JPS6030294B2 - Method for heat treatment of fractions containing glycoproteins that promote differentiation and proliferation of human granulocytes - Google Patents

Description

Detailed Description of the Invention The present invention relates to a glycoprotein (hereinafter referred to as HGIGP) that acts on bone marrow cells in human bone marrow isolated from the urine of healthy people and promotes the differentiation and proliferation of these cells into forehead granulocytes. ) is concentrated, the protein content is adjusted to at least 70 to 9 in 1, and heat treated at a temperature of 50 to 7,000 for 8 to 3 incubations, and mixed into the fraction. The present invention relates to a method for heat treatment of a fraction containing P in HG, which is characterized by inactivating viruses that are at risk of being contaminated.

HGIGP is a substance that exists in trace amounts in human urine, and has the function of acting on human bone marrow cells to differentiate and proliferate only granular cells (hereinafter referred to as biological activity).

The co-researcher of the present invention previously isolated and purified this substance from human urine, identified the substance, made it possible to manufacture it using a reproducible manufacturing method, and discovered its use as a therapeutic agent for human leukopenia. A patent application was filed (Japanese Patent Application No. 53-31999. Hereinafter referred to as Prior Application 1). However, it is known that viruses such as hepatitis and endemic diseases are present in human urine, and pharmaceuticals manufactured from human urine are not administered to humans as medicines without undergoing virus removal or inactivation treatment. If there is a risk of contracting a viral infection, the HGIG of Prior Application 1
There is also a risk of this in the method for producing P. In order to avoid such risks, the virus is usually measured in advance using an immunoassay method, and raw materials containing high concentrations of viruses are excluded, thereby achieving a certain degree of effectiveness in preventing viral infections. However, this method cannot be used in industrial production methods that handle urine from tens of thousands of people at once. On the other hand, the problem of viral infections is also included in individual human serum protein preparations obtained by fractionating dish clumps.

It has previously been found that by subjecting albumin preparations to a heat treatment of 60 oo for 1 hour, viral infections can be prevented without altering the albumin.
Safe in clinical use. Since it was discovered that preparations subjected to heat treatment for 60 oo for one feeding time are effective in preventing post-administration viral infections, this method has been applied to other human serum protein preparations. For materials to which the heat treatment method can be applied, the material itself must be stable to this treatment.

Therefore, various stabilizers have been discovered to enable this heat treatment.There are substances that cannot withstand heat treatment without a stabilizer, but can make heat treatment possible in the presence of a stabilizer. . Amino acids, sugars, and the like are generally used as stabilizers for human serum proteins at physiologically isotonic or lower concentrations. This 60% also applies to preparations containing HGIGP.
Although heat treatment at 1° C. is desirable for inactivating viruses, this treatment also reduces the biological activity of HGIGP. The present inventors conducted repeated research to improve the thermal stability of HGIGP, and as a result of examining the processing conditions, the purified HGI
GP discovered that heating stability could be improved by adding human blood albumin or human placental albumin, and filed a patent application (Tokugan 1986-80695, hereinafter referred to as Prior Application 2).However, the heating stability of Prior Application 2 The treatment method involves adding albumin to once-purified HGIGP and heating it, which is not a desirable method. Therefore, the present inventors thought that urinary protein contained in human urine might enhance the heat stability of HGIGP in the same way as albumin, and conducted repeated research.
By heating a fraction containing HGIGP in a so-called semi-purified state in which human urine protein is present at a concentration of 709' or higher, deactivation of the biological activity of HGIGP due to heating can be almost completely prevented. I discovered that. An object of the present invention is to provide a method for heat treatment of a fraction containing HGIGP, which renders viruses inhospitable by heat treatment without adding any additives. Next, the method of the present invention will be explained in detail.

One ounce of fresh urine collected from a healthy person usually contains 30 to 50
Contains mg of protein.

This human urine is processed by the known ultrafiltration method, ion exchanger adsorption method, silica gel adsorption method, salting out method, desalting method, etc., or a combination of these methods, and the proteins contained in the human urine are analyzed. A fraction containing HGIGP, which is concentrated with protein in concentrated human urine, is separated, and this fraction is concentrated under reduced pressure to reduce the protein content to at least 7.
Adjust to 9 of 0, preferably 9 of 100 to 150. H
When the protein content of the GIGP-containing fraction is 1/70, the biological activity of P in HG is greatly deactivated by heating, as is clear from Experiment 1 described below, which is undesirable. The prepared HGIGP-containing fraction is then heated at a temperature of 50-70°C, preferably 55-65°C, for 8-3 feeding hours, preferably 8-12 hours to inactivate the virus.

The P-containing fraction of HG thus obtained was purified, and purified HGIG free of active virus was obtained.
P can be produced. Next, embodiments of the present invention will be described.

'1} One aspect of the present invention is as follows.

