JPH0740025B2 - Polymer particles for affinity chromatography - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention has a specific number average particle size and a narrow particle size distribution,
In addition, the present invention relates to a polymer particle for affinity chromatography in which the inside of the particle has a three-dimensional network structure.

[Problems to be Solved by Conventional Techniques and Inventions] Conventionally, polymer particles have been produced and used as a packing material for chromatography, a carrier for immobilizing an enzyme, a carrier for affinity chromatography, a base material for an ion exchange resin, etc. Has been done.

A dispersion method and a spray method are known as methods for producing spherical polymer particles.

In the dispersion method, a dilute solution of a polymer dispersed in a dispersion medium containing a surfactant is solidified by volatilizing the solvent from the dilute solution of the polymer (see JP-A-56-24430). Polymer particles can be obtained by gradually adding a coagulant of small droplets to the mixture and solidifying it (see Japanese Patent Laid-Open No. 57159801). This method yields particles having a broad particle size distribution and requires washing with an organic solvent as well as water to remove the solvent, dispersion medium and surfactant from the solidified droplets.

Another method known as a dispersion method is a method in which a polymerizable monomer is dispersed in a dispersion medium and then polymerized to obtain polymer particles, and the particles obtained by such a method also have a wide particle size distribution. There is. When these particles are magnified and observed with an electron microscope, it can be seen that finer spherical particles aggregate to form particles. It is thought that this structure is the cause, but when the suspension of particles obtained by this method is stirred with a magnetic stirrer or the like, a large amount of minute polymer waste is generated.

In the spray method, polymer particles are obtained by spraying a polymer solution into a coagulant. This particle also has a wide particle size distribution and a relatively large particle size (Japanese Patent Laid-Open No. 52-12).
(See Japanese Patent No. 9788).

However, when many polymer particles with a small particle size are included, pressure loss increases when used as a column-shaped adsorbent with good adsorption efficiency, especially plasma proteins etc. are selectively removed from blood. When used for such a purpose, problems such as hemolysis occur, and the selectivity becomes poor because the molecular weight cut-off is not sharp.

When the particle size of the polymer particles is increased in order to improve the above-mentioned problem related to pressure loss, the specific surface area (total surface area of particles per unit volume) becomes small, which causes a problem that the adsorption rate decreases.

Although polymer particles having a narrow particle size distribution and less problems as described above have also been produced (Japanese Patent Laid-Open No. 57-102905), these particles do not have a three-dimensional network structure, but fine primary particles are aggregated. However, the mechanical strength is not always sufficient and the primary particles are detached.

[Means for Solving the Problems] The present invention has been made to solve the problems such as the wide particle size distribution of polymer particles, which is the cause of the above-mentioned problems, and has a number average particle size of 300 to 600 μm. The present invention relates to polymer particles for affinity chromatography in which the content of all particles is within ± 10% of the number average particle diameter and the inside of the particles has a three-dimensional network structure.

[Examples] There is no particular limitation on the type of polymer constituting the polymer particles for affinity chromatography of the present invention, and any polymer can be used as long as it can produce polymer particles.

Specific examples of such polymers include, for example, cellulose, cellulose derivatives, cellulosic polymers such as regenerated cellulose, silk-based polymers such as silk fibroin,
Chitin polymers such as chitosan, natural polymers such as collagen, agarose, alginate, carrageenan, gelatin, starch; acrylonitrile polymers such as polyacrylonitrile, acrylonitrile-styrene copolymer, acrylonitrile-vinyl sulfonic acid copolymer. (Meth) acrylate polymers such as polymethylmethacrylate, methylmethacrylate-hydroxyethylmethacrylate copolymer, polymethylmethacrylate stereocomplex, hydroxyethylmethacrylate-styrene copolymer; polystyrene, styrene-butadiene copolymer, styrene-chloro Styrenic polymers such as methyl styrene copolymers; vinyl alcohols such as polyvinyl alcohol and ethylene-vinyl alcohol copolymers Rimmer; other polyamides, polyesters, polyethers, polyurethanes, etc. condensation polymer such as polysulfone, synthetic polymers capable of preparing a polymer solution by dissolving in a solvent suitable for each polymer.

