JPS6364407B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for purifying immunoglobulin from human placenta extract. Human immunoglobulin contains various antibodies and is used for the prevention and treatment of infectious diseases, and is currently one of the useful plasma fraction preparations. Plasma, the raw material for immunoglobulin, is
Plasma is obtained from fresh whole blood, expired blood for transfusion, or collected during plasmapheresis, but there is a shortage of plasma in all countries, and placenta is said to be the third raw material. Currently, they are being supplemented with blood plasma from other people. However, placenta-derived plasma fractions contain dyes,
Impurities such as proteolytic enzymes, hormones, blood type substances, and alkaline phosphatase are mixed in, and are expected to cause various problems such as side effects when used clinically, so they must be completely removed during the purification stage. It is necessary to keep it. Taylor et al. reported a method for purifying immunoglobulin and albumin from placental blood using the cold ethanol method (Journal of the
Journal of the American Chemical Society
society): Vol. 78, pp. 1356-1358, 1956),
The impurities contained in these purified products have not been clarified, and the ethanol fractionation method is problematic in that it often denatures proteins. Boros et al. have reported that lipids, fibrinogen, and hemoproteins can be removed by treating crude immunoglobulin obtained from placental blood with chloroform (Development in Biological Standard Disease). Developments in Biology
standardization, S. KARGER): Volume 27, 102~
106, 1974), Pla et al. reported that chloroform can be used to remove 99.6% of the pigment when purifying albumin derived from placental blood (Development in Biological Standardization). (Developments in
Biological standardization, S. KARGER): No.
27, pp. 115-122, 1974). However, since chloroform is a plasminogen activator, proteolytic enzymes activated by chloroform treatment are detected. The present inventors also found that proteolytic enzymes present in placental blood are activated by chloroform treatment, activated by streptokinase, and inhibited by 0.5 molar aminocapric acid, and that lysine-cephalose We have observed that plasmin is adsorbed by affinity chromatography using plasmin, and based on these results, we believe that it is mainly plasmin. It is known that immunoglobulin decomposition occurs when human immunoglobulin preparations are stored at room temperature or 4°C for a long period of time. This is particularly noticeable in preparations derived from placental blood. In severe cases, degradation occurs within several weeks upon storage at 4°C, resulting in changes in antibody content and affecting clinical efficacy. Therefore, it is necessary to remove proteolytic enzymes as early as possible during the purification process. As a method for removing other impurities, it is known that blood type substances can be efficiently separated by gel filtration using Sepharose 4B (Noboru Hiyama: Protein Nucleic Acid Enzyme: Vol. 14, pp. 658-666, 1969). It is difficult to remove impurities such as alkaline phosphatase and pigments other than blood group substances. Therefore, it has not been possible to obtain highly pure and stable immunoglobulin using conventional general purification methods. The present invention almost completely removes impurities from human placental blood, resulting in high purity and less denaturation of immunoglobulin.
The purpose is to provide a method for purifying immunoglobulin with a high yield, and in a method for purifying immunoglobulin from crude immunoglobulin obtained from a human placenta extract, treatment with a chloroform-ethanol mixture or chloroform is performed. The present invention relates to a method for purifying immunoglobulin, which is characterized in that the immunoglobulin is purified by adding an adsorbent to the immunoglobulin, followed by purification using a cation exchanger. As a starting material for the present invention, placental blood or a placental extract obtained by cutting the placenta into small pieces and adding physiological saline to extract blood components can be used. placenta 1
When extracted using 1 part physiological saline per kg, the protein concentration in the placental extract was 76.0 mg/kg.
ml, protein components are albumin 18.3%, immunoglobulin 4.3%, dye has an absorbance of 115.0 at a wavelength of 403 nm, blood type substances are type A and B, and alkaline phosphatase (hereinafter referred to as ALP) is 560 K-A units. /dl,
Contains proteolytic enzymes and hormones. The method for measuring each of the above components is as follows. The protein concentration was calculated as the amount of protein (mg) in 1 ml by the microkjeldahl method. Serum components were measured by electrophoresis using a cellulose acetate membrane, followed by staining (Ponceau 3R).
