JPS5929197B2 - Protein collection method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for collecting proteins from an aqueous solution containing proteins using an ultra-P filter material made of polyacrylonitrile or a copolymer mainly composed of acrylonitrile as an adsorbent. It is.

An adsorption method using an adsorbent exists as a method for collecting and purifying useful proteins, such as enzymes and certain hormones, from an aqueous solution containing trace amounts of these proteins. As these adsorbents, heavy metals, barium sulfate, silicic acid and its salts, and ion exchangers having various functional groups are known. However, some adsorbents have low adsorption capacity, or conversely, adsorb strongly, making subsequent elution operations difficult. The adsorption mechanism of proteins to adsorbents is not clear, but in most cases it is thought that proteins are adsorbed through a kind of chemical bond with the functional groups of the adsorbent, or physically adsorbed due to the shape of the adsorbent. It is being

The present inventors conducted various studies on the relationship between the shape of the adsorbent and the protein adsorption capacity, and found that semipermeable membranes or ultrafiltration membranes made of acrylonitrile polymer have a high adsorption capacity (per unit weight) for proteins. The present invention was completed based on the discovery that it can be easily eluted. The present invention provides a method for collecting proteins from an aqueous solution containing proteins using an adsorbent, in which a semipermeable membrane or an ultrafiltration material made of polyacrylonitrile or a copolymer containing 30% by weight or more of acrylonitrile is used as the adsorbent. This is a protein collection method characterized in that it is used. The details of the present invention will be described below.

The method for producing the ultrafiltration material of the present invention generally involves dissolving a polymer in its solvent to prepare a polymer solution, shaping it into an appropriate form, and then coagulating or gelling it in a non-solvent. You can get it.

For example, methods for manufacturing flat ultrafiltration membranes are described in JP-A-50-90579 and JP-A-48-86163. Hollow fiber ultrafiltration membranes are disclosed in JP-A-49-6552, JP-A-48-57268, and the like. The ultrafiltration membranes obtained by these methods generally have a thickness of several tens of microns to several hundred microns, and have interconnected pores inside the membrane, allowing water to pass through it easily.
Used as a membrane.

In other words, if an aqueous solution is passed through these membranes, if the size of a certain solute is larger than the pore size of the membrane's pores, it will not pass through the membrane;
On the other hand, small solutes pass through the membrane. Therefore, if the pore size in the membrane is extremely large or extremely small, it will be difficult for solute molecules to penetrate into the membrane, making it unsuitable for the adsorption membrane of the present invention. In the case of an ultra-f membrane, it is made up of a surface layer containing pores with a minute pore size and an internal porous layer having pores with a relatively large pore size, and it is difficult to define the pore size of the membrane unconditionally.
The water permeation rate of the membrane reflects the pore size of these surface and inner layers, and in this sense, the performance of the membrane outside the bale is first determined by the water permeation rate. The ultraviolet membrane used in the present invention has a water permeability constant Lp (7/Cr!I.i.atm) of 0.
．． A range of 0.01 or more and 0.60 or less is desirable. Furthermore, in the present invention, which utilizes the adsorption function of the ultraviolet membrane, a membrane without a dense surface layer is preferable. Furthermore, in the case of a membrane in which a surface dense layer exists, it is effective to perform adsorption under pressure from the porous layer side on the back surface, and to perform desorption by applying pressure from the dense layer side. These ultrafon membranes have a very large internal surface area due to their porous structure and act as excellent adsorbents.

For example, the surface area of acrylic fibers is less than 1 I/V due to the dense structure inside the fibers, whereas these ultrafilter membranes have a total surface area of several tens of ml/f. In order to be excellent as an adsorbent, simply having a large surface area is not enough.

That is, there must be some kind of interaction between the adsorbent and the substance to be adsorbed to ensure good adsorption. As a result of intensive studies on this point, the present inventors discovered that polyacrylonitrile or a copolymer containing 30% by weight or more of acrylonitrile exhibits excellent adsorption ability for proteins. Moreover, it was found that unadsorbed proteins were easily eluted with acid or acrylic water. Cellulose acetate membranes are commercially available as ultrafiltration membranes with large surface areas, but these membranes hardly adsorb proteins. In the adsorption operation using an ultrafon membrane, the membrane may be simply brought into contact with an aqueous protein solution at normal pressure.

