JP2005349268A - Substance separation and purification process utilizing diffusion through porous film - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an industrially applicable process utilizing diffusion through a film as a process for separating and purifying a physiologically active substance under mild conditions. <P>SOLUTION: A film having a mean pore diameter of 5 to 100 nm and a porosity of 30 to 90% is used. A steady-state diffusion process utilizing diffusion through the insides of the pores of the film is applied, and the obtained diffused liquid is concentrated by the film in a manner of parallel filtration. It is possible to separate and purify physiologically active substances on an industrial scale by applying a plurality of the steady-state diffusion processes to the residual of the solution before subjection to the diffusion filtration. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

    The present invention relates to a substance separation method used in a separation / purification step when producing a physiologically active substance or the like under mild conditions. More specifically, the present invention relates to a method for separating and purifying a substance using a substance diffusion phenomenon through pores in a porous membrane.

    Ultracentrifugation, various types of chromatography, adsorption, dialysis, and precipitation using ethanol or ammonia sulfate as methods for separating and purifying physiologically active substances such as proteins and glycoproteins, or substances that are unstable and easily denatured against heat and chemicals Separation is employed. Microfiltration membranes and ultrafiltration membranes are used for solid-liquid separation. Large-scale processing is difficult with methods other than sedimentation separation, and all are batch processing, making it difficult to incorporate a production line as a continuous process. Various chromatographies are widely used as separation and purification methods because of their high performance. However, the applied liquid is clean and contains only a limited number of substance types in the liquid, and its versatility as a single type of chromatography carrier is small.

    On the other hand, the membrane filtration method is suitable for solid-liquid separation, but the membrane is clogged, it is difficult to maintain stable filtration performance, and the pressure on manufacturing cost is great, so it is applied only to limited solutions. Yes. (Patent Documents 2 and 3) When the size of the molecules to be separated is reduced, the average pore size of the applied ultrafiltration membrane is reduced, and accordingly, the filtration amount per effective filtration area of the membrane is reduced. In addition, dialysis is the only method for separating and purifying substances in a liquid using a diffusion phenomenon through a membrane, and a large-scale continuous dialysis process has hardly been put to practical use. The biggest reason why membrane separation utilizing membrane diffusion is not utilized is the extremely small movement rate of the substance, which is 1 / 1,000 or less in the case of filtration. In gas membrane separation, a method of increasing the selective permeability of gas molecules in a method of embedding a special carrier in a pore is known. (Patent Document 1)

    In the present invention, the porous film means a film in which the presence of pores is recognized by a field emission scanning electron microscope, a film having an average pore diameter of 5 nm or more and a porosity of 30% or more. Diffusion means mass transfer using a concentration gradient as a driving force, and pore diffusion means diffusion mainly through diffusion in the porous membrane. The diffusion of a substance in a conventional membrane is diffusion defined as dissolution diffusion by diffusion through a substrate of a material constituting the membrane. To distinguish between pore diffusion and dissolution diffusion, the apparent activation energy of diffusion may be measured. In the case of the former diffusion, the energy is 0 to 4 kcal / mole, and the latter is 8 to 50 kcal / mole. The steady pore diffusion method means diffusion in which the difference in the concentration of the material between the membrane surface and the membrane back surface is kept substantially constant in time in the diffusion through the pores in the membrane, so that the diffusion rate is kept constant. In diffusion conventionally used in dialysis or the like, this concentration difference changes with time, the diffusion rate decreases with time, and a steady state cannot be achieved.

      The liquid to be diffused is a liquid before applying the steady-pore diffusion method, and the diffusion liquid is a solution containing a substance that has passed through the membrane obtained by the diffusion method and containing a so-called membrane-permeable substance. The substance concentration inside is lower than the substance concentration in the liquid to be diffused. (Non-patent document 1) The residual liquid of the diffusion liquid means a solution at the outlet on the film surface on the diffusion liquid side after applying the steady hole diffusion method to the diffusion liquid. Parallel filtration is a type of filtration in which a solution is allowed to flow along the membrane surface, and is also referred to as cross-claw filtration or tangential filtration.

The average pore diameter is given by (viscosity, film thickness, filtration rate / transmembrane differential pressure, porosity). Here, the filtration rate is the filtration rate of pure water per square meter, measured in units of ml / min, the film thickness is in microns, the viscosity is in centipoise, the transmembrane pressure is in mmHg, and the porosity is in dimensionless units. It is. The average pore diameter at this time is in nm units. The porosity is given by:
Porosity = (1-membrane density / material polymer density)
The density of the film is calculated by (film weight / film area * film thickness). The density of the material polymer is the density of the membrane when the porosity is 0%, and this has already been given in the literature. A multilayer structure film is a layer having a thickness of 10 to 1000 nm when observed with an electron microscope from the cross-sectional direction of the film. When observed from the surface of the film, a network or a gap between particles is formed as a hole, and particles are fused. It is a film in which the appearance is observed.