Absorption at a wavelength of 540 nm was measured and quantified using a densitometer. Immunoglobulin (hereinafter referred to as IgG)
Quantification was determined by one-way radial immunodiffusion method. The content of pigments such as hemoglobin was calculated from the absorbance at a wavelength of 403 nm. Proteolytic enzymes were measured by fibrin plate method. Plasmin activity was measured by preparing a plate using bovine fibrinogen and thrombin, placing 0.03 ml of the sample on the plate and leaving it at 37°C for 18 to 20 hours. Those that showed dissolution were marked as (10) and those that did not show (-). In addition, plasminogen activity was measured in 0.5 ml of sample.
Add 0.05ml of 1000 units/ml streptokinase.
After reacting at 37°C for 10 minutes, the same measurement was performed using the plate method. Also, when preparing the plate, add 1000 units/ml of streptokinase and 500 units/ml of urokinase.
It was confirmed that 0.03 ml was added and no dissolution was observed. Blood group substances can be determined by the method of Cohen et al. (Vox Sanguinis).
21, pp. 311-318, 1971) using serum for anti-A and anti-B determination, and expressed as the final dilution factor based on the hemagglutination inhibition reaction. ALP measurement was performed using an ALP measurement reagent according to the King-King method. All samples were dialyzed against 0.02M phosphate buffer, and the ALP content is expressed in K-A units per dl, 5K-A units/dl.
More than 5 K-A units/dl was indicated as positive (+), and less than 5 K-A units/dl was indicated as negative (-). Among the hormones in the sample, gonadotropin (hereinafter referred to as HCG) in particular was determined by the hemagglutination inhibition reaction with HCG-sensitized red blood cells of an HCG measurement reagent for pregnancy diagnosis. All samples were subjected to the test after being absorbed with 2% sheep blood cells, and positive results were indicated as (+) and negative results as (-). In addition, the sensitivity of the HCG measurement reagent is
HCG is 1 international unit. Crude immunoglobulin can be obtained from placenta extract by known means such as ammonium sulfate precipitation. Add 130 g (0.68 M) of ammonium sulfate per placenta extract to remove the resulting precipitate, and then add 136 g (1.40 M) of ammonium sulfate per 1 supernatant to collect the resulting precipitate. This precipitate contains mainly globulin and the supernatant contains mainly albumin. Dissolve the precipitate in distilled water and add 266 g of ammonium sulfate per solution again to bring the pH to 7.0.
Collect the precipitate that has been generated by correcting the area, and collect the precipitate.
After dissolving in 0.02M phosphate buffer (PH7.2), it was dialyzed against 0.02M phosphate buffer (PH7.2) to obtain crude immunoglobulin. The recovery rate of each component in the crude immunoglobulin obtained by ammonium sulfate precipitation method is as shown in Table 1.
The protein recovery rate is approximately 20%, but the IgG recovery rate is approximately 90%.
% and can be recovered in good yield.

[Table] Regarding impurities, the pigment decreased to approximately 1/30, but blood type substances, HCG, and ALP remained as shown in Table 2 and were easily detected.

[Table] Although plasmin was not detected, it existed in the form of plasminogen. The dye remaining in the crude immunoglobulin can be almost completely removed by treatment with chloroform/ethanol. Chloroform 3.5% (V/V) and ethanol 1.5% (V/V) were added to the crude immunoglobulin.
V) was added at 4°C or lower, stirred or shaken at 4°C or lower for 60 minutes, and then centrifuged (8000 rpm, 30 minutes). The resulting supernatant was subjected to solvent removal under reduced pressure, and further 0.02M
Dialyze against phosphate buffer (PH7.2). The concentration of the organic solvent used here is preferably 3.25 to 10.5% (V/V) in consideration of protein denaturation. Although it is possible to treat with chloroform alone, it is possible to remove the pigment more efficiently by adding ethanol at the same time, and as shown in Table 3, the ratio of chloroform to ethanol is 1.75 to 7.0.
