JPH0429727A - Polymer porous membrane for removing lipoprotein - Google Patents

Abstract

PURPOSE:To obtain a polymer porous membrane removing lipoprotein from plasma or serum by setting the ratio of the filtering speed of a 5wt.% human serum albumin aqueous solution and that of pure water to 1/50 or more and the inhibition coefficient of gold colloid particles having a particle size of 30nm to 0.3 or more. CONSTITUTION:A polymer porous membrane is characterized by that the ratio (Jp/Jw) of the filtering speed (JP) of a 5wt.% human serum albumin aqueous solution and the filtering speed (Jw) of pure water is set to 1/50 or more, and the inhibition coefficient (R) of gold colloid particles having a particle size of 30nm is set to 0.30 or more. By the use of this polymer porous membrane, lipoprotein can be removed from plasma. Further, lipoprotein can be recovered in high yield without almost generating the denaturation of plasma protein other than plasma lipoprotein or the lowering of physiological activity, and plasma can be filtered at high filtering speed.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a porous polymer membrane for removing lipoproteins contained in protein-containing aqueous solutions, particularly plasma and serum. Preferred applications of the present invention include: (1) secondary filter in plasma exchange therapy for hyperlipidemic patients; (2) acquisition of low lipoprotein plasma from porosity plasma and high lipid plasma at blood collection and blood donation sites.

(3) Removal of lipoproteins from raw plasma, intermediate products, and preparations in the plasma fractionation process.

(4) Bedside filter for infusion (5) Heter-propiolactone method, Solvent Deterge
Removal of lipoproteins as a pretreatment to improve production efficiency in the virus inactivation process of plasma and blood products using chemicals such as the nt method.

[Conventional technology]

Lipoproteins are complexes of lipids and proteins. That is,
Since lipids are hydrophobic, they cannot exist independently in plasma or serum, but instead exist in the blood in the form of lipids surrounded by proteins and some phospholipids. Among lipids, cholesterol, phospholipids, and triglycerides have special bonds with apoproteins and exist in the form of lipoproteins, and free fatty acids mainly bind to albumin, which circulate together in the blood and are utilized by the living body. Ru.

Lipoproteins are divided into several types based on their lipid composition and role in lipid metabolism, and their names differ depending on the measurement method. In ultracentrifugation, the chylomicron (CM) density (g/ ml ) 0.95 or less, : Very low density lipoprotein (VLDL) density (g/mA') 0.95
1-1.006; Intermediate type (IDL) density < g/7
nl) 1.006-1.019 low density lipoprotein (LD
L) Density (g/ynl) 1.019-1.063
, high-density lipoprotein (HDL2) density (g7/7nl) 1
．． 063-1.125. ; high-density lipoprotein (HDL)
3) Classified as having a density (g/ml) of 1.125 or higher (see Haruo Nakamura, Serum Lipid Abnormalities and Their Treatment, p. 23, Nanzando).

The density of lipoprotein particles in plasma is determined by the ratio of proteins and lipids that make up the lipoprotein; the more lipids there are, the lower the density and the larger the diameter of the particles. The diameter of lipoprotein particles is 80 nm or more for chylomicron and 30-80 nm for VLDL.
m, IDL, LDL, HDL, etc. are said to be 30 nm or less. On the other hand, in electrophoresis, preβ-1βα-
Three fractions of lipoproteins were observed, ~'LDL, LD, respectively.
Compatible with L and HDL.

Lipids and lipoproteins are useful and indispensable for living organisms, but their constant presence in the blood at higher than necessary concentrations is not good for living organisms and can lead to adverse effects such as hyperlipidemia. . Conventionally, there is an adsorption method as a method for physically removing lipids from blood. For example, an adsorbent in which dextran sulfate is immobilized on porous cellulose particles (Posoba LA-40, manufactured by Kanekabuchi Chemical Industry Co., Ltd.) selectively adsorbs low-density lipoproteins and is used for the treatment of high-cholesterol plasma.

However, there is still no practical example of removing lipids and lipoproteins by membrane filtration.