Japanese Patent Application Laid-Open No. 06-091130 “High-Pressure Promoting Membrane for Selective Separation and Use thereof” A specific component in a gas is facilitated and transported by embedding a promoter liquid containing a carrier dissolved in a solvent in pores in the porous membrane. Japanese Patent Application Laid-Open No. 06-044954 “Battery and Selective Permeation Membrane Used in the Battery” Permeation of water vapor is blocked by the porous permeable membrane, and only oxygen is efficiently permeated to give an excellent battery. JP-A-11-195410 “Lithium Secondary Battery” By using a polyolefin microporous membrane as a battery separator, depletion of electrolyte between electrode groups is prevented. Rumiko Fujioka, Masako Yoshida, Chizu Yoshimura, Tomoko Yamamura, and Seiichi Manabe “Trial Manufacture and Separation Characteristics of a New Membrane Separator Using Differences in Liquid Diffusion Coefficient between Particles” Bulletin of Faculty of Human Environment, Fukuoka Women's University, Vol. 29, 13-20, 1998

      In the present invention, the film diffusion phenomenon can be utilized industrially. At that time, the characteristics of this membrane diffusion are: (1) Clogging of the membrane is unlikely to occur (2) Separation and purification based on the difference in diffusion rate is possible (3) Sieve mechanism that is central in membrane filtration mechanism The object of the present invention is to propose a method for eliminating the drawbacks of membrane diffusion, that is, the slowness of mass transfer and the low concentration of the substance in the diffusion solution, while maintaining the characteristics that occur as they are.

    The greatest feature of the present invention is that a steady pore diffusion method is used as a method for separating materials. Conventionally, diffusion of a membrane is movement by a so-called dissolution and diffusion mechanism in which a substance dissolves in a polymer matrix that is a material of the membrane and diffuses in the membrane after dissolution. The diffusion coefficient in this mechanism is about 10 −10 square centimeters / second. Therefore, it is slow so that it is difficult to use industrially. On the other hand, in the pore diffusion, the diffusion coefficient is about 10 −6 square centimeter / second, which is about 1/10 of the filtration rate, which is an industrially usable value. Due to diffusion, clogging into the hole occurs only to a negligible extent. Steady diffusion allows a diffusion mechanism to be incorporated as part of a continuous process. In order to realize steady diffusion, it is preferable that the concentration gradient through the membrane is constant and clogging does not occur. Reasons why steady-state pore diffusion has not been used in the past are as follows: (1) Filtration occurs due to the pressure required to flow the liquid. (2) Since there is no material separation data due to pore diffusion, how the difference in diffusion coefficient is used for separation. It is unknown whether it contributes (3) Since the characteristics of the pores used for pore diffusion are unknown, it is possible to obtain a porous membrane that meets this purpose.

    As a result of studying a diffusion device in which pore diffusion is dominant, operating conditions, and pore characteristics, the present inventor (1) a pump for feeding a liquid to be diffused into the membrane surface and a residual liquid of the liquid to be diffused as a film (2) Make the pumping speeds of both pumps practically coincide with each other (3) Porosity with an average pore diameter of 3 nm to 200 nm Porous polymer membranes with a porosity of 10% or more and 90% or less can achieve diffusion with dominant pore diffusion. (4) The diffusion coefficient in the aqueous solution in which a plurality of substances having different pore diffusion coefficients are mixed is independent. In many cases, the value is close to or slightly decreased. In other words, it was clarified that substances with different diffusion coefficients can be separated from each other. This patent was reached for the first time by obtaining the experimental results of (1) to (4).

    The greatest feature of the present invention is that a porous membrane having specific pore characteristics is used, and a method for separating and purifying a substance in liquid using the pore diffusion phenomenon is provided. That is, the porous material having an average pore diameter of 5 nm or more and 100 nm or less and a porosity of 30% or more and 90% or less is highly hydrophilic in the sense that it is easy to regenerate the membrane and prevent clogging of the pores due to adsorption. It is desirable to be a molecule. Specific examples include regenerated cellulose membranes, polysulfone membranes, polyacrylonitrile membranes, and cellulose derivative membranes. If the average pore diameter is less than 5 nm, the contribution by the dissolution / diffusion mechanism is large, and the diffusion coefficient is too small. From the viewpoint of preventing infectious particles such as viruses from passing through the membrane, the average pore size is preferably 75 nm or less, and the average pore size is preferably 10 nm or more and the porosity is preferably 50% or more from the viewpoint of the diffusion rate. The upper limit of the porosity is 90% or less. If it exceeds 90%, the mechanical properties of the film are remarkably deteriorated, and the probability of occurrence of pinholes is also increased. The porosity is desirably 80% or less from the viewpoint of film strength, ease of handling, and prevention of pinholes.