A dilute acid or alkali aqueous solution is added to fresh urine collected from a healthy person to adjust the pH to 6-9, preferably 7-8, and then centrifuged to remove unmelible substances contained in the urine. Remove. The urine thus obtained is brought into contact with an adsorbent containing silicon, such as silica gel, silica gel-magnesium silicate, diatom, silica glass, bentonite, etc., to elute the adsorbed components. Elution is preferably at pH 9
The above alkaline aqueous solution is used. The alkaline aqueous solution used for elution is not particularly limited, but preferably 0.3-1.
An aqueous solution of 8M concentration is used. The pH of the eluate thus obtained was adjusted to 7-8, and the active substance was salted out by adding a neutral salt such as ammonium sulfate to 70% saturation.
A fraction containing GP is obtained. When this fraction is dissolved in water, the protein concentration in the aqueous solution is adjusted to at least 70 9' by adjusting the amount of water dissolved. Next, this aqueous solution is heated at a heating temperature of 50 to 7,000 ℃, preferably 55 to 6,500 ℃ for 8 to 3 hours, preferably 8 to 1 hour to inactivate the virus. Purification of HGIGP from this heat-treated fraction is carried out as follows.

The heat-treated aqueous solution is passed through a molecular separation filter to reduce the molecular weight to 10.
000 or less are removed and brought into contact with a cation exchanger (for example, carboxymethyl exchange group-containing dextran, carboxymethyl cellulose, phosphocellulose) to adsorb and remove impurities contained in the solution.

The contacting is carried out at approximately neutrality, the HGIGP-containing fraction and the ion exchanger are preferably at pH 6-8, preferably at a pH of 0.01
Equilibrated with ~0.19 M inorganic salt buffer.
At this time, most of the HGIGP passes through. This is concentrated and then equilibrated with a dilute buffer of pH 6-8, contacted with an equilibrated anion exchanger (e.g. DEAE cellulose) to adsorb HGICP, and an inorganic salt of 0.1-0.3M, e.g. The salt concentration is varied using a sodium chloride solution, and elution is carried out by a so-called linear concentration gradient elution method. At this time, HOIGP is eluted at a salt concentration of 0.1M or more, but complete separation is difficult. The elution fractions with a salt concentration of 0.1 to 0.3 M are collected, and desalted and concentrated if necessary. In addition, before this step of elution by changing the salt concentration, HG
IGP is adsorbed on an anion exchanger, and 0.1 to 0.3
It may be purified by elution with an aqueous solution having a salt concentration of M.

The fractions were then subjected to molecular chromatography with 1
Highly crosslinked polymer gels with a water absorption of 0 to 20, such as Cephadex G-150, Biogel P-1
Pass the liquid through a column packed with 00 to eliminate the effective substances in the fraction.
．． 05 to 0.1 molar salt buffer, and the relative eluate volume is 1.11 to 1.60, preferably 1.11 to 1.60.
The fraction having a concentration of 1.45 is separated, the active ingredients are collected, and further desalted and concentrated or freeze-dried (the above method is hereinafter referred to as method A).

) As described above, the biological activity of HGIGP is not deactivated,
Heat-treated, virus-free, more purified HG
IGP content is obtained. {2) Other aspects of the present invention are as follows. A dilute acid or alkaline aqueous solution is added to fresh urine collected from healthy people to adjust the pH to 6-9, preferably 7-81, and then centrifuged to remove insoluble matter contained in the urine. remove. The urine supernatant obtained here is brought into contact with a silicon-containing adsorbent, such as silica gel, silica gel-magnesium silicate, diatom, silica glass, bentonite, etc., to dissolve the adsorbed components. Elution is preferably performed with an alkaline aqueous solution having a pH of 9 or higher. The alkaline aqueous solution used for elution is not particularly limited, but preferably an aqueous solution such as ammonium hydroxide or sodium hydroxide having a concentration of 0.3 to 1.8 is used. The pH of the eluate thus obtained is adjusted to 7-8, and a neutral salt such as ammonium sulfate is added to 70% saturation to salt out the active substance to obtain a crude fraction containing HGIGP. Next, this phase fraction is dissolved in a small amount of water, and low-molecular components with a molecular weight of 10,000 or less are removed using a molecular separation filter. Phosphosel.

(base) to adsorb and remove impurities contained in the solution. The contact is carried out in near neutrality, with H
The GIGP-containing fraction and the ion exchanger are equilibrated with an inorganic salt buffer to pH 6-8, preferably 0.01-0.15M. At this time, most of the HGIGP passes through.
Concentrate the solution containing the passed HGIGP and adjust the protein concentration to at least 70%
The virus is inactivated by heating at a heating temperature of 0.0, preferably 55-6500°C for 8-3 hours, preferably 8-1 hour. Purification of HGIGP from this heat-treated fraction is carried out as follows. The heat-treated fraction was adjusted to pH 6.
~8 dilute buffer and contacted with an equilibrated anion exchanger (e.g. DEAE cellulose), as described below in A.
(This method will be referred to as method B hereafter.