When the polymer constituting the polymer particles is, for example, a cellulosic polymer, polymer particles having characteristics such as nonspecific adsorption of blood cell components and plasma proteins are relatively small, so that plasma proteins from blood can be obtained. Particles can be obtained which can be suitably used for applications such as selective removal of In addition, when a polymer capable of introducing or cross-linking other groups such as an ion-exchange group such as a styrene-butadiene copolymer or a styrene-chloromethylstyrene copolymer is used, the affinity Particles that can be used as polymer particles for affinity chromatography of the present invention, which is a carrier of a packing material for chromatography, a base material for an ion exchange resin, a particle having high mechanical strength, or a base material for ion exchange resin having high mechanical strength Particles that can be used for Further, when a polymer having an active hydroxyl group such as polyvinyl alcohol or ethylene-vinyl alcohol copolymer is used, particles which can be used as a carrier for affinity chromatography having a small pressure loss can be obtained.

The polymer particles of the present invention are spherical particles (which is a concept that includes not only substantially spherical particles but also rotators having an elliptical shape with a minor axis / major axis up to about 0.8) and a number average particle diameter. (In the case of an elliptical rotating body, the volume average particle diameter, that is, it is calculated as the cube root of the value obtained by multiplying the square of the major axis by the minor axis) is 30.
It is in the range of 0 to 600 μm, and all particles are within ± 10% of the number average particle size.

When the number average particle size is less than 300 μm, for example, it is used as a polymer particle for an adsorbent for selectively removing plasma proteins from blood, particularly as an adsorbent for adsorbing and removing plasma proteins by a direct blood perfusion method. This causes a large pressure loss such as spilling, and causes problems such as easy hemolysis. On the other hand, if it exceeds 600 μm, the specific surface area becomes small, the adsorption speed becomes slow, and the adsorbed and removed amount of the unwanted substances becomes small relative to the circulating amount of blood.

If the ratio of particles within ± 10% of the number average particle size is not all, when used as an adsorbent for the adsorbent as described above, pressure loss increases or hemolysis occurs. It becomes easier and easier.

The state of the surface of the polymer particles of the present invention is not particularly limited, and may have a skin layer, may have a network structure, or may be in an intermediate state between the skin layer and the network structure. However, the inside has a three-dimensional mesh structure.

The three-dimensional network structure is a polymer of finer particles (polymers) obtained by polymerizing droplets of a polymerizable monomer as described above, while it literally has pores like sponges. It means a structure in which surfaces are three-dimensionally continuous structure or fibers are three-dimensionally continuous structure.

The size of the mesh and the porosity that form the three-dimensional network inside the polymer particles are not particularly limited, but the size of the mesh is preferably about 0.1 to 10 μm, and the inside has a cavity with a diameter of more than 10 μm. You may have a part locally. The porosity is preferably about 50 to 95%.

When the size of the mesh of the mesh structure is less than 0.1 μm, not only the adsorbing speed of the unwanted substances in blood decreases when used as an adsorbent for an adsorbent, but also the adsorptive removal of these unwanted substances. Will not be performed sufficiently. On the other hand, if it exceeds 10 μm, the particles tend to be deformed or crushed during transportation, such as when packed in a column and used, and the mechanical strength of the particles tends to be insufficient. is there.

When a skin layer is present on the surface of the polymer particles, a skin layer having a thickness of about 0.1 to 10 μm is usually obtained. Thus, the polymer particles having a skin layer on the surface of the polymer particles have a relatively small pore size on the surface, so that the exclusion limit molecular weight is about 1,000,000 or less, which is suitable as a chromatography particle or a base material particle for an ion exchange resin. become.