[Problem that the invention seeks to solve]

The first object of the present invention is to provide a porous polymer membrane that removes lipoproteins from plasma and serum. The second object of the present invention is to create a porous polymer pore in which the concentration and physiological activity of albumin, gushichipurine, etc., which are useful proteins present in plasma and serum, remain almost unchanged even after filtration when lipoproteins are removed. The goal is to provide a membrane. A third object of the present invention is to provide a porous polymer membrane that is less likely to be clogged, has a high filtration rate, and has a large filtration capacity when performing filtration to remove lipoproteins.

[Means for solving problems]

The present invention provides a filtration rate (Jp) of a 5% by weight human serum albumin aqueous solution and a filtration rate (Jw) of pure water for removing lipoproteins from an aqueous solution containing proteins, human or animal plasma or serum. ) ratio (Jp/Jw) is (115
0) or more, and the rejection coefficient (R) of colloidal gold particles with a particle size of 30 nm is 0.3 or more, and the average pore size 2re measured by electron microscopy is a high It is characterized by a gradual decrease from the inner wall surface of the molecular porous membrane toward the outer wall surface of the polymer porous membrane through which the protein-containing aqueous solution filters out, and the average pore diameter of the inner wall surface of the polymer porous membrane as measured by electron microscopy is 2re. But 1100
nm to 1000 nm, and the average pore diameter of the outer wall of the porous polymer membrane measured by electron microscopy is 15 nm to 150 nm.
The present invention relates to a polymer porous membrane characterized in that the porous membrane is in the nanometer range.

The porous polymer membrane used in the present invention has a filtration rate (Jp) of a 5% by weight human serum albumin aqueous solution and a filtration rate (Jp) of pure water (
The ratio (Jp/Jw) of Jw) is (1150) or more and the rejection coefficient (
R) is required to be 0.3 or more. In order to efficiently recover proteins, it is necessary to consider the ratio of Jp and Jw.

Both the lipoproteins to be removed and useful proteins in plasma other than lipoproteins are proteins, so during filtration it is necessary to select a membrane material that has a low adsorption of proteins to the membrane. As a result of studies using various porous polymer membranes with different (Jp/Jw), the present inventors found that as (Jp/Jw) increases, lipoprotein removal and protein recovery become more efficient. They also found that there was little change in protein composition before and after filtration.

From this point of view, (Jp/Jw) needs to be (1150) or more, preferably (1/20) or more,
More preferably it is (1/10) or more. (Jp/Jw
) is less than (1150), not only is the efficiency of lipoprotein removal and protein recovery low, but also a protein cake layer is formed on the surface of the porous membrane over time, resulting in a decrease in these efficiencies. If the albumin permeability of the porous polymer membrane (the albumin permeability after 10 minutes when an aqueous solution containing 5% albumin dissolved in it is filtered through the porous polymer membrane) is 80% or more, a protein cake layer is formed. This makes it possible to achieve a protein transmittance of 70% or more in the liquid. Furthermore, by washing and filtrating with an aqueous solution or a buffer solution, a protein recovery rate of 80% or more can be achieved.

Albumin adsorption rate of porous polymer membrane (albumin concentration 50
If the amount of albumin adsorbed at 25°C from a physiological saline solution containing 0 ppm and 0.9% by weight of sodium chloride is less than 100 μg/bo, even if the protein concentration is 1% by weight or more,
It has been found that lipoproteins and other plasma proteins can be efficiently separated.

In plasma, lipoproteins exist in various densities and particle sizes depending on the lipid content. From lipoproteins with particle diameters of 30 nm or more, such as chylomicrons and VLDL, which have high lipid content, to LDL with particle diameters of 30 nm or less
, HDL lipoproteins are present. In addition, the concentration of lipoproteins varies from person to person, and there is plasma with a high concentration such as Chikang plasma. It is difficult to separate lipoproteins with a particle size of 15 nm or less from globulin and blood coagulation factors, which are plasma proteins other than lipoproteins, by filtration. It has been found that 50% or more of lipoproteins in plasma can be removed by filtration using the porous polymer membrane having a rejection coefficient (R) of colloidal gold particles of 30 nm in diameter of 0.3 or more.

