JPH03228671A - Porous regenerated cellulose membrane for removing mycoplasma and removal of mycoplasma - Google Patents

Abstract

PURPOSE:To obtain the title porous regenerated cellulose membrane having high removal ratio of mycoplasma, having specific average pore diameter range by water filtration speed method, specific membrane thickness and pore structure capable of being approximated by multi-layer structure membrane substantially having multi-stage filtration function. CONSTITUTION:The above-mentioned porous regenerated cellulose membrane for removing mycoplasma has 30-100nm average pore diameter 2rf by water filtration speed method and <=20mum membrane thickness (d) in a range shown by the formula. The membrane has pore structure capable of being approximated by multi-layer structure membrane substantially having multi-stage filtration function. By using the membrane, mycoplasma can be removed from animal plasma, serum, an aqueous solution of salt or an aqueous solution containing protein or amino acid. In the removal, mycoplasma can be removed in high removal ratio such as >=99.999%. In the case of a solution containing protein, permeability of protein is >=80% and recovery ratio can be increased >=90% by filtration followed by washing with a buffer solution, etc., and filtration.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a porous polymer membrane for removing mycoplasma present in an aqueous salt solution or an aqueous solution containing proteins or amino acids, and a method for removing mycoplasma using the porous membrane. . Here, a porous membrane is a membrane in which pores are clearly observed when observed using an electron microscope at a magnification of 100,000 times. A film in which pores are not clearly observed on the surface of the film when observed under an electron microscope at 100,000 times magnification is not included. In other words, a porous polymer membrane has an average pore diameter of 10n according to the water filtration rate method.
It can also be said to be a film with a diameter of m or more.

Preferred application examples of the present invention include the following uses.

(1) Removal of mycoplasma from animal-derived body fluids such as plasma and serum of animals such as cows and horses.

(2) Removal of mycoplasma from serum-containing or serum-free media for cell culture.

(3) Removal of mycoplasma from drugs or aqueous solutions containing proteins used in genetic engineering, etc.

(4) Preventing mycoplasma from entering the vaccine manufacturing process.

The present invention can be widely used in medicine, biochemistry, animal husbandry, bioindustry including biochemical industry, and the like.

[Conventional technology]

Mycoplasma is the smallest microorganism that can grow in an artificial cell-free culture medium, and it can be used in humans, cows, horses, pigs, sheep, goats, turkeys, etc.
It infects all animals including chickens, pigeons, dogs, cats, rats, and mice. It has no cell wall, is pleomorphic, and the smallest self-proliferating unit is 125-250 nIl, with the largest one reaching 600-900 nI11. Cell culture is an essential operation in biochemical, medical, and pharmaceutical research and production in bioindustry, but when mycoplasma enters the cultured cell system, the properties of the cell membrane change, resulting in cell The morphology of cells may change, or abnormalities may occur in the cell's metabolic system or chromosomes.

Therefore, mycoplasma contamination can have a major impact on research and production in the bioindustry field.

Mycoplasma can be introduced into a cell culture system in a variety of ways, including through air or droplets, biological materials such as cells and tissues that have already been infected and will become infected, and contamination from the culture medium.

As a method for removing or inactivating mycoplasma,
(B) Heat sterilization method using high temperature and high pressure steam, (II)
There are methods such as mixing mycoplasmacidal compounds (usually antibiotics) into the culture system. However, the heat sterilization method has problems such as denaturation of proteins mainly in the cell culture system, which reduces the physiological activity of proteins and prevents cells from growing sufficiently.
Heat sterilization cannot be applied to media and serum whose composition cannot be heat sterilized, and although some success has been achieved in some cases by adding chemicals (antibiotics for mycoplasma), complete inactivation is not always possible. However, problems still remain regarding the side effects of additives on cells.

