JPS59118718A - Recovery of igm - Google Patents

Abstract

PURPOSE:To prepare IgM easily, in high yield, by separating albumin and IgG fraction from blood plasma, extracting IgM from the above residue, adding polyethylene glycol to the extracted liquid, and recovering the produced precipitate. CONSTITUTION:Albumin and IgG fraction are separated from blood plasma by Cohn's ethanol fractionation and acrinol fractionation. The obtained residue is dispersed in a buffer solution such as acetic acid buffer solution having 4- 7 pH, or water, physiological saline water, etc., stirred, and separated into solid and liquid to extract IgM. The extracted liquid is adjusted to the protein concentration of 1-5% and pH of 4.3-6, preferably 4.5-5, mixed with polyethylene glycol, stirred at 10-20 deg.C, and left to stand for about a day, and the produced precipitate is recovered. The obtained precipitate is treated with dextran sulfate and calcium chloride to obtain IgM free from foreign materials.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for efficiently recovering IgM, which is a type of human immunoglobulin.

Albumin and IgG are produced on an industrial scale using human plasma as a raw material, but IgM is not mass-produced because the content of IgM in plasma is small.

The present inventors have developed a new method for easily obtaining IgM at a high yield from the residue of albumin and IgG fractions separated from plasma, which had previously been discarded.

Conventionally, several methods for obtaining IgM have been reported.
For those using plasma as a starting material, for example, the method of treating Cohn's fraction
There are several methods, including separation using ion exchange chromatography (No. 6), but all of them involve long purification steps, which pose problems in terms of both operation and yield.

As a result of intensive studies to develop a method to obtain IgM at low cost and easily, the present inventors discovered that albumin and IgM can be obtained from plasma.
It was discovered that polyethylene glycol has an extremely large purifying effect on the residue obtained by separating the gG fraction, and based on this finding, the present invention was completed.

That is, the present invention relates to a method for recovering IgM, which is characterized by adding polyethylene glycol to an extract obtained by extracting IgM from a residue obtained by separating albumin and IgG fractions from plasma, and collecting the generated precipitate.

The method for separating albumin and IgG fractions from plasma is: +
-7(7) x Tanol fractionation method, acrinol fractionation method,
There are many types of buildings, but the type does not matter. This couple-
It is not necessarily the case that the residue after separating the fractions must contain IgM.

Examples of such residues include fractions ① and IV-1 in the case of Cohn's ethanol fractionation method, and precipitate ③ in the case of the acrinol fractionation method.

IgM can be extracted from the residue by following a known method, for example, by suspending the residue in a buffer such as acetate buffer or phosphate buffer with a pH of about 4 to 7, or in water, physiological saline, etc. After stirring at 5° C. for about 4 hours, solid-liquid separation may be performed.

The extract has a protein concentration of 1 to 5% and a voice of 43 to 5%.
After adjusting the temperature to about 6, preferably about 45 to 50, add polyethylene glycol.

This pH adjustment may be performed after adding polyethylene glycol. It goes without saying that prior to adding polyethylene glycol, other pretreatments such as rinsing may be performed as necessary.

Polyethylene glycol has a molecular weight of 2000-1000
A value of about 0 is good, and a value of about 6000 is particularly preferable. The amount added is 3 if the molecular weight is 6000.
~10%, particularly approximately 4 to 7φ is appropriate. When using substances with other molecular weights, tests may be conducted in advance to determine the appropriate amount to be added.

After adding polyethylene glycol, the mixture is stirred and mixed at 10 to 20°C, and then left to stand for about day and night to collect the formed precipitate. The recovery method may be any conventional solid-liquid separation method, such as centrifugation.

The resulting precipitate still contains large amounts of lipoproteins, denatured proteins, and certain unstable substances, or these impurities can be easily removed by treating the precipitate with dextran sulfate and calcium chloride. can be removed.

The treatment method is to first dissolve the precipitate to a protein concentration of about 2 to 25%, adjust the pH of this solution to about 5 to 8, and adjust the pH to 0.00.
Dextran sulfate of about 1-003% and 0.01-0
．． Add about 1M calcium chloride. after that,
The precipitate may be removed by stirring and centrifuging for about 30 minutes.

The method of the present invention allows IgM to be obtained easily and in high yield from residues that were conventionally discarded, and requires only one treatment with polyethylene glycol, followed by treatment with, for example, dextran sulfate and calcium chloride. High purity IgM can be recovered at an unprecedentedly high yield.

Next, the amount of addition of PH and polyethylene glycol, and the amount of I
Measurement of improvement in gM recovery and purity 1. The results of the reconnaissance experiment are shown below.

The experimental method involved adding 25ml of a paste of Cohn's fraction (1) that had been frozen and stored at -20°C to 75m7 of a 0.1M sodium acetate buffer solution with a pH of 4.0. was added, stirred for 5 hours, and centrifuged to obtain a 1 gM extract. This extract was adjusted so that the protein concentration was 18 and the pH was as shown in the table below, and polyethylene glycol (PEG) 6000 was added to the solution so that the concentration was as shown in the table below.

After stirring for 2 hours at 10-20°C, the mixture was left to stand day and night, centrifuged, and the supernatant liquid was collected and analyzed, and the results shown in the table below were obtained.

The upper row of each number in the table represents (total protein amount of supernatant solution/total protein amount of stock solution) x 100, and the lower row represents (IgM amount of supernatant solution/IgM amount of stock solution) x 1o.

It represents. As is clear from the table, P14 is 43~
About 6, especially about 45 to 50 is good, PEG 6000
The appropriate concentration was about 3 to 10 parts, especially about 4 to 7 parts.

Example 1 Cohn fraction stored frozen at 20°C ■1000.
9 to 40 O, ], M sodium acetate buffer 3, β
was added and stirred at 5°C for 4 hours. This is 100 cells. ! 7 was added and filtered to obtain an extract. The extract has a pH of 4.5-0. IM sodium acetate buffer was added to bring the protein concentration to 18%, and 10 NNa
The pH was adjusted to 45 with OH. To this, polyethylene glycol 6000 was added at a concentration of 5 w/v %, stirred at 15° C. for 2 hours, and allowed to stand overnight. The precipitate was collected by centrifugation, and the precipitate was dissolved in 0.1 M phosphate buffer (pH 6.5). Add 0.0% of carunium chloride to this solution. Add dextran sulfate to 0.0 OIM and dextran sulfate to 1% OIM, and incubate at room temperature for 30 minutes.
Stirred for 0 minutes. The resulting precipitate was centrifuged and weighed 1 g.
Obtain M solution.

This 1 gM solution contained 28 g of IgM, and the yield from the raw material was 65%. Also, IgM
The protein purity of the protein was 85%, and it was confirmed by radioimmunodiffusion that it contained 75% of IgA and macroglobulin 1.

Patent applicant: Fuji Organ Pharmaceutical Co., Ltd.

Claims (1)

[Claims] Ig characterized in that polyethylene glycol is added to an extract obtained by extracting IgM from a residue obtained by separating albumin and IgiG fractions from plasma, and the resulting precipitate is collected.
How to collect M.