JPH02104533A - Production of high-purity bovine immunoglobulin - Google Patents

Abstract

PURPOSE:To produce high-purity bovine immunoglobulin with simple operation by desalting whey or concentrated whey protein and contacting with a cation exchange material within a specific pH range. CONSTITUTION:Whey or a concentrated whey protein having a protein concentration of >=0.5wt.% is desalted to an electrical conductivity of <=2mS/cm. The desalted liquid is adjusted to pH 4.5-5.5 and made to contact with a cation exchange material (preferably alkali metal-type) at 0-40 deg.C to effect the adsorption of the immunoglobulin. The ion exchange material is recovered, washed with water adjusted to pH 4.5-5.5 to completely wash off the unadsorbed whey protein and eluted with a salt solution to obtain a high-purity immunoglobulin containing >=55% of immunoglobulin based on the protein. The whey or the concentrated whey protein left after the recovery of the immunoglobulin can be utilized as an acid whey as it is or as a concentrated acid whey protein.

Description

[Detailed description of the invention] [Field of industrial use] The present invention relates to a method for producing highly purified immunoglobulin.

[Previous days and prior art] Immune globulin in cow's milk, especially immunoglobulin found in colostrum, is a very important substance that confers immunity to calves, and on the other hand, it has been shown to have specificity. It was also known as having However, according to recent research, l
Since it was discovered that this effect is effective not only in calves but also in humans, there has been a strong demand for the establishment of this industrial recovery method, and various research and development efforts have been made. It is being done.

Traditional methods for obtaining immunoglobulins from milk or colostrum are very cumbersome. Of course, the most common method is to first desorb it by centrifugation, then lower the pH to precipitate and separate it, and remove it.
H was recovered and centrifuged again. In this method, the precipitate is removed, then vA'Pa ammonium is added to precipitate the immunoglobulin, the resulting precipitate is dissolved in water, and the ammonium sulfate is removed by dialysis to obtain the immunoglobulin.

European Patent Application No. 85005 describes a method for obtaining immunoglobulins from colostrum. In this method, colostrum or whey is first subjected to electrophoresis to collect both immunoglobulin-enriched fractions, and then fractionated by chromatography on an ion exchanger to obtain an immunoglobulin fraction.

Furthermore, in JP-A No. 61-68429, milk or colostrum is acidified, casein is precipitated and filtered, low-molecular substances are removed from the obtained liquid by ultrafiltration, and immunoglobulins are obtained by [r11 yield]. A method is disclosed.

[Problems to be Solved by the Invention] Among the conventional techniques mentioned above, the general method of No. 1M has a complicated process, and since Oma's ammonium sulfate is added to the milk, it is necessary to remove the immunoglobulin from the milk. is difficult to use and is not suitable as an industrial method. In the second method, it is necessary to first perform electrophoresis, which requires special equipment and is not suitable for industrial human processing. The third method is basically a whey protein recovery method, and is a special method that uses colostrum with a high immunoglobulin content as a raw material to obtain a product with a high immunoglobulin content. However, it cannot be called an industrial method due to the availability of raw materials.

[Object of the Invention and Summary of the Invention] Therefore, the object of the present invention is to provide a novel industrial production method for high-purity bovine immunoglobin purine that improves the drawbacks of the above-mentioned prior art regarding the separation and purification of immunoglobin purine from milk. Our goal is to provide the following.

The present invention uses whey or whey protein concentrate, which is commercially available in large quantities, to adsorb immunoglobin to a cation exchanger, and obtains highly pure bovine immunoglobulin through a simple desorption operation. This achieves the objective L above.

That is, the present invention provides a method for producing high-purity tl immunoglobulin [1 purine] by recovering immunoglobulin from whey or whey protein concentrate from which casein and fat have been removed from milk. %w/w or more, desalinate so that the electrical conductivity is 2 mS/ctn or less,
The desalted solution is adjusted to pH 4.5 to 5□5 and brought into contact with a cation exchanger at a temperature of 0 to 40°C to adsorb immunoglobulin, and then the ion exchanger is collected, I) 84. 5
A method for producing high-purity bovine immunoglobulin purine containing at least 55% w/w of immunoglobulin in protein, which is characterized by washing with water adjusted to ~5.5 and then desorption with a salt solution. It is.

[Detailed description of the present invention 1 Hereinafter, the technical configuration of the present invention will be explained in detail.

As a raw material, use is made of fermented whey or acid whey obtained by removing casein and fat from milk in which immunoglobulins have not been deactivated, or a whey protein concentrate obtained by ultrafiltration of these. Normally, 100 d of milk contains immunoglobulin at a temperature of about 60°C, and accounts for about 10% w/w of whey protein.

Using a whey protein-containing solution containing 0.5% w/w or more of these whey proteins, remove the whey proteins by ion exchange m'ig gas dialysis method, dialysis, etc. as necessary so that the electrical conductivity is 218/cIR or less. Salt. Then, the desalted solution was adjusted to pH 4.
, adjust to 5 to 5.5. Hydrochloric acid for pH adjustment.

Any acid agent such as lactic acid or citric acid can be used. When the electrical conductivity exceeds 2 mS/Cl11, or when I)H is lower than 4.5 or higher than 5.5, the immunoglobulin concentration in the obtained protein becomes low.

