JPS6115684A - Wholly porous support coated with spacer molecules - Google Patents

Abstract

PURPOSE:The titled support, which is suitable for use as a precursor of a filler for high-speed affinity chromatography, is adjusted to certain levels in alcoholic hydroxyl content, water retention and specific surface area to facilitate the separation and purification of substances from living bodies. CONSTITUTION:A wholly porous support coated with molecular spacer which has 1-14mmol/g of alcoholic hydroxyls originating from vinyl alcohol units, a water retention of 0.5-4g/g and a specific surface area of 5-1,000m<2>/g and a crosslinked structure of copolymer with spacer molecules covalently bonding to the base polymer. The spacer molecules have 2 or more hydrophilic groups, linear structure, but no strongly ionic groups. The copolymer is crosslinked using a crosslinking monomer with a triazine ring.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a fully porous carrier with spacer molecules. More specifically, the present invention relates to a fully porous, hard spacer molecule-attached carrier suitable primarily as a precursor for a filler for high-speed affinity chromatography.

[Conventional technology]

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, 1) method using solubility difference, l) method using charge difference, R1) molecular size,
Examples include a method that utilizes a difference in shape, and a method that utilizes a difference 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 ( Co-authored by Part Chibata, Tetsuya Tosa, and Yushi Matsuo, “Affini Teikuro 1 Togerahui” (see Kodansha).

Conventionally, for affinity chromatography, 1. t-
Many have been used in which diaminohexane or the like is immobilized as a spacer molecule. In particular, agarose has been widely used (even under the trade name Sepharose, Pharmacia, 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.

[Problem that the invention seeks to solve]

By the way, it is preferable to immobilize the ligand molecules on the carrier by a method using covalent bonding. The ligand molecule may be covalently bonded directly to the active group on the carrier, or alternatively, a linear molecule called a spacer molecule is covalently bonded to the active group, and then the ligand is attached to the end of the spacer molecule. Molecules may also be covalently bonded. In order to effectively perform affinity chromatography, it is preferable to insert a spacer molecule between the carrier and the ligand molecule. For this purpose, a spacer molecule having a functional group at the end that can covalently bond with the ligand molecule is immobilized.
A carrier with so-called spacer molecules is required.

1) A spacer molecule with an appropriate molecular length is 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. i) A ligand molecule can be covalently attached to the end of a spacer molecule immobilized on a carrier without losing its biological affinity; li) The cis-spacer density can be varied depending on the purpose. ■) When carrying out chromatography with a ligand molecule bound, the non-specific adsorption of the carrier should be slightly reduced so that only the target substance is specifically adsorbed. 1. The carrier is not destroyed during the immobilization operation; vl) It is porous; 1. (vll) When performing chromatography, characteristics such as resistance to changes in solvents, denaturing agents, temperature, and non-rotation during storage are desired. Affinity chromatography is often carried out 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, ethylene oxide sterilization, heat sterilization, or 1-m 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. It is expected to be effective for

[Means for solving problems]

Therefore, when used as a precursor for a packing material for high-speed affinity chromatography (hereinafter referred to as packing material), 1
As a result of intensive studies to develop a particularly effective carrier, it was found that the carrier has an alcoholic hydroxyl group of 1.0 to 440 mmol, l.
The amount of water that can be held is 0.2 to μOf/f, and the specific surface area is 3
-1000-, and it has been found that a carrier with spacer molecules made of a crosslinked copolymer having a structure in which spacer molecules are covalently bonded is suitable, and the present invention has been completed.

The spacer molecule-attached carrier of the present invention has an alcoholic hydroxyl group of 1.0 to 4 AO mmol/? Included within the range of Among the alcoholic hydroxyl groups, hydroxyl groups derived from vinyl alcohol units are preferred. By containing hydroxyl groups within this range, the filler obtained from the spacer molecule adduct has hydrophilic properties and exhibits hydrophobic adsorption and distribution of many water-soluble substances in water. The amount of hydroxyl groups is practically ↓ 1.
j = / 1. It is preferable that it be in the range of Omm+)Vf.

The amount of hydroxyl groups can be determined from the amount of acetic anhydride consumed by reacting the hydroxyl groups with acetic anhydride, or by measuring 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.