That is, it is possible to cut a flat membrane or hollow fiber into small pieces and bring them into contact with the raw water while stirring, or to wind the flat membrane in a spiral shape and pack it into a column to allow the raw water to flow down. The most effective method is to send raw water under pressure from one side of the ultrafiltration membrane to selectively adsorb proteins, and then send the eluate under pressure from one side or the other of the membrane, allowing the protein to be adsorbed inside the membrane. This is a method to elute proteins. When hollow fibers are used, the latter method becomes even more effective since a membrane support is not required. The adsorption capacity of the membrane of the present invention increases near the isoelectric point of the protein to be adsorbed.

Therefore, it is desirable to perform the adsorption operation with the pH adjusted to be as close to isoelectric as possible. Proteins to which the membrane of the present invention exhibits particularly excellent adsorption have a molecular weight of 3000 or more, and include insulin, α-chymotrypsin, lysozyme, urokinase, albumin, glucose, oxidase, amylase, and asparakinase. Urokinase will be explained as an example.

Urokinase is an enzyme that exists in trace amounts in human urine and activates blasminogen in the blood to produce blasmin, which has fipurin-dissolving ability, and is widely used as a therapeutic drug for various thromboses. Therefore, methods for efficiently collecting this enzyme from human urine are attracting attention, and many patents have been proposed for adsorbent materials. A method for specifically separating and purifying urokinase using acrylic synthetic fibers as an adsorbent is disclosed in Japanese Patent Publication No. 10232/1982, but as shown in the following examples, the method according to the present invention does not involve the above-mentioned known method. The acquisition rate of urokinase is several times higher than that of the method.
The present invention will be explained in more detail below using Examples.

Example 1 A flat membrane-like ultraswept membrane was obtained by the method described in JP-A-5090579 using a copolymer containing 9.1 wt% acrylonitrile and 9 wt% vinyl acetate.

The thickness of this flat membrane was 120 μm, and the surface area value determined by the nitrogen adsorption method was 54 i/f. Also, the water permeability constant Lp of the membrane is 0
．． 065 (y/Cd.mi.atm). This membrane was cut into 5 mu square pieces, added to fresh urine at a ratio of 1 y/l, and stirred for 3 hours. Thereafter, the film-like adsorbent was collected, transferred onto a Buchner funnel, and washed with water. The adsorbent was then contacted with a 4% aqueous ammonia solution to elute the protein from the adsorbent. Ammonium sulfate was added to the eluate to achieve 60% saturation, and stirring was continued for 1 hour. After standing still, the resulting precipitate was collected by centrifugation, dissolved in a small amount of water, and then dialyzed against water using a dialysis membrane. Summer. The urokinase activity of the obtained protein aqueous solution was expressed as P
lOug's fibrin method (BiOchem.BiOph
ys. Acta Vol. 24, p. 278), 45
00 (Urlit no ψ protein). In addition, the acquisition rate was calculated from the amount of fresh urine as a raw material and its urokinase activity, and the acquisition rate was 95%. Comparative Example 1 A similar experiment to Example 1 was conducted using acrylic fibers (Bonnell, manufactured by Mitsubishi Rayon Co., Ltd.), barium sulfate, and carboxymethyl cellulose instead of the ultrafiltration membrane as the adsorbent.

The acrylic fiber was cut into 511 lengths,
The sample was then immersed in a benzene-methanol mixed liquid to remove the oil before use. Barium sulfate and carboxymethyl cellulose were used as powdered reagents as they were. Urokinase activity and acquisition rate determined in the same manner as in Example 1 are shown in Table 1. Acrylic fiber is the best in Table 1,
It is inferior to the membrane of Example 1 in terms of yield and purity. Example 2, Comparative Example 2 Various commercially available proteins were dissolved in ion-exchanged water to a concentration of 0.05%, and the pH of the solution was adjusted to be near the isoelectric point or in the acidic range.

To 20 ml of this solution was added the polyacrylonitrile semipermeable membrane used in Example 1 and, for comparison, a commercially available cellulose acetate ultrafiltration membrane (Lp=0.055) cut into 5111 squares, 100η, and heated at room temperature. The mixture was gently stirred for 24 hours. Thereafter, the membrane was taken out and washed with distilled water, and the protein adsorbed on the membrane was measured by spectrophotometry, and its quantification was performed using a calibration curve prepared in advance. The results are shown in Table 2, which shows that the acrylonitrile semipermeable membrane exhibits excellent adsorption ability for proteins. Example 3, Comparative Example 3 Various copolymers with different compositions were synthesized by known methods using other vinyl monomers copolymerizable with acrylonitrile.