The DI preparation solution is then contacted with a cation exchanger to adsorb immunoglobulin. Any form of counterion can be used as the counterion of the cation exchanger, but preferably an alkali metal form is used. I) Regarding the method of contacting the H adjustment solution with the ion exchanger, an appropriate method such as a batch stirring method or a continuous column method can be adopted, taking into consideration the liquid passing characteristics of the ion exchanger used. The mixing volume ratio of the pl+ adjustment liquid and the ion exchanger is determined according to the adsorption capacity of the ion exchanger, and a sufficient amount of the pH adjustment liquid is brought into contact with the mixture. The temperature during contact between the pH adjusting solution and the ion exchanger should be between 0 and 4°C, as immunoglobulins tend to gradually denature at temperatures exceeding 40°C.
It is necessary to set the temperature to 0°C. The contact time between the pH adjustment solution and the ion exchanger is determined by taking into consideration the temperature during contact, the contact method to be employed (batch method or continuous column method), etc.

Next, the ion exchanger that has been brought into contact with the DI + adjustment solution is separated, washed with water adjusted to pH 4.5 to 5.5, and after thoroughly washing off unadsorbed whey protein particles, according to a conventional method, The immunoglobulin adsorbed to the ion exchanger is desorbed using a salt solution such as common salt with high ionic strength or a salt solution with increased pH. The pH of the washing water is 4.5-5.
This is to prevent the adsorbed immunoglobulin from being desorbed and lost during washing.

Under the conditions of the present invention, immunoglobulins are selectively adsorbed from whey proteins, so that a sludge containing at least 55% w/w of immunoglobulins in the proteins of the desorbed salt solution can be obtained.

Hereinafter, the immunoglobulin recovery solution obtained as in step F consists of protein, salts, and water, and removal of salts and water can be carried out according to a conventional method. That is, after salts are removed to a desired extent by ultrafiltration, electrodialysis, or dialysis, water can be removed by freeze-drying, spray-drying, etc. to easily form a powder. .

[Example] Example 1 Indeion 3 having an ion exchange group of sulfopropyl group (Phoenix Chemicals! 11)
Fill the column with 500mff1, add 22% of 10% saline
After passing through L, the solution was washed with water to prepare a Na-type ion exchanger. Cheese whey was concentrated by ultrafiltration and then diluted to give a protein concentration of 0.6% w/w t-1.5n after electrical conduction.
+S/Cm, and then the pH was adjusted to 5.0 with hydrochloric acid. Next, the previously adjusted Ha type ion exchanger 500- was added to the pH-adjusted 15Ql and stirred at a temperature of 4°C for 3 hours to adsorb immunoglobulin. After a predetermined time, the ion exchanger was collected by filtration, and then the pH was adjusted to 5.0.
It was washed with 1M acetic acid-sodium acetate buffer and packed into a column. Next, the adsorbed components were desorbed by passing 5% w/w saline solution thereinto to obtain 9 in the recovery liquid 2.0.

The recovered solution had a protein concentration of 0.90% w/w and the following protein composition.

Albumin 2.0% α-globulin 15% β-globulin 2.5% Immunoglobulin 78% (of which 1i) G approximately 85%) Ii) M approximately 10% Others 2,5x Example 2 Ion exchange of carboxymethyl group 31I! CH having a group
-t' Pearl 650G (manufactured by Toyo Soda) 500d
was packed into a column with an inner diameter of 10α, passed through a saline solution, and then washed with water to form the Na form. , hydrochloric acid casein whey was desalted by ion exchange GL electrodialysis method, and the protein m degree was 0.55% w/
Electrical conductivity g 1.213/ca+ at w, fill 4
．． A liquid No. 7 was obtained.

Next, the desalted solution was passed through the ion exchange column at a temperature of 4° C. and a flow rate of 41/h for 10 hours. After fluid passage, p114
．． 71. :El set, ri0.0IH7'ji acid-acetic acid t
5% w
/w of saline solution to remove the adsorbed components, and collect the recovered liquid 2.
Obtained 0Ky.

The recovered solution had a protein concentration of 1.05% w/w, and the immunoglobulin purity in the protein was 65%.

[Effect of the Invention 1 According to the present invention, the following effects can be obtained, and the present invention is excellent in industrial effects.

(1) Using whey or whey protein concentrate that is commercially available in large quantities, the immunoglobulin contained therein can be effectively separated and recovered.

(2) Highly purified immunoglobulin can be obtained through a simple process.

(3) Whey or BoI-protein condensate after immunoglobulin recovery can be reused as it is as acid whey or as acid whey protein concentrate.

Claims (1)

[Claims] (1) In a method for producing high-purity bovine immunoglobulin by recovering immunoglobulin from whey or whey protein concentrate from which casein and fat have been removed from milk, whey containing a protein concentration of 0.5% w/w or more is used. was desalted to have an electrical conductivity of 2 mS/cm or less, and the desalted solution was adjusted to pH 4.
．． 5 to 5.5 and brought into contact with a cation exchanger at a temperature of 0 to 40°C to adsorb immunoglobulin, then the ion exchanger was collected, and water adjusted to pH 4.5 to 5.5 was used. 1. A method for producing n-purity immunoglobulin containing 55% w/w or more of immunoglobulin in protein, which comprises washing with water and then removing it with a salt solution.