JPH01258739A - Affinity carrier - Google Patents

Abstract

PURPOSE:To increase compressive strength of an affinity carrier in a packed state by forming the affinity carrier from cellulose particles having each specified value of particle size in the wet state, crystallinity measured by X-ray diffraction method, and total volume of pores having pore sizes within a specified range, etc. CONSTITUTION:After neutralizing fine particles of coagulated viscose contg. 5-60wt.% (expressed in terms of cellulose) cellulose xanthate with an acid, generated fine cellulose particles are separated from mother liquid and used as the affinity carrier. The carrier consists of spherical or elliptical particles having <=500mum particle size in the wet state and 5-45% crystallinity measured by the X-ray diffraction method. Further, the particles dried at critical point have the maximum pore volume in the region between 0.006-1mum pore size measured by a mercury porosimeter method, wherein the total pore volume in the same region is at least 0.05 milliliter/g.

Description

[Detailed description of the invention] (Industrial application field) FIELD OF THE INVENTION The present invention relates to affinity carriers.

More particularly, it relates to a porous 7-finity carrier consisting essentially of regenerated cellulose.

(Prior technology) Affinity technology utilizes the specific affinity between biological substances for the purpose of specifically adsorbing, separating, and purifying trace substances produced in biological systems, or removing specific components from plasma preparations. Separation techniques are widely used.

As affinity carriers used for these purposes, affinity materials that do not non-specifically adsorb proteins, lipids, etc. other than the target are suitable; conventionally, cross-linked particles such as agarose, dextran, and polyacrylamide have been mainly used. ing. However, since these crosslinked particles have low strength, they are limited to use under low pressure conditions and have the disadvantage that separation requires a long time. In order to solve these drawbacks and perform separation and purification in a short time under high pressure conditions, agarose-based carriers with a high degree of crosslinking, polyvinyl alcohol-based carriers,
Although cellulose-based carriers and the like have been developed, one that is fully satisfactory in all aspects of performance such as the amount of ligand introduced, carrier strength, and non-specific adsorption has not yet been developed.

(Problems to be Solved by the Invention) An object of the present invention is to provide a novel affinity carrier.

Another object of the present invention is to provide a novel affinity carrier having pores and pore distribution that exhibit excellent performance as an affinity carrier.

Still another object of the present invention is to provide an affinity carrier that exhibits high pressure resistance when packed in a column, and therefore allows the liquid to be processed to be passed under pressure and processed at a high flow rate, and has a large processing capacity. It's about doing.

Still another object of the present invention is to provide a small affinity carrier that can be introduced in a large amount and has non-specific absorption.

Further objects and advantages of the present invention will become apparent from the description below.

According to the present invention, the above objects and advantages of the present invention are as follows: (a)
It consists essentially of spherical or prolong spherical particles with a wet particle size of 500 μm or less, (b) the crystallinity as determined by X-ray diffraction is in the range of 5 to 45%, and (c) the critical point dry state Regarding particles, in terms of the relationship between pore diameter and pore volume measured by mercury porosimeter method,
The maximum value of the pore volume is in the section with a pore diameter of 0.006 to 1 μm, and the total volume of the pores in the same section is at least 0.05 mC/
(d) the amount of albumin that can be introduced is at least 30 mg;
/g, and (e) a pressure resistance when wet is at least 5 kg/cm''.

The porous microcellulose particles of the present invention consist essentially of spherical or elongated spherical particles having a wet particle size of 500 μm or less. The wet particle size is measured according to the method described below. The porous microcellulose particles of the present invention preferably have a particle size of 3 to 400 μm, more preferably 10 to 400 μm.
It has a particle size of 300 μm.

Further, the porous microcellulose particles of the present invention are substantially composed of spherical or elongated spherical particles. As used herein, the term "spheroidal" is a concept that includes particles whose projected view or plan view has a shape such as an ellipse, an elongated circle, a peanut shape, or an oval shape. The porous microcellulose particles of the present invention are spherical or elongated as described above, and are therefore different from particles that are rounded or irregularly shaped.

Secondly, the porous microcellulose particles of the present invention have a crystallinity in the range of 5 to 45% by X-ray diffraction, preferably in the range of 10 to 43%, and more preferably in the range of 10 to 43%. It is in the range of 20-40%.

Thirdly, the porous microcellulose particles of the present invention have a pore size of 0.006 to 1 in the relationship between pore size and pore volume measured by a mercury porometer method for particles upon critical drying.
The maximum value of the pore volume is in the mμ section, and the total volume of the pores in the same section is at least 0°05 m<2/g.

Critical drying is carried out by a specific method that will be described later.According to critical drying, the pore shape and distribution of the dried particles closely reproduces the wet state, so the pore volume when wet is Critical drying is extremely meaningful for affinity carrier particles where pore size is important.

In the porous microcellulose particles of the present invention, the total volume of pores in the same section is preferably in the range of 0.1 to 3 mQ/g, more preferably in the range of 0.12 to 2.5 mQ/g. .

