JPS60248180A - Method and apparatus for purification of kallidinogenase - Google Patents

Abstract

PURPOSE:To prepare highly pure kallidinogenase, with a simple operation, in high efficiency, by passing a stock solution containing kallidinogenase successively through two liquid chromatographic apparatuses packed with an anion exchange resin as a column filler. CONSTITUTION:The autodigested liquid of swine pancreas stored in the raw material tank is treated with a coagulant in the coagulation tank 1, and the filtrate discharged from the filtration tank 2 is passed through the column 3 packed with an anion exchange resin to effect the first-stage separation. The product is desalted and concentrated by the separation membrane 4, subjected to the second-stage separation by the column 5 packed with an anion exchange resin, desalted and concentrated by the separation membrane 9, and freeze- dried by the freeze-drier 10 to obtain the highly purified product 12.

Description

DETAILED DESCRIPTION OF THE INVENTION 3.1 Technical Field The present invention relates to a method for efficiently removing impurities from various tissues and body fluids of mammals containing kallidinogenase and purifying kallidinogenase to a high degree of purity.

3.2 Prior art and its problems Kallidinogenase is a type of proteolytic enzyme produced in animal bodies, and acts on kininogen to liberate kinin, which is a substance with cardiovascular effects. Therefore, this kallidinogenase has physiological activities such as peripheral vasodilatory action and is widely used as a therapeutic agent for cardiovascular diseases.

As a pharmaceutical, kallidinogenase obtained from pig pancreas is mainly used.

In addition to kallidinogenase, there are many other mixed proteins that coexist in the pancreas. In particular, contamination with kininase, which decomposes kinin, significantly reduces the action of kallidinogenase as a drug. Therefore, methods for extracting and separating kallidinogenase that do not contain these coexisting enzymes have been studied for a long time.

Conventionally, methods for purifying kallidinogenase include ammonium sulfate salting out,
Methods used include a combination of solvent precipitation, ion exchange chromatography, gel filtration, etc., or affinity chromatography using an expensive reagent, kallidinogenase inhibitor.

However, purification by ammonium sulfate salting out and solvent precipitation requires a lot of time and effort, and results in large sample losses. In the case of ion-exchange chromatography, the method of adsorbing and separating the weakly basic anion-exchanged cellulose as a packing material has disadvantages in the physical robustness of the ion exchanger and its susceptibility to microbial contamination. The method using an ion exchange resin has the drawback that elution of the target product is slow and it takes a large amount of solvent and a considerable amount of time to completely elute and separate the product.

In a method using gel filtration, it is difficult to remove impure proteins having a molecular weight comparable to that of kallidinogenase contained in a crude kallidinogenase solution.

In the case of affinity chromatography, the natural trypsin inhibitors used therein, such as ovomucoid trypsin inhibitor and soybean trypsin inhibitor, are expensive, so this method is also inappropriate as a means for industrial use.

As shown in E above, all of these methods are too complicated to operate as industrial production methods, are too expensive for mass production, have insufficient purification efficiency, and cannot obtain high purity products. Many problems remain.

3.3 Purpose of the present invention In view of the current situation as described in Section I, the present invention was made with the aim of providing a method that is easy to operate, rapid, and inexpensively purifies high-purity kallidinogenase. be.

3.4 Structure of the present invention The present invention relates to a method for high purity purification of kallidinogenase, in which a flocculant is used to facilitate the filtration of autolyzed fluid obtained from pig pancreas, and the filtrate is treated with an anion exchange resin. The fraction obtained is desalted and concentrated using a membrane, and then subjected to the second separation using a column packed with anion exchange resin. The present invention provides a method and apparatus for obtaining a highly purified product by desalting and concentrating a sample using a membrane and freeze-drying the sample.

In conventional purification of kallidinogenase, kallidinogenase is separated using a column using a soft packing material such as cellulose, Sephadex gel, or polyacrylamide gel.

