JP2562576B2 - Method for separating albumin and globulin - Google Patents

Description

TECHNICAL FIELD The present invention relates to a rapid separation method of albumin using chromatography.

Recently, from the viewpoint of both effective use of blood and prevention of side effects due to blood transfusion, rather than using whole blood as it is as preserved blood, it is divided into the smallest possible components and only the components necessary for the patient are infused and other components are infused. Has been rapidly used as a component preparation, that is, to be used for another patient in need thereof.

Albumin preparations are one of the most widely used ingredient preparations, according to the Biological Standards of the Ministry of Health, Labor and Welfare
There are "heated human plasma protein" and "human serum albumin",
It is useful for preventing edema and ascites, as an emergency treatment for hemorrhagic shock and burns, and for infusion during plasma exchange.

The demand for albumin preparations far exceeds its supply, and this imbalance is likely to become more serious in the future. As a matter of course, since the supply amount is limited, there is an eager desire to develop an efficient purification method.

On the other hand, albumin is also extremely important in the field of clinical examination.

In general, albumin is often decreased in many diseases, and the amount ratio of albumin and globulin, so-called A / G ratio, is often decreased. For example, decrease of albumin associated with liver dysfunction such as liver cirrhosis, chronic hepatitis, liver cancer, acute hepatitis, liver atrophy, multiple myeloma, macroglobulinemia, malignant lymphoma, collagen disease, liver cirrhosis, chronic infection, syphilis, joint It reflects the decrease in globulin associated with rheumatism and malaria. For this reason, the A / G ratio is one of the most frequently performed health examination (screening) items.

(Problems to be Solved by Conventional Techniques and Inventions) Conventional fractionation methods for serum proteins include salting-out method, corn (Co
hn) Fractionation method, polyethylene glycol method, etc. Among them, the corn method using cold ethanol developed in the 1940s is most widely used. Nevertheless, this method has many drawbacks. First, the use of high concentrations of ethanol leads to the denaturation and inactivation of many valuable biologically active plasma proteins. Further, this method is complicated and requires a long time, and can be carried out only in a batch system.

Although the separation method using a semipermeable membrane has high operability, there is still no method having a satisfactory separation ability.

Recently, gel filtration method and ion exchange chromatography method have been developed.

For example, a method in which albumin is isolated from a plasma product by combining a cation exchanger and an anion exchanger (Japanese Patent Application Laid-Open No. S60-187242).
52-47915) has been developed, but clotting factors I, II, VII, VI
It can be applied only to the plasma fraction that does not substantially contain II, IX and X, and the major portion of IgG, and the operation cannot be said to be convenient.

On the other hand, on the other hand, a method for separating plasma proteins by molecular sieve chromatography using a hydrophilic carrier (JP-A-54-4681).
3) is being developed. According to this method, α, β, γ
Mutual separation of each globulin and albumin of (3) is better than that of the conventional method, but separation of albumin and other components is insufficient.

For example, a part of IgG and almost all of transferrin are mixed in the albumin fraction. Further, it takes 30 minutes or more to separate them.

On the other hand, the A / G ratio is conventionally measured by a salting-out method, a paper electrophoretic method, a cellulose acetate membrane method, a diffracting method, etc., but the normal values by the respective measuring methods are 1.0 to 1.5 and 1.6 to respectively.
There are large variations of 1.9, 1.6 to 2.4, and 1.5 to 2.4. Therefore, the development of a reliable standard method for A / G ratio measurement is awaited.

(Means for Solving Problems) As a result of intensive research to overcome the above-mentioned problems, the present inventors have found that albumin and other components can be rapidly and easily produced from albumin-containing body fluids or plasma products. The present invention has been completed by finding a method for efficiently separating the above.