When the surface of the polymer particles has a network structure, the network structure usually has pores having a pore size of 0.01 to 5 μm. Thus, when the surface of the polymer particles has a network structure, it becomes suitable as a particle for chromatography having an exclusion limit molecular weight of about 1,000,000 or more, or a carrier for immobilized enzyme.

Next, the production of the polymer particles of the present invention will be described in the case of using a cellulosic polymer.

Cellulose-based particles, which is one type of the polymer particles of the present invention, are prepared by dissolving a cellulose-based solution in which cellulose, a cellulose derivative or the like is dissolved in, for example, the apparatus and method (vibration method and dry-wet method) described in JP-A-62-191033. It can be produced by applying a method in combination with a coagulation method).

The solvent used when preparing the cellulose-based solution is a solvent for cellulose, such as an aqueous solution of copper ammonia, a mixed solution of dimethylsulfoxide and paraformaldehyde, an aqueous solution of calcium thiocyanate, and acetic acid which is a typical cellulose derivative. Examples of the solvent for cellulose include dimethyl sulfoxide, dimethylformamide, dimethylacetamide, N-methyl-2-pyrrolidone and aceto.

In these solvents, whether or not a skin layer is formed on the surface of the obtained cellulosic particles, what is the thickness of the skin layer when it is formed, and the size of the pores of the internal network structure In order to adjust the size,
Methanol, ethanol, ethylene glycol, propylene glycol, glycerin, water, inorganic salts, polyethylene glycol, polyvinylpyrrolidone, etc. may be added.

A cellulosic solution of about 5 to 20% (weight%, the same applies hereinafter) prepared in this manner is disclosed in, for example, JP-A-62-19.
Using a device such as the one described in Japanese Patent No. 1033, small droplets having a substantially uniform size are ejected into the gas phase, and the droplets are caused to fly over a flight distance that makes them substantially spherical and then brought into contact with a coagulant. The cellulosic particles produced in this manner are substantially spherical particles.

The coagulant is composed of a non-solvent for the polymer, but it is preferable that the coagulant has a surface tension that dissolves in the solvent forming the droplets and naturally wets the droplets. Specific examples of such a coagulant include water, a mixed solution of water and the good solvent or non-solvent, a mixed solution of water and a surfactant, and the like.

Generally, the concentration of the polymer in the polymer solution is high, the proportion of the non-solvent is low, and the use of a coagulating liquid with a strong coagulation force such as water can form a skin layer. The size of the network of the network structure inside the particles can be reduced by agglomerating the particles.

The use of a coagulating liquid having a low polymer concentration in the polymer solution, a non-solvent ratio, and a relatively weak coagulation force can form particles having a network structure up to the surface.

In the above description, polymer particles were produced using a cellulosic polymer, but when using other polymers, a polymer solution prepared by dissolving the polymer in a suitable solvent as described above is used, and the non-solvent of the polymer is used. It can be used as a coagulating liquid to produce polymer particles in the same manner as described above.

Solvents, non-solvents and coagulants for polymers are described, for example, in "Chemical Handbook, Chemical Handbook, Maruzen Co., Ltd. (1984)", "J.
andrup & EMImmergut, editors: John Wily & Sons, N
ew York, Polymer Handbook
(1975) ”or the like.

If there is a site for introducing or cross-linking another group such as an ion-exchange group in the polymer particles obtained in this way, then if another group is introduced or cross-linked. Good.

The polymer particles of the present invention thus obtained have a specific average particle size and a specific particle size distribution, and since the inside of the particle has a three-dimensional network structure, it is suitable for use as a carrier for affinity chromatography. When it is used for this purpose, it is excellent in pressure loss, adsorption rate, separation rate, selectivity and the like.

The above-mentioned excellent characteristics are exhibited even when the polymer particles are used as a packing material for chromatography, an enzyme-immobilizing carrier, a base material for ion exchange resins, and the like.

