JPS5843372B2 - IgA purification method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for purifying IgA (human serum immunoglobulin A) in high yield on an industrial scale.

IgA is an immunoglobulin that exists in the blood.
Like L, it has various antibody activities, plays an important role in preventing infection in vivo, and is widely used clinically for the prevention and treatment of inoculated diseases.

IgA is present in serum in large amounts next to IgG, and its concentration in normal human serum is 60 to 490 .mu./di.

For this reason, IgA is separated and purified from human serum, human plasma, or their byproducts.

There are already many reports on IgA purification methods, such as the first method, which uses ion exchange adsorption and immunoadsorbent adsorption, and the method of Litman et al. (Biochem, Biophys, Acta). , 26
3, 89.

1972) A second method, Pejaudier L., using caprylic acid precipitation and DEAE cellulose adsorption.
) et al.'s method (VoxSang, 23, 165, 1972
) The third method is the method of Schwick (H, G, Vo.
xSang, 23, 1972) There is a fourth method, a method using a zinc acetate precipitation method and a polyethylene glycol fractionation method (US Pat. No. 3,808,189).

The first method is effective for obtaining high-purity IgA on a laboratory scale, but is unsuitable for industrial production, and the other three methods all use human serum, human plasma, or these methods. Although it is suitable for producing IgA from by-products on an industrial scale, improvements are desired in terms of purity and yield.

The inventors have continued research on IgA production methods for many years and have completed a method for economically and efficiently purifying IgA on an industrial scale.

In the present invention, IgA is extracted from a paste of corn fraction (1) with distilled water, sodium sulfate is added to the extract under alkaline conditions to a saturation concentration of 14 to 15% W/V, and the resulting insoluble matter is removed. After adjusting the pH of the liquid to 5.5 to 6.5,
Ammonium sulfate was added to a saturation concentration of 9 to 12% W/V, the resulting precipitate was collected, the salt was removed from this precipitate, the pH was adjusted to 5.2 to 5.3, and the resulting precipitate was removed. It collects IgA.

The present invention will be described in detail. Human serum or human plasma may be used as the starting material for IgA, but active ingredients such as fibrinogen, albumin, or globulin can be fractionated from these and the remaining fraction, which was conventionally discarded, can be used as the starting material. It is cheap.

As a waste fraction of the plasma fraction, a paste of Cohn's fraction (J, Am,
chem, Soc, 68.

459.1946) is advantageous in that it contains a large amount of IgA.

However, this paste contains large amounts of lipoproteins, macroglobulins, certain denatured proteins, etc., which makes it difficult to extract and purify IgA, especially on an industrial scale.

In order to remove lipoproteins, macroglobulins, etc. from the corn fraction (■), there is a method of extracting IgA and then removing it with zinc acetate (as described in the above US patent specification), but the simplest and most reliable method is to extract only IgA in the first step. The purpose is to specifically extract the

The quality of the IgA extraction effect can be determined by using the one-way immunodiffusion method.
This is done by quantifying A, and whether or not undesirable substances such as denatured proteins and lipoproteins have been removed can be easily determined by visually observing whether the resulting extract is clear or cloudy. can be distinguished.

100 g of paste of Cohn fraction ① was suspended in a physiological condition solvent or distilled water, centrifuged to remove insoluble matter, and the intensity of cloudiness of the resulting supernatant was observed to determine the amount of IgA extracted. When the relationships were summarized, the results shown in Table 1 were obtained.

The intensity of the cloudiness was arbitrarily divided into five levels from +5 to +1, and the smaller the value, the closer to clearness it was.

As a result, IgA can be extracted well using IJum solution, glycine solution, and phosphate buffer, but the resulting supernatant is cloudy, and when extracted with distilled water, the amount of IgA extracted is However, since a clear supernatant can be obtained, it is easy to carry out the purification treatment in the subsequent steps.

Cohn fraction ■ contains a large amount of IgA, but it also contains a large amount of ■ml, which is the same as the proportion in blood, and approximately three times as much ■ml as IgA is mixed into the extract. ing.