The relationship between the pore diameter and pore volume of the porous microcellulose particles of the present invention is measured by a mercury porosimeter method. Using the obtained relationship, the area of the inner surface of the hole can be calculated.

The porous microcellulose particles of the present invention preferably have an inner surface area of 15 to 400
m2/g, more preferably 25-350 m2
/g.

Fourthly, the porous microcellulose particles of the present invention can introduce albumin in an amount of at least 30 mg/g. The amount of albumin that can be introduced is preferably at least 50 mg/g.

Finally, the porous microcellulose particles of the present invention have a pressure resistance of at least 5 mg/cm''. A preferred pressure resistance is at least 20 kg/C112, and a more preferred pressure resistance is at least 60 kg/cm''. be.

The porous microcellulose particles of the present invention further include X
In the line diffraction diagram, the diffraction angle (2θ) is 20°0±0.3°
It is preferable to have two clearly distinguishable peaks at 21.8±0.3°.

Various ligands can be introduced into the affinity carrier made of porous microcellulose particles of the present invention.

Examples of the ligands to be introduced into the affinity carrier of the present invention include antibodies that bind to various antigens, protein A that binds to immunoglobulin IgG, peptides that have affinity for enzymes, modified proteins, and peptides, depending on the purpose.
Examples include receptor proteins for amino acids, coenzymes, vitamins, lipids, steroids, and hormones, pigments, polynucleotides, saccharides, and glycoproteins such as lectins.

These ligands can be used, for example, by introducing active groups into porous fine cellulose particles using cyanogen bromide, by oxidizing porous fine cellulose particles with periodic acid to generate aldehyde groups as active groups, A method of introducing a halogenated acetyl group by treating microcellulose particles with bromoacetylbromide, a method of reacting porous microcellulose particles with cyanuric chloride to produce cyanuric cellulose, a method of converting porous microcellulose particles into evinrolhydrin, etc. A method of introducing epoxy groups by reacting with The epoxidized porous microcellulose particles thus produced are reacted with ammonia to introduce amino groups, or the bromide cyanide particles produced by the method described above are reacted with diaminoethane, etc. to introduce amino groups, and then glutaraldehyde is introduced. Examples include a method of introducing an aldehyde group by reaction. The method of introducing a ligand into the affinity carrier of the present invention is not limited to the above method, but is selected depending on the type, property, chemical structure, etc. of the ligand. - Generally, it is desirable to apply the methods used for polysaccharide-based carriers such as those described above.

Since the reactive groups introduced into the carrier by these methods react with the ligand under mild conditions, the ligand can be easily bound to the carrier. The amount of ligand introduced is appropriately selected depending on the purpose and separation conditions.

In actual affinity separation, the required amount of ligand binding does not necessarily mean that a larger amount is better; there is an optimal amount, and the amount cannot be determined unconditionally depending on the target, but the amount that can be introduced with active groups is large. Therefore, if necessary,
The feature is that the binding amount can be set within a wide range.

In addition, although the affinity carrier of the present invention varies slightly depending on its manufacturing method, it has a high strength and has a pressure resistance of 5 kg/cm when packed in a column and used as described above.
Therefore, one of its major features is that it can be used at higher flow rates at a lower cost.Furthermore, another feature is that the non-specific adsorption of components other than those to be separated is extremely low.Especially for ions. Even under conditions of low strength, there is almost no non-specific adsorption of proteins, etc., so there is an advantage that high-performance separation and purification is possible.

The porous microcellulose particles of the present invention have excellent performance as an affinity carrier as described above, and are also excellent in terms of economy because they are made from inexpensive and widely available cellulose as a raw material.

As shown in the Examples below, good results were also obtained in separation and purification steps under pressurized conditions in which ligands were actually introduced.

According to the present invention, the porous microcellulose particles of the present invention can be produced, for example, as follows.

That is, in the first step, coagulated viscose fine particles containing 5 to 60% by weight of cellulose xanthate in terms of cellulose are prepared, in the second step the coagulated viscose fine particles are neutralized with acid, and then in the third step, the coagulated viscose fine particles are produced. The cellulose particles are separated from the mother liquor. The coagulated viscose fine particles used in the first step are prepared by: (A) preparing an aqueous alkaline polymer solution of cellulose xanthate and a first water-soluble polymer compound other than the cellulose xanthate, and (B) preparing the above alkaline polymer. mixing the aqueous solution and a second water-soluble anionic polymer compound to produce a fine particle dispersion of the aqueous alkaline polymer solution; (C) heating the dispersion or dispersing the dispersion with cellulose xanthate; It can be produced by coagulating the cellulose xanthate in the dispersion into fine particles containing the first water-soluble polymer compound in Section 2 by mixing with a coagulant.

