JPS6241210B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to molecular sieve chromatography,
The present invention relates to a method for separating plasma proteins by using a hydrophilic carrier and passing a solution containing plasma proteins through an eluent consisting of a buffer solution and a neutral salt solution.

More specifically, the present invention relates to a method of separating plasma proteins by molecular sieve chromatography in an eluent by utilizing the properties of plasma proteins as polymer electrolytes.

Blood consists of a formed component consisting of red blood cells, white blood cells, and platelets, and a non-formed component called plasma. The main components of plasma are water and protein, and also contains sugars, lipids, enzymes, hormones, electrolytes, etc.

Plasma proteins electrophoretically contain albumin, α,
It consists of β and γ-globulin and fibrinogen, and α, β and γ-globulin are aggregates of several types of proteins.

Conventional methods for separating these plasma proteins include (1) fractional precipitation by adding neutral salts such as ammonium sulfate, organic solvents such as cold ethanol, and water-soluble polymers such as PEG; (2) ion exchange, adsorption, molecular sieves, etc. Chromatography (3), electrophoresis (4), which utilizes differences in charge, and immunoadsorption, which utilize differences in molecular properties such as solubility, charge, and molecular size, are known. , one or a combination of these methods may be used. Some of these methods have drawbacks such as destruction of the higher-order structure of plasma protein molecules or formation of aggregates due to room temperature operation. In addition, many methods have unfavorable protein recovery rates or require complicated operations. On the other hand, mixtures with different molecular sizes based on molecular weight and molecular spread,
By passing through a column that uses dextran gel as a carrier, which has a three-dimensionally cross-linked network structure, molecules larger than the network cannot enter the gel and are eluted as they are, while smaller molecules enter the network. , according to the size and shape of the molecule,
The so-called molecular sieve effect, in which particles are eluted in order from the largest to the largest, is widely used and is called molecular sieve chromatography (also called gel permeation, gel permeation chromatography, etc.). This molecular sieve chromatography is simple and effective for separating physically and chemically weak substances such as plasma proteins because it can be operated under mild conditions. However, this method requires a long time for separation and is less effective than other separation methods.
It is not suitable as a large-scale protein separation method and has not been widely used.

As a result of intensive research to improve these shortcomings, the present inventors have found that protein components in plasma can be quickly, easily, and efficiently fractionated, and at the same time,
Dimers and 3-dimers of each protein contained in albumin and γ-globulin preparations manufactured by the Cohn method etc.
The present invention has been completed by discovering a method that can effectively remove and separate aggregate-contaminated proteins such as polymers.

That is, the present invention uses a specific hydrophilic carrier instead of a conventionally used carrier in molecular sieve chromatography, and allows a solution containing plasma proteins to pass through an eluent consisting of a buffer solution and a neutral salt solution. , provides a method for quickly, simply and efficiently separating and fractionating plasma proteins.

The present invention will be explained in detail below.

The hydrophilic carrier used in the present invention is one molecule of a silane binder per silanol group of silica gel described in Japanese Patent Application No. 52-34742 previously filed by Toyo Soda Kogyo Co., Ltd., one of the applicants of the present invention. It has a fully porous structure in which hydrophilic groups are chemically bonded to the bonded carrier, that is, silica gel, and has a high pressure resistance.
It has a mechanical strength of 300Kg/ ^cm2 or less and a particle size of 5
A hydrophilic carrier having a spherical shape of ~100μ is used.

Then, by passing a solution containing plasma proteins through the eluent of the column packed with the hydrophilic carrier,
It separates and fractionates plasma proteins without precipitation or coagulation.

In the present invention, PH4 to 9, preferably PH5
~7 and ionic strength 0.01~1.0, preferably 0.05~
An eluent consisting of a 0.5% buffer solution and a neutral salt solution is used.

The buffer solution can be arbitrarily selected from a buffer solution exhibiting a pH range of 4 to 9, but Tris buffer, phosphate buffer, and acetate buffer are particularly preferred, and the neutral salt solution is a commonly used neutral salt solution. Although it can be arbitrarily selected from among the above, aqueous solutions of NaCl and KCl are particularly preferred.