Therefore, without losing IgA from this extract, Ig
Removal of G becomes necessary to increase the purity of IgA.

As a method to separate IgG mixed with IgA,
Peziaudale (mentioned above) uses a DEAE-cellulose adsorption method, but this method requires preliminary operations such as pre-washing and equilibration of the DEAE-cellulose, so it is too complicated to carry out on an industrial scale and is not preferred.

The inventors selected the salting-out method as a simple method suitable for industrial scale, and after studying various salting-out agents, found that sodium sulfate could most effectively remove ml.

Next, an experiment to find the optimal conditions will be explained in detail.

The most commonly used salting-out agent is ammonium sulfate, and this and the sodium sulfate used in the present invention are added to the IgA extract for salting out, and the resulting insoluble matter is removed and remains in the supernatant. The amount of ml and the amount of IgA were quantified by one-way immunodiffusion.

The results are summarized in Table 2. Figure 1 is a graphical representation of the results in Table 2, with the amount of salting out agent added on the horizontal axis and the remaining amount of IgA and ■ml on the vertical axis.The solid line represents the remaining amount of IgA when sodium sulfate is added. The solid line B connects the remaining amount of ■ml, and the dotted lines C and D connect the remaining amount of IgA when ammonium sulfate is added and the remaining amount of ■ml, respectively.

As can be seen from Figure 1, adding 14 qb of sodium sulfate salts out only about 20 qb of IgA, but about 6 qb of IgG.
Part 2 can also be removed by salting out, and if the amount of sodium sulfate added is increased to 15 part, approximately 90 ml of Ig will be removed.
The abundance of A becomes relatively dominant.

On the other hand, as the amount of ammonium sulfate added increases,
ml is salted out, but IgA is also salted out in parallel, ■m
It can be seen that there is no specificity as a salting-out agent for 1.

The above results ■ Sodium sulfate 14-1 as a salting-out agent for ρ
It has been found that it is most effective to add at a saturation concentration of 5W/■.

Next, the inventors added sodium sulfate to the IgA extract to a saturation concentration of 15 to investigate the pH conditions during salting out with sodium sulfate. The weight was determined.

In Figure 2, the horizontal axis shows pH, and the vertical axis shows IgA and the residual amount (rru?/di!) of 1 ml.
The remaining amount of IgA at 6,7°9.10 was connected with a solid line E, and the remaining amount of ■ml was connected with a dotted line F.

As can be seen from Fig. 2, on the acidic side, the target IgA is salted out together with IgG, and on the neutral side, IgA is salted out at the same time.
is less likely to be salted out, but it is not an effective condition as the removal of ■ml is small, and when it becomes alkaline, ■ml is specifically salted out, so it can be seen that the pH at the time of salting out is better on the alkaline side.

Even though ammonium sulfate specifically salts out IgG on the alkaline side, it is not practical because it decomposes on the alkaline side to generate ammonia gas.

Although not shown in FIG. 1, salting out with sodium chloride was also ineffective, as was ammonium sulfate.

As a result of the above, IJum (sulfuric acid) is specific as a salting-out agent, and the pH during salting-out is preferably on the alkaline side, with a pH of 8.
We found that ~10 was most effective.

The pH of the supernatant enriched with IgA is returned to neutral, and the IgA
Ammonium sulfate is added in order to both concentrate and remove coexisting albumin, etc.

As a result of the inventors' research, the most favorable conditions in this case are as follows:
At pH 5.5 to 6.5, the ammonium sulfate saturation concentration was 9 to 12.

In this step, almost all IgA is precipitated, and albumin, α2 globulin, etc. remain in the supernatant, improving the purity of IgA.

The IgA precipitate is collected by centrifugation or filtration, suspended in cold water, and dialyzed against cold water at 4°C for 16 hours.

The purity of IgA in the dialysate obtained here is between 40 and 50, and it can be used as a medicine as it is, but
It lacks long-term stability because it still contains macroglobulin, lipoprotein, etc.

