JPS6045852B2 - Method for producing placental gonadotropic hormone - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing placental gonadotropin hormone (HCG).

HCG is a glycoprotein hormone with a molecular weight of about 30,000 to 40,000, produced in the placenta and contained in pregnant women's urine.

Many methods have been published for collecting HCG from pregnant women's urine, but the most representative one is the benzoic acid adsorption method [Katman et al., J. Biol, Chem, 98739 (1932)].
, Kaolin adsorption method [Brodlurg et al. 9Pro, So
c, Exptl, Biol.

&Med, 71228 (1947)]. Since none of these methods can selectively adsorb HCG, there are drawbacks such as a large amount of different substances being mixed in the HCG product and only products with low unit activity being obtained. The inventor has conducted extensive research in order to establish a more advantageous manufacturing method that overcomes the drawbacks of conventional methods.

First, attempts were made to collect HCG by contacting pregnant woman's urine with an ion exchange resin or ion exchanger, but when using raw urine, HCG was collected.
There are almost no ion exchange resins or ion exchangers that adsorb CG well, and even if it does, the eluent may be Λ-ammonia water, IN sodium chloride aqueous solution, 50% alcohol,
No elution rate exceeded 40% using various elution methods using 40% alcohol or 10% AcNH.

In addition, when using 2-3 times diluted urine, some ion exchange resins and exchangers may adsorb HCG, but no elution rate exceeds 70% using various elution methods. The specific activity was only 100 to 5001 U/mg (protein), and it was found that this method was not industrially advantageous, as it required a large amount of expensive adsorbent. Therefore, with respect to known adsorbents conventionally used in the production of HCG, urinary H
The optimum pH for CG adsorption was investigated, adsorption was carried out at that pH, and the results shown in Table 1 were obtained by elution with Shuichi ammonia water.

The specific activity was measured using acetone powder as the eluted component for those with an adsorption rate of 10 or more. As is clear from Table 1, the kaolin type had a good adsorption rate, but the elution rate and specific activity were poor.

Therefore, the present inventors conducted tests similar to those described above for a wider range of adsorbents and obtained the results shown in Table 2.

1) Kaolin-based material manufactured by Japan Activated White Clay 2) Made by Hayashi Kasei 3) Same 4) Made by Shiraishi Kogyo 75) Tomita Pharmaceutical Co., Ltd. Dimagnesium aluminate silicate, that is, known kaolin is better than many of the adsorbents mentioned above. is excellent, and also has A as an HCG adsorption factor in kaolin.
l.

O3, SiO2, MgO, etc. are possible, but alumina,
It was found that silica and magnesia alone do not adsorb HCG. Subsequently, similar tests were conducted on synthetic adsorbents with various compositions, and as shown in Table 3, synthetic zeolite, alumina hydrate, aluminum hydroxide, and sodium bicarbonate molecular compound 7 adsorbed HCG. Magnesium silicate has poor adsorption and elution rates.
Synthetic aluminum silicate (aluminosilicate) of the present invention
Only kaolin showed excellent results in all aspects of adsorption, desorption rate, and specific activity, and was found to be far more effective than kaolin.

In each of the above tables, the symbols for adsorption, elution rate, and specific activity indicate the following numerical values. The present invention has been completed based on the above-mentioned findings, and the urine of pregnant women is treated with weakly acidic acid with the formula Al2O.
3.? Components in the urine were adsorbed by contacting with an adsorbent made of aluminum silicate containing IO2. This is a method for producing placental gonadotropin hormone, which is characterized in that the active ingredient is collected from the residual liquid or the eluate obtained by eluting the adsorbed substance with an aqueous solution containing a lower alcohol and a salt soluble therein.

In the method of the present invention, pregnant women's urine may be used as it is, but the pH of pregnant women's urine may be adjusted to 8-9, preferably 8.5, and impurities such as mucopolysaccharides are precipitated and removed by filtering or other means. It is preferable to use something. Formula used as adsorbent. Al2O3・Cf:)IO2・Bo.

Aluminum silicate (aluminosilicate, trade name KYOWARD 700 manufactured by Kyowa Chemical Industry) having 0 is Al2O3
It is a synthetic aluminum silicate powder containing 9 to 13% SiO2 and 58 to 64% SiO2, and the particle size is not particularly limited as long as the object of the present invention can be achieved, but the particle size may be within the following preferred range depending on the adsorption and desorption operation method. There is.

The contact between the urine and the adsorbent and the elution of the adsorbed material from the adsorbent can be carried out by any of the batch method, batch-column method, and column method. The preferred particle size of the adsorbent is 100 to 200 mesh in a batch method, and 50 to 100 mesh in a batch-column method and a column method in which contact with urine is performed in a batch method and elution is performed in a column.

When bringing the urine and the adsorbent into contact, the urine is preferably slightly acidic, particularly preferably at a pH of about 3-5. If the pH exceeds 5, HCG adsorption tends to be inhibited. The amount of adsorbent used and the contact time are not particularly limited, but in practice, the amount added is preferably 0.5-1.0 g of adsorbent per 1 e of urine, and the contact time is preferably about 2 hours.

The alkaline aqueous solution used for elution is preferably moderately alkaline, with PHLO. 5-12 is preferred.