    As a method for producing a porous membrane, a wet or dry membrane production method can be employed. In this method, microphase separation usually occurs during film formation. A multilayer structure film can be produced by successively generating microphase separation in the film thickness direction. The multilayer structure film has excellent fine particle removability and a higher diffusion rate than other structure films having the same average pore diameter and porosity.

A feature of the present invention is that a steady hole diffusion method is employed. In order to keep the concentration difference of the liquid through the membrane constant (required condition for maintaining the steady state), the liquid to be diffused is made to flow at a constant speed along the surface of the membrane, and the diffusion liquid is made constant along the back surface of the membrane. Run at speed. It is desirable to set the flow direction at this time in the same direction because mass transfer due to filtration hardly occurs. If the strain rate of the liquid to be diffused on the film surface is 1 / second or more, deposition of substances on the film surface can be prevented. Since an extreme increase in strain rate causes inactivation of the physiologically active substance in the liquid to be diffused, the strain rate has an optimum value depending on the tissue of the liquid to be diffused.

      The concentration of the target substance in the diffusion liquid is about 1/10 of the concentration in the diffusion liquid. In the present invention, a process of concentrating the diffusion liquid by parallel filtration is employed in order to increase the substance concentration in the diffusion liquid. In the membrane concentration step by parallel filtration, the filtration conditions are mild and not accompanied by a phase change, so that the bioactive substance in the diffusion liquid hardly changes. The membrane material used for parallel filtration is most preferably a hydrophilic membrane module currently used for artificial kidneys. That is, there are an artificial kidney made of regenerated cellulose having an average pore diameter of about 3 nm and an artificial kidney made of polysulfone having an average pore diameter of 4 nm. Since the diffusion solution is generally clean, clogging of the artificial kidney for concentration can be prevented by setting the strain rate of the diffusion solution to 3 / second or more and 300 / second or less.

      By adopting the method of the present invention, it becomes possible to separate and purify physiologically active substances and the like. Membrane separation was considered optimal for separation and purification of thermally, mechanically, and chemically unstable substances, but industrially, there are many obstacles to membrane separation as described above. The membrane diffusion method, which reflects the characteristics of membrane separation most, has hardly been industrially developed. The present invention succeeded in greatly improving the small separation rate, which was the greatest drawback of membrane diffusion, and improved the membrane diffusion to a level that can be used industrially.

      The present invention is carried out by taking as an example the case where albumin is concentrated and separated from a solution (diffusion liquid) that may contain hepatitis C virus in an aqueous albumin solution (concentration of 5% by weight) having a molecular weight of about 50,000. The best mode for this is shown. A multilayered film of about 150 layers is used with a regenerated cellulose porous film having an average pore diameter of 35 nm. The porosity is 60%, and the porosity in water is 80%. The membrane is a hollow fiber membrane with an inner diameter of 330 microns, a thickness of 30 microns, and an effective diffusion area of 100 square centimeters. The length of the hollow fiber membrane is 8 cm. In order to increase the effective diffusion area, a method of increasing the number of hollow fiber membranes may be used, and the length of the hollow fiber membranes may be increased by increasing the size of the inner diameter. A hollow fiber membrane is formed into a cylindrical module. The diffused liquid part is connected to the outer cylinder inlet of the hollow fiber membrane module, the hydrostatic pressure is set to 30 to 100 centimeters of water, and a screw cock for flow rate control is attached to the outer cylinder outlet of the module. An interlocking pump is installed at the inlet / outlet of the module corresponding to the inlet and outlet of the hollow part inside the hollow fiber membrane. If the flow rate by the interlocking pump is set to 1.5 ml / min, the strain rate is 1.2 / sec. On the other hand, the flow speed of the diffusion liquid portion is set to a value close to the flow speed on the diffusion liquid side. The concentration of albumin in the diffusion solution is 0.5% by weight, and hepatitis C virus is below the detection limit in this solution. This diffusion solution is subjected to parallel filtration at a strain rate of 5 / sec into a hollow fiber for artificial kidney made of regenerated cellulose having an average pore diameter of 3 nm. When the transmembrane pressure difference is 200 mmHg, an aqueous solution with an albumin concentration of 10% by weight is recovered. The remaining liquid of the liquid to be diffused can be further subjected to steady pore diffusion using the same circuit as the next liquid to be diffused to increase the albumin recovery rate. It is possible to increase the recovery rate of albumin to 90% or more by continuing this separation and purification process.