Further, the coagulated viscose fine particles used in the first step of the present invention are obtained by: (A) preparing an alkaline polymer aqueous solution of cellulose xanthate and the other first water-soluble polymer compound; and (B) preparing the above-mentioned (C) mixing an alkaline polymer aqueous solution and a water-soluble polyethylene glycol or polyethylene glycol derivative having a number average molecular weight of 1,500 or more to produce a fine particle dispersion of the alkaline polymer aqueous solution at a temperature of 55° C. or higher; The cellulose xanthate in the dispersion may be further heated to a temperature equal to or higher than the temperature at which the dispersion was produced, or the dispersion may be mixed with a coagulant for cellulose xanthate. It can be produced by coagulating it into fine particles containing a water-soluble polymer compound.

As described above, the first method and the second method include the step (A) of preparing an alkaline polymer aqueous solution of cellulose xanthate and a first water-soluble polymer compound, and a fine particle dispersion of an alkaline polymer aqueous solution. The method consists of a step (B) of producing cellulose and a step (C) of producing cellulose-containing microparticles, which are basically the same steps.

The first method and the second method are different in that the second polymer compound used in the above step (B) is anionic in the first method, but nonionic in the second method. do. First, a first method for producing coagulated viscose fine particles used in the present invention will be described below.

According to the first method, as described above, an aqueous alkaline polymer solution of cellulose xanthate and a first water-soluble polymer compound other than cellulose xanthate is prepared in step A, and fine particles of the aqueous alkaline polymer solution are dispersed in step B. A liquid is generated, and in step C, fine particles containing the first water-soluble polymer compound are generated. Step A of preparing an alkaline polymer aqueous solution of cellulose xanthate and a first water-soluble polymer compound other than cellulose xanthate is to simultaneously dissolve cellulose xanthate and a first water-soluble polymer compound other than cellulose xanthate in water or an alkaline aqueous solution. Alternatively, cellulose xanthate is first dissolved in water or an alkaline aqueous solution, and the first water-soluble polymer compound is dissolved in the obtained viscose, or the first water-soluble polymer compound is dissolved in water or an alkali aqueous solution. This can be carried out by dissolving cellulose xanthate in an aqueous solution and then dissolving cellulose xanthate in the solution.

The above-mentioned dissolution can be carried out, for example, by mixing using a kneader or a high-viscosity stirring blade.

Cellulose xanthate may be obtained as an intermediate in the rayon manufacturing process or cellophane manufacturing process, and for example, cellulose concentration is 33% by weight and alkali concentration is 16% by weight.
And cellulose xanthate with a monovalent value of about 40 is suitable.

As the first water-soluble polymer compound, for example, a nonionic or anionic polymer compound is preferably used. Examples of the nonionic first water-soluble polymer compound include polyethylene glycol, polyethylene glycol derivatives, and polyvinylpyrrolidone. These polymer compounds have, for example, a number average molecular weight of 400 or more, and preferably have a number average molecular weight of 600 to 400,000.

As a polyethylene glycol derivative, for example, only the hydroxyl group at one end of polyethylene glycol has 1-1 carbon atoms.
A water-soluble compound or an A-B-A' type block copolymer (A , A' are the same or different and represent a polyethylene oxide block, and B represents a polypropylene oxide block) are preferably used. More specifically,
For example, polyethylene glycol monomethyl ether, polyethylene glycol monolauryl ether, polyethylene glycol monocetyl ethyl polyethylene glycol monomethyl phenyl ether, polyethylene glycol monononylphenyl ether; polyethylene glycol monoacetate, polyethylene glycol monolaurate; and polyoxyethylene block-polymer Examples include oxypropylene block-polyoxyethylene block.

The anionic first water-soluble polymer compound preferably has, for example, a sulfonic acid group, a phosphonic acid group, or a carboxylic acid group as an anionic group. These anionic groups may be in the form of free acids or salts.

The first water-soluble polymer compound having a sulfonic acid group as an anionic group has a sulfonic acid group such as vinyl sulfonic acid, styrene sulfonic acid, methylstyrene sulfonic acid,
It can be derived from monomers such as allylsulfonic acid, methallylsulfonic acid, acrylamide metalpropanesulfonic acid or salts thereof.

Similarly, the first water-soluble polymer compound having a phosphonic acid group as an anionic group can be derived from monomers such as styrene phosphonic acid, vinyl phosphonic acid, or salts thereof.

Furthermore, water-soluble polymer compounds having carboxylic acid groups as anionic groups could be derived from monomers such as acrylic acid, methacrylic acid, styrene carboxylic acid, maleic acid, itaconic acid, or salts thereof.

For example, the first water-soluble polymer compound having a carboxylic acid group can be prepared by polymerizing sodium acrylate alone or by mixing it with other copolymerizable monomers such as methyl acrylate according to a method known per se. It is supplied as a homopolymer or copolymer containing polymerized units of sodium acrylate. Furthermore, for example, a water-soluble polymer compound having a sulfonic acid group can be produced by sulfonating a styrene homopolymer.

The same applies to the case where the sulfonic acid group is derived from a monomer other than styrene sulfonic acid, and the case where the sulfonic acid group and the carboxylic acid group are respectively derived from the above monomers.

The first water-soluble anionic polymer compound preferably contains at least 20 mol % of polymerized units of the above-mentioned monomers having anionic groups. Such preferred polymeric compounds include copolymers and homopolymers.