At this time, it was discovered that albumin and globulin, which are useful proteins contained in plasma, can also be recovered by filtration at a high yield.

The rejection coefficient (R) of colloidal gold particles with a particle diameter of 30 nm is
If it is less than 0.3, the lipoprotein removal rate decreases to less than 30%. The inhibition coefficient (R) is 0.3 or more, preferably 0.4 or more, and when it is 0.4 or more, the lipoprotein removal rate is 30% or more.

If the purpose is simply to remove lipoproteins, the inhibition coefficient (R
) is 3.0 or more, the removal rate of lipoproteins becomes 80% or more, but at the same time, the permeability of globulin, which is a useful protein in plasma, decreases.

When actually filtering plasma, the issues are filtration speed and filtration capacity. In plasma, lipoproteins exist in various particle sizes depending on the lipid content. Particle diameter is 30n, such as chylomicron and VLDL.
From lipoproteins of m or more, 30n such as LDL and HDL
There are less than m proteins. In particular, lipoproteins such as chylomicron, which have a high lipid content, low density, and a particle size of 80 nm or more, tend to clog the pores of the membrane, reducing the filtration rate or making filtration extremely difficult. As a result of extensive research, the present inventors have found a solution to this problem by making the average pore diameter of the inner wall of the membrane larger than the average pore diameter of the outer wall, and using polymer pores whose pore diameter gradually decreases from the inner wall toward the outer wall. Invented membrane. That is, the average pore diameter of 200 by electron microscopy is
It is characterized in that the filtration of the protein-containing aqueous solution gradually decreases from the inner wall surface of the porous polymer membrane into which the aqueous solution containing protein flows in toward the outer wall surface of the porous polymer membrane through which the aqueous solution containing protein flows out. The average pore diameter 2π determined by electron microscopy is
Range of 1100nm to 1000n, preferably 70nm
~500 nm, and the average pore diameter 2re of the outer wall surface of the porous polymer membrane measured by electron microscopy is 15 nm ~ 150 nm.
Polymer porous membranes having a thickness in the range of m, preferably in the range of 25 nm to 80 nm are suitable. If the average pore diameter of the inner wall surface of the porous polymer membrane is 1100 nm or less, clogging is likely to occur (,
When it is 11000n or more, the strength of the film decreases. When the average pore diameter of the outer wall surface of the porous polymer membrane is 15 nm or less, the permeability of plasma proteins other than lipoproteins decreases, and when it is 150 nm or more, the lipoprotein rejection rate decreases. The concentration of lipoproteins in plasma varies from person to person; some plasmas have high concentrations, such as porous plasma, and the concentrations of the various lipoproteins that are its components also vary. Enlarging the membrane area or multi-stage filtration is also possible, but both are unfavorable from an economic standpoint. By employing the porous polymer membrane of the present invention, plasma proteins other than lipoproteins can be recovered after filtration without changing their composition from before filtration. Although conventional membranes on the market can remove lipoproteins, albumin and globulin, which are useful proteins in plasma, are also removed at the same time, and the filtration time is long (
The filtration rate is an important factor in plasma filtration because this causes denaturation of proteins in the plasma and impairs the usefulness of the proteins during filtration. From an economic standpoint, it is desirable that the filtration capacity, that is, the amount of liquid that can be processed per unit filtration area, be large.

The average pore diameter of the porous polymer membrane as a whole is preferably 30 to 1100 nm as determined by the water filtration rate method. Examples of the form of the porous membrane include a flat membrane, a tubular membrane, and a hollow fiber membrane. The film thickness is preferably 10 to 200 μm. More preferably, it is 20 to 80 μm. In the case of hollow fiber membranes, the inner diameter is preferably 100 μm to 1 mm. Materials for the polymer membrane include, for example, polyvinyl alcohol, ethylene/vinyl alcohol copolymer, regenerated cellulose, polyurethane,
Examples include mucopolysaccharide, low-substituted cellulose acetate, low-substituted cellulose butyrate, cellulose sulfate, polymethyl methacrylate, polyacrylic acid, polystyrene, and the like.