Next, in the present invention, the state of the plasma protein solution passed through the eluent will be explained.

Fibrinogen is one of the plasma proteins. When blood leaves the body, it clots, and this is caused by fibrinogen turning into fibrin and forming blood clots. Therefore, also in chromatography, fibrin is precipitated and attached to the carrier.

In order to prevent this phenomenon, it is preferable to perform pretreatment to remove fibrin, and in some cases, serum, which is plasma after fibrinogen has been removed, can be used as it is.

In addition, a state in which a water-soluble polymer or a neutral salt is coexisting, fibrinogen is removed in advance, and the molecular form of the protein is changed due to interaction between the water-soluble polymer or neutral salt remaining in the aqueous solution and plasma protein, Alternatively, in order to further enhance the separation effect, one of the aforementioned separation methods can be used as a pretreatment means for the plasma sample.

Furthermore, in order to separate specific protein components in plasma, pretreatment of concentration using water-soluble polymers, neutral salts, or alcohols is performed once or twice, and water-soluble polymers and It can be used even if neutral salts or alcohols remain.

Therefore, the solution containing plasma proteins in the present invention is an aqueous solution, an aqueous solution containing a water-soluble polymer, a neutral salt, or an alcohol.

Water-soluble polymers include polyethylene glycol,
polyvinyl alcohol, polyvinylpyrrolidone,
Examples include polyacrylic acid, polymethacrylic acid, carboxymethyl cellulose, hydroxyethyl cellulose, methyl cellulose, gelatin, and starches, with polyethylene glycol, hydroxyethyl cellulose, and hydroxyethyl starch being particularly preferred.

In addition, neutral salts include NaCl, KCl, NH ₄ Cl,
Mention may be made of Na ₂ SO ₄ , K ₂ SO ₄ and (NH ₄ ) ₂ SO ₄ . Examples of alcohols include alcohols having 2 to 5 carbon atoms.

Molecular sieve chromatography is performed using the eluent and plasma protein-containing solution as described above, and the hydrophilic carrier is stainless steel.
It is used packed in various types of columns, including those packed into columns of 0.75 (φ) x 60 cm (length), but when collecting a large amount of samples, 20 (φ)
Columns with a large diameter (φ) and length of ×60 cm (length) can also be used. In general, a hydrophilic carrier manufactured by Toyo Soda Kogyo Co., Ltd. (product name: TSK
- Using one or a combination of columns packed with GEL2000SW, 3000SW, 4000SW) provides good protein recovery and separation efficiency. In some cases, it is also possible to increase the fractionation efficiency by using a combination of several columns packed with gels with different exclusion molecular weights.

The flow rate of the eluent can also be varied, and for example, the pressure of the liquid pump can be adjusted to any flow rate between 0.1 and 500 ml/min. In carrying out the present invention, before adding a sample into the column, it is necessary to wash and equilibrate the gel by passing a buffer solution in an amount several times the volume of the carrier. Since water-soluble substances in the sample can clog the liquid delivery system and carrier, they are preferably removed in advance by passing through a filter with a pore size of 0.2 to 10μ.

Further, in order to eliminate air dissolved in the eluent, it is preferable to perform depressurization or ultrasonic operation.

As explained above, the present invention uses a hydrophilic carrier to pass a solution containing plasma proteins through an eluent consisting of a buffer solution and a neutral salt solution, and detects the eluate to determine the protein concentration. The composition groups of a sample can be quickly, easily, and efficiently separated and fractionated.

Hereinafter, the present invention will be explained in detail with reference to Examples and Comparative Examples.

Example 1 Serum was filtered using a filter having a pore size of 0.2 μ, and 30 μl of the serum was filtered using a hydrophilic carrier [trade name manufactured by Toyo Soda Kogyo Co., Ltd.] using a HLC-802 high performance liquid chromatograph manufactured by Toyo Soda Kogyo Co., Ltd.
TSK-GEL3000SW (7.5mmID x 60cm x 2)] was added to the column packed with pressure 90Kg/cm ² , 1
0.02M phosphate buffer PH6.0 (+
Elute with 0.15M NaCl, ionic strength 0.17).
As a result, the eluate could be separated into fractions based on five consecutive peaks and one separate peak, as shown in FIG. 1, by ultraviolet absorbance measurement at a wavelength of 280 nm. Cellulose acetate electrophoresis and agar immunoelectrophoresis revealed five peaks in the order of elution: the first peak was α ₁ - and α ₂ -globulin, the second peak was α 1 -globulin, the third peak was β-globulin, and the third peak was α ₁ -globulin. The 4th peak was confirmed to be γ-globulin, and the 5th peak was confirmed to be albumin and β-globulin. The required separation time was within 50 minutes.