The water-soluble anionic polymer compound preferably has a number average molecular weight of at least 5,000, more preferably 10,000 to 3,000,000.

The water-soluble first anionic polymer compound used in step A is not limited to the vinyl type polymers mentioned above,
Other examples include carboxymethyl cellulose, sulfoethyl cellulose, and salts thereof such as Na salts.

According to the first method, as described above, first, in step A, an alkaline polymer aqueous solution is prepared. The aqueous polymer solution preferably has a cellulose concentration derived from cellulose xanthate of 3 to 15% by weight, more preferably 5 to 12% by weight.
The alkaline concentration is preferably adjusted to 2 to 1.
It is adjusted to 5% by weight, more preferably 5 to IO% by weight. Further, the amount of the first water-soluble polymer compound is preferably adjusted to 0.03 to 5 parts by weight per 1 part by weight of cellulose.

According to the first method, the alkaline polymer aqueous solution prepared and prepared in step A is then mixed with a second water-soluble anionic polymer compound in step B.

For mixing, any means capable of producing a fine particle dispersion of an aqueous alkaline polymer solution can be used. For example, mechanical stirring using stirring blades, baffles, etc., ultrasonic stirring, or mixing using a static mixer can be carried out alone or in combination.

The second water-soluble anionic polymer compound is preferably used as an aqueous solution, more preferably as an aqueous solution in which the second polymer compound has a concentration of 0.5 to 25% by weight, particularly preferably 2 to 22% by weight. , used. The aqueous solution preferably has a viscosity of 3 centipoise to 50,000 centipoise, particularly 5 centipoise to 30,000 centipoise at 20°C.

The alkaline polymer aqueous solution and the second water-soluble anionic polymer compound are 0.3 to 100 of the second polymer compound per part of cellulose IIi in the alkaline polymer aqueous solution.
Parts by weight, more preferably 1 to 45 parts by weight, particularly preferably 4 to 20 parts by weight, are used and mixed. The mixing is preferably carried out at a temperature lower than the boiling point of carbon disulfide contained in the aqueous alkaline polymer solution, more preferably in the range of 0 to 40°C.

According to the research of the present inventors, when an acid-decomposable inorganic salt such as calcium carbonate is present as a dispersant in an amount of 0.5 to 5% by weight in the alkaline polymer aqueous solution in Step A, It has become clear that the morphology of the fine particles in the fine particle dispersion produced in the second step is maintained stably and well.

Examples of the second water-soluble anionic polymer compound include the same compounds as the above-mentioned examples of the anionic first water-soluble polymer compound.

The second water-soluble anionic polymer compound may be the same as or different from the first water-soluble polymer compound.

According to the present method for producing coagulated fine particles, the fine particle dispersion of the aqueous alkaline polymer solution produced in step B is then coagulated in step C.

The coagulation reaction described above is desirably carried out while adding a mixing operation to the produced dispersion.

Coagulation by heating can be advantageously carried out at a temperature higher than the boiling point of carbon disulfide contained in the alkaline polymer aqueous solution, for example, at a temperature of 50'' to 90°C. In the case of coagulation using a coagulant, the temperature is raised to such a temperature. Not necessary, usually 0-40°C
The coagulation can be carried out at a temperature of . As the coagulant, for example, lower aliphatic alcohols, alkali metal or alkaline earth metal salts of inorganic acids, and combinations thereof with the third water-soluble polymer compound are preferably used. The lower aliphatic alcohol may be linear or branched; for example, aliphatic alcohols having 1 to 4 carbon atoms such as methanol, 1so-propanol, loobanol, and n-butanol are preferred. used. Examples of alkali metal salts of inorganic acids include Na, CI, Na
, SO, and salts such as K, SO, are preferable, and alkaline earth metal salts include, for example, Mg5O,
Mg salts such as, Ca salts such as CaCl2 are preferred.

As the third water-soluble polymer compound, for example, nonionic and anionic polymer compounds are preferably used. It is particularly desirable to use the same third water-soluble polymer compound as the second anionic polymer compound used in step B.

Examples of the third water-soluble polymer compound will be understood from the above-mentioned examples of the first water-soluble polymer compound.

The coagulant as described above is used in a proportion of, for example, about 20 to 300% by weight based on the cellulose in the viscose.

Next, a second method for producing coagulated viscose fine particles used in the present invention will be explained.

According to the second method, as described above, an alkaline aqueous polymer solution of cellulose xanthate and a first water-soluble polymer compound other than cellulose xanthate is prepared in step A, and fine particles of the alkaline aqueous polymer solution are prepared in step B. A dispersion liquid is produced, and in step C, fine particles containing cellulose are produced. In this respect, it is basically the same as the first manufacturing method described above, as described above. Step A of preparing an alkaline polymer aqueous solution of cellulose xanthate and a first water-soluble polymer compound other than cellulose xanthate is carried out in the same manner as the method described in the description of the first manufacturing method above. For example, the xanthate and other first water-soluble polymer compounds used are the same as those described in the first production method.