The amount of hydroxyl groups when the dried carrier with spacer molecules /f is reacted with /mmol/acetic anhydride is /mmol
/? shall be.

In the following physical property measurements, if the functional group at the end of the immobilized spacer molecule reacts under the measurement conditions, the cough functional group should be protected and measured in the same way as when measuring the amount of hydroxyl groups. You can learn about physical properties.

The spacer molecule covalently bonded to the spacer molecule-attached carrier is 0.00/~3000 pmoVf, preferably
It exists in the range of 0.00/~J j 011moVf. If the spacer molecules are bound in this range, a packing material suitable for the purpose of high-speed affinity chromatography can be produced.

The amount of immobilized spacer molecules can be determined by, for example, when the spacer molecules are amino acids or peptides, after hydrolyzing the spacer molecule-attached carrier with tN hydrochloric acid, etc., the amount of amino acids in the supernatant is measured using an amino acid analyzer, etc. 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 spacer molecule-attached carrier preferably have λ 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 ligand molecule immobilized at the end of the spacer molecule and one target substance. Usually, the spacer molecule has one 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 the number of carbon atoms/~IO. Examples of typical spacer molecules include:
6-aminohexane powder, β-alanine, 1. A-diaminohexane, bis(3-aminopropyl)amine, glycine, glycylglycine, glycylglycylglycine,
Examples include succinamide ethylamine, succinamide butylamine, aminoethanol, and co-2p-diaminodiethyl ether.

A single spacer molecule or two or more types of spacer molecules may be bound to the spacer molecule-attached carrier.

The spacer molecule-attached carrier of the present invention is made of a crosslinked copolymer. Although the crosslinked structure is not particularly limited, a structure crosslinked by a crosslinkable monomer unit having a triazine ring, or a structure formed by bonding epichlorohydrin or a bisepoxy compound to a hydroxyl group is preferable. Among these, a structure crosslinked with 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 represents a structure formed by polymerization or copolymerization of monomers represented by the following formulas (4) and (B).

lζ3 (5) fB) (However, R1, R2 and R3 are all independently -C
Particularly preferred are triallylisocyanurates in which Hz CH=CHt.

The proportion of crosslinkable monomer units in all monomer units forming the carrier with spacer molecules is within the range V expressed by the following formula (1).
C6 is preferred.

b O, Oj□≦O≠ (1) a + n b (where a is the molar fraction of monomer units excluding crosslinkable monomer units in the carrier with spacer molecules, and b is the crosslinkable monomer unit) (The molar fraction of body units, n is the number of functional groups such as polymerizable vinyl groups in the 7 molecules of the crosslinkable monomer.) If necessary, the left side of equation (1) is more preferably 0.2.

In order to balance the separation ability and strength of the packing material obtained from the carrier with spacer molecules, it is necessary to maintain the water retention amount within an appropriate range.
It is necessary. Conventionally, fillers obtained from cross-linked agarose or cross-linked dextran have high water content and low mechanical strength, as described above, and this tendency is particularly large for fillers with large pore sizes. The filler obtained from the spacer molecule-attached carrier of the present invention has a water content of 0.00% regardless of the pore size. ! -IAOf/f, preferably O15-3
．． 0f/f range, and has sufficient strength to be used as a packing material for high-speed affinity chromatography. The water holding capacity of a carrier with spacer molecules is expressed as the amount per weight of the carrier with spacer molecules.The copolymer that has been sufficiently swollen in water is placed in a centrifuge tube equipped with a filter, and the water adhering to the surface of the copolymer is centrifuged. After that, the copolymer is dried and the weight can be determined from the change in weight before and after drying.

The specific surface area of the carrier with spacer molecules is usually 5 to 100.
0rr? /f range. That is, the spacer molecule-attached carrier of the present invention has permanent pores, and since it is hard, the pores shrink somewhat when dried, but maintain their swollen state.

Although there are various methods for measuring the specific surface area, in the present invention, it was determined by the most common BET method using nitrogen gas.

In addition, the sample used for specific surface area measurement must be sufficiently dry. The carrier of the present invention has high hydrophilicity and is difficult to dry.
It is best to dry under reduced pressure at temperatures below ℃.

