilized can be obtained by reacting the active group with a ligand molecule. Examples of the ligand molecules include enzymes, substrates, competitive inhibitors, amino acids, oligopeptides, polypeptides, antigens, antibodies, and the like. Due to the above characteristics, the use of the packing material obtained from the carrier with spacer molecules of the present invention makes it possible to easily separate, purify, and remove biological substances, and also enables high-speed affinity chromatography. It has made a significant contribution to the field. The present invention will be explained in more detail in the following examples, but the present invention is not limited to the examples at all. [Example] Example 1 100 g of vinyl acetate, 45.4 g of triallyl isocyanurate, 80 g of n-butyl acetate, 40 g of decalin and 3.4 g of 2,2'-azobisisobutyronitrile
800ml of water in which a small amount of polyvinyl alcohol and sodium phosphate are dissolved.
3 equipped with a reflux condenser, nitrogen inlet tube, and stirring bar.
After stirring thoroughly in a three-necked flask, 65
Suspension polymerization was carried out by heating at 75°C for 18 hours and then at 75°C for 5 hours to obtain a granular copolymer. Next, after filtering, washing with water, and then extracting with acetone, it was placed in a 5-three-necked flask equipped with a reflux condenser, a nitrogen inlet tube, and a stirring bar with methanol 2 in which 65 g of caustic soda was dissolved.
After stirring at 15° C. for 20 hours to carry out a saponification reaction of the copolymer, the particles were filtered, washed with water, and further dried. Put 10g of the particles in a 500ml beaker, add 100ml of dioxane dried with molecular sieve 4A,
3.24 g of 1,1'-carbonyldiimidazole was added and the reaction was carried out at room temperature for 15 minutes with stirring to obtain an activated carrier having an imidazolyl carbamate group as an active group. The activated carrier was washed with the above-mentioned dry dioxane and then filtered by suction. The activated carrier was added to 200 ml of a 1M sodium carbonate aqueous solution (PH 10.0) containing 26.2 g of 6-aminohexanoic acid, and the mixture was reacted at 4°C for 25 hours with shaking to form 6-aminohexanoic acid as a spacer molecule. A carrier with immobilized spacer molecules was obtained. The average particle size of the carrier with spacer molecules is 9.0 μm, the hydroxyl group density is 4.9 mmol/g, the carrier with spacer molecules,
The amount of immobilized spacer molecules is 40μmol/
g・Carrier with spacer molecules, water holding capacity is 1.9g water/
g・Carrier with spacer molecules, specific surface area is 42m ² /
g. It was a carrier with spacer molecules attached. Example 2 After carrying out the saponification reaction of the granular copolymer obtained in Example 1, 10 g of particles were placed in a 500 ml beaker, and 100 ml of acetone dried with molecular sieve 4A and 1.1'-carbonyldiimidazole were added. 4.05g
was added and the reaction was carried out for 15 minutes at room temperature while stirring.
An activated carrier having an imidazolyl carbamate group as an active group was used. 6-aminohexanoic acid as a spacer molecule was reacted with the activated carrier in the same manner as in Example 1 to obtain a spacer molecule-attached carrier on which 6-aminohexanoic acid was immobilized. The amount of immobilized 6-aminohexanoic acid was 119 μmol/
g. It was a carrier with spacer molecules attached. Example 3 2 g of the dried carrier with spacer molecules obtained in Example 1 was placed in a 50 ml beaker, and further
0.2M2-(N-morpholino)ethanesulfonic acid aqueous solution (PH4.75) 15ml, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
0.29 g was added and stirred at room temperature for 30 minutes. After adding 25.8 mg of p-aminobenzamidine hydrochloride to this suspension and adjusting the pH to 4.75, the suspension was transferred to a 100 ml Erlenmeyer flask and stirred at room temperature for 24 hours to use 6-aminohexane as a spacer molecule. An adsorbent having p-aminobenzamidine as an acid and a ligand molecule was obtained. The obtained adsorbent is water, 0.05N
1M sodium chloride aqueous solution containing sodium hydroxide, 1M sodium chloride aqueous solution containing 0.05N hydrochloric acid,
Washed with water. Collect the washing liquid and analyze its 292nm
The amount of immobilized p-aminobenzamidine calculated from the absorbance at is 13 μmol per 1 g of dry adsorbent.
It was hot. This adsorbent was packed into a Pyrex glass column (inner diameter 6 mm x length 10 cm), and an aqueous solution (PH7.4) containing 50 mM sodium phosphate and 100 mM sodium chloride was used as the mobile phase at room temperature at a flow rate of 0.5 ml/
When 0.5 μg of bovine tribusin was injected at min, no protein with trypsin activity was eluted. Then the mobile phase was 50mM sodium phosphate, 100mM
When switching to an aqueous solution (PH7.4) containing sodium chloride and 20mM 6-aminohexanoic acid, components mainly composed of two proteins with trypsin activity were eluted. In addition, as a mobile phase liquid pump, KHU26
1/2 (Kyowa Seimitsu Co., Ltd.), the detector used is RF-530 (Shimadzu Corporation) for protein detection, excitation wavelength 285 nm,
Detection wavelength 340nm, FD-110 for enzyme activity detection
(JASCO Corporation) Excitation wavelength 365nm, detection wavelength
460nm was used. For detection of trypsin activity, 7-amino-4-, which is released by decomposition of the synthetic substrate t-ptoxycarbonyl-L-glutamyl-L-lysyl-L-lysine-4-methylcoumaryl-7-amide by trypsin, is used. The fluorescence of methylcoumarin was used. Example 4 An aqueous solution (PH7.4) containing 50mM sodium phosphate and 100mM sodium chloride was used as a mobile phase in the column packed in Example 3 at room temperature and at a flow rate of 0.5ml/min.
A mixture of 10 μg of human plasminogen (Midori Juji) activated with 15 U of high molecular weight urokinase (Midori Juji) at 37°C for 3 minutes was injected. Plasminogen, which has no enzyme activity, was only eluted in the pass-through area, but when the mobile phase was subsequently changed to an aqueous solution (PH7.4) containing 50mM sodium phosphate, 100mM sodium chloride, and 20mM 6-aminohexanoic acid. , a protein with plasmin activity was eluted. Note that the same equipment, synthetic substrates, etc. as in Example 3 were used.

Claims (1)

[Claims] 1. It has alcoholic hydroxyl groups derived from vinyl alcohol units from 1.0 to 14.0 mmol/g per polymer weight, the amount of water that can be retained is from 0.5 to 4.0 g/g, and the specific surface area is from 5 to 1000 m ² /g. It has a structure in which spacer molecules that are linear, have two or more hydrophilic functional groups, and do not have strong ionic groups are covalently bonded.
A fully porous carrier with spacer molecules for use as a packing material for high-speed affinity chromatography, which is made of a crosslinked copolymer crosslinked with a crosslinkable monomer unit having a triazine ring.