Step B of producing a fine particle dispersion of an alkaline polymer aqueous solution includes an alkaline polymer aqueous solution and a number average molecular weight of 1500.
This is carried out by mixing the above water-soluble polyethylene glycol or polyethylene glycol derivative.

As mentioned above, the high molecular weight polyethylene glycol or polyethylene glycol derivative used has a number average molecular weight of 1.500 or more, preferably 1.50.
It has a number average molecular weight of 0 to 400,000.

As a polyethylene glycol derivative, for example, only the hydroxyl group at one end of polyethylene glycol has 1-1 carbon atoms.
8 alkyl group, a phenyl group substituted with an alkyl group having 1 to 18 carbon atoms, or a water-soluble compound blocked with an acyl group having 2 to 18 carbon atoms or an A-B-A' type block copolymer (A, (A' are the same or different and represent a polyethylene oxide block, and B represents a polypropylene oxide block) are preferably used. More specifically, for example, polyethylene glycol monoacetate J,
Polyethylene glycol monolauryl ester, polyethylene glycol monocetyl ethyl; polyethylene glycol monomethyl phenyl ether, polyethylene glycol monononylphenyl ether; polyethylene glycol monoacetate, polyethylene glycol monolaurate; and polyoxyethylene block-polyoxypropylene block- Examples include polyoxyethylene blocks.

Among polyethylene glycol and its derivatives, polyethylene glycol is more preferred, and has a number average molecular weight of 6,
000 to 200.000 is more preferable, and the number average molecule! Those having a number average molecular weight of 8,000 to 100°,000 are particularly preferred, and those having a number average molecular weight of 10,000 to 30,000 are particularly preferred.

The polyethylene glycol derivative is preferably 1.500
It has a number average molecular weight of ~16.000.

According to the second method, in step B, an alkaline aqueous polymer solution and a water-soluble high molecular weight polyethylene glycol or a derivative thereof are first mixed. For mixing, any means capable of producing a fine particle dispersion of an alkaline aqueous polymer solution can be used. The specific means are as described in the description of the first manufacturing method above.

The water-soluble high molecular weight polyethylene glycol or its derivative is preferably used as an aqueous solution, more preferably the concentration of the polyethylene glycol or its derivative is from 0.5 to
60% by weight, particularly preferably 5-55% by weight, especially 10% by weight
It is used as a ~40% by weight aqueous solution.

The alkaline polymer aqueous solution and polyethylene glycol or polyethylene glycol derivative are 1 to 30 parts by weight, more preferably 2 to 28 parts by weight of polyethylene glycol or polyethylene glycol derivative per 1 part by weight of cellulose.
Parts by weight, particularly preferably 4 to 24 parts by weight, especially 8 to 16 parts by weight
Parts by weight are used and mixed. Although there is no particular restriction on the temperature during mixing, it is desirable to carry out the mixing at a temperature lower than the temperature at which a fine particle dispersion of the aqueous alkaline polymer solution is produced. The fine particle dispersion of the aqueous alkaline polymer solution is produced at a temperature of 55°C or higher. 55°
At temperatures lower than C, it is not possible to obtain a fine particle dispersion of the basic aqueous alkaline polymer solution capable of providing the desired fine cellulose particles.

According to the second method, the fine particle dispersion of the aqueous alkaline polymer solution produced in step B is then solidified in step C.

Further, the coagulation reaction is carried out at a temperature equal to or higher than the temperature at which the dispersion is produced.

Both coagulation by heating and coagulation using a coagulant are preferably 6
It is carried out at a temperature of 0°C to 90°C, but it can also be coagulated with a coagulant at a temperature below 60°C.

The coagulant and its usage ratio are the same as described in the description of the first manufacturing method above.

When a combination of polyethylene glycol or its derivatives is used as the coagulant, the addition of the coagulant can prevent the concentration of polyethylene glycol or its derivatives in the system from decreasing. It has the advantage that coagulation can be carried out stably.

The coagulated viscose fine particles obtained by the first method and the second method as described above consist essentially of spherical to long spherical particles with an average particle diameter of 400 μm, and have a cellulose component of 5.
~60% by weight (in terms of cellulose). The cellulose component of the coagulated viscose fine particles referred to here is obtained by washing and replacing the water and water-soluble polymer compound adhering to the surface of the fine particles with excess n-hexane, and drying at 50°C for 60 minutes to obtain the adhering n-hexane. is removed; after that, the fine particles are heated at 105°
C Dry for 13 hours to determine the cellulose component. In addition, if a water-soluble polymer compound is contained, the contained polymer compound is removed by washing with water in advance when determining the cellulose component. The polymer compound in the coagulated viscose fine particles was removed using 0.5 to 2% by weight of caustic soda at a temperature of 20 to 20%.
Performed at 30°C.

According to the present invention, the coagulated viscose fine particles from which the polymer compound has been removed in the first step are then neutralized with acid in the second step to convert the viscose into cellulose.