On the other hand, removal by filtration has been adopted as a method for removing mycoplasma. However, with currently commercially available polyvinyl chloride-propylene copolymer membranes (manufacturer's nominal pore size: 0.22 μm) or polyvinylidene difluoride membranes (manufacturer's nominal pore size: 0.1 μm), cells can be removed by one filtration operation. It is impossible to completely remove mycoplasma from culture medium solutions. Therefore, 2
The reality is that filtration is repeated three or more times to increase the removal rate of mycoplasma. Furthermore, when these commercially available membranes are used to filter an aqueous protein solution, clogging occurs due to protein adsorption to the membrane, and the filtration rate is significantly reduced. 1-5k due to such a decrease in filtration speed
A pressure of g/c- is required, and as the pressure increases, the membrane permeability of proteins decreases significantly. It is highly possible that the mycoplasma itself is deformed by the applied pressure, or that the mycoplasma removal rate of the membrane decreases as the applied pressure increases.

[Problem to be solved by the invention]

The purpose of the present invention is to provide a method for removing mycoplasma from an aqueous protein solution that has a high removal rate of mycoplasma, a high recovery rate of protein from animal plasma or serum, or an aqueous solution containing proteins or amino acids, and which can filter a large amount in a short time. To provide a porous polymer membrane for removing mycoplasma and a method for removing mycoplasma.

When recovering proteins by removing mycoplasma from an aqueous protein solution, the required level of mycoplasma removal is much higher than the level of membrane permeation of proteins. For example, when removing mycoplasma that coexists with proteins using a polymer membrane, the membrane permeability of proteins (=(protein concentration in furnace solution/protein concentration in stock solution) X100) (χ) is 1 to 99%. Generally speaking, the higher the protein permeability, the better. On the other hand, mycoplasma removal rate (=(1-(mycoplasma concentration in solution F/mycoplasma concentration in stock solution))]X10
0 ) (χ) is 99.99 to 99.999999%
or more is a problem. If the purpose is only to remove mycoplasma, reducing the average pore diameter of the membrane will improve the mycoplasma removal rate to some extent. However, when the solution contains proteins, reducing the average pore size causes clogging of the membrane with proteins, which reduces the permeability of proteins and the permeation rate of solutions containing proteins. A problem arises in that protein composition changes greatly.

[Means to solve the problem]

In order to achieve the purpose of the present invention, in the present invention, the average pore diameter 2 by the water filtration rate method is 30 nm or more and 1100 nm or more.
n or less and the film thickness d is 20 μm or more, it is in the range of the following formula, (1000/7) (2 stones -) + 10≦d≦(1500
0/7)(25)-14 (unit: μm) and a porous regenerated cellulose membrane for mycoplasma removal having a pore structure that can be approximated by a multilayer structure membrane having substantially a multistage filtration function, and the membrane Provided is a method for removing mycoplasma from animal plasma or serum, an aqueous salt solution, or an aqueous solution containing proteins or amino acids using the method.

The first feature of the present invention is that it employs a porous regenerated cellulose membrane. The regenerated cellulose membrane adsorbs very little protein present in an aqueous solution, and the slightly adsorbed protein is reversibly adsorbed and desorbed, and almost no change in the physiological activity of the protein due to adsorption is observed. do not have. Regarding protein adsorption, among regenerated celluloses, ammonium-produced cellulose is particularly susceptible to denaturation of proteins due to adsorption, or regenerated cellulose and proteins in structures obtained by coagulating with a solution containing 20% by weight or more of an organic solvent (e.g., acetone). There is little interaction with Therefore, ammonium ammonium regenerated cellulose is particularly preferred for achieving this purpose.

The second feature of the present invention is that it has a specified pore structure. That is, it has a pore structure that can be approximated by a multilayer structure membrane having substantially a multistage filtration function. Here, the multilayer structure membrane having substantially a multistage filtration function is explained as follows.

(a) In the plane parallel to the surface or back surface (inner wall surface or outer wall surface in the case of a hollow fiber shape) of the porous polymer membrane, the cellulose aggregates form a network-like structure, and the shape is relatively circular to elliptical. It has a so-called closed shape (a shape that can be observed as a hole).

In the same plane, the pore size distribution and pore shape are almost the same regardless of the location, and in terms of the filtration performance of this plane,
-Can be approximated by a screen filter.