The shape of the spacer molecule-attached carrier of the present invention is not particularly limited, and can be in any shape such as granules, membranes, threads, and lumps depending on the method of use. When used as a precursor for a filler for high-speed affinity chromatography, granular pellets are preferred.

Although the particle size in that case is not particularly limited, it is usually in the range of 3 to 000 pm in terms of weight average particle size. When used as a packing material for high-speed affinity chromatography, 3~,
It is preferably in the range of 20 pm, more practically 3 to jpm.

Next, an example of a method for manufacturing the spacer molecule-attached carrier of the present invention will be described.

In the spacer molecule-attached carrier of the present invention, for example, the 10-chain ester group of a copolymer consisting of a vinyl carboxylic ester and a crosslinkable monomer having a triazine ring is converted into a hydroxyl group by saponification or transesterification reaction. After conversion, it can be obtained by reacting a spacer molecule with an active group of an activated carrier obtained by reacting a part of the hydroxyl group with an activating reagent.

Here, carboxylic acid vinyl ester refers to a compound having one or more polymerizable carboxylic acid vinyl ester groups, such as vinyl acetate, vinyl prepionate, vinyl butyrate,
Selected from vinyl valerate and vinyl pino diphosphate, either alone or in combination of 211 or more. Among them, vinyl acetate and vinyl propionate are particularly preferred from the viewpoint of ease of polymerization, transesterification, saponification, and availability.

However, the cross-linking monomer having a triazine ring is represented by the above formula (Alt + 81. Among them, R+, R2 and R1 are all CH2C
Triallylisocyanurate in which H=CH2 has good copolymerizability with vinyl acetate and high stability against transesterification and saponification, and is therefore preferred as a crosslinking agent.

Polymerization to obtain a copolymer consisting of carboxylic acid vinyl ester and a crosslinkable monomer having a triazine ring can be carried out by a conventional polymerization method such as suspension polymerization, bulk polymerization, or emulsion polymerization. Suspension polymerization is preferred when obtaining a spacer molecule adduct as a precursor of a filler for high-speed affinity chromatography.

The use of monomers other than carboxylic acid vinyl ester and crosslinkable monomers having a triazine ring in combination to the extent that they do not significantly affect the physical properties of the copolymer can be advantageous for the spacer molecule-attached carrier of the present invention. There is no problem with that.

In addition, when copolymerizing carboxylic acid vinyl ester and a crosslinkable monomer having a triazine ring, the copolymer obtained by adding seven or more organic solvents that dissolve the monomer to the monomer can be used. The pore size, pore size, or pore size distribution of the pores is controlled while forming the coalescing permanent pores. Specifically, organic solvents that dissolve monomers include aromatic hydrocarbons such as toluene and xylene, aliphatic hydrocarbons such as heptane, octane, cyclohexane, and decalin, and n-acetic acid.
Aliphatic esters such as butyl, 1so-butyl acetate, n-hexyl acetate, azide, dioctyl pinate, aromatic esters such as dimethyl phthalate, dioctyl phthalate, methyl benzoate, butanol, heptatool, octatool, etc. alcohol, etc. When carrying out suspension polymerization, a solvent that is difficult to dissolve in water is preferred. These organic solvents are used in an amount of 20 to 300 parts by weight based on 700 parts by weight of the monomer. In particular, when producing a support with spacer molecules as a precursor for a packing material for high-speed affinity chromatography that requires mechanical strength, it is preferable that the amount of organic solvent be in the range of 30-/'00 parts by weight. .

In order to control the pore size and pore size distribution of the carrier with spacer molecules, a linear polymer or rubber that is soluble in the monomer mixture is added to the monomer mixture in order to increase the flexibility of the carrier with spacer molecules. Good too. Refers to linear polymers and rubbers that dissolve in monomer mixtures, such as polyvinyl acetate, polystyrene, chloroprene rubber, butadiene rubber, etc.
It is used in an amount of 20 parts by weight or less, preferably 70 parts by weight or less, based on ioo parts by weight of the monomer.

The type and amount of the initiator used during polymerization can be arbitrarily selected depending on the polymerization method. In normal suspension polymerization and bulk polymerization, common radical polymerization initiators, such as J, 2
'-Azobisin butyronitrile 1.2. ．． Azo-based initiators such as 2'-azobis-(λ, dimethylvaleronitrile) and peroxide-based initiators such as benzoyl peroxide and lauroyl peroxide can be used.