% to 3.25 to 10.5%.

[Table] Furthermore, the operating temperature should be adjusted to avoid protein denaturation.
It is preferable to operate at a temperature that does not freeze by 10% or less. As shown in Table 1, the recovery rate of each component of immunoglobulin purified by chloroform/ethanol treatment is approximately 3% for protein, and 95% of the pigment contained in crude immunoglobulin has been removed. It was done. However, not only blood type substances, HCG, and ALP remained even after the chloroform/ethanol treatment as shown in Table 2, but also proteolytic enzyme activity due to plasmin activated by chloroform was detected. Furthermore, it is clear that streptokinase still exists in the form of plasminogen, since the protease activity was enhanced. The proteolytic enzyme activity contained in immunoglobulin after chloroform/ethanol treatment was determined by acid clay,
It can be completely removed by treatment with an adsorbent such as kaolin. Add adsorbent to immunoglobulin after chloroform/ethanol treatment at 2.5-20% (W/V)
Mix and stir at 4℃ or 37℃ for 18 hours.
After adsorption for 4 hours, centrifugation (8000 rpm, 10
The supernatant was obtained as adsorbent-treated immunoglobulin. The adsorbents used here include acid clay whose main component is silicate, activated clay, kaolin,
Talc etc. or activated carbon can be used, but
When adsorbing at 4°C for 18 hours, acid clay is suitable because it can remove plasmin and plasminogen activity at a concentration of 10% (W/V) as shown in Table 4, and adsorption is performed at 37°C for 4 hours. in case of,
Acid clay has a concentration of 3% (W/V), and kaolin has a concentration of 5
% (W/V) concentration.

[Table] When acid clay is used as an adsorbent, it can be used in a wide range of PH during adsorption from PH5 to PH9, it can be treated near neutrality, it can be treated in a short time, and the amount is small. Acid clay is most suitable as an adsorbent because it can completely remove proteolytic enzyme activity in small amounts. Acidic clay 3% (W/V), 37℃
As shown in Table 1, the recovery rate of each component of immunoglobulin treated for 4 hours was 2.3% for protein recovery and 51.0% for IgG, which was not much different from that in the case of chloroform/ethanol treatment. As shown, not only protease activity but also HCG can be completely removed. However, blood group substances and ALP cannot be removed even by adsorbent treatment. blood group substances that cannot be removed by adsorbent treatment;
Impurities such as ALP and some dyes can be completely removed by ion exchange chromatography using a cation exchanger. Examples of the cation exchanger include cation exchange cellulose such as carboxymethyl-cellulose, cation exchange cellulose such as carboxymethyl-cephadex,
Or cation exchange resin is good. For example, carboxymethyl-cephadex (hereinafter referred to as CM-cephadex) is equilibrated with 0.02M phosphate buffer (PH7.2) and made into a 3.6 x 21 cm column.
50 ml of acid clay-treated immunoglobulin was adsorbed onto the column, and unadsorbed proteins were washed away with approximately 1000 ml of 0.02M phosphate buffer (PH7.2) to collect the unadsorbed fraction (F-).
Then, the protein adsorbed on the column was eluted with 0.2M phosphate buffer (PH7.2) to obtain a 0.2M phosphate buffer eluted fraction (F-). The flow rate of the phosphate buffer was approximately 80 ml per hour. The absorbance pattern at a wavelength of 280 nm of the eluate of CM-Sephadex column chromatography of acid clay-treated immunoglobulin is the unadsorbed fraction (F-) as shown in Figure 1.
There are two peaks: and the elution fraction (F-).