The obtained copolymer was dissolved in dimethylformamide to a concentration of 20 wt%, and this solution was cast onto a glass plate to a uniform thickness with an applicator, and the solution was poured onto a glass plate at 20°C.
It was immersed in water to form a gel. The obtained film was heat-treated in a constant length state in hot water at 75°C and air-dried. The surface area of the air-dried membrane was measured by nitrogen adsorption method. The membrane was also attached to an ultrafilter, and the water permeation rate was determined at a pressure of 1 atmosphere. Using each membrane, the adsorption amount of α-chymotrypsin was determined in the same manner as in Example 2. The results are shown in Table 3. Example 4 Using the acrylonitrile copolymer used in Example 1,
Hollow fibers were manufactured using a wet spinning method.

The outer diameter of the hollow fiber is 700μ and the inner diameter is 500μ.The water permeation rate of the hollow fiber membrane was determined by sending pressurized water from the outside of the hollow fiber and measuring the amount of water obtained from the hollow part, which was 0,085 (7/
Cd. mm. ATM). The hollow fibers were bundled to form the hollow fiber module shown in FIG. 1, and the apparatus shown in FIG. 2 was used to collect urokinase contained in human urine by adsorption. The details of the operation will be explained below. In FIG. 2, fresh human urine collected from an adult male is put into a stock solution tank 1 and sent to a module 3 via a valve 4 by a pump 2.

Most of the urine that has passed through the module returns to the stock solution tank 1 via the valve 5. At this time, adjust the valve 5 to keep the pressure inside the module constant (at least normal pressure). A portion of the urine passes through the hollow membrane and returns to the stock solution tank via the valve 6, T.
At this time, urokinase in urine is adsorbed to the membrane. As such circulation operation continues, the liquid permeation rate of the membrane gradually decreases due to the adsorption of urokinase. When the liquid permeation rate drops to 10% of the initial water permeation rate, the operation is stopped, the urine in the stock solution tank is discarded, and water is added. Switch the valve 4 to the module and stock solution tank side, and further switch the valve 7 to only the module direction.

Thereafter, the pump is activated to send water through valve 7 to module 3, and through valve 6 back to the stock solution tank. In this case as well, the valve 6 is adjusted to maintain a constant pressure (at least normal pressure) inside the hollow fiber. A portion of the water pressurized by the pump passes through the membrane from the inner wall of the hollow fiber, reaches the outer wall of the hollow fiber, flows out of the hollow fiber, and returns to the stock solution tank via valves 4 and 5. By this operation, impurities adhering to the inside and outside of the hollow fibers are removed and the module is cleaned. After thoroughly cleaning the inside of the module,
The water in the stock solution tank was replaced with 4% ammonia water, which is an eluate for urokinase, and the operation was performed in the same manner as the cleaning operation to desorb the urokinase adsorbed to the membrane. The liquid permeation rate of the membrane recovers as the operating time increases, reaching 90% of the initial water permeation rate.
Circulating operation was stopped when the temperature reached . A urokinase solution was recovered in the same manner as in Example 1 from the obtained dilute ammonia water containing urokinase. The acquisition rate of urokinase obtained by treating 30' urine using a module with a membrane area (hollow fiber outer surface area) of 1.2 I was 95%.
The activity was 4600 (Unit/amount of protein).

[Brief explanation of drawings]

FIG. 1 is a cross-sectional view of a hollow fiber ultrasonic module. 101 is a hollow fiber, 102 is an adhesive, 103, 104,
105 and 106 are module entrances and exits. 107 is an outer cylinder of the module. Figure 2 shows a protein adsorption device using the hollow fiber module (Figure 1). 1 is the stock solution tank, 2 is the pump, 3 is the hollow fiber module,
4, 5, 6, and 7 are valves.

Claims (1)

[Scope of Claims] 1. A method for collecting proteins from an aqueous solution containing proteins using an adsorbent, wherein the adsorbent is a semipermeable membrane made of polyacrylonitrile or a copolymer containing 30% by weight or more of acrylonitrile. A protein collection method characterized by using an ultrafiltration material. 2. The protein collection method according to claim 1, which uses a membrane-like substance as the semipermeable membrane or ultrafiltration material. 3. The protein collection method according to claim 1, wherein a hollow fiber is used as the semipermeable membrane or ultrafiltration material. 4. The process according to claim 1, wherein the semipermeable membrane or ultrafiltration medium is contacted with an aqueous solution containing protein under pressure, and then the protein adsorbed to the semipermeable membrane or ultrafiltration medium is desorbed. Protein collection method.