Next, in the third step, the cellulose fine particles produced from the mother liquor are separated. After separation, desulfurization, pickling, water washing, or methanol washing can be carried out if necessary; or after that, or after the above separation, heat treatment can also be carried out.

Desulfurization can be carried out using an aqueous alkali solution such as caustic soda or sodium sulfide. If necessary, in order to remove residual alkali, it is then pickled with dilute hydrochloric acid or the like, and then washed with water or methanol.

The present invention will be described in further detail with reference to Examples below, but before that, the measurement method and the like in the present invention will be described.

(1) Average particle size: Measured by microscopic observation when wet.

(2) Crystal form and crystallinity: X-ray diffraction measurements are performed using particles that have been air-dried after substitution with methanol.

The diffraction angle 2θ of type ① cellulose is around 20° and 21.8
It can be identified because it has two peaks near '. The degree of crystallinity is defined by the following formula from the X-ray diffraction pattern.

However, K-0,896 (incoherent scattering correction coefficient of cellulose) a, b, c are the areas surrounded by the diffraction curve and the base straight line between the diffraction angle 2θ-5″ to 45°, Each corresponds to the following measurements:

a: amorphous starch, b: air scattering, C: sample, (3) Pore diameter-pore volume measurement: After replacing the water in the water-wet particles with ethanol, the water was completely replaced with inamyl acetate and this was removed to the carbon dioxide critical point. Measure with a mercury porosimeter using dried particles.

(4) Amount of active group introduced: pHII
The cellulose particles activated under the conditions described above were added to a 0.12M borax buffer (pH 9,0) containing bovine serum albumin (BSA) in an amount equal to the dry weight of the cellulose particles (System Note B).
(SA concentration: 2.5% by weight) and reacted at 4°C for 20 hours.

Next, the amount of bound BSA per dry particle is determined by measuring the amount of BSA remaining in the solution, and this is calculated as the amount of active group introduced (
wt/wt).

(5) Internal diameter 4III installed vertically as a standard measurement method for particle wet pressure resistance
111 Length 150mm stainless steel column (pore diameter 2μ
1.88 mff of cellulose fine particles, which had been pre-swelled in water for 18 hours, were filled into a sintered filter (equipped with a sintered filter) using water at a flow rate of 0 and 1 me/win from top to bottom. In this case, when water flows through the ram from top to bottom, the pressure loss value at the point where the slope of the tangent line (pressure/flow speed) is eight times the slope of the flow rate Q me/min on the flow velocity-pressure loss curve is calculated. Particle wet pressure resistance.

Example 1 500g of pulp made from softwood at 2060.18% by weight
It was immersed in 20Q of caustic soda solution for 1 hour and squeezed to 2.8@. The temperature was raised from 25°C to 50°C and the mixture was ground for 1 hour for aging, and then 35% by weight of carbon disulfide (175 g) was added to the cellulose and sulfurized at 25°C for 1 hour to form cellulose xanthate. did. After dissolving the xanthate in a caustic soda aqueous solution, polyethylene glycol (
250 g of flakes having a molecular weight of 4,000) were added and dissolved to prepare an aqueous alkaline polymer solution of cellulose xanthate and polyethylene glycol. The alkaline polymer aqueous solution has a cellulose concentration of 9.1% and a caustic soda concentration of 5.1%.
4% by weight, polyethylene glycol 4.6% by weight, viscosity 7600 centipoise.

60 g of the above-prepared aqueous alkaline polymer solution and an aqueous solution of sodium polyacrylate as an anionic second polymer compound (polymer concentration 12% by weight, molecular 850,000; manufactured by Nippon Tsumugi Co., Ltd.; trade name: Jurimer AC-1ON) ) and 2 g of calcium carbonate as a dispersant were put into a 500 mQ flask to make the total amount 300 g.

At a liquid temperature of 30°C, use a laboratory stirrer (manufactured by Yamato Scientific Co., Ltd.: MODEL LR-51B, rotating blade 7cm) 6
After stirring at 00 rpm for 10 minutes to purify the fine particles of the aqueous alkaline polymer solution, the liquid temperature was raised from 30°C to 70°C in 15 minutes while continuing to stir, and maintained at 70°C for 30 minutes. The microparticles containing polyethylene glycol were coagulated. While continuously stirring, the mixture was neutralized and regenerated with 100 g/Q sulfuric acid to obtain a cellulose fine particle molecular liquid. The above dispersion is passed through a sifter using the IG4 type to separate cellulose particles containing polyethylene glycol from the mother liquor, and then washed with a large excess of water to remove polyethylene glycol from the particles to obtain porous cellulose particles. Ta.

The physical properties of the porous particles thus obtained are as follows.

Average particle size 72 μm1 Pore size distribution 50-3000 people, pore size total volume 1.41 mQl gs〃 Area
117 m”/g.

in the same section Maximum value of differential curve: 1200 people, Amount of active group introduced: 260 mg/g.