). This method B also yields a more purified HGIGP-containing product equivalent to that of method A. {3' Still other aspects of the present invention are as follows. A dilute acid or alkaline aqueous solution is added to fresh urine collected from healthy people to bring the pH to 6.
-9, preferably 7-8, and then centrifuged to remove insoluble matter contained in the urine. The condition of the urine obtained here is treated with an adsorbent containing silicon, such as silica gel,
Post-corrosion is performed on silica gel, magnesium silicate, diatomite, silica glass, bentonite, etc., and the adsorbed components are eluted. Elution is preferably performed with an alkaline aqueous solution having a pH of 9 or higher. The alkaline aqueous solution used for elution is not particularly limited, but preferably an aqueous solution of ammonium hydroxide, sodium hydroxide, or the like having a concentration of 0.3 to 1.8 M is used. The pH of the eluate thus obtained is adjusted to 7-8, and a neutral salt such as ammonium sulfate is added to 70% saturation to salt out the effective substance to obtain a fraction containing HGIGP. Next, this fraction is dissolved in a small amount of water, and low-molecular components with a molecular weight of 10,000 or less are removed using a molecular separation filter, and a cation exchanger (e.g., carboxymethyl exchange group-bonded dextran, carboxymethyl cellulose, phosphocellulose) is removed. The solution is brought into contact with the solution to adsorb and remove impurities contained in the solution.

The contact is carried out in approximately neutral conditions, and the HGIGP-containing fraction and ion exchanger are added to baits 6 to 8 preferably at a concentration of 0.01 to
Equilibrated with 0.19K inorganic salt buffer. At this time, most of the HGIGP passes through. This is concentrated, equilibrated with a dilute buffer solution of pH 6-8, and contacted with an equilibrated anion exchanger (e.g. DEAE cellulose) to adsorb HGIGP and 0.1-0.3 M inorganic salt. Varying the salt concentration, for example using a sodium chloride solution,
Elution is performed by a so-called linear concentration gradient elution method. At this time, HG
Although IGP elutes at a salt concentration of 0.1 M or higher, complete separation is difficult. This 0.1~0. Collect the elution fractions according to arc 4 salt concentration. Concentrate the eluted fraction, adjust the protein concentration to at least 70 qo, and heat at a heating temperature of 50 to 70 qo, preferably 55 to 65 ° C., for 8 to 3 hours, preferably 8 to 12 hours, Inactivate the virus. Purification of HGIGP from this heat-treated fraction is carried out as follows. For the purpose of molecular muscle chromatography, the heat-treated fractions are passed through a column filled with a highly cross-linked gel having a water absorption of 10 to 20 cm, and then treated in the same manner as method A (the previous method was replaced with This will be referred to as the C method hereafter.

). This method C also yields a more purified HGIGP-containing material equivalent to methods A and B described above. (4- Furthermore, other aspects of the present invention are as follows.

Fresh urine collected from healthy people is centrifuged to remove impurities, and ultrafiltration reduces the molecular weight to 100.
The fraction below 0 degrees Celsius is removed and the resulting concentrated urine is concentrated under reduced pressure to adjust the protein content to at least 70 claws per kite. Next, this HGIGP-containing fraction was divided into 50-70oo,
Preferably 8 to 3 field hours at a heating temperature of 55 to 65 do.
It is desirably heated for 8 to 1 hour to inactivate the virus. Purification of HGIGP from this heat-treated fraction is carried out in the same manner as the method B described above (this method is hereinafter referred to as method D). This method D also yields a more purified HGIGP-containing material equivalent to methods A, B, and C described above.

In addition, in the above methods A, B, C, and D, the relative eluate volume is a numerical value expressed by Ve/Vo, where Ve indicates the volume of solvent required to elute the substance in the column, and Vo is It shows the amount of solvent outside the gel particles in the column.

The powdered substance obtained by freeze-drying the heat-treated fraction obtained by the method of the present invention contains 600-9 of 800 proteins and 3-5 of HGIGP per powder.

When using this powdered substance as a medicine,
It is dissolved in sterile physiological saline, sterile water, sterile isotonic solution for injection, etc., and administered to patients with leukopenia by intravenous, intramuscular, or subcutaneous injection. The effective dose is 9 or more, preferably 0.75 HGIGP per lk9 body weight per day.
It is 9 of 75-2.24. Since this HGIGP-containing substance does not have the risk of contaminating active viruses, it can be used as a drug obtained from human urine as a safe leukopenia treatment without the risk of viral infection. Said A
To further purify the HGIGP-containing material obtained by methods B, C, and D, the crude material is treated with a dilute buffer containing 1.0-2.0 M salt, such as phosphate buffer pH 6.
0 to 8.0, preferably pH 6.0 to 7.0, and adsorbed on a sugar-affinity adsorbent, such as Concanavalin A-Sepharosel (manufactured by Fine Chemical Laboratory), which has been equilibrated in advance with the buffer. ,
Next, a 1.0-2.0M salt-added buffer containing 20mM to 100mM of healthy substances, such as Q-methyl-D-glucoside, pH 6.0-8.0, preferably pH 6.0-7.
．． The HOIGP fraction is eluted at 0, and the HOIGP fraction is collected, desalted if necessary, and concentrated or freeze-dried.