Therefore, if a packing material with a small particle size is used, the packing material will be deformed and the column pressure will become extremely high unless the flow rate is made very small.

In general, the separation performance improves as the particle size is reduced, but for this type of filler, it is difficult to simultaneously increase the separation performance and separation speed for the reasons mentioned above.

In this invention, a column packed with a hard packing material having a small particle size for high-performance liquid chromatography (high-speed LC) is used, so that high-performance separation can be achieved in a short time.

The configuration of the present invention will be explained in detail below.

FIG. 1 is a block diagram showing the configuration of the present invention: 1-coagulation tank, 2-filtration tank, 3-first liquid chromatography, 4-separation membrane, 5-second liquid chromatography,
9- Separation membrane, l〇- Freeze dryer, 11- Raw material tank, 12-
This is the product tank.

Next, the contents of each process will be explained.

(1) The autolyzed fluid of pig pancreas is treated with a flocculant. At that time, a caotine-based flocculant is used, a normal acid solution is added dropwise to adjust the pH to 4.0 to 7.0, preferably pH 4.5 to 5.0, and the flocculant is added at a concentration of 50 to 500 pp.
m, preferably 100 to 300 ppm, and stir well. After a few minutes, the agglomeration effect appears and separation becomes possible with a nylon mesh. The recovery rate of kallidinogenase at this time is 80% or more.

(2) After removing aggregates and further filtration to remove particles larger than 2 fiLm, (3) this filtrate is injected into a column packed with anion exchange resin to adsorb kallidinogenase. The injection volume is limited to 30 times the column volume.

Next, 0.001 to 0.03+sol, preferably 0.
01-0.02mol (7) Ammonium acetate (OH
The inside of the column is washed by flowing 15 to 150 times, preferably 30 to 50 times, the column volume of C00NH4) into the column. Next, o, e~2. Omol, preferably 0.
By flowing ammonium acetate of 8 to 1.2+io+, a fraction having a kallidinogenase specific activity of about IEU/A280 is obtained from an amount equal to the volume of the column.

(4) This fractionated liquid is desalted and concentrated using a separation membrane and used for the next purification. In addition, the column is next 2.0 to 4, Omo
1 of ammonium acetate was flowed several times the column volume to elute the components strongly adsorbed to the column, and further, 0.1 to 0.03 mol of ammonium acetate, which was used for washing the inside of the column, was flowed several times the volume. Equilibrate the column and prepare it for the next purification of kallidinogenase.

(5) Separate kallidinogenase and impurities from the desalted and concentrated fraction by liquid chromatography. The column is filled with anion exchange resin,
As an eluent, pH 4.5 to 8, preferably pH 6 to 8.
ammonium acetate, from 0.1 mol to 2 mol
I, preferably from 0,2 intestine O1 to 1 mol Wl
& is elevated and perform a gradient to separate kallidinogenase and impurities.

The specific activity of kallidinogenase, which is about the same as the volume of the column, is 20.
Obtain a fraction of ~4 (IEU/^280).

(6) Desalt and concentrate this fraction using a separation membrane, (7)
A dry product of kallidinogenase is obtained using a freeze dryer.
A purified product of 200 klJ/mg or more can be obtained.

For each operation in F, instead of ammonium acetate, p
Buffer solutions having an H range of 4.5 to 8.5, such as potassium dihydrogen phosphate, sodium hydroxide, lactic acid, and sodium lactate, can be suitably used.

3.5 Example 1: 1 kg of porcine pancreas was added with 3 g of water, allowed to stand for autolysis, and then 25 wt % of acetone was added as a sample.

The flocculation state and recovery rate after filtration when 0.2% cationic flocculant (Sumifloc FC-250) was added to a 25 m sample are shown.

When the pH was in the range of 4.5 to 5.0, both the aggregation state and the recovery rate were good.