Next, the polymer particles of the present invention will be described based on Examples, but it goes without saying that the present invention is not limited to these Examples.

Example 1 Cellulose diacetate was dissolved in a mixed solution of dimethyl sulfoxide / propylene glycol in a weight ratio of 4/6 so that the concentration thereof was 12.5%.

5 cm in front of the nozzle with a width of 2 cm at a distance of 2 cm.
An average flat plate-shaped electrode having a size of m and a length of 25 cm in the advancing direction of the droplet was installed, and a DC voltage of 800 V was applied between the electrode and the nozzle. From the orifice with a diameter of 250 μm provided in this nozzle, the solution held at 145 ° C. was discharged at a linear velocity of 7.8 m / sec while vibrating at 3850 Hz to form uniform droplets of the solution, After flying about 3m, at 23 ℃
Particles of cellulose diacetate were obtained by infiltrating into 10% methanol aqueous solution and coagulating.

The obtained cellulose diacetate particles were put into a 0.6% aqueous solution of caustic soda at 50 ° C., stirred for 2 hours, collected, neutralized and washed with water to obtain regenerated cellulose particles which were almost 100% regenerated.

When the number average particle diameter of the obtained regenerated cellulose particles was measured by the following method, it was 430 μm, and all the particles were within the average particle diameter ± 5%.

After replacing the liquid in the obtained regenerated cellulose particles with ethanol, carbon dioxide gas critical point drying (using Hitachi Co. Ltd. critical point dryer HCP-2) was performed, and gold was vapor-deposited, followed by scanning electron When observed with a microscope, the surface has a thickness of about 0.
There was a skin layer of 2 μm, and the inside was a porous three-dimensional network having a pore size of about 0.2 to 2 μm.

The regenerated cellulose particles were packed in a column having an inner diameter of 14 mm and a length of 7 cm, and fresh blood of cattle added with 30 units / ml of heparin was kept warm at 37 ° C., and the pressure loss was measured by gradually increasing the flow rate. However, the pressure loss increased almost linearly as the linear velocity increased, but even at 8 cm / min, 50 mmHg
However, this value remained almost unchanged even after continuous operation for 1 hour, and direct blood perfusion was possible.

The regenerated cellulose particles were reacted with epichlorohydrin and then with n-hexylamine to obtain n-hexylamine-immobilized cellulose particles. When the selectivity of these particles was measured by the following method, the adsorption rates for lysozyme and albumin were 73% and 8%, respectively.

(Number Average Particle Size and Particle Size Distribution) An optical microscope image of several hundred particles (about 500 to 1000 particles) is processed and obtained using an image processing device (Luzex II manufactured by Nireco Co., Ltd.).

(Selectivity) Lysozyme and albumin were dissolved in 0.025M phosphate buffer adjusted to pH 7.4 to a concentration of 3 mg / ml and 7.3 mg / ml, respectively, and the sedimentation volume was obtained for each of 6 solutions. Then, the cellulosic particles to which a ligand having adsorptivity to plasma white matter was fixed so that the ratio was 1 volume were added, and the mixture was shaken at 37 ° C for 2 hours, and then the concentration of the supernatant was measured to determine the adsorption of each. Find the rate.

(Determination of possibility of direct blood perfusion) Fresh blood of cattle containing 30 units / ml of heparin was kept warm at 37 ° C, and the flow rate was gradually increased to a particle-filled column with a length of 7 cm and an inner diameter of 14 mmφ. Flow was performed until the linear velocity was 8 cm / min, and it was determined that blood perfusion was possible if the pressure loss did not exceed 100 mmHg during this period.

Example 2 Polyacrylonitrile was mixed with dimethyl sulfoxide / propylene glycol in a weight ratio of 70/3 so that the concentration of polyacrylonitrile was 9%.
It was dissolved in a mixed solution of 0.