-Furthermore, in order to purify the resulting fraction by aerophoresis, it is subjected to conditioning zone electrohydration using, for example, acrylamide gel, agar gel pH 7.0 to 9.0 as a support, and then cooled. HGIGP from the support by dilute salt solution
It is also possible to collect, desalt, concentrate or freeze-dry.

The HGIGPs obtained by further purifying the HGIGP-containing products obtained by methods A, B, C, and D described above by the method of Reference Example 5 described below are all white or slightly brown in color, tasteless, and odorless. It is a powder that is slightly hygroscopic and has the following physical and chemical characteristics,
The substance was the same as that of Prior Application 1.

[1- Molecular Weight] The molecular weight of HGIGP treated by the method of the present invention was measured by the sodium dodecyl sulfate polyacrylamide gel electroplating method, and it was found to be about 85,000.

Further, as a result of gel filtration using Sephadex G-150, the molecular weight of HGIGP was 75,000 to 90,000. Therefore, the molecular weight of HGIGP should be 75,000 to 900.
00 seems to be the most reliable range. [
2} Solubility The solubility of HGIGP in various solvents is shown in Table 1.

Table 1 In addition, it is easily dissolved in dilute salt solutions, such as phosphate solutions, trisaminomethane solutions, and has a pH value of
It dissolves in the range of ~12.

(3) pH of pHHGIGPI% aqueous solution is 5.0~
6.0, indicating acidity.

{41 Specific optical rotation The optical rotation was measured at 2000 using a 0.25% aqueous solution of HGIGP, and the specific optical rotation [d] color o was determined as 0 to ±
It was in the range of 40.

'5) Infrared absorption spectrum The infrared absorption spectrum of HGIGP obtained by the KBr tablet method is as shown in FIG.

HGIGP has characteristic absorption values as shown in Table 2. Table 2 {6} Isoflea point polyacrylamide gel isoelectric focusing method pol cr
yiamide gel isoelecmc rank rc
As a result of measuring the isonic point of HGIGP by Satoshi (Satoshi), the isonic point was pH 4.7 and 0.2.

Table 3 shows the results of testing the color reaction of HGIGP dissolved in water.

Table 3: Temperature-stable HGIGP was dissolved in water at a concentration of 1% (by weight) and
When heated at 0±0.5 qC for 30 minutes, its biological activity was completely lost.

[9} The constituent amino acid HGIGP of the protein portion was hydrolyzed by a conventional method, and the amino acid composition of the protein portion was analyzed using an automatic amino acid analyzer.

The results are shown in Table 4. From Table 4 Table 4 ~HGIG
The protein part of P is composed of 10,000 amino acids, with large amounts of acidic and neutral amino acids, and extremely small amounts of basic amino acids.

Another feature is that linear amino acids such as aspartic acid, threonine, serine, glutamic acid, and glycine (alanine, valine, and leucine) account for more than 70% of the total amino acids. , vol. 227, p. 680, 1
97), the relative mobility was 0 by dew pneumophoresis using sodium dodecyl sulfate polyacrylamide gel.
．． A single band is shown at position 25.

HGIGP, trypsin inhibitor (molecular weight 2150
0), ovalupmin (molecular weight 43,000), human serum albumin monomer (molecular weight 65,000) and human serum albumin dimer (molecular weight 130,000) were simultaneously spread,
The molecular weight of HGIGP was determined from the relative mobility of these substances with known molecular weights and that of HGIGP, and the result was approximately 850.
00 (Figure 2). In Figure 2, a, b, c,
d indicates trypsin inhibitor, ovalbumin, human serum albumin monomer, and human serum albumin dimer, respectively, and the arrow indicates HGIGP. (11) Ultraviolet absorption The ultraviolet absorption spectrum of a 0.1% aqueous solution of HGIGP measured in one skin cell is as shown in Figure 3.
It showed maximum absorption at the touch and terminal absorption at wavelengths below 25 skin.

Also, the absorbance value (E) of HGIGP at 28 points is 3-
It was 8. (12) Neutral sugars constituting the healthy part were determined by the phenol-sulfuric acid method, and sialic acid was determined by Warren's thiobarbic acid method (Jom female lof).
Biological Chemistry, vol. 234,
(1971, 1959), amino sugars are converted into E1so
n-Morgan method (Biochemica I Jo
Nr Misaki 1, vol. 27, p. 1824, 1933).

The amount of neutral sugar was expressed as a glucose equivalent amount. As a result, neutral sugar 10.0-13.0%, sialic acid 3%
．． The sugar content was 0 to 7.0%, and the amino sugar content was 1.0% or less, and the total sugar content was 13.0% to 20.0%. (13)
The composition ratio of protein and carbohydrates was quantified by the semi-micro Kjeldahl method and was 75-85%.
2) As stated, the amount of carbohydrates is 13.0% to 20.0%.