(b) The mutual arrangement of the pores within the plane of this sheet is substantially disordered, or if the porous membrane is a hollow fiber, the pores move in the direction of the fiber axis due to the flow of particles composed of regenerated cellulose. Orientation is slightly recognized.

(c) Within this plane, a specified pore size distribution, average pore diameter, and in-plane porosity can be measured from an electron micrograph.

(cl) The average pore diameter, pore diameter distribution, and in-plane porosity are all substantially different between the membrane surfaces (inner wall surfaces in the case of hollow fibers) at different distances in the membrane thickness direction. There is essentially no correlation. That is, these pore properties are given as a function of distance from the membrane surface.

(e) When the cross section of the membrane is observed using an electron microscope from the membrane thickness direction, particles composed of regenerated cellulose are continuous along the direction parallel to the membrane surface (circumferential direction in the case of hollow fibers), and some of the particles are A rod-shaped deformed form is observed. When a porous film with a layered structure is fractured in liquid nitrogen and its cross section is observed under an electron microscope, it reveals deposits of particles with a diameter of 0.05 to 2 μm (average particle size is 232 (μII+)). It is approximated by The calendar number of the layered structure is given by 6''d/23z. d is the membrane thickness (wall thickness in the case of hollow fibers) (μm). When the number of layers is 10 or more, the mycoplasma removal rate becomes extremely high.

A membrane having the characteristics (a) to (e) above substantially has a multistage filtration function during filtration.

The third feature of the present invention is that it has a specified combination of average pore diameter 2rt and film thickness d. In other words, the average pore diameter (2 pores) determined by the water filtration rate method is 30 nm or more and 100 na+
Below, the film thickness d Cam unit) is 20 μva or more (1
) is within the range of Eq.

(1000/7) (2)+10≦d≦(15000
/7) (2rr) 14
(1) When the diameter of the two peaks is 30 nm or less, protein permeability decreases rapidly. In particular, when human plasma or bovine plasma contains many types of components, and especially when the concentration of components that form particulates in an aqueous solution, such as neutral fat, is high, the permeability of proteins decreases more rapidly. In terms of protein permeability, filtration rate, and filtration capacity, the larger the two stones, the better. However, when the amount of sweat exceeds 1100n, the transmembrane differential pressure becomes 0.
There are some Mycoplasma species that have a removal rate of 99.99% or less when filtered at 1 atmosphere or less. Therefore, in order to increase the removal rate of mycoplasma to 99.99% or more, 2■ is 100n.
Must be less than or equal to s+. On the other hand, when 2 is constant and the film thickness d is varied, increasing d increases the mycoplasma removal rate and also increases the filtration capacity. However, the filtration rate decreases. Therefore, to increase the filtration rate, the number of stones is increased.

However, an increase of 2■ decreases the mycoplasma removal rate. Therefore, the relationship expressed by equation (1) can be empirically obtained between d and 2 diaphragms that can be used under practically usable filtration conditions.

In order to increase protein permeability, filtration rate, and filtration capacity, ammonium ammonium regenerated cellulose is used as the material, and the apparent porosity (Prρ) by the density method is 1.
It is preferably 5% or more and 60% or less. Prρ is 6
When it exceeds 0%, the mycoplasma removal rate decreases rapidly. If the amount of solution to be filtered is extremely small,
Hollow fibers are preferred as the shape of the porous membrane, and hollow fibers are preferred for industrially carrying out large-scale filtration under certain conditions.

In many cases, not only mycoplasma but also viruses are mixed into the protein-containing aqueous solution to be treated. In this case, it is important to remove both. In order to remove both by filtration and maintain a high protein filtration rate, it is preferable to provide a more specific pore structure. That is, both the average pore diameter in a plane parallel to the membrane surface observed by an electron microscope and the in-plane porosity Pre by electron microscopy decrease monotonically from the inner wall surface to the outer wall surface. Viruses can also be removed by adding the feature of a hollow fiber membrane consisting of pores with a two-neelon-like void capillary-like structure in the vicinity of the outer wall surface. Here, the neuron-like void-cabillary-like structure is a pore with a diameter of 50 to 500 nm (this is called a void).
A pore structure whose basic unit is a pore structure with a shape similar to a neuron, which is composed of a large number of tubular pores with a diameter of 10 to 50 nm (called capillaries) around this void. means. A feature of this structure is that it is only possible to connect the voids via capillaries. The existence of such pores can be confirmed by: 1) heating the polymeric monomer using a hollow fiber and then polymerizing it; 2) cutting it into a thin film after polymerization to prepare a sample for electron microscopy; The regenerated cellulose of the hollow fiber material is dissolved and removed by immersion in a cheap solution, and the resulting electron microscopy sample is observed at high magnification (more than 50,000 times) using a field emission scanning electron microscope. (The pore image obtained by this method is called a pore negative image.)