Transesterification or saponification of the copolymer obtained by polymerization is carried out using an acid or alkali in water, alcohol, or a mixture thereof as a solvent. However, in order to obtain a filler with sufficient mechanical strength, the conversion rate of ester groups to hydroxyl groups must be 9%, that is, the reaction rate must be between /θ and 0.
It is best to control it so that it is within the range of The reaction rate can be controlled by selecting appropriate conditions by changing the reaction solvent, reaction temperature, or reaction time.

Examples of methods for obtaining activated carriers by introducing active groups into copolymers obtained by saponification and/or transesterification include the following methods. Saponification and/
For example, 1. /'-carbonyldiimidazole is reacted to obtain an activated support having an imidazolyl carbamate group.

The amount of imidazolyl carbamate group bonded is 1. It can be controlled by the amount of /'-carbonyldiimidazole, reaction temperature, etc.

As a method for reacting the active group on the activated carrier obtained as described above with a spacer molecule and immobilizing the spacer molecule by covalent bonding, the following method can be mentioned. A spacer molecule-attached carrier on which spacer molecules are immobilized can be obtained by adjusting a solution of spacer molecules to an appropriate volume, adding the above-mentioned activated carrier to the solution, suspending the solution, and stirring.

〔Effect of the invention〕

The spacer molecule support of the present invention is hard, and when used, for example, as a packing material for high-speed affinity chromatography, the eluent can be passed at a high flow rate, making rapid analysis possible.

Furthermore, the carrier with spacer molecules of the present invention is stable over a wide pH range, and has the advantage that it can be stably used without deterioration even under alkaline conditions, which cannot be applied to carriers having a silica gel skeleton.

Furthermore, since the carrier with spacer molecules 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.

Based on the above-mentioned characteristics, using the packing material obtained from the spacer molecule-attached carrier 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 Hinyl acetate 1009. ) Really in cyanurate Hiroma 4! t1 n-butyl acetate IOf, decalin p.

t and hi, 2. j'-Azobisisobutyronitrile 3
．． After putting the mixed liquid consisting of 2 and water rθOd in which a small amount of polyvinyl alcohol and sodium phosphorus solution dissolved into a three-hole flask equipped with a reflux condenser, a nitrogen inlet tube, and a stirring bar, stir thoroughly. , at 45℃/hour,
Further, suspension polymerization was carried out by heating at 7°C for 5 hours to obtain a granular copolymer. Next, after filtration for 1 hour, washing with water, and extraction with acetone, the mixture was stirred for 20 hours at 13°C in a JD three-necked flask equipped with a reflux condenser, a nitrogen inlet tube, and a stirring bar, together with two volumes of methanol in which caustic soda TJF had been dissolved. After carrying out a saponification reaction of the polymer, the particles were passed through, washed with water, and then dried.

The particles were placed in a beaker of l09tJOθ- and dried with molecular sheep≠A dioxane/θθ-11. /′
- Add 3.2119 carbonyldiimidazole and conduct the reaction at room temperature for lj 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 dry dioxane and then filtered with suction. The activated carrier is 2-aminohexanoic acid 2
7M sodium carbonate aqueous solution containing t, 2f* (PH10
,0)J,0 What is the average particle size of the carrier?
, oprnz hydroxyl group density has been m moV for a long time?・Carrier with spacer molecules, the amount of immobilized spacer molecules is uO
pmxrl/f・Carrier with spacer molecules, water retention capacity is 1.
1 water/f・Carrier with spacer molecules, surface area is 1I2
dl?・Used as a carrier with spacer molecules.

Example λ The granular copolymer obtained in Example 1 was subjected to a resaponization reaction and then placed in a beaker containing 10 ft-600 particles of acetone/00d, dried with Molecular Sheep IA.
1. /'-Carbonyldiimidazole Hiroshi θ21 was added thereto and a /Y separation reaction was carried out at room temperature with stirring to obtain an activated carrier having an imidazolyl carbamate group as an active group. t- as a spacer molecule in the same manner as in Example 1.
Aminohexanoic acid was reacted with the activated carrier to obtain a spacer molecule-attached carrier on which t-aminohexanoic acid was immobilized. The amount of immobilized 6-aminohexanoic acid was //μrno1/f/spacer molecule-attached carrier.