(1 core Elemental analysis The results of elemental analysis are as follows.
Carbon 42.3-47.3%
Hydrogen 5.7-7.8% Nitrogen 9.6-14.3% Oxygen 34.4-39.4% Sulfur 0.2% or less Next, test examples and examples of the method of the present invention will be shown. This will be explained in further detail below.

However, the content of HGIGP in the HGIGP-containing fraction obtained by the method of the present invention is extremely low and cannot be used for testing as it is. Therefore, the HGIGP content purified by the method described later as a reference example was used to test the effect. I did this. [Experiment 1] The influence of heat treatment on the protein concentration in an aqueous solution was tested as follows.

m Sample 'a - Test sample Human urine was passed through a silica gel column in the same manner as in Example 1, and powdered ammonium sulfate was added to the eluate to give a concentration of 70%.
The mixture was saturated and a precipitate formed.

This precipitate was added at 10, 50, 60, 70, 80, 1 per 1M.
Dissolved in water at a ratio of 9:00, 150, 200, 0.1
M Tris-HCl buffer was added to adjust the pH to 7.0.
This aqueous solution was then heated at 60°C for 1 hour, immediately cooled with ice water, and treated in the same manner as in Reference Example 1.
Freeze-dried powders were obtained (sample Nos. 9 to 16). {b}
Control samples Powders produced in the same manner as the test samples without heat treatment (Samples Nos. 1 to 8).

(c) Purified sample A sample in which the powder produced in the same manner as the control sample was purified by the method of Reference Example 5 (Sample No.
17-24).

Rigid heating purified sample Purified HG produced by the same method as Example 1 of the previous application 1
IGP powder per 1, 10, 50, 60, 70, 8
Powder (Samples No. 25 to 32) was dissolved in water at a ratio of 0, 100, 150 and 200, heated at 6000 for 1 hour, cooled on ice, and lyophilized.

The production activity of the above 32 children's samples was measured as follows. '2) Measurement of biological activity Colony formation of jaw granulocytes in mouse bone marrow cells in vitro was performed as a parameter.

That is, in a plastic culture dish with a diameter of 35 skin, it contained 20% beef tallow fetal serum, a 0.1-board sample adjusted to the same HGIGP concentration, 0.3% agar, and 7.5 × 1 M mouse bone marrow cells. McCoy's vessel medium was added, the total volume was adjusted to 1', and cultured for 7 days in humidified air containing 5% CO2. After culturing, the cells were examined under an inverted microscope, and cell aggregates with a size of 5 or more were counted as the number of colonies. The percentage of the number of granulocyte colonies in the test sample relative to the number of granulocyte colonies in the control sample with the same protein concentration was calculated and used as the biological activity recovery rate.

The results are shown in Table 5. Table 5 As is clear from Table 5, when the protein concentration in the test sample group was 70:9/skin or higher, the biological activity recovery rate was 85% or more, and when the concentration was less than this, the biological activity recovery rate was poor.

Therefore, in producing HGIGP from human urine by the method of the present invention, the protein concentration in the aqueous solution must be at least 70:9 when heat treatment is performed. On the other hand, in the case of purified HGIGP, the bionada was reduced in mollization by heating at any concentration. In addition, method B (Example 2
), C method (Example 3) and ○ method (Example 4) also had the same biological activity as in Experiment 1 above.

(Experiment 2) The following test was conducted regarding virus inactivation by heat treatment.

100ml of human urine was passed through a silica gel column in the same manner as in Example 1, and powdered ammonium sulfate was added to the eluate to make it 70% saturated, yielding a precipitate of about 40ml.

The resulting precipitate was divided into four equal parts of 10 parts each, dissolved in 50 parts of water, and further distilled water was added to adjust the total volume to 100 parts.
The protein concentration of solution K is 100 9'). Then, add variola virus, mumps virus, measles virus, vesicular stomatitis virus, chikungunya virus, Japanese encephalitis virus, rubella virus, poliovirus, and Coxsatskiiurus n-urus to these solutions. It was added as having a virus infectivity titer of 1 x 1 to 1 x 1 pi/0.2 secretion (measurement method will be described later).

Next, the two solutions were heated without heating, and the other two solutions were heated at 6000 for one hour, cooled on ice, and treated in the same manner as in Reference Example 1 to prepare HCIGP-containing powder.
The infectious titer of the virus was measured for each of these powders, and the reluctance of the virus by heating was tested. The infectious titer of the virus was measured using the following cells. Pox virus is H
It was subcultured in eLa cells, and the focus forming unit (F
OCUS Mermingrnit: Vimlogy, 3 Mothers, pp. 174-179, 196 Gunou) was measured. VERO cells for passage of mumps virus, measles virus, and Japanese encephalitis virus, and HeL cells for passage of poliovirus, Coxsatsky virus, and echovirus.
The infectivity of these cells was determined using 50% tissue culture infectivity (50% tissue culture infectivity) using each cell as a guideline for cell degeneration caused by the virus.
vedose: edited by the National Institute of Preventive Health Alumni Association, ``Virus Experimental Studies, Hamaruzen, 1967.'' In this case, all observations were conducted for 10 days.