The material that makes the aqueous solution alkaline can be selected as appropriate within the range that achieves the purpose, but preferred examples are Bo's ammonia water and △
Examples include aqueous sodium monocarbonate solution (PHll). Various methods can be used to collect HCG from the eluate obtained by eluting the adsorbent from the adsorbent with an alkaline aqueous solution.

For example, the eluate may be adjusted to be weakly acidic with dilute hydrochloric acid or the like, and HCG may be precipitated by salting out with ammonium sulfate or the like, or acetone may be added to precipitate it. Elution can also be carried out using an aqueous solution containing a lower alcohol and a salt soluble therein.

Examples of lower alcohols used include methanol, ethanol, and isopropanol. Examples of salts soluble in lower alcohols include ammonium acetate and ammonium formate. It is better that the alcohol concentration in the aqueous solution is not too high.

When the alcohol concentration reaches about 70%, HCG hardly elutes. Furthermore, when the salt concentration increases, for example when the concentration of ammonium acetate exceeds 15%, HCG tends to be denatured. An example of a preferred aqueous solution is one containing about 40% alcohol and about 10% ammonium acetate. If a lower alcohol is added to the eluate obtained by elution with the above lower alcohol and aqueous solution containing a soluble salt, HCG
precipitates.

If the amount of alcohol added is small, the yield of the target product will be poor,
If the amount is too large, impurities will increase in the precipitate, so it is usually preferable to add about three times the amount of alcohol to the eluate. HCG collected as precipitate as described above
L can generally reach 2000-30001 U/Mg (protein), which corresponds to a specific activity of 5-1@ compared to that by conventional methods.

Next, the present invention will be explained in more detail with reference to Examples.

7 Example 1 50e of pregnant woman's urine from 2nd month to 6th month of pregnancy was adjusted to pH 8.5 with 4N sodium monohydroxide solution. After removing the precipitate, the pH was adjusted to 3.5 with Samichi hydrochloric acid. Synthetic aluminum silicate (
Keyword 7 concave manufactured by Kyowa Chemical Industry, 200 meters)
Add 250 g and leave in contact for 2 hours.

After standing still, separate the supernatant and adsorbent, wash the adsorbent with 5.0e water, and wash the adsorbent with 5.0e water. - Elute with aqueous ammonia 3.0e. The eluate was adjusted to pH5. Ammonium sulfuric acid was added to the solution from which the precipitate formed as O was removed to a concentration of 50% to precipitate HCG.
The obtained HCG is 15000001U1 specific activity is 200
01 U/Mg (protein), yield was 91.2%.

Example 2 Pregnant woman urine 10e from 2nd month to 6th month of pregnancy was collected in Example 1.
Similarly, adjust the pH to 8.5, remove the precipitate, and adjust the pH to 3. with dilute hydrochloric acid.
5.

100g of synthetic aluminum silicate (50-100 mesh) similar to Example 1 with a diameter of 10Cr! Pack into l column,
The above treated urine flows down at a rate of 2e/hour, and the water is 1.0e/hour.
After washing with 40% ethanol/10% ammonium acetate solution 1.0', HCG was precipitated by adding 3 times the volume of 95% ethanol to 500 ml of the eluate. recovered HC
G is 2960001U1 specific activity is 30001U/Mg (
protein), the yield was 85%. Example 3 Pregnant woman urine 50e from 2nd month to 6th month of pregnancy was treated with pH 8.5 in the same manner as in Example 1.
Synthetic aluminum silicate (50-100 mesh) as in Example 1 and adjusted to pH 3.5 with dilute HCl
g and leave in contact for 2 hours.

After standing still, separate the adsorbent from the supernatant, wash the adsorbent with 5.0e of water, pack it into a column with a diameter of 10cm, further wash with 5.0e of water, and add 5.0e (1) 2N sodium monocarbonate. Elutes in aqueous solution. Eluate 3.0e was adjusted to pH 5.0 with dilute HCl. O
After centrifuging the resulting precipitate, ammonium sulfate was added to 50%
A precipitate of HCG was obtained. Yield: 90.
2% specific activity 20001 U/Mg (protein). Example 4 100e of urine from 2nd month to 6th month of pregnancy was pH-treated in the same manner as in Example 1.
8.5 and adjusted to pH 3.5 with dilute hydrochloric acid, and 500 g of synthetic aluminum silicate (200 mesh) as in Example 1.
and leave in contact for 2 hours.

After standing still, separate the supernatant and adsorbent, wash with 5.0e of water, and add 40% methanol/10% ammonium acetate solution 5.0'
Elutes with 12.0e of methanol was added to 4.0e of the eluate to obtain a precipitate of HCG. The amount of HCG recovered was 250
00001U1 specific activity is 27001U/Mg (protein)
The yield was 85%.

Claims (1)

[Claims] 1 Pregnant woman urine is slightly acidic and the formula Al_2O_3・9SiO_2
The components in urine are adsorbed by contacting with an adsorbent made of aluminum silicate containing -xH_2O, the adsorbed substances are eluted from the adsorbent with an alkaline aqueous solution, and the eluate is made weakly acidic to remove the resulting precipitate. 1. A method for producing placental gonadotropin hormone, which comprises collecting an active ingredient from a liquid or an eluate obtained by eluting the adsorbed substance with an aqueous solution containing a lower alcohol and a salt soluble therein.