Example 3 The spacer molecule-attached carrier obtained in Example 1 was dried, and λ2 was obtained! Place in an O-beaker and add 0.2M2-
(N-morpholino)ethanesulfonic acid aqueous solution (pI (1
A7j)/jd, l-ethyl-5-(3-dimethylaminopropyl)carbodiimide hydrochloride O, was added, and the mixture was stirred at room temperature for 3θ minutes. p-aminopenzamidine hydrochloride 2119 was added to this suspension, and the pH was adjusted to 1-7.
After the suspension was adjusted to J, the suspension was transferred to a 10θ Erlenmeyer flask and stirred at room temperature for 2 q hours to obtain an adsorbent having l-aminohexanoic acid as a spacer molecule and p-aminobenzamidine as a ligand molecule. Ta. The obtained adsorbent was mixed with water, 0. It was washed in the following order: 7M aqueous sodium chloride solution containing OjN sodium hydroxide, 7M aqueous sodium chloride solution containing OO-N hydrochloric acid, and water. The amount of immobilized p-aminobenzamidine calculated from the absorbance at 2f and 2nm after collecting the washing liquid was calculated as follows: dry adsorbent/g/3μ
It was mot.

This adsorbent was packed into a Pyrex glass column (inner diameter 6 x length 70 m), and an aqueous solution containing jOrrM sodium phosphate and 100 mM sodium chloride (pH 7 μ
) as the mobile phase at room temperature and at a flow rate of 0. When bovine trypsin o and zpt were injected at jtrt/km, no protein with trypsin activity was eluted. Then add the mobile phase to
An aqueous solution containing rrM sodium phosphate, 1100rr sodium chloride, and 20mM &-amine hexanoic acid (
When pT(7,+) K was removed, a component mainly composed of λ proteins with trypsin activity was eluted.

The mobile phase pump is KHU, 2A% (Kyowa Seimitsu ■), and the detector is RF-
! 30 (@Shimadzu Corporation) Excitation wavelength, 213 nm.

Detection wavelength: 34' OnIns FD for detection of enzyme activity
-/10 (JASCO Corporation ■) The excitation wavelength was 3tjnm, and the detection wavelength≠AOnm was used. For detection of trypsin activity, the synthetic substrate t-butoxycarbonyl-L-glutamyl-L
The fluorescence of 7-amitouge methylcoumarin, which is released by the decomposition of -IJ sil-L-lysine-gumethylcoumaryl-7-amide with trypsin, was utilized.

An aqueous solution containing j0ITIM sodium phosphate and 100mM sodium chloride (pT
(74') t-mobile phase, room temperature, flow rate Oj 47m
So human plasminogen (Green Cross) 10μt polymer okiya urokinase (Green Cross) /! The activated mixture was injected using U for 3 min at 37 °C. Plasminogen, which has no enzymatic activity, was eluted only in the pass-through area, but the mobile phase was then changed to 10mM sodium phosphate, 100mM sodium chloride, and 20μt.
M l=-Aqueous solution containing aminohexanoic acid (pH 74'
), proteins with plasmin activity were eluted. The same materials as in Example 3 were used for the tube, equipment, synthetic substrate, etc.

Claims (4)

[Claims] (1) 1.0 to 1.0 alcoholic hydroxyl groups per polymer weight
14.0 mmol/g, and the amount of water that can be retained is 0.
5-4.0g/g, specific surface area 5-1000m^3/g
A fully porous spacer molecule-attached carrier made of a crosslinked copolymer having a structure in which spacer molecules are covalently bonded. (2) The fully porous carrier with spacer molecules according to claim 1, wherein the alcoholic hydroxyl group is a hydroxyl group derived from a vinyl alcohol unit. (3) The fully porous carrier with spacer molecules according to claim 1, wherein the spacer molecules have two or more hydrophilic functional groups, are linear, and do not have strong ionic groups. (4) The fully porous carrier with spacer molecules according to claim 1 or 2, wherein the crosslinked copolymer is crosslinked with a crosslinkable monomer unit having a triazine ring.