The vesicular stomatitis virus was passaged using FL cells and chikungunya virus VERO cells, and the infectivity was determined using each cell in plaque form.
1 unit: the above-mentioned "Virus Experimental Science") was measured.

Rubella virus was passaged in BHK-21 cells, and blank forming units were measured using vERO cells.

As a result, virus infectivity was observed in the two samples that were not heat-treated, whereas the virus infectivity was completely lost in the two heat-treated samples, and the method of the present invention showed that virus infectivity was observed. It was found that it was inactivated. This result suggests that the infectivity of viruses other than those used in this experiment can also be inactivated by the method of the present invention. Example 1 New urine collected from healthy people was added with 10% sodium hydroxide to adjust the pH'
Adjust this temperature and heat to 1500°C while cooling to 0°C. p. m
．． The mixture was centrifuged using a continuous centrifuge to remove impurities and obtain a supernatant.

Next, this condition was adjusted to pH 7 with 10% hydrochloric acid, and the solution was passed through a column (10×80 skin) filled with silica gel, and the components adsorbed on the silica gel were eluted from the column with 40 sleeves of 5% aqueous ammonia.

The pH of the eluate thus obtained was adjusted to 7.5 with 1N sulfuric acid, powdered ammonium sulfate was added to the solution to make it 70% saturated, and the mixture was left overnight at 0°C.
The precipitation in the evening was the rule.

This precipitate was dissolved in 200ml of water, and further distilled water was added to adjust the total volume to 400ml or more, and the protein concentration was adjusted to 100% or more. This solution was heated at 6000 °C for 1 time,
Thereafter, the mixture was cooled on ice to obtain a heat-treated HGIGP-containing fraction.
Reference Example 1 The insoluble matter produced in the heat-treated HGIGP-containing fraction obtained in Example 1 was removed, placed in a dialysis tube (manufactured by Pisking), and added to a 0.09 phosphate buffer (pH 6.
5), the buffer was added to the dialysis solution to adjust the total volume by 10%, and CM Sephadex C was equilibrated in advance with 0.08M phosphate buffer (pH 6.5). -50 ion exchange column (4.0 x 40), impurities were adsorbed on the ion exchange resin, and a filtrate was obtained.

10 pieces of this permeate were concentrated using a Diaflow Hollow Fiber Concentrator (manufactured by Famicom, DC-3 type), and the concentrated solution was mixed with 0.1M ToIJ-HCl buffer (pH 7) in the same manner as above.
．． 0) overnight at 5° C., and the buffer solution was added to the dialyzed solution to make three adjustments.

This solution was equilibrated and activated with the buffer beforehand.
Pass the liquid through an AE cellulose column (4.0 x 40c),
After thoroughly washing the column with 0.1M Tris-HCl buffer (pH 7.0), elution was performed with 0.1M Tris-HCl buffer (pH 7.0) containing 0.3M NaCl. The eluate was then dialyzed against 0.1M Tris-HCl buffer (pH 7.0) to obtain a dialysis solution. This dialyzed solution was again passed through a DEAE cellulose column (4.0 x 40 arc) equilibrated and activated with the buffer solution, and eluted using a linear concentration gradient elution method of 0.1M to 0.3M sodium chloride. , collect fractions that have the effect of promoting differentiation and proliferation of forehead granulocytes, dialyze this fraction against distilled water, freeze-dry, and add a small amount of this powder to 0.1
It was dissolved in M Tris-HCl buffer (pH 7.0) and dialyzed against the buffer to obtain a dialysis solution.

Next, 20 volumes of this dialysed fluid was developed on a Sephadex G-150 column (4.0 x 60 columns) equilibrated with 0.1 M Tris-HCl buffer (pH 7.0), and the relative eluate volume was 1 .11-1.45 fractions were collected, this fraction was thoroughly dialyzed against distilled water, the dialyzed fluid was freeze-dried,
About one powder was obtained containing about 15 parts of HGIGP.

This powder was tested for biological activity and virus activity in the same manner as in Experiments 1 and 2 above. the result,
The biological activity was almost the same as that without heat treatment, and no virus activity was detected. Example 2 400ml of fresh urine collected from a healthy person was mixed with 10% sodium hydroxide to adjust the pH to 8, and heated to 1500ml while cooling to 0°C. p. m. The mixture was centrifuged using a continuous centrifuge to remove impurities and obtain a supernatant.

Next, this supernatant was adjusted to pH 7 with 10% hydrochloric acid and passed through a column (10×80 arc) filled with silica gel, and the components adsorbed on the silica gel were eluted from the column with 40 sleeves of 5% aqueous ammonia.

The pH of the lacquer solution thus obtained was adjusted to 7.5 with 1N sulfuric acid, powdered ammonium sulfate was added to this to make it 70% saturated, and the mixture was left overnight at 0°C, and the precipitate formed was heated in a furnace. Separated.