Example 2 The eluent used in Example 1 was mixed with 0.05M Tris-HCl buffer PH7.8 (+0.15M NaCl, ionic strength
The same operations and measurements as in Example 1 were carried out except that 0.2) was changed. As a result, it was confirmed that the sample was divided into the same number of peaks as in Example 1, and almost the same separation effect was observed.

Example 3 0.05M acetate buffer PH5.0 (+0.25M
Serum was separated using the same procedure as in Example 1 using NaCl (ionic strength: 0.3). Although the sample was separated into the same number of peaks as in Example 1, it was confirmed that the separation effect between the fourth and fifth peaks was superior to that in Example 1, as shown in FIG.

Example 4 After serum is centrifuged at low temperature to remove formed components, the plasma portion is separated. The pH of this plasma was adjusted to 8.0 with 1N sodium hydroxide, and 50% was added while cooling and stirring.
Add polyethylene glycol 4000 solution to give a final concentration of polyethylene glycol of 12%. Centrifuge at low temperature, collect the supernatant, and adjust the pH to 4.6 with 1N hydrochloric acid.
Add polyethylene glycol to the polyethylene glycol while stirring and cool.
Add 4,000 strips to give a final concentration of 25% and collect the precipitate after centrifugation at low temperature. Dissolve the precipitate in distilled water and adjust the pH
Set it to 7.0. Filter it using a filter, add 500ml into a column packed with the same carrier as in Example 1, and add 0.02M phosphate buffer in the same manner as in Example 1.
Elution was performed at PH7.0 (+0.25M NaCl ionic strength 0.4). A fixed amount of the eluate was collected using a fraction collector, and each fraction was analyzed by cellulose acetate electrophoresis and agar immunophoresis. It was confirmed that the largest peak was albumin contaminated with only a very small amount of β-globulin.

Example 5 Fractions that precipitate at a final concentration of 12% according to the polyethylene glycol precipitation method of Example 4 are collected by centrifugation and dissolved in 0.02M phosphate buffer pH 7.0.
Add 50% polyethylene glycol 4000 solution to this solution with stirring to give a final concentration of polyethylene glycol of 40%, remove the fibrinogen-rich precipitate formed by centrifugation, and add polyethylene glycol particles to the supernatant with stirring. Add to make a final concentration of 12%. The resulting precipitate is collected by centrifugation and dissolved in 0.8-0.9% physiological saline to a protein concentration of approximately 5%.

Add it to a column packed with the same carrier as in Example 1, and add 0.05M acetate buffer in the same manner as in Example 1.
Elution was performed at PH5.0 (+0.25M NaCl, ionic strength 0.3). Ultraviolet absorbance measurement at a wavelength of 280 nm revealed albumin contamination on the shoulder of the main peak γ-globulin fraction, as well as a small amount of contaminant protein.

These could be removed by fractionating only the main peak.

Example 6 Saturated ammonium sulfate is added to the plasma at room temperature while stirring to a final concentration of 0.20 saturation. The resulting precipitate is centrifuged, the supernatant is collected, and saturated ammonium sulfate is added to give a final concentration of 0.35 saturation. The resulting precipitate is removed by centrifugation and dissolved in 0.8-0.9% physiological saline to a protein concentration of approximately 2%. The solution was added to a column packed with the same carrier as in Example 1, and the same procedure as in Example 1 was performed to obtain 0.05M
Acetate buffer PH5.0 (+0.05M NaCl ionic strength
0.1). As shown in Figure 3, several types of contaminant proteins were included in addition to the main peak of γ-globulin, but these were removed and highly pure γ-globulin could be separated.