After aggregation, the aggregates are removed using a nylon mesh, followed by filtration. If direct filtration is performed without using a flocculant, the filter paper will become severely clogged and filtration will take a long time.

2. This filtrate is injected into a column (inner diameter 20 mm, length 500 mm) filled with anion exchange resin to adsorb kallidinogenase. Next, 0.02 mol of ammonium acetate (pH=8.0) 31 is poured for washing, and then 1 mol of ammonium acetate (pH-8.0) is poured to elute the kallidinogenase. At this time, the absorbance is continuously measured using an ultraviolet absorption monitor (wavelength: 280 nm), and the portion with increased absorption is fractionated. This is illustrated in Figure 2. Approximately 200 ml of aliquots were obtained.

The table below shows the measured kallidinogenase activity values (measured by an enzymatic chemical method).

The recovery rate was 80%, and the specific activity (EU/A280) was about 30 times higher.

Figures 3.4 and 5 show changes in recovery rate and transmittance when purification conditions were changed using this method.

Figure 3 shows the relationship between the concentration of cleaning solution and recovery rate, Figure 4 shows the relationship between the amount of cleaning solution and its transmittance at 280cm, and Figure 5 shows the relationship between the amount of cleaning solution and its transmittance at 280cm.
The figure shows the relationship between the salt concentration of the kallidinogenase eluate and the recovery rate.

3. Desalt this fractionated liquid using a separation membrane until the conductivity becomes 1 mS/cm or less, and then add acetic acid at pH 8.0 using a column (inner diameter 20 m + W, length 250 m + 11) filled with anion exchange resin. A gradient was performed using an ammonium eluent whose concentration was gradually increased from 0.19 mol to 0.85 mo+ to separate kallidinogenase and obtain 100 mJ1 of a fraction.

The recovery rate was 88% and the specific activity (EU/A280) was 4.
It rose to nearly 0 times. Separation time is approximately 3 hours.

The recovery rate was 88% and the specific activity (EU/A280) was 4.
It rose to nearly 0 times. Separation time is approximately 3 hours.

FIG. 6 shows its chart, and FIG. 7 shows its gradient I. At this time, ammonium acetate buffer having a pH of 6.0 was used as the eluent. Flow rate is 2+nl/sin
I went there. Detection was performed by absorbance of 28On++ ultraviolet light. At the same time, for kallidigenonase, the E of the eluate
Detection was performed by measuring U/ml.

Incidentally, FIG. 8 shows the relationship between the ammonium acetate concentration and the retention ratio at a pH of 6.0.

In the retention ratio, '' indicates the degree of retention in the column, and is expressed by the following formula.

k'=(t-t)/l.

T O Here, t is the retention time of the component that is not adsorbed, and t is the retention time of the target component.

For separation of kallidigenonase, k' ranges from 1 to 10.

In order to completely elute quickly eluting components from the column without eluting kallidigenonase, use a concentration (÷0.2M) where k' is several tens of digits.

After separating kallidigenonase, to elute the slowly eluting components from the column, use a concentration of k′ et 0 (6xx).
Use. (Since kallidigenonase is separated into two components A and B, two straight lines are obtained.) This aliquot was desalted and dried in a freeze dryer.
Almost pure kallidinogenase of tkU/mg was obtained. This value was measured using the increased blood flow measurement method [H, Moriy
a, K, Yamazaki and H, Fukus
hin+a.

J.Bjochem,,58,201(113f35)
].

3. Effects of the present invention In the method for purifying kallidinogenase and the device thereof of the present invention, high purity kallidinogenase could be obtained by using two columns.

Furthermore, since a packing material for high performance liquid chromatography is used, the separation speed is fast, and since high performance liquid chromatography is used, automation of the process is easy.

As a result, the time required for purification was reduced to a fraction of the conventional method, making it possible to significantly reduce costs.