The alcoholic hydroxyl group and anion exchange group directly bonded to the main chain are 3 to 9 meq / g and 0.
Body fluid or plasma containing albumin by liquid chromatography using a mobile phase having a pH of 7.0 to 9.5 and an ionic strength of 0.3 to 1.0, using a rigid all-porous granular cross-linked copolymer having 1 to 3.0 meq / g as a stationary phase. A short-time method for separating albumin and globulin, characterized in that an eluting solvent is passed at a high flow rate to separate albumin and globulin within 8 minutes from the product, and albumin and globulin are separated within 8 minutes. Therefore, it provides a method for separating albumin from other components from a body fluid or plasma product containing albumin in a much shorter time than in the conventional method, and simply and more efficiently. is there.

 Hereinafter, the present invention will be described in detail.

The hard, all-hard-hard, granular crosslinked copolymer (hereinafter, simply referred to as a gel) used as a stationary phase in the present invention has at least an alcoholic hydroxyl group bonded to the main chain. The alcoholic hydroxyl group directly bonded to the main chain is, for example, a hydroxyl group obtained by saponifying a polymer of vinyl acetate or vinylene carbonate. The amount of the hydroxyl group is 3 to the dry weight of the gel.
It should be in the range of 9mq / g. Here, the amount of hydroxyl groups is
For example, it can be determined by reacting the hydroxyl group with acetic anhydride and measuring the amount of acetic anhydride consumed or the weight change of the gel.

Further, the gel of the present invention has an ion exchange group other than the hydroxyl group. For example, weakly basic anion exchange groups such as amino groups and diethylamino groups, strong basic anion exchange groups such as quaternary ammonium groups, weakly acidic cation exchange groups such as carboxyl groups and phosphoric acid groups, and sulfonic acid groups. Strongly acidic cation exchange groups are preferred. Of these, a basic anion exchange group is preferable, and a weak basic anion exchange group such as a diethylamino group is particularly preferable. Also, the amount of ion-exchange group is
It is preferably in the range of 0.1 to 3.0 meq / g. The amount of ion exchange groups can be determined by a usual method for measuring the exchange capacity of ion exchange resins.

The above-mentioned hydroxyl group and ion-exchange group may be distributed throughout the gel, or may be distributed only in the pore forming portion, that is, the pore surface.

The cross-linked structure of the cross-linked copolymer of the present invention is not particularly limited, for example, triallyl isocyanurate, diallyl isocyanurate or triallyl cyanurate and the like by a cross-linkable monomer unit having a tricyanurate ring or triazine ring Examples thereof include crosslinked structures. Among them, a structure crosslinked with triallyl isocyanurate is preferable.

The hard gel used in the present invention has high mechanical strength,
Moreover, it is a gel having a structure in which pores are distributed inside. Unlike a soft gel such as Sephadex described above, such a gel substantially maintains a pore structure when swollen even in a dry state, and therefore has a large specific surface area in a dry state. The gel of the present invention usually has a dry gel weight of 2 m
^It has a specific surface area of ² / g or more, preferably 5-1000 m ² / g. On the other hand, the specific surface area of the soft gel when dried is a small value of 1 m ² / g or less. The gel having a specific surface area within the range of the present invention is
Since it has a high mechanical strength, when it is used as a carrier for chromatography, the eluting solvent can be passed through at a high flow rate, which enables rapid separation and analysis. The pore size in the gel may be at least large enough to allow albumin to penetrate. The size of the pore is dextran,
It can be estimated by a known method from the calibration curve obtained by performing gel permeation chromatography using a standard sample of known molecular weight such as polyethylene glycol.

The particle size of the gel of the present invention is usually in the range of 1 to 2000 μm. When used as a packing material for high performance liquid chromatography, the average particle size is preferably in the range of 2 to 15 μm. Larger particle sizes may be used for the purpose of separating large samples.

An example of the hard gel of the present invention is disclosed in JP-A-59-8261.

Separation of albumin is carried out by so-called liquid chromatography in which the gel is usually packed in a stainless steel or glass column, but it may be carried out as thin layer chromatography.

The pH of the mobile phase used in the chromatography of the present invention is 6.
It is 5 to 10.0. More preferably, it is 7.0 to 9.5. The ionic strength of the mobile phase is 0.3 to 1.0.