Regenerated cellulose porous hollow fiber membrane having an average pore diameter of 35 nm and a porosity of 60% in an aqueous solution in which deoxyribonucleic acid (DNA) having a molecular weight of 14 million and acid dye Acid orange7 (molecular weight 350.32) are dissolved (inner diameter 330 microns, film thickness 30 microns) Steady hole diffusion using an effective diffusion area of 100 square centimeters was performed at 25 ° C. The flow rate of the diffusion liquid was 2 ml / min, and the flow rate of the diffusion liquid was 2.2 ml / min. The diffusion solution was subjected to parallel filtration using a regenerated cellulose hollow fiber module having an average pore diameter of 3 nm at a strain rate of 3 / sec and a transmembrane pressure difference of 100 mmHg. As a result, the common logarithm of the diffusion coefficient (25 ° C.) in a single aqueous solution of DNA and Acid orange 7 was −8.214 square centimeter / s and −6.850 square centimeter / s, respectively, but −9. 364 or less and −6.618 square centimeter / s, only Acid orange 7 diffuses into the diffusion solution, and both are completely separated, and this separation efficiency exceeds the prediction from the diffusion coefficient of each component alone. It was a level. The final concentration rate of Acid orange 7 was about 3 times the value in the first mixture. The apparent activation energy of diffusion of Acid orange 7 was 0.468 kcal / mole, and it was confirmed that diffusion in the film occurred due to pore diffusion.

      Porous diffusion was carried out in the same manner as in the example using an equivalent mixed aqueous solution of Acid blue 1 and Acid orange 7 having a molecular weight of 544.68. The logarithms of the diffusion coefficients (25 ° C.) when used alone were −7.059 and −6.833 square centimeters / s in the mixed aqueous solution, which were −7.104 square centimeters / s and −6.850 square centimeters / s, respectively. It can be seen that both components independently diffuse the pores. However, the ratio of the diffusion coefficient indicating the degree of separation efficiency is 1 and 25, and it is necessary to further separate the diffusion liquid by pore diffusion. When this diffusion liquid was used as the liquid to be diffused and the number of holes was expanded, the diffusion coefficient ratio was 1.28. Therefore, the apparent diffusion coefficient ratio was 1.6 due to the two-stage hole diffusion. That is, it can be seen that the separation between the components becomes more effective by performing the pore diffusion in multiple stages.

A saturated aqueous solution of one type of dispersion paint Disperse orange 3 (molecular weight 242.24) and one type of optical brightener chromotere (molecular weight 300.27) was separated by pore diffusion in the same manner as in Example 1. The logarithm of the diffusion coefficient in the case of a single component was −7.777 square centimeter / s for Disperse orange 3 and −6.463 square centimeter / s for chromotail. In the mixed component solution, the former was −6.583 and the latter was −9.672 or less, and both were completely separated. Therefore, as a system to which pore diffusion can be applied, there are cases in which separation by pore diffusion becomes possible because the state of association of molecules in the solution changes due to the interaction between mixed components.

The present invention can be used for industries that require separation and purification under mild conditions (eg, pharmaceutical industry, food industry), particularly separation and purification of physiologically active substances such as proteins and DNA. Further, in the industry handling colloidal systems, it can be incorporated into industrial processes as a method for purifying and separating specific fine particles including colloidal particles.

Claims (4)

      In the process of separating and purifying a substance having a molecular weight of 100 by the steady pore diffusion method using a porous membrane having an average pore diameter of 5 nm to 100 nm, a porosity of 30% to 90%, parallel filtration of the diffusion liquid is performed. A method of separating and purifying a target substance by concentrating the target substance and performing a separation and purification step of a steady pore diffusion method over a plurality of times in the liquid to be diffused.       The method for separating and purifying a substance according to claim 1, wherein the porous membrane is a multilayer structure membrane, and the membrane used for parallel filtration is a membrane module for an artificial kidney.       In Claims 1 and 2, the strain rate of the liquid to be diffused on the membrane surface in the steady pore diffusion method is 1 / second or more, and the strain rate of the diffusion solution on the membrane surface in parallel filtration is 3.0 / second or more, And a method for separating and purifying a substance, characterized by being 300 / sec or less 3. The porous membrane according to claim 1, wherein the average pore diameter of the porous membrane is 10 nm to 75 nm, the porosity is 50% to 80%, the material of the membrane is a hydrophilic polymer, and parallel filtration Separation and purification method, characterized in that the membrane material for filtration is a hydrophilic polymer