This precipitate was dissolved in 5% aqueous ammonia, placed in a dialysis tube (manufactured by Visking), thoroughly dialyzed against 0.09M phosphate sanitary solution (pH 6.5), and the buffer solution was added to the dialysis solution. Add liquid to adjust the total volume to 10 so
CM equilibrated with 8M phosphate buffer (pH 6.5)
Cephadex C-50 ion exchange column (40 x 40
The liquid was passed through the skin, and the impurities were adsorbed on the ion exchange resin to obtain a passed liquid.

Ten millimeters of this passed-through solution was concentrated using a diaflow hollow fiber concentrator (manufactured by Amicon, DC-3 top type), and the protein concentration was adjusted to 70.9'.

This concentrated solution was then heated at 60°C for 1 hour and cooled on ice to obtain a heat-treated HGIGP-containing fraction. Reference Example 2 The insoluble matter generated in the heat-treated HGIGP-containing fraction obtained in Example 2 was removed, and the mixture was placed in a dialysis tube (manufactured by Visking Co., Ltd.) and added to 0.1 M Tris-HCl buffer (pH 7).
．． 0) overnight at 5°C, and the buffer solution was added to the dialyzed solution to adjust the concentration to 1.

This solution was equilibrated and activated with the buffer beforehand.
The liquid was passed through an AE cellulose column (4.0 x 40 arcs) and 0.
After thoroughly washing the column with 1M Tris-HCl buffer (pH 7.0), elution was performed with 0.1M Tris-HCl buffer (pH 7.0) containing 0.3M NaCl. The eluate was then dialyzed against 0.1M Tris-HCl buffer (pH 7.0) to obtain a dialysis solution. This dialyzed fluid was again equilibrated with the buffer solution and activated with a DEAE cellulose column (4
．． 0x40 arc) and from 0.1M to 0. It was exported by a linear concentration gradient elution method using $M of common salt to proceed with the fractionation that has the effect of promoting the differentiation and proliferation of forehead granulocytes.Powdered ammonium sulfate was added to this fraction to achieve 70% saturation, and the precipitate was collected.
This precipitate was dissolved in a small amount of 0.1M Tris-HCl buffer (pH 7).
．． 0) and dialyzed against the buffer to obtain a dialysis solution.

Next, 20M of this dialysate was developed on a Sephadex G-150 column (4.0 x 60 arcs) equilibrated with 0.1M Tris-HCl buffer (pH 7.0) in advance, and the relative eluate volume was 1. 11-1.45 fractions were collected, this fraction was thoroughly dialyzed against distilled water, the dialyzed fluid was freeze-dried,
Approximately 450 particles of powder containing approximately 13.5 female HGIGPs were obtained.

This powder was tested for biological activity and virus activity in the same manner as in Experiments 1 and 2 above.

As a result, the biological activity was almost the same as that without heat treatment, and no virus activity was detected. Example 3 10% sodium hydroxide was added to 400 ml of fresh urine collected from a healthy person to adjust the pH to 8, and the mixture was heated to 1500 ml while cooling to 0°C. p. m. The mixture was centrifuged in a continuous centrifuge to remove insoluble matter and obtain a supernatant.

Next, this upper groove was adjusted to pH 7 with 10% hydrochloric acid, and the liquid was passed through a column (10 x 80 skin) filled with silica gel, and the components adsorbed on the silica gel were eluted from the column with 40 sleeves of 5% aqueous ammonia.

The pH of the eluate thus obtained was adjusted to 7.5 with 1N sulfuric acid, powdered ammonium sulfate was added to this to make it 70% saturated, and the mixture was left at 0°C overnight, and the precipitate formed was separated in a furnace. did.

This precipitate was dissolved in 5% ammonia water, placed in a dialysis tube (manufactured by Visking), thoroughly dialyzed against 0.05M phosphate buffer (pH 6.5), and added to the dialysis solution. Add liquid to adjust the total volume to 10 ml, and make it 0.
CM equilibrated with 05M phosphate buffer (Palisade 6.5)
Cephadex C-50 ion exchange column (4.0 x 4
0 arc), impurities were adsorbed on the ion exchange resin, and a permeate was obtained.

10% of this passed-through liquid was concentrated using a Diaflow Hollow Fiber Concentrator (manufactured by Amicon, DC-3 top type), and the concentrated liquid was converted into a 0.1M Tris-HCl buffer (pH 7.
0) overnight at 5° C., and the buffer solution was added to the dialyzed solution to make a final adjustment. This solution was equilibrated and activated with the buffer beforehand on a DEAE cellulose column (4.
0x40) and 0.1M Tris-HCl buffer (
After thoroughly washing the column with 0.1M Tris-HCl buffer (pH 7.0) containing 0.3M sodium chloride, elution was performed with 0.1M Tris-HCl buffer (pH 7.0). The eluate was then dialyzed against 0.1M Tris-HCl buffer (pH 7.0) to obtain a dialysis solution. This dialyzed fluid was again equilibrated and activated with the buffer solution D
Pass the liquid through an EAE cellulose column (4.0 x 40),
It was eluted using a linear concentration gradient elution method using 0.1M to 0.3M common salt, and a fraction having the effect of promoting the differentiation and growth of jaw granulocytes was collected, and powdered ammonium sulfate was added to this fraction to give a concentration of 70%.
The precipitate was collected and dissolved in 0.1M Tris-HCl buffer (pH 7.0) to bring the protein concentration to 15.
Adjusted to 0 evening/desk. This solution was then heated at 6000 to 1
The mixture was heated for a while and cooled on ice to obtain a heat-treated HCIGP-containing fraction. Reference Example 3 The heat-treated HGIGP-containing fraction obtained in Example 3 was filtered to remove the waste, and dialyzed against 0.1M Tris-HCl buffer (pH 7.0) to obtain a dialyzed fluid. Ta.