In addition, ethanol, methanol, acetonitrile,
By adding a small amount of water-soluble organic solvent such as ethylene glycol, the shape of the chromatogram becomes sharp,
The recovery rate of protein can be increased.

Usually, during the chromatography, the composition of the mobile phase may remain unchanged without change, but if necessary, it may be changed stepwise or continuously.

The albumin-containing body fluid or plasma product of the present invention refers to albumin among body fluids of humans and non-human animals such as plasma, serum, placental serum, placenta extract, and cerebrospinal fluid, and their fractions. Say what it contains.

(Examples) Examples of the present invention will be shown below, but the scope of the present invention is not limited by these examples.

Example 1 Albumin was separated by liquid chromatography under the following conditions. The column (Asahipak
ES-502N) is a packed column for high performance liquid chromatography having a stationary phase of a hard, totally porous granular crosslinked polymer having an alcoholic hydroxyl group directly bonded to the main chain and an ion exchange group (diethylamino group).

Column: Asahipak Chemicals Co., Ltd. Product name Asahipak ES-502N Inner diameter 7.6 mm, Length 10 cm Mobile phase: 50 mN Bis-Tris HCl + 500 mM NaCl + 10% ethanol (pH 7.0) Pump: JASCO Corporation Product name TRI ROTAR-V (Flow rate) : 1.0ml / min) Detector: JASCO Corporation, trade name UVIDEC-100-VI (wavelength: 280mm) Temperature: 35 ℃ (Result) Serum (Ortho Diagnostic Systems Co., Ltd. normal control) Example of separation of serum)
As shown in the figure. As can be seen from the chromatogram, the separation of albumin and globulin was completed in about 4 minutes.

Example 2 Mobile phase: 50 mM ethanolamine-HCl + 500 mM NaCl + 4%
Serum was separated under the same conditions as in Example 1 except for ethanol (pH 9.5).

(Results) The obtained chromatogram is shown in FIG. As can be seen, the separation of albumin and globulin was completed in about 5 minutes, and the separation was extremely good.

Example 3 Separation of albumin was carried out in
It was performed under the conditions shown in.

Column: saponified gel obtained by copolymerizing vinyl acetate and triallyl isocyanurate as a cross-linking agent with aqueous caustic soda solution, specific surface area per dry weight of gel is 55 m ² / g, hydroxyl group density Is 6.5 meq / g and the ion-exchange group is diethylaminoethyl group is 0.45 meq / g. Packed gel with inner diameter of 7.6 mm and length of 50 mm. Mobile phase: 50 mM ammonium acetate-HC1 + 250 mM MgCl ₂ (pH
8.0) (Results) When the serum of a healthy person was analyzed under the above conditions, albumin was successfully separated from the serum protein containing the globulin component within 5 minutes as shown in FIG.

Example 4 Separation of albumin was performed in
It was performed under the conditions shown in.

Column: saponified gel obtained by copolymerizing vinyl acetate and triallyl isocyanurate as a cross-linking agent with aqueous caustic soda solution, specific surface area per dry weight of gel is 55 m ² / g, hydroxyl group density Is 4.5 meq / g, ion-exchange group is 0.80 meq / g of diethylaminoethyl group is packed in gel with inner diameter of 7.6 mm and length of 50 mm. Mobile phase: 50 mM ammonium acetate-HC1 + 100 mM NaCl (pH
8.0) (Results) When the serum of a healthy person was analyzed under the above conditions, albumin could be well separated from the serum protein containing the globulin component within 5 minutes as shown in FIG.

Comparative Example 1 Albumin was separated from Example 1 except for the conditions shown below.
It carried out on the conditions shown in.