3.7 Definition Definitions of terms used in this specification are as follows.

"High performance liquid chromatography" filler is a hard liquid chromatography filler with a particle size of 5 to 50 m, a degree of swelling in water (volume in water/volume in dry state) of 1 to 1.5. Refers to fillers for use in

[Brief explanation of drawings]

FIG. 1 is a block diagram showing the configuration of the present invention. 1 - aggregation tank, 2 - filtration tank, 3 - first liquid chromatography, 4 - separation membrane, 5 - second liquid chromatography,
9- Separation membrane, 10- Freeze dryer, 11-, JL tank, 1
It is a 2-9 product tank. FIG. 2 is an explanatory diagram of how to use the ultraviolet absorption monitor. Figure 3 is a diagram showing the relationship between cleaning solution concentration and recovery rate in the first liquid chromatography, Figure 4 is a diagram showing the relationship between the amount of cleaning solution and transmittance, and Figure 5 is a diagram showing the relationship between eluent salt concentration and recovery rate. Figure 6 is a chart showing the time course of fractionation in the second liquid chromatography, Figure 7 is its gradient, and Figure 8 is a diagram showing the relationship between ammonium acetate concentration and k' at pH=8. In addition, for Figure 6, horizontal axis: retention time (minutes), vertical axis: solid line, dotted line for absorbance at a wavelength of 280 nm, activity value per unit of kallidinogenase (EU/ml), for Figure 7, horizontal axis: retention time. (min) Vertical axis: Salt concentration of ammonium acetate buffer (moles) In relation to Figure 8. Horizontal axis: salt concentration (mol) of ammonium acetate buffer; vertical axis: retention ratio (k') of kallidinogenase. Applicant Showa Denko Co., Ltd. 9-ya Hiroshi Agent Patent Attorney Sei Kikuchi - Figure 2 Figure 3 Figure 5 Acetic acid buffer attitude (p) 16.0)

Claims (1)

[Claims] 1. A purified product of kallidinogenase is obtained by sequentially passing a raw material solution containing kallidinogenase through two liquid chromatography devices using an anion exchange resin for high performance liquid chromatography as a column packing material. A method for purifying kallidinogenase, characterized in that it obtains. 2. To the raw material solution containing kallidinogenase, (1) add a flocculant to flocculate it (2) remove large particles and filter it; 3) purify it by first liquid chromatography; and 4) transfer the fractionated liquid to a separation membrane. 5) This fraction is purified by second liquid chromatography. 6) This fraction is desalted and concentrated using a separation membrane. 7) Lyophilization is carried out to dry and purify kallidinogenase. A method for purifying kallidinogenase to obtain the product. 3. (1) aggregation device (2) filtration device (3) first liquid chromatography device (4) desalting concentration device (5) first device for purifying the raw material solution containing kallidinogenase and obtaining the dried purified product. An AA system for the purification of kallidinogenase characterized by having a two-liquid chromatography device (8), a desalting concentration device (7), and a freeze-drying device in series. 4. The method according to claim 2, wherein the flocculant is cationic. 5. The flocculant is cationic, and P during flocculation operation
3. The method according to claim 2, wherein H is 4.0 to 7.0 and the agglomerate concentration is 50 to 500 ppm. 6. The apparatus according to claim 3, wherein the first column packing material for liquid chromatography is an anion exchange resin for high performance liquid chromatography. 7. The apparatus according to claim 3, wherein the second column packing material for liquid chromatography is an anion exchange resin for high performance liquid chromatography. 8. The apparatus according to claim 3, wherein the flocculant is a cationic one, and the first and second column packing materials for liquid chromatography are anion exchange resins for high performance liquid chromatography. 9. The device according to claim 3, wherein the separation membrane has a molecular weight cutoff of 500 to 10,000, preferably 1,000 to 5,000. However, the packing material “for high performance liquid chromatography” is
It refers to a hard packing material for liquid chromatography with a particle size of 5 to 50 ILm and a swelling ratio of charcoal in water (volume in water/volume in dry state) of 1 to 1.5.