Example 7 A systematic method for fractionating human plasma proteins using low-temperature ethanol was developed by Cohn et al. (EJCohn: Journal of American
Chemical Society 68 :459 (1946)) It is still widely used industrially.

A commercially available albumin solution (20% aqueous solution) produced by this method was diluted with distilled water to a 5% aqueous solution.
According to the method of Example 1, it was added to a column packed with carrier products TSK-GEL3000SW and 2000SW manufactured by Toyo Soda Kogyo Co., Ltd., and the eluate was mixed with 0.05M acetate buffer PH5.0 (+0.25M NaCl, ionic strength 0.3), and the peaks shown in Figure 4 were observed when the samples were passed sequentially. The last large peak is DL-acetyltryptophan, which is used as a stabilizer. It was confirmed that this method can completely remove aggregates of albumin dimers or more.

Example 8 Commercially available human immunoglobulin (15% aqueous solution) produced by Cohn's method as in Example 7 was
Diluted with 0.9% physiological saline to a 2% aqueous solution, added to a column packed with the same carrier as in Example 1 according to the method of Example 1, and added 0.5M acetate buffer PH as the eluent.
5.0 (+0.25M NaCl, ionic strength 0.3), two contaminant proteins were observed in addition to the main peak of γ-globulin as shown in Figure 5, and these were removed to obtain highly pure γ-globulin. - It was possible to separate globulins.

Comparative Example 1 Column packed with cross-linked dextran gel (trade name: Cephadex G-200) (gel bed volume: 460 ml)
After adding 30μ of human serum to 0.1M Tris-HCl buffer PH8.0 (+0.2M
Elute with NaCl, ionic strength 0.25). The eluate was collected in 5 ml portions, subjected to Folin Lowry reaction, and its absorbance (750 mμ) was measured. The results are shown in FIG.

The A peak is α, β-lipoprotein, α and β-globulin, and the B peak is γ-globulin,
The C peak was confirmed to be albumin.

As can be seen from this figure, when cross-linked dextran is used for separation, the separation is not sufficient and also takes a very long time.

[Brief explanation of the drawing]

1 shows a chromatogram of plasma proteins obtained in molecular sieve chromatography. Figures 1 and 2
The figure and FIG. 6 are chromatograms of each protein component in the separated plasma. Figures 3, 4 and 5 are chromatograms of specific protein components in concentrated plasma. 1...α ₁ - and α ₂ -globulin, 2...
α ₁ -globulin, 3...β-globulin, 4...
...γ-globulin, 5...albumin and β-
Globmin, 6...Stabilizer, A...α/β-lipoprotein and α/β-globulin, B…γ-globulin, C…albumin.

Claims (1)

[Scope of Claims] 1. In molecular sieve chromatography, a hydrophilic carrier is used and a buffer solution and a neutral salt solution are used.
9. A method for separating plasma proteins, which comprises separating plasma proteins by passing a solution containing plasma proteins through an eluent having an ionic strength of 0.01 to 1.0. 2 The hydrophilic carrier is a fully porous carrier with chemically bonded hydrophilic groups using silica gel as the base material, and has a pressure resistance of 300
It has a mechanical strength of Kg/ ^cm2 or less and a particle size of 5 to 5.
The method according to claim 1, wherein the spherical shape is 100μ. 3. The method according to claim 1 or 2, wherein the buffer is Tris buffer, phosphate buffer, or acetate buffer. 4 Neutral salt solutions include NaCl, KCl, NH ₄ Cl,
(NH ₄ ) ₂ SO ₄ The method according to claim 1 or 2, wherein (NH 4 ) 2 SO 4 is used. 5. The method according to any one of claims 1 to 4, wherein the solution containing plasma proteins is an aqueous solution. 6. The method according to any one of claims 1 to 4, wherein the solution containing plasma proteins is an aqueous solution containing a water-soluble polymer. 7. The method according to any one of claims 1 to 4, wherein the solution containing plasma proteins is an aqueous solution containing a neutral salt. 8. The method according to any one of claims 1 to 4, wherein the solution containing plasma proteins is an aqueous solution containing an alcohol.