Macroglobulin and lipoprotein that are mixed in this process have a considerably different molecular weight from IgA, so these impurities can be separated in the laboratory by gel filtration, but gel filtration is required to perform it on an industrial scale. However, the disadvantage is that the processing capacity for one time is small and it takes a long time.

The inventors continued their research on removing impurities and simply changed the pH.
We have found conditions that allow contaminants to be removed in a short time and on an industrial scale simply by adjusting the

Add a small amount of IN (hydrochloric acid or hydroxylated) to the IgA fraction subjected to cold water dialysis to change the pH, let it stand for about 30 minutes, centrifuge to obtain a supernatant, and determine the amount of IgA and protein. The purity of IgA was calculated from the amount and the relationship with the width was determined.

Figure 3 shows the pH on the horizontal axis and the purity ring on the vertical axis.
Simply adjusting the pH to 5.3 significantly increases the purity of IgA, reaching a purity of 75 at pH 5.2.

By adding sodium chloride or glycine to the IgA concentrate obtained in this way and performing sterilization filtration, an IgA solution that can be safely used as a medicine can be produced on an industrial scale.
IgA powder is produced by freeze-drying the gA solution.

The yield, purity, and stability of IgA are compared between the present invention and conventional methods (the second to fourth methods), and the results are summarized in Table 3 to clarify the superiority of the present invention.

The yield of IgA according to the present invention is higher than that of the second method, the purity is clearly higher than that of the third and fourth methods, and the long-term stability is also very good.

In addition to the excellent yield, purity, and stability, the present invention is easy to process, making it suitable for industrial scale and extremely useful as an industrial purification method for IgA.

Example 9 Add cold distilled water 5001 to paste 50 of fraction 1 obtained from normal human plasma by Kohn's cold alcohol fractionation method, and make a uniform suspension using a mixer.

Next, insoluble matter in the suspension is removed using a Sharpless centrifuge to obtain a clear extract 5001.

A sodium hydroxide solution of IN was added to this extract, and p
After adjusting H to 9.0, add anhydrous sodium sulfate to 14
．． In addition to the saturation concentration of 54W/V, maintain the liquid temperature at around 20°C.

The precipitate was removed using a Sharpless centrifuge or an R-filter, and the clear supernatant was collected and its pH was adjusted with IN hydrochloric acid.
After adjusting to 6.0, ammonium sulfate was added to 76g/
Add to the ratio of l.

Most of the IgA is salted out in this process, so remove the supernatant using a Sharpless centrifuge or a strainer, dissolve the collected precipitate in cold distilled water, and dissolve the IgA in cold distilled water overnight. Perform dialysis.

The purity of IgA at this stage was 41.

Next, 0.5N hydrochloric acid or 0.5N sodium hydroxide was added to this dialysate to adjust the pH to 5.3±0.05, and precipitated impure proteins were removed using a Sharpless centrifuge. , obtain a clear IgA solution.

Glycine is added to this IgA solution at a ratio of 10 parts per protein and sterilized by filtration to obtain a sterile IgA solution.

The purity of IgA is 76 parts, and the yield is 4801 rLl as a 10 parts solution.

[Brief explanation of the drawing]

Figure 1 shows the concentration of salting-out agent and IgA and IgG in the extract.
Figure 2 is a diagram showing the relationship between the remaining amount of IgA and IgG, and Figure 3 is a diagram showing the relationship between the pH of a saturated solution of IJum 15 and the remaining amount of IgA and IgG. FIG. 2 is a diagram showing the relationship between purity.

Claims (1)

[Claims] 1 Extract IgA from the paste of Cohn fraction ■ with distilled water, add sulfuric acid to the extract under alkaline conditions) Add IgA to a saturation concentration of 14 to 154 W/V, remove the resulting insoluble matter, and remove the supernatant. After adjusting the pH to 5.5 to 6.5,
Ammonium sulfate was added to a W/V saturation concentration of 9 to 12, the precipitate was collected, the salt was removed from the precipitate, the pH was adjusted to 5.2 to 5.3, the resulting precipitate was removed, and IgA
1. A method for purifying IgA, which comprises recovering.