Hollow fibers having the pore structure shown in the present invention, particularly preferable pore structure characteristics, are capable of (a) generating microphase separation before coagulation during spinning, and (b) particles generated by the separation (the polymer-rich phase becomes particles). (c) This phase separation occurs simultaneously along the front and back surfaces of the membrane (inner wall surface and outer wall surface in the case of hollow fibers), and this phase separation occurs in the membrane thickness direction ( advance in the wall thickness direction).

In order to suitably carry out (a) to (c), it is necessary to strictly control the composition and temperature of the stock solution and coagulation solution, and to suppress the tension in the fiber length direction as much as possible when spinning hollow fibers. .

The method for producing hollow fibers of the present invention will be explained using ammonium ammonium regenerated cellulose hollow fibers as an example. A preset solution with a cellulose concentration in the range of 3-9% is prepared. Undissolved components and air bubbles are removed from the spinning dope, and the components that become the core during microphase separation are removed as much as possible through more precise furnace treatment. The temperature of the spinning stock solution is strictly controlled at a set temperature of 15 to 30°C (usually within ±0.4°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 stock solution passes through the gas for a short distance and immediately passes through the coagulation bath. In this case, a solution composed of components similar to those of the hollow agent is used in the coagulation bath. However, the liquid composition and temperature of the coagulation bath must be strictly controlled. Microphase separation occurs in the spun stock solution due to the hollow agent and the liquid in the coagulation bath. After microphase separation, the diameter of the regenerated cellulose particles inside the final hollow fiber after coagulation/regeneration/water washing/drying is 50-1100.
Niall within the range of 0n.

In order to efficiently filter and remove mycoplasma mixed into a solution using the membrane of the present invention having a multilayer structure, it is necessary to reduce the pressure difference between the membranes as much as possible during filtration. In order to remove all mycoplasma with a removal rate of 99.99% or more, the transmembrane pressure must be within 1 atmosphere. When the transmembrane pressure difference exceeds 1 atm, a small amount of mycoplasma begins to be released from the furnace when the amount of filtration exceeds a certain amount. Further, when the object to be filtered is animal plasma or serum, an aqueous salt solution, or an aqueous solution containing proteins or amino acids, vertical filtration (dead-end filtration method) is practically preferable as the filtration method. Vertical filtration allows filtration of a large amount in a short time.

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

Protein concentration: In the case of albumin, the transmittance at a wavelength of 280 nm in the ultraviolet absorption spectrum is measured and calculated using a predetermined calibration curve. The total protein in the solution before and after filtration is determined by measuring the absorbance at 540 nm using a color reaction using Biuret's reagent.

Albumin permeability Two serum albumin is dissolved in pure water at a concentration of 5% by weight. Using the obtained solution, the transmembrane pressure difference 2
At 00++ oaHg, the effective filtration area of the membrane is 1M.
When Ol is filtered, the transmittance is calculated from the albumin concentration (C8 and cr, respectively) before filtration and in the furnace solution using the following formula.

Transmittance (χ) = (c, /co) xtoo (χ) (2
) Structure of porous polymer membrane: Regenerated cellulose porous membrane is made of resin (
For example, after embedding in acrylic resin), the
Samples with a thickness of 0.5 to 1 μm are sequentially cut out along the film thickness direction from the surface (outer wall surface in the case of hollow fibers) using a diamond knife attached to a model (Model M8800). After deembedding the sample section with a solvent (for example, chloroform), an electron micrograph of each section is taken. From the photograph of each section, the average diameter distribution function N (r) within the layer of interest is measured. Here, N(r) is the number of holes that exist in the layer of interest with a rough diameter of r to r+dr.N(r)d
It is a general diameter frequency distribution function defined as denoted by r. Average pore diameter 2'
i? and in-plane porosity Pre are given by the following equations, respectively.