5 cm in front of the nozzle, with a width of 5c at a distance of 2 cm.
A parallel plate electrode having a size of m and a length of 10 cm in the droplet ejection direction was installed, and a DC voltage of 800 V was applied between the electrode and the nozzle. From the orifice with a diameter of 120 μm provided in this nozzle, the solution kept at 103 ° C. was discharged at a linear velocity of 6 m / sec while applying a vibration of 1000 Hz to form a uniform droplet of the solution, and the solution was blown in the air. After flying 3m, at 23 ℃
Particles of polyacrylonitrile were obtained by infiltrating into a 0.1% neutral detergent (polyoxyethylene sorbitan monolaurate) aqueous solution and coagulating.

When the number average particle size of the obtained polyacrylonitrile particles was measured by the same method as in Example 1, it was 460 μm and all the particles were within the average particle size ± 6%.

The obtained polyacrylonitrile particles were vacuum dried at room temperature, gold was vapor deposited and then observed with a scanning electron microscope. As a result, there was a skin layer with a thickness of about 0.1 μm on the surface, and the inside had a pore size of about 0.2-1 μm. It had a uniform porous three-dimensional network.

When the pressure loss was measured in the same manner as in Example 1 using the polyacrylonitrile particles, the pressure loss increased almost linearly with the increase of the linear velocity, but even at 8 cm / min, 3
Even when continuously operated for 1 hour at 0 mmHg, this value hardly changed, and direct blood perfusion was possible.

Example 3 Polymethylmethacrylate was dissolved in a mixed solution of dimethyl sulfoxide / propylene glycol in a weight ratio of 68/32 so that the concentration was about 17%.

5 cm in width, 5 cm in front of the nozzle and 2 cm apart
An average flat plate-shaped electrode having a length of 10 cm in the droplet ejection direction was installed, and a DC voltage of 800 V was applied between the electrode and the nozzle. From the nozzle with a diameter of 120 μm provided in this nozzle, the solution kept at 101 ° C. was drawn at a linear velocity of 7.1 m / sec.
It was ejected while applying vibration of 30 Hz to form uniform droplets of the solution, and after flying in the air for about 3 m, it was heated to 0.1 at 23 ° C.
% Neutral detergent (polyoxyethylene sorbitan monolaurate) was allowed to infiltrate and coagulate to obtain polymethylmethacrylate particles.

The number average particle diameter of the obtained polymethylmethacrylate particles was measured by the same method as in Example 1 to find that it was 470 μm.
At this point, all the particles were within the average particle size ± 8%.

The obtained polymethylmethacrylate particles were vacuum-dried at room temperature, and after depositing gold, they were observed with a scanning electron microscope. As a result, there was a skin layer with a thickness of about 0.2 μm, and the inside had a pore size of about 0.2-0.5. It had a uniform porous three-dimensional network structure of μm.

When the pressure loss was measured using the polymethylmethacrylate particles in the same manner as in Example 1, the pressure loss increased almost linearly with the increase of the linear velocity, but even at 8 cm / min, the pressure loss was 30 mmHg, which was 1 This value did not change even after standing for a time, and direct blood perfusion was possible.

Example 4 Polystyrene was added to N-methyl-concentration to a concentration of 15%.
Weight ratio of 2-pyrrolidone / propylene glycol is 68/3
It was dissolved in a mixed solution of 2.

5 cm in front of the nozzle with a width of 2 cm at a distance of 2 cm.
An average flat plate-shaped electrode having a size of m and a length of 10 cm in the droplet ejection direction was installed, and a DC voltage of 800 V was applied between the electrode and the nozzle. From the orifice with a diameter of 100 μm provided in this nozzle, the solution kept at 124 ° C. was discharged at a linear velocity of 7.6 m / sec while vibrating at 850 Hz to form uniform droplets of the solution, and the air was blown into the air. After flying about 3m, 23 ℃
Of 0.1% neutral detergent (polyoxyethylene sorbitan monolaurate) was infiltrated and coagulated to obtain polystyrene particles.