In general, hydrophilic polymers with a low albumin adsorption capacity and hydrophobic polymers made hydrophilic are preferred, and cellulose regenerated by cuprammonium method, polymers whose main component is polyvinyl alcohol, and partially saponified cellulose are preferred. preferable. Regenerated cellulose produced by the copper ammonia method is most suitable from the viewpoint of having little effect on living organisms due to filtration. As the aqueous protein solution, human or animal plasma or serum is preferably employed in the present invention, but the present invention can also be applied to protein solutions containing lipoproteins.

When carrying out the method for removing lipoproteins of the present invention, there are two methods: a method in which plasma is filtered while flowing in parallel along the membrane surface of a porous polymer membrane (parallel filtration), and a method in which plasma is brought into contact with the membrane under an almost stationary state. There is a method of filtration (vertical filtration). In order to efficiently achieve the object of the present invention, parallel filtration is preferable when the amount of the solution to be filtered is relatively large.

Vertical filtration is preferred when the amount of plasma to be filtered is relatively small and filtration is required for a short time.

In order to further increase the recovery rate of plasma proteins other than lipoproteins, it is preferable to provide the porous polymer membrane with a special pore structure as described below. That is, the pore structure of the porous membrane is a network structure, and the membrane has a multilayer structure in which networks are laminated in the thickness direction. Here, the network structure is a structure in which polymers form a network-like aggregate, and pores correspond to the meshes. A multilayer structure means that (a) when a porous polymer membrane has a hollow fiber shape on the front or back surface, it has an independent pore size distribution in a plane parallel to the inner wall surface or outer wall surface, independent of the location in the plane of interest; (b) The mutual positional relationship within this plane is substantially disordered, or the porous membrane is a hollow fiber. In the case of , the regularity of arrangement only in the direction of the fiber axis is observed, (C) a certain specified pore size distribution, average pore size, and in-plane porosity can be measured within this plane, and (d) from the membrane surface. The average pore diameter,
Either the pore size distribution or the in-plane porosity decreases depending on the distance from the outer wall of the membrane, and there is virtually no correlation between the pores between each layer. A porous membrane with a multilayer structure is broken in liquid nitrogen, and when the cross section is observed with an electron microscope, it has a diameter of 0.05 mm.
It is approximated by a deposit of ~2 μm particles.

A membrane having the characteristics of the pore structure shown in the present invention (a) generates microphase separation, (b) particles generated by the separation (in most cases, the particles are the polymer-rich phase), The diameter of the dilute polymer phase (sometimes it becomes particles) is 50 nm or more1
000 nm or less, and (C) conditions in which the composition and temperature of the stock solution and coagulation solution are strictly controlled so that the separation occurs simultaneously along the front and back surfaces of the film and progresses in the film thickness direction. (d) The average pore diameter of the membrane at the membrane outer wall surface and membrane inner wall surface is controlled by the coagulation liquid composition and hollow agent liquid composition.

The method for manufacturing the porous membrane according to the present invention will be explained using a cuprammonium regenerated cellulose porous membrane as an example. A solution with a cellulose concentration set within the range of 2 to 10% is prepared. Undissolved components and air bubbles are removed from the spinning dope. The spinning stock solution is 1
Strictly controlled to a set temperature between 0 and 40°C (usually ±0
．． (within 5°C).

The spinning dope is discharged from the outer spinning opening of the annular spinning opening. A coagulating agent (abbreviated as hollow agent) whose temperature and composition are strictly controlled is discharged from the central spinning opening of the annular spinning opening. The discharged spinning stock solution immediately passes through the coagulation bath. In this case, a liquid composition different from that of the hollow agent is used in the coagulation bath. The liquid composition and temperature of the hollow agent and coagulation bath must be strictly controlled. Microphase separation occurs between the hollow agent and the liquid in the coagulation bath. Particles are generated in the stock solution by controlling the composition of the coagulation bath, the composition of the hollow agent, the length of the coagulation bath, the spinning speed, the discharge speed of the hollow agent, and the winding speed.
The final particle diameter inside the hollow fiber can be determined within the range of 1000 nm. The hollow fiber thus obtained is regenerated with dilute sulfuric acid, washed with water, and dried under tension.