2 r@ =5:'2rN(r)dr/ 5r2N(r
) dr (3) Pre (% display) = (πS r ”N
(r) d r)
Create pure water from which fine particles are removed.

The pure water was maintained at a constant pressure of 200 μHg at a transmembrane pressure difference (ΔP) of 20° C., and the filtration rate Jv was measured.

However, the unit of Jv is (M1/min). When the effective filtration area of the porous polymer membrane used in the measurement is A (Cij), and the porosity of the porous membrane obtained by the apparent density method is Pre, the two hills are given by the following equation.

2 rf =2. o (Jv・d−η/ΔP・A・Pr
e)"" (5) where d is the film thickness (in μ-units) and η is the viscosity of pure water (in centipoise). Pre is the apparent density ρ when swollen with water, and the density ρ of cellulose = 1.561
It is calculated using the following formula using g/d.

Pre(χ)-(1-ρ,,/ρp)xlOO(6) Mycoplasma quantification method: Measured by the CFU method using the ability to form colonies on an agar medium as an index.

〔Example〕

Example 1 A copper ammonia solution (the weight ratio of cupric ammonia/water was 3.5.
7/6.3/90.1) dissolved at 4.9% by weight in 2
After multiple filtration, the mixture was defoamed to obtain a spinning stock solution. Add this spinning dope to 25.
While controlling the temperature at 0±0.4°C, a multiple mesh filter was installed just before the spinneret, and the outer spinning outlet of the annular spinning outlet (outside diameter 2.5°C) was installed.
5 mmφ, inner diameter 1.5 mmφ) at a rate of 2.3 d/min. - Power water/acetone/ammonia weight ratio 10
We adopted a solution (hollow agent) whose composition was strictly controlled at 0.0151.410.91, and heated it at 25.0±0.4°C.
4 from the central spinning port (outer diameter 0.4 mm) while controlling the temperature
．． It was discharged at a rate of 2 m/min.

After the discharged filament was airborne for a distance of approximately 5M, water/acetone/ammonia (weight ratio 100.0/60.0/1
．． 0) strictly controlled composition of 25.0±0.4°
5. into a mixed solution of C; It was wound up at a speed of 1 minute. At this time, the shape of the coagulation bath, the flow of the coagulation liquid in the coagulation bath, etc. are controlled so that no tension is exerted in the yarn length direction. Immediately after being discharged, the transparent blue-colored fibrous material gradually underwent microphase separation, followed by coagulation, and a fibrous structure was formed. After that, 25.0±0.4
It was regenerated at a constant length set at a shrinkage rate of 5% with a 2% by weight aqueous sulfuric acid solution at ℃, and then washed with water. After replacing the water in the obtained hollow fibers with Metatsuru, the fibers were vacuum dried at 20.0°C. The thus obtained hollow fiber had an outer diameter of 380 μm, a membrane thickness (wall thickness) of 42 μM, and an inner diameter of 295 μm. According to scanning electron microscopy observation of the inner and outer wall surfaces of the hollow fiber, both wall surfaces have a network structure and a structure in which the network is deposited. Further, in the negative image of the hollow fiber, the vicinity of the outer wall surface is composed of pores having a neuron-like void-cabinary-like structure. 2 and Pre decrease monotonically from the inner wall surface to the outer wall surface. However, only the outermost layer has a particularly large value of 2G and Pre. Particle diameter 2S is 0.14 μm, 21t is 72 r++
++, P re was 43%. The albumin transmittance was 99.5% or more. A cylindrical mud application module with an effective overarea of 0.02 nf was assembled by bundling 250 of these hollow fibers to form a mycoplasma removal filter module. Mycoplasma
aorale 5. A stock solution to be subjected to filtration was prepared by diluting a solution containing 1×10′ CFU/d twice with phosphate buffered saline. Sample liquid 500d to 200mm
Vertical filtration under Hg pressure. Filtration was performed only once.