The absorbance pattern at a wavelength of 403 nm, which indicates the elution of dyes, and the elution pattern of blood group substances are based on the unadsorbed fraction (F-
), and these peaks are not observed in the elution fraction (F-). The recovery rate of each component in the 0.2M phosphate buffer (PH7.2) elution fraction (F-) is shown in Table 1. Although the protein recovery rate is significantly lower, the recovery rate of IgG is good at 32%. Therefore, it is possible to completely remove blood group substances and ALP, which could not be removed by conventional operations (Table 2). For comparison, when immunoglobulin that had not been treated with acid clay was treated in the same manner using a CM-Sephadex column, the absorbance at a wavelength of 280 nm and the wavelength
The absorbance at 403nm and the pattern of blood group substances are
It is almost the same as the case of acid clay-treated immunoglobulin, and blood group substances and ALP are not detected in the 0.2M phosphate buffer (PH7.2) elution fraction, but proteolytic enzymes and HCG are detected because acid clay is not treated. The activity is not completely removed and still exists. Based on the above, in order to remove impurities such as pigments, proteolytic enzymes, hormone blood group substances, ALP, etc. from crude immunoglobulin obtained from placenta extract, and to obtain high-purity purified immunoglobulin in a high yield, crude It is necessary to treat the immunoglobulin with chloroform/ethanol and then an adsorbent, followed by cation exchange chromatography. The 0.2M phosphate buffer (PH7.2) elution fraction (F-) of CM-Sephadex ion exchange chromatography performed as cation exchange chromatography was added to 0.01-0.1M ethylenediamine acetate buffer. After dialysis, purified immunoglobulin can be obtained by lyophilization. The protein recovery rate of purified immunoglobulin was 0.7% of the placenta extract, and the IgG recovery rate was 32% of the placenta extract.
The purity of the purified immunoglobulin was analyzed using a Chiselius electrophoresis apparatus using sodium diethylbarbiturate buffer (PH 8.6, ionic strength 0.1), and as shown in Figure 2, it was found to be almost pure immunoglobulin, with a purity of 99.8. It was %. In addition, when measured by one-way radial immunodiffusion method, more than 95% is IgG, and similarly, immunoglobulin (IgA) and immunoglobulin M were measured by one-way radial immunodiffusion method.
(IgM) content was 2% or less. Analysis by disc electrophoresis uses a 5% polyacrylamide gel, runs a current of 5 mA per gel tube for 210 minutes in Tris-glycine buffer (PH8.3), and then stains with Amidoblack 10B. It was decolored with acetic acid and measured with a densitometer. The result was a single broad band. Furthermore, analysis by SDS-gel electrophoresis was performed using a 7.5% gel, the sample was treated with 2-mercaptoethanol/SDS, and a phosphate buffer (PH7.2) containing 0.1% SDS was applied at 8 mA per gel tube. After electrophoresis by applying a current for 360 minutes, measurement was performed using a densitometer that had been stained and decolored in the same manner as above. As a result, bands of IgG molecules with molecular weights of around 50,000 and around 20,000 were mainly migrated. These disk electrophoresis and SDS
- From the results of gel electrophoresis, it is clear that the purified immunoglobulin obtained by this method is no different from plasma-derived immunoglobulin in terms of homogeneity and mobility, and has not undergone any denaturation. . The stability of the purified immunoglobulin was examined by immunoelectrophoresis using the Graber method after storage at a protein concentration of 150 mg/ml for 2 months at 37°C under aseptic conditions. Purified immunoglobulin shows no change in migration pattern before and after storage, which may be due to residual proteolytic enzymes such as plasmin.
Degradation of IgG molecules does not occur at all. Furthermore,
No change in the properties of purified immunoglobulin was observed even when purified immunoglobulin was stored at 4°C for 1 year, and even under the harsh conditions of heating purified immunoglobulin at 57°C for 4 hours, precipitation or No changes in properties such as gelation were observed. Therefore, since the immunoglobulin purified by this method has almost all proteolytic enzymes removed, the IgG is not subject to degradation and is stable, and can withstand long-term storage. The diphtheria antitoxin titer of the purified immunoglobulin was measured by the rabbit intradermal reaction method according to the method of Jensen. Standard diphtheria antitoxin (0.02 unit/ml) and test toxin were mixed and diluent was added to make a total volume of 2 ml to serve as a control group, and the sample and test toxin were mixed and prepared to form a sample group.