When manufacturing hollow fibers, a multilayer structure membrane obtained by taking measures to prevent tensile force from acting particularly in the fiber length direction is suitable for the purpose of the present invention.

Below, methods for measuring various physical property values measured in the present invention will be summarized.

Filtration rate of pure water and 5% by weight human serum albumin aqueous solution: filtration rate of pure water and 5% by weight human serum albumin aqueous solution
Filter at 0°C with a transmembrane pressure of 200+nmHg.

The average filtration rate at 0.511rd filtration rate is calculated and is defined as the filtration rate (-7 minutes). The human serum albumin aqueous solution is prepared by dissolving human serum albumin in pure water to a concentration of 5% by weight. The concentration of albumin is calculated by measuring the transmittance at a wavelength of 280 nm in the ultraviolet absorption spectrum and using a predetermined calibration curve.

Rejection coefficient (R) of colloidal gold particles: The rejection coefficient (R) of colloidal gold particles is calculated by the following formula. Rejection coefficient (R) =
pog+o(Co/C+), where C0 is the concentration of colloidal gold particles in the stock solution before filtration, and C7 is the concentration of colloidal gold particles during filtration.

30 nm colloidal gold particles were prepared in Nature (Vol.
, 241.2022, January, ry 1973)
“Controlled Nucleation for
rthe Regulation of the Pa
rticle 5ize in Mon.

disperse Gold 5uspensions
It was prepared by the reduction reaction of HAuCl4 with sodium citrate based on the paper of ``The particle size of monodisperse particles is measured using an electron microscope.

The concentration of gold colloid particles in the stock solution is approximately 1011 particles/
d, and the number of particles during filtration was determined by the absorbance method (wavelength 530 nm).
).

Average pore diameter by electron microscopy 2re: After embedding the polymer porous membrane in a resin (for example, acrylic resin),
II 8800 model) to a thickness of 0 from the surface (outer wall surface in the case of hollow fibers) along the film thickness direction.
．． Cut out samples of 5 to 1 μm in order.

After deembedding the sample section with a solvent (eg, chloroform), an electron micrograph of each section is taken.

The hole radius is r−r+d per 1 ctJ of the section of interest.
The number of holes present in r is denoted as N(r)dr. The average pore diameter 2π is given by the following equation.

Average pore diameter (2r+; nm unit) by water filtration rate method: Pure water is filtered in advance using a filter with an average pore diameter of 0.2 μ to prepare pure water from which fine particles have been removed. 20% of this pure water
The filtration rate Jv is measured at a constant transmembrane pressure (ΔP) of 200 mmHg at °C. However, the unit of Jv is (ml
/min). If the effective area of the porous polymer membrane used in the measurement is A(rrr), and the porosity of the porous membrane obtained by the apparent density method is Prρ, then the 2-layer is given by the following equation.

2 r+ =2.0(Jv-d・η/ΔP-A-Prρ
) 1″ where d is the film thickness (in μm), η is the viscosity of pure water (in centipoise), the porosity Prρ is the apparent density when swollen with water, ρaW, and the density of the polymer is ρp1 For cellulose, op = 1 Using .561g/J, Prρ(X)
-(1-ρaw/ρp)×100.

Lipoprotein determination method: VLD by heparin Ca precipitation/nephelometry
Measure L.

Quantitative method for β-lipoprotein: Measure by electrophoresis.

Quantitative method of albumin: Measure by BCG method.

Globulin quantification method: γ-globulin is measured by electrophoresis.

〔Example〕

Next, the present invention will be explained in more detail with reference to Examples.