Column: Non-surface area per gel dry weight of 65 m ² / g, hydroxyl group density obtained by saponifying gel obtained by copolymerizing vinyl acetate and triallyl isocyanurate as a cross-linking agent with aqueous caustic soda solution. Column with a diameter of 7.6 mm filled with 6.0 meq / g gel (no ion-exchange group introduced) Mobile phase: 50 mM ammonium acetate-HC1 + 250 mM MgCl ₂ (pH
8.0) (Results) When serum of a healthy person was analyzed under the above conditions, as shown in FIG. 5, the elution times of serum proteins containing albumin and globulin were almost the same, and they could not be separated.

Comparative Example 2 Albumin was separated from Example 1 except for the conditions shown below.
It carried out on the conditions shown in.

Column: Non-surface area per gel dry weight of 65 m ² / g, hydroxyl group density obtained by saponifying gel obtained by copolymerizing vinyl acetate and triallyl isocyanurate as a cross-linking agent with aqueous caustic soda solution. Column with a diameter of 7.6 mm and a length of 50 mm filled with a gel having a pH of 6.0 meq / g and an ion exchange group of diethylaminoethyl group of 0.05 meq / g.

Mobile phase: 50mM ammonium acetate -HC1 + 250mM MgCl ₂ (pH
8.0) (Results) When the serum of a healthy person was analyzed under the above conditions, as shown in FIG. 6, the elution times of serum proteins containing albumin and globulin were almost the same and could not be separated.

Comparative Example 3 Separation of albumin was performed in Example 1 except for the conditions shown below.
It carried out on the conditions shown in.

Mobile phase: 50 mM ammonium acetate-HC1 (pH8.0) (Results) When serum of healthy subjects was analyzed under the above conditions, globulin was eluted within 2 minutes after the start of analysis, but albumin was not eluted after 60 minutes. I could not confirm.

(Effect of the Invention) In the method for separating albumin and globulin by the chromatography of the present invention, in addition to the alcoholic hydroxyl group directly bonded to the main chain, a hard all-porous granular cross-linked copolymer introduced with an ion exchange group is used. As stationary phase, pH 6.5-10.
0, by using a mobile phase with an ionic strength of 0.05 to 1.5, by utilizing physical differences such as molecular weight, isoelectric point and hydrophilicity / hydrophobicity of albumin and globulin, and suppressing their adsorption to gel as much as possible. , Albumin and globulin can be efficiently separated in a short time. Moreover, during the chromatography
Since the composition of the mobile phase may remain constant without changing, the operation is extremely simple.

As a comparative example of the present invention, the above-mentioned ion exchange chromatography method (JP-A-52-47915) and molecular sieve chromatography method (JP-A-54-46813) can be mentioned. The former contains a coagulation factor and IgG. Separation and operability are problematic, with the proviso that it can only be applied to the missing plasma fraction. The latter still has problems in resolution and speed.

As described above, the separation of albumin and other components (globulin) in the present invention is far superior to any conventional method in all respects such as separability, rapidity, and convenience. ing.

[Brief description of drawings]

FIG. 1 is a chromatogram showing the result of separating healthy human serum according to Example 1, and FIG. 2 is a chromatogram showing the result of separating healthy human serum similarly according to Example 2.
FIG. 4 is a chromatogram showing the result of separating healthy human serum according to Example 3, FIG. 4 is a chromatogram showing the result of separating healthy human serum according to Example 4, and FIG. FIG. 6 is a chromatogram showing the results of separation, and FIG. 6 is a chromatogram showing the results of separating healthy human serum according to Comparative Example 2.

Claims (1)

(57) [Claims] 1. An alcoholic hydroxyl group and an anion exchange group directly bonded to the main chain are each contained in an amount of 3 to 9 meq / dry weight of gel.
g, and 0.1-3.0 meq / g hard all-porous granular cross-linked copolymer as a stationary phase, pH 7.0-9.5, ionic strength 0.3-
By liquid chromatography using a mobile phase of 1.0, in order to separate albumin and globulin from albumin-containing body fluid or plasma product within 8 minutes, an eluting solvent is passed through at an alternating current speed to separate albumin and globulin for 8 minutes. A method for short-time separation of albumin and globulin, which is characterized in that the separation is carried out within.