Leave it for a minute, then inject 0.1ml into the rabbit skin for 40~
After 48 hours, intradermal redness was measured and the diphtheria antitoxin units in the sample were calculated. The diphtheria antitoxin value of the purified immunoglobulin is 0.025 per 1.5 mg of protein, which is a relatively high diphtheria antitoxin value.
In addition, purified immunoglobulin was used at a protein concentration of 150 mg/ml.
No decrease in diphtheria antitoxin titer was observed even after storage at 37°C for 2 months under sterile conditions. The anti-complement properties of purified immunoglobulin were measured by Kabat &Mayer's 50% hemolysis method and compared with immunoglobulin derived from venous blood. 100CH ₅₀ samples in 1 ml of guinea pig complement
Add 1 ml of each sample and add 1 ml of gelatin veronal buffer to a total volume of 5 ml, leave at 37°C for 60 minutes, measure the amount of remaining complement, and calculate the difference from the complement value of the control without adding the sample. value. As shown in Table 5, the anti-complement property of placenta-derived purified immunoglobulin was 41.9.
The immunoglobulin derived from venous blood had a value of 32.5, and although the purified immunoglobulin derived from the placenta showed a slightly higher value, it is thought that there is almost no difference between the two.

[Table] As described above, the method of the present invention is a very useful method because it allows highly purified and highly stable immunoglobulin to be purified in high yield with relatively simple operations. Next, the present invention will be explained in more detail with reference to Examples. Example 1 a Preparation of placenta extract 120 cryopreserved human placentas were cut into small pieces, and physiological saline was added at a ratio of 1 part per 1 kg of placenta.
After stirring for a minute, centrifuge to separate the supernatant.
Ninety placental extracts were obtained. b Preparation of crude immunoglobulin (ammonium sulfate precipitation method) Ammonium sulfate per 1 placenta extract
After adding 130g (0.68M) and dissolving the mixture, allowing it to stand for 2 hours, it was centrifuged to obtain a supernatant. Furthermore, supernatant 1
136 g (1.40 M) of ammonium sulfate was added and dissolved, and after standing for 2 hours, centrifugation was performed to obtain a precipitate. After dissolving the precipitate in distilled water, ammonium sulfate was added and dissolved again at a ratio of 266 g per 1 solution, the pH was corrected to around 7.0, and the mixture was allowed to stand for 2 hours.
The resulting precipitate was collected by centrifugation. The precipitate obtained by repeating the ammonium sulfate precipitation method twice was dissolved in 0.02M phosphate buffer (PH7.2) and dialyzed against the same buffer to obtain crude immunoglobulin. c Chloroform/ethanol treatment Crude immunoglobulin was cooled to 4°C, and a mixture of chloroform and ethanol in a ratio of 7:3 was added to the crude immunoglobulin at a ratio of 5% (V/V) at 4°C. After shaking for a minute, centrifugation (8000 rpm, 30 minutes) was carried out, the solvent was removed from the obtained supernatant under reduced pressure, and 0.02M phosphate buffer (PH
7.2) to obtain chloroform/ethanol-treated immunoglobulin. d Treatment with adsorbent Add acid clay at a ratio of 3% (W/V) to the chloroform-ethanol treated immunoglobulin, mix and stir, adsorb at 37°C for 4 hours, and then centrifuge (8000 rpm, 10 minutes). The supernatant was obtained as acid clay treated immunoglobulin. e CM-Sephadex ion-exchange chromatography Equilibrate CM-Sephadex with 0.02M phosphate buffer (PH7.2), prepare a column with a height of 3.6 cm and a length of 21 cm, and add 50 ml of acid clay-treated immunoglobulin.
was adsorbed onto the column, and unadsorbed protein was washed away with approximately 1000 ml of 0.02M phosphate buffer (PH7.2).
Then, it was eluted with 0.2M phosphate buffer (PH7.2). The flow rate of phosphate buffer is 80ml per hour.