Next, 20% of this dialysate was developed on a Sephadex G-150 column (4.0 x 60 columns) equilibrated with 0.1M Tris-HCl buffer (pH 7.0), and the relative eluate volume was 1 .11-1.45 fractions were collected, this fraction was thoroughly dialyzed against distilled water, the dialyzed fluid was freeze-dried,
Approximately 500 females of powder containing approximately 15 females of HGIGP were obtained. This powder was tested for biological activity and virus activity in the same manner as in Experiments 1 and 2 above. As a result, the biological activity was almost the same as that without heat treatment, and no virus activity was detected. Example 4 50 pieces of fresh urine collected from healthy people were made neutral by adding 10% aqueous sodium hydroxide solution, and centrifuged in a refrigerated continuous centrifuge (1000 p.m.) to remove insoluble matter. Remove the supernatant and immediately diaphragm.

- Fiber concentrator (manufactured by Amicon, DC-30 type,
The urine was concentrated for 1 pm (equipped with a molecular weight 10,000 or less permeable membrane), water was added to make the solution 50 pm, and concentrated again to obtain 3 pm concentrated urine. Next, the concentrated urine is concentrated using a rotary vacuum concentrator, and the protein content is about 50% by weight.
got '. The concentrate was then heated in a hot water bath at a temperature of 6000±0.5 for 1 hour and immediately cooled to obtain a heat-treated HGIGP-containing fraction. Reference Example 4 Above Example 4
Insoluble matter generated in the heat-treated HGIGP-containing fraction obtained by
．． DEAE which had been equilibrated in advance with 0.1M Tris-HCl buffer (pH 7.0).
Pass the liquid through a cellulose column (4.0 x 40) to
After adsorbing IGP onto DEAE cellulose, 0.1 M
After thoroughly washing the column with Tris-HCl buffer (pH 7.0), it was discharged with 0.1 M Tris-HCl buffer (pH 7.0) containing 0.3 M NaCl.

Thereafter, approximately 62 pieces of powder containing HGIGP were obtained in the same manner as in Reference Example 1.

This powder was tested for biological activity and viral activity in the same manner as in Experiments 1 and 2 above.

As a result, the biological activity was almost the same as that without heat treatment, and no virus activity was detected. Reference Example 5 Concanaparin A was prepared by dissolving the powder 200-9 obtained in Reference Example 1 in 0.02M phosphate buffer (pH 7.0) containing 1.0M common salt and equilibrating with the buffer in advance.
- Pass the liquid through a column containing Sepharose Hime 100M, 1.
0.02M phosphate buffer (pH 7.0) containing 0M NaCl
) and then 50mM Q-methyl-D.
- Contains glucosides and 1.0M common salt.

Elute with 02M phosphate buffer (pH 7.0), collect fractions that have an effect on differentiation and proliferation of forehead granulocytes by measurement using the method described below, dialyze this against distilled water, and collect the dialyzed fluid. Lyophilized.

Furthermore, 9 of about 50 of the freeze-dried powder obtained here was added to 10
0.129 M Tris-HCl buffer containing % glycerin (
Cooling water was passed through a preparative electrophoresis device (Fuji Riken, Fujikarabar-O type) using an 8% acrylamide gel (pH 8.9, 2 peaks x 25 sides). Then, a current of 10 mA was applied to perform electrophoresis, and a fraction with a relative mobility of 0.46 was collected with 0.029 M Tris-glycine buffer (pH 8.3) and dialyzed against distilled water.
The dialyzed fluid was freeze-dried to obtain HGIGP 9 of about 10.

Brief Description of the Drawings The drawings show HGIG purified from the virus-inactivated HGIGP fraction obtained according to the present invention.
Figure 1 shows the infrared absorption spectrum, Figure 2 shows the relationship between mobility and molecular weight measured by dew electrophoresis, and Figure 3 shows the ultraviolet absorption spectrum. show.

Figure 1 Figure 2 Figure 3

Claims (1)

[Claims] 1. Concentrate a fraction of human urine containing glycoproteins that promote the differentiation and proliferation of human granulocytes, adjust the protein content to at least 70 mg per ml, heat at a temperature of 50 to 70°C for 8 to 30 hours, and A method for heat treatment of a fraction containing a glycoprotein that promotes the differentiation and proliferation of human granulocytes, the method comprising inactivating viruses present in the fraction.