Example 2 The porous microcellulose particles obtained in Example 1 were packed into a stainless steel column with an inner diameter of 4 mm and a length of 15 cm according to the standard measurement method for particle wet pressure resistance described above, and the flow rate and pressure drop were measured when water was passed through the column. We measured the relationship between

For comparison, similar measurements were performed on a commercially available agarose filler. The results are shown in Figure 1.

The porous microcellulose particles of the present invention have excellent wet pressure resistance and can withstand increased pressure loss due to scale-up and use at high flow rates.

In FIG. 1, curves 1 and 2 are for a known crosslinked agarose filler and a known highly crosslinked agarose filler, respectively, and curve 3 is for the cellulose fine particles of the present invention.

Examples 3 to 8 Various porous cellulose particles were prepared in the same manner as in Example 1, except that the molecular weight and amount of polyethylene glycol (PEG) added were changed as shown in Table 1.

The pore volumes of these particles all have pore diameters of 0.08 to 0.
It has a maximum value between 2μm and a pore size of 0°06~1p.
The total volume of section I11 was 1.1-1'-8 mQ/g.

A ligand was introduced into the obtained particles by the following method.

A 100 ml reaction vessel was equipped with a stirrer and a glass calomel electrode for pH measurement. 4m suspension of various porous gels
Add 27 mL of distilled water 25% BrCN 24 mQ to Q,
The reaction was carried out at 20° C. for 6 minutes while keeping the pH value at 10.5-11 with IN sodium hydroxide. The reaction mixture was then transferred to a glass filter and 4°C 30++ cQ O, 1M
-NaHCO, and then washed with a 0.12M borax buffer (pH 9,0) at 4°C to obtain activated cellulose particles.

Next, 0.00 mg containing 50 mg of bovine serum albumin (BSA).
Activated particles (apparent volume f) in 2 mQ of 12M borax buffer
k0, 4 mQ> was added, and the mixture was stirred and mixed at 4°C for 20 hours. After the reaction, 50mM phosphate buffer (pH 6).

8). The amount of BSA introduced into the porous particles is determined by the amount of BSA in the supernatant of the reaction mixture and in this washing solution.
S! It was determined by measuring the absorbance at 11°.

Furthermore, the wet pressure resistance was measured in the same manner as in Example 2.

Table 1 shows the wet pressure resistance of these particles and the amount of BSA introduced.

Table 1 1) The amount of PEG added indicates weight % relative to cellulose.

2) The amount of BSA introduced indicates the amount of protein introduced per dry carrier weight.

Example 9 4 mQ of the filler suspension of Example 1 was activated according to the method of Example 3, and protein A from Staphylococcus aureus was introduced. The amount of protein A used for coupling was 1
omg. The protein A-introduced gel prepared in this manner retained 30 mg of protein A per gram of dry weight. This protein A-introduced gel was heated to an inner diameter of 4 m.
m, flow rate 10rnQ in a stainless steel column with a length of 75mm.
/min for 1 hour to prepare an affinity column. When 1 ml of mouse ascites fluid from which cell debris had been removed by centrifugation was injected into this affinity column and IgG was separated and purified, 9.8 mg of IgG could be obtained. The chromatogram is shown in FIG.

Moreover, the separation conditions in this case are as follows.

Separation conditions A: Binding buffer: 3M NaC (1, 0, 1M glycine (pH 8, 9), B: Elution buffer: O, 1M citric acid (pH 3, 0),
Flow rate: l, 6 mQ/cm'', min After the binding buffer was passed for 1 hour, the elution buffer was passed for 1 hour.

Detection: ABSza. , Temperature: 4° C.1 Example 10 The packing material 4mQ of Example 1 was activated according to the method of Example 3, and an anti-AFP monoclonal antibody was introduced therein. The amount of AFP antibody used for coupling was 4 mg. The AFP antibody-introduced gel prepared in this manner retained 5 mg of anti-AFP monoclonal antibody per 1 g of dry weight.

This AFP antibody-introduced gel was packed into a stainless steel column with an inner diameter of 4 mm and a length of 75 mm at a flow rate of 10 mQ/min for lhr to obtain an affinity column. 200 ng of AFP was diluted with 200 μα of human serum and injected into the column to recover AFP. The amount of AFP recovered was quantified by the sandwich method using a peroxidase-labeled anti-AFP antibody. The amount of AFP recovered was 162 ng, and the recovery rate was 81%. The separation conditions are as follows.

Separation conditions binding buffer: O, 1MPBS pH 7,2,
Elution buffer: 0, IM glycine HCQ
Buffer pH 2.5, Temperature: 37° C1. After passing the binding buffer at 0.5 m (2/hr for 3 hours), elution buffer 1 m (2/hr) was passed for 3 hours.

Detection: ABS2110x Example 11 60 g of cellulose fine particles obtained in Example 1 (Dry
) was stirred in an 8 wt.% aqueous solution of caustic soda containing 20 wt.% of shrimp chlorohydrin at 60°C.
Crosslinking was carried out for 3 hours. Subsequently, it was separated from the mother liquor using a glass filter, and then neutralized with 5% by weight hydrochloric acid to obtain crosslinked cellulose fine particles.