In addition, vertical filtration under a pressure of 800 Hg and 200 d
Filtration was carried out while circulating at a flow rate of /min. As a comparative example, vertical filtration was carried out at a pressure of 200 mmHg using a polyvinylidene difluoride membrane with a nominal pore size of 0.1 times m. 50% of the furnace liquid at each predetermined filtration rate.
μ! The cells were then inoculated into test dishes, and mycoplasma was quantified by colony formation method. The results are summarized in Table 1.

Table 1 *1 Vertical filtration 200 anal Hg (filtration pressure) *2 Vertical filtration 800 anal Hg *3 Vertical filtration 200 mm) Ig *4 Vertical filtration 200 mnHg *5 Ammonium ammonium method regenerated cellulose *6 Borivinylidene difluoride Example 2 Add 10% fetal bovine serum by weight to the standard medium for cell culture (MEM) solution, and add 2.
Add AIDS-causing virus HIV to a concentration of 4XIO7CFU/d, or add 5.5X10'P of AIDS-causing virus HIV.
It was added so that it became FU (plaque forming unit)/d. These solutions 801d were subjected to the same porous membrane and filtration conditions as in Example 1. Table 2 was obtained by summarizing the mycoplasma concentration in the furnace fluid and the protein recovery rate.

(Left below) Table * 1 *2 *3 *4 *5 *6 [Effect] Vertical filtration 200-8g (filtration pressure) Vertical filtration 800-8g Vertical filtration 200■Hg Vertical filtration 200■Hg Ammonium copper method regeneration Cellulose polyvinylidene difluoride According to the present invention, mycoplasma mixed in animal plasma, serum, cell culture medium solution, and other solutions can be removed with a high removal rate, for example, 99.999% or more, and when the solution contains protein. This can be achieved by achieving a protein transmittance of 80% or higher and a recovery rate of 90% or higher by washing with a buffer solution or the like. In addition, since the filtration rate is high and the decrease in filtration rate over time is small,
Suitable for removing mycoplasma from proteins that may deteriorate over time. Furthermore, even if there is a risk of virus contamination, it becomes possible to remove not only mycoplasma but also viruses at the same time.

Claims (1)

[Claims] 1. Average pore diameter 2@r_f@ is 30n by water filtration rate method
A porous regenerated cellulose membrane for removing mycoplasma, which has a membrane thickness d of m or more and 100 nm or less, a membrane thickness d of 20 μm or more, which is within the range of the following formula, and which has a pore structure that can be approximated by a multilayer structure membrane having substantially a multistage filtration function. (1000/7) (2@r_f@)+10≦d≦(15
000/7) (2@r_f@)-14 (unit: μ
m) 2. In the regenerated cellulose membrane according to claim 1, the porosity (Prρ) measured by the apparent density method is 15% or more and 60% or less, and the substance constituting the membrane is made of ammonium chloride regenerated cellulose. A porous regenerated cellulose membrane for removing mycoplasma from an aqueous solution containing proteins, characterized by: 3. Both the average pore diameter 2@r_e@ by electron microscopy and the porosity Pre by the same method decrease monotonically from the inner wall surface to the outer wall surface, and there is a neuron-like void-cabillary-like structure near the outer wall surface. Claim 1 characterized in that it is a hollow fiber membrane composed of pores with
and/or removing the mycoplasma described in paragraph 2;
and a porous regenerated cellulose membrane that also removes viruses. 4. Mycoplasma mixed in an aqueous solution can be removed by ultrafiltration using the porous membrane according to the first and/or second and/or third claims at a transmembrane pressure of 1 atm or less. How to remove mycoplasma. 5. The aqueous solution to be filtered is animal plasma or serum,
5. The mycoplasma removal method according to claim 4, wherein the method is an aqueous solution of a salt or an aqueous solution containing proteins or amino acids, and the filtration method is essentially vertical filtration.