A copper ammonia solution (with a weight ratio of copper ammonia/water of 3.0
7/6.3/90.1) at 4.8% by weight,
After filtration, the mixture was defoamed to obtain a spinning stock solution. This spinning dope is 25.0
2.37 rLl from the outer spinning spout (outer diameter 2.5 mmφ, inner diameter 1.5 mmφ) of the annular spinning spout while controlling the temperature at ±0.5°C.
/min. On the other hand, we adopted a solution (hollow agent) whose composition was strictly controlled at a water/acetone/ammonia ratio of 100.0150.410.53 (weight ratio), and
．． While controlling the temperature at 0 ± 0.5°C, the central spinning spout (outside diameter 0.
4 mmφ) at a rate of 4.1 J/min.

The discharged filamentous material was mixed with a water/acetone/ammonia ratio of 100.
．． It was directly introduced into a mixed solution at 25.0±0.5°C whose composition was strictly controlled at 0/32.010.21 (weight ratio) and wound up at a speed of 6.0 m/min in the solution. . Immediately after being discharged, the transparent blue-colored fibrous material gradually underwent microphase separation, followed by coagulation, and a fibrous structure was formed. Thereafter, it was regenerated at a constant length with a 2% by weight sulfuric acid aqueous solution at 25.0±0.5°C, and then washed with water. The obtained hollow fibers were vacuum dried at 20.0°C. The outer diameter of the hollow fiber thus obtained was 380 μm, the membrane thickness was 40 μm, and the inner diameter was 30 μm.
The water filtration rate pore size was 80 nm. According to scanning electron microscopy observation of the inner and outer walls of the hollow fibers, both wall surfaces had a network structure, and the network was deposited.The average pore diameter 2re of the inner wall surface of the membrane was 387 nm, and The average pore size 2re was 47 nm. The (Jp/Jw) of this film was (1/10), and the rejection coefficient (R) of 30 nm gold colloid particles was 1.9. By bundling 250 of these hollow fibers, the effective area is 0.
A cylindrical filtration module of 02rn' was assembled and used as a lipoprotein removal filter.

Transmembrane pressure of human plasma is 200mmHg using the filter of the present invention.
It was filtered using a vertical filtration method.

In a similar manner, porous polymer membranes with water filtration rate pore sizes of 30, 40, 50 and 60 nm were made, filters were assembled, and fresh human plasma was filtered.

The results are shown in Table 1.

Margins below [Effects] By using the porous polymer membrane of the present invention, lipoproteins can be removed from plasma. Furthermore, plasma proteins other than plasma lipoproteins can be recovered in high yields with almost no denaturation or decrease in physiological activity, and plasma can be filtered at a high filtration rate.

Patent applicant: Asahi Kasei Industries, Ltd.

Claims (1)

[Claims] Filtration rate of 1.5% by weight human serum albumin aqueous solution (J
The ratio (Jp/Jw) of p) and the filtration rate (Jw) of pure water is
(1/50) or more, and the inhibition coefficient (R) of colloidal gold particles with a particle size of 30 nm is 0.3 or more, a polymer for removing lipoproteins from an aqueous solution containing protein. Porous membrane. 2. The average pore diameter 2@r_e@ determined by electron microscopy is determined from the inner wall surface of the porous polymer membrane into which the protein-containing aqueous solution flows.
2. The porous polymer membrane according to claim 1, wherein the filtration of the protein-containing aqueous solution gradually decreases toward the outer wall surface of the porous polymer membrane from which the protein-containing aqueous solution flows. 3. The average pore diameter 2@r_e@ of the inner wall surface of the porous polymer membrane measured by electron microscopy is in the range of 100 nm to 1000 nm, and the average pore diameter 2@r_e@ of the outer wall surface of the porous polymer membrane measured by electron microscopy is 15 nm to 1000 nm. The porous polymer membrane according to claim 1, wherein the porous polymer membrane has a wavelength of 150 nm. 4. The lipoprotein according to claim 1 and/or 2 and/or 3, wherein the aqueous solution containing the protein to be filtered is human or animal plasma or serum. Polymer porous membrane for removal.