Obtained. The physical properties of the obtained crosslinked particles are shown.

Average particle size: 80μm1 Crystallinity:
29%, pore diameter showing maximum value of pore volume:
0.3Lpm, amount of active group introduced: 3
00 mg/g, Example 12 4 mQ of the suspension containing filler with an apparent volume of 2 + 11 + 2 obtained in Example 11 was activated according to the method of Example 3,
10 mg of horseradish peroxidase (manufactured by Sigma, RZ-3) was introduced into this. The thus prepared peroxidase-introduced gel retained 29 mg of peroxidase (1θ equivalent) on a dry basis.

20ml of this peroxidase-introduced gel with jacket
0.1M phosphate buffer (pH 7,2) 41T in the reactor
The mixture was suspended in lα, and the temperature inside the container was set at 10°C.

Here, horseradish peroxidase (manufactured by Sigma, R
After adding 5° OmQ of antiserum obtained by inoculating rabbits with Z-3) and stirring and mixing for 3 hours, the gel suspension was filtered through a glass filter and added with O.1M phosphate buffer (pH 7, 2)
Washed 5 times with 1 OmQ. Return the washed gel to the above reactor and add 4m of O, 1M glycine buffer (pH 2,5).
After adding Q and stirring and mixing for 1 hour, the filtrate was collected by glass filter filtration to obtain 6 mg of anti-peroxidase antibody.

Embodiments of the invention are as follows.

1. The affinity carrier according to claim 1, which has a wet particle size in the range of 3 to 400 mμ.

2. The affinity carrier according to claim 1, which has a wet particle size in the range of 10 to 300 mμ.

3. The affinity carrier according to claim 1, which has a crystallinity in the range of 10 to 43%.

4. The affinity carrier according to claim 1, which has a degree of crystallinity in the range of 20 to 40%.

5. The affinity carrier according to claim 1, which has two clearly distinguishable peaks at diffraction angles (2θ) of 20.0±0°3° and 21.8±0.3° in an X-ray diffraction diagram. .

6. The 7-finity carrier according to claim 1, wherein the total volume of the pores in the pore diameter range of 0.006 to 1 μm is in the range of 0.1 to 3 mρ/g.

7. Claim 1, wherein the total volume of the pores in the pore diameter range of 0.006 to 1 μm is in the range of 0.12 to 2-5 mQ/H.
Affinity carriers as described in Section.

8. The area of the inner surface of the pore calculated from the relationship between the pore diameter and pore volume in the pore diameter range of 0.006 to 1 μm is 15 to 400
2. The affinity carrier of claim 1 in the range m''/g.

9. The area of the inner surface of the pore calculated from the relationship between the pore diameter and pore volume in the pore diameter range of 0.006 to lpm is 25 to 350.
Affinity carrier according to claim 1, in the range of m''/g.

10. The amount of albumin that can be introduced is at least 50 mg/g
The affinity carrier according to claim 1.

11. Pressure resistance when wet is at least 20 kg/cm2
The affinity carrier according to claim 1.

12. The affinity carrier according to claim 1, which has a pressure resistance when wetted of at least 60 kg/cm2.

[Brief explanation of the drawing]

FIG. 1 shows the relationship between the flow rate and pressure drop of the porous microcellulose particles of the present invention. FIG. 2 is an example of an affinity chromatogram using the porous microcellulose particles of the present invention as an affinity carrier. Oto 20 Highlights (mL) Procedural amendment May 24, 1988 Director General of the Patent Office Kunio Ogawa 1, Indication of the case Patent Application No. 85350 of 1985 2, Name of the invention Affinity carrier 3, Amendment Relationship with the patent applicant case (095) Kanebo Co., Ltd. (1 other person) 4.
Agent 〒107 5. Date of amendment order (voluntary) 6. Column 7 of "Detailed explanation of the invention" of the specification subject to the amendment. Details of the amendment as shown in the attached sheet. (1) "mμ" on page 6, line 1 of the specification is corrected to 1μm. (2) 'mg/m'" on page 7, line 7, rkg/c
(3) rme on page 30, line 18 and page 31, line 1.
/min" is corrected to rmρ/m1nj. (4) “25%” on page 34, line 16 is changed to “2°5%”
I am corrected. (5) rm(2/cm”, m1n, page 37, line 14)
Correct J to rmQ/cm”・m1nJ.

Claims (1)

[Claims] 1. (a) consists essentially of spherical or oblate particles with a wet particle size of 500 μm or less, and (b) has a crystallinity in the range of 5 to 45% as measured by X-ray diffraction. (c) Regarding the particle at critical point drying, in the relationship between pore diameter and pore volume measured by mercury porosimeter method, the maximum value of pore volume is in the pore diameter range of 0.006 to 1 μm, and the maximum value is in the same range. (d) the total volume of the pores is at least 0.05 ml/g; and (d) the amount of albumin that can be introduced is at least 30 mg/g.
and (e) a pressure resistance when wet is at least 5 kg/cm^2.