The mixture was fractionated into 9 ml portions. 0.2M phosphate buffer (PH7.2) elution fraction
0.01M ethylenediamine acetate buffer (PH8.0)
After dialysing against the immunoglobulin, purified immunoglobulin was obtained by freeze-drying. Example 2 a Preparation of placenta extract 10 cryopreserved human placentas were cut into small pieces, and placenta 1
Physiological saline was added at a ratio of 1 part per kg, stirred for 60 minutes, and then centrifuged to separate the supernatant.
4.8 placental extracts were obtained. b Preparation of crude immunoglobulin (ammonium sulfate precipitation method) Ammonium sulfate per 1 placenta extract
After adding 130g (0.68M) and dissolving the mixture, allowing it to stand for 2 hours, it was centrifuged to obtain a supernatant. Furthermore, supernatant 1
136 g (1.40 M) of ammonium sulfate was added and dissolved, and after standing for 2 hours, the mixture was centrifuged to obtain a precipitate. After dissolving the precipitate in distilled water, ammonium sulfate was added and dissolved again at a ratio of 266 g per 1 solution, the pH was corrected to around 7.0, and the mixture was allowed to stand for 2 hours.
The resulting precipitate was collected by centrifugation. The precipitate obtained by repeating the ammonium sulfate precipitation method twice was dissolved in 0.02M phosphate buffer (PH7.2) and dialyzed against the same buffer to obtain crude immunoglobulin. c Chloroform/ethanol treatment Crude immunoglobulin was cooled to 4°C, and a mixture of chloroform and ethanol in a ratio of 7:3 was added to the crude immunoglobulin at a ratio of 5% (V/V) at 4°C. After shaking for a minute, centrifugation (8000 rpm, 30 minutes) was carried out, the solvent was removed from the obtained supernatant under reduced pressure, and 0.02M phosphate buffer (PH
7.2) to obtain chloroform/ethanol-treated immunoglobulin. d Treatment with adsorbent Add kaolin at a ratio of 4% (W/V) to the chloroform-ethanol treated immunoglobulin, mix and stir, adsorb at 37°C for 4 hours, and then centrifuge (8000 rpm, 10 minutes). The supernatant was obtained as kaolin-treated immunoglobulin. e CM-cellulose ion exchange chromatography CM-cellulose was dissolved in 0.02M phosphate buffer (PH
7.2), prepare a column with a height of 3.6 cm and a length of 21 cm, adsorb 50 ml of acid clay-treated immunoglobulin onto the column, and wash away unadsorbed protein with approximately 1000 ml of 0.02 M phosphate buffer (PH 7.2). , and then eluted with 0.2M phosphate buffer (PH7.2). The flow rate of the phosphate buffer solution was 80 ml per hour, and fractions were divided into 9 ml portions. 0.2M phosphate buffer (PH7.2) elution fraction
0.01M ethylenediamine acetate buffer (PH8.0)
After dialysis against 100 mg of white purified immunoglobulin, 1000 mg of white purified immunoglobulin was obtained by lyophilization.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram showing the elution pattern of column chromatography for purification of crude immunoglobulin treated with acid clay using a carboxymethyl-sephadex column in the immunoglobulin purification method of the present invention;
FIG. 2 is a diagram showing the electrophoresis images of placenta extract and immunoglobulin purified by the method of the present invention by Chiselius electrophoresis.

Claims (1)

[Scope of Claims] 1. A method for purifying immunoglobulin from crude immunoglobulin obtained from a human placenta extract, which involves treatment with a chloroform/ethanol mixture or chloroform, followed by treatment with an adsorbent, and subsequent treatment with cations. A method for purifying immunoglobulin, characterized by purifying it using an exchanger. 2. The method for purifying immunoglobulin according to claim 1, wherein the adsorbent is acid clay, activated clay, kaolin, talc, or activated carbon. 3. The method for purifying immunoglobulin according to claim 1 or 2, wherein the cation exchanger is a cation exchange resin, cation exchange cellulose, or cation exchange Cephadex.