When the number average particle diameter of the obtained polystyrene particles was measured by the same method as in Example 1, it was 440 μm and all the particles were within the average particle diameter ± 5%.

The polystyrene particles obtained were vacuum dried at room temperature, evaporated with gold, and then observed with a scanning electron microscope. As a result, there was a skin layer with a thickness of about 0.2 μm on the surface, and the inside had a pore size of about
0.2 to 2 μm porous three-dimensional network and approx. 20 μm
It had m cavities locally.

When the pressure loss was measured using the polystyrene particles in the same manner as in Example 1, the pressure loss increased substantially linearly with an increase in the linear velocity, but even at 8 cm / min, the pressure loss was 25 mmH.
With g, this value remained almost unchanged even after continuous operation for 1 hour, and direct blood perfusion was possible.

Comparative Example 1 In the same manner as in Example 1 except that the conditions for forming uniform droplets were changed, the number average particle size was 290 μm, and 100% of the particles were within ± of the number average size.
Regenerated cellulose particles within 5% and having substantially the same sectional structure as in Example 1 were obtained. When the pressure loss was measured using these particles in the same manner as in Example 1, it was already over 100 mmHg at the linear velocity of 5 cm / min.

Comparative Example 2 In the same manner as in Example 2 except that the conditions for forming uniform droplets were changed, the number average particle size was 290 μm, and 100% of the particles were within ± 5% of the number average particle size. Polyacrylonitrile particles having the same sectional structure were obtained. When the pressure loss was measured using this particle in the same manner as in Example 1, the linear velocity was 5 cm / min.
By the way, it was already over 100mmHg.

Comparative Example 3 In the same manner as in Example 3 except that the conditions for forming uniform droplets were changed, the number average particle size was 274 μm, and 100% of the particles were within ± 7% of the number average particle size. Polymethylmethacrylate particles having the same sectional structure were obtained. When the pressure loss was measured using this particle in the same manner as in Example 1, the linear velocity was 5c.
It was already over 100 mmHg at m / min.

Comparative Example 4 In the same manner as in Example 4 except that the conditions for forming uniform droplets were changed, the number average particle size was 280 μm, and 100% of the particles were within ± 5% of the number average particle size. Polystyrene particles having the same sectional structure were obtained. When the pressure loss was measured using these particles in the same manner as in Example 1, it was already over 100 mmHg at the linear velocity of 5 cm / min.

Comparative Example 5 The number average particle size is 450 μm, and 89% of the particles have a number average particle size of 10
When the pressure loss was measured in the same manner as in Example 1 by using commercially available regenerated cellulose particles within the range of 2%, the linear velocity was 2 cm /
The pressure loss already exceeded 100 mmHg at min, and after that the pressure loss further increased, so the experiment was stopped.

From the above results, it is understood that the pressure drop during blood perfusion is directly related to the particle size and distribution of polymer particles, rather than the type of polymer. This result indicates that the polymer particles have high surface energy (cellulose).
This is a result different from the conventional wisdom that pressure loss becomes smaller in the case of a polymer having a small surface energy (polymethylmethacrylate or polystyrene), and even a polymer particle having a small surface energy can be directly adsorbed for blood perfusion. This shows that it can be used as particles.

EFFECTS OF THE INVENTION The polymer particles for affinity chromatography of the present invention have a specific number average particle size and a specific particle size distribution, and since the inside of the particles has a three-dimensional network structure, it is a filler for affinity chromatography. For example, when it is used for selective adsorption and removal of plasma protein from blood by direct blood perfusion method, no particles are generated, pressure loss is small, and problems such as hemolysis are less likely to occur. And other effects are achieved.

Claims (1)

[Claims] 1. The number average particle size is in the range of 300 to 600 μm,
Polymer particles for affinity chromatography in which all particles are within ± 10% of the number average particle size and the inside of the particles has a three-dimensional network structure.