JPS5957160A - Separation of humor component - Google Patents

Abstract

PURPOSE:To enable a quick separation of components in a humor with a high recovery rate by using for a humor chromatograph column a filler obtained by saponification and other treatments of a suspension polymer particle between vinyl ester calboxylic acid and a crosslinking monomer having an isocyanurate ring. CONSTITUTION:Vinyl ester calboxylic acid such as vinyl acetate and vinyl propionic acid and a monomer having more than two polymerizing group in isocyanurate ring are dissolved into a solvent such as decane and dibutyl eter which dissolves both monomers but hard to dissolve into water. After the addition of polymerization initiator, the solution undergoes a suspension polymerization in a water having a suspension stabilizer dissolved to obtain a particulate polymer. The polymer thus obtained subjected to a saponification and ester conversion reaction and cleassified to obtain a particle which fills a liquid chromatograph column. When this column is used, components in a humor, for example, immunoglobulin and other protein can be separated with a high recovery rate with a higher separation performance at a high speed through an eluent containing 0.01-0.4M/l of a buffer basic agent comprising a weak acid and weak acid salt.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for separating body fluid components by liquid chromatography, and more specifically, to a liquid chromatography method using a molecular sieve with high hydrophilicity and appropriate micropores as the primary separation mechanism. This paper relates to an innovative separation technology for body fluid components that uses a combination of a graphic column and an eluent with a suitable composition to perform molecular sieving and distribution functions at the same time.

In recent years, liquid chromatography has rapidly become faster and has many advantages over conventionally widely used fractional precipitation methods and electrophoresis methods, such as ease of operation, rapidity, and separation performance. It is also expected to be used as a separation technology. Liquid chromatography is performed using a Gelper I J-Yonk 1-+71 Glassoi- (above,
(abbreviated as C), ion-exchanged chromatography, distribution chromatography, and adsorption L17 chromatography. Gl) C is U-mography based on the so-called molecular sieving action, which utilizes the size of the fine JL of a packing material (hereinafter referred to as gel) to sequentially elute and separate large solute molecules. Suitable for separating plasma proteins. Chromatography based on other separation mechanisms is a type of chromatography in which gels have an affinity for retaining solutes, such as adsorption or distribution, and separation is performed by utilizing the difference in affinity between the amounts of solutes. Suitable for separating low molecular weight components such as amino acids and various amines.

Separation of body fluid components using GPC also has the advantage that pretreatment for removing proteins, such as chromatography, which is used for other separation mechanisms, is not soluble and the operation is simplified. In recent years, many research reports have been made, such as Journal
l of Polymer 5eicence :Po
Lymer Letters [1dition, V
ol 17+ 335 (1979) shows an example of serum separation using a 1rIJ fast GI) C column, which is known to many researchers. In the experimental example, globulin components such as fooglopurin and albumin are separated into fraction 1, but even higher separation performance is desired.

As mentioned above, GPC is effective in principle for separating body fluid components that contain high molecular weight substances such as proteins, but on the other hand, it is difficult to separate solutes based on their molecular size.
This structure makes it difficult to separate substances with similar molecular weights, such as plasma proteins and amino acids. In liquid chromatography, if it were possible to combine the high-molecular-weight substance separation function of GPC with the high-separation function of partition and adsorption chromatography, and eliminate the complicated operations of partition and adsorption chromatography, it would be possible to achieve a revolutionary separation of body fluid components. Becomes a separation technology. In recent years, attempts have been made to achieve the above-mentioned separation function by introducing ion exchange groups into high-speed GPC gels to simultaneously cause molecular sieving action and adsorption or distribution action, thereby simplifying pretreatment and improving separation performance. However, since this method introduces functional groups that have a strong affinity for solutes, such as ion exchange groups, the composition of the eluent must be adjusted during the separation process. There may be complications such as having to change the mind, or there may be problems with separation.
There are still problems with IT performance.

In order to solve the problems in the separation technology of body fluid components using liquid chromatography mentioned above, the unsuccessful researchers developed gel and solution 1i! +1 After comprehensively examining both aspects of the fluid, we have completed an unprecedented and superior method for separating body fluid components. According to the present invention, at least a carboxylic acid vinyl ester monomer, a crosslinkable monomer having an isocyanurate ring, and a melting medium that dissolves both of the monomers but is difficult to dissolve in water are used. A liquid chromatography column packed with a granular filler obtained by saponification or transesterification of a copolymer obtained by suspension polymerization of a mixed solution containing a buffer containing at least a weak acid and a weak acid salt. The base and sulfate coexist and the buffer base is 0.01 to 0.4 M/7! A method for separating body fluid components is provided, which comprises injecting and separating a body fluid sample while passing through a /8R solution containing a concentration of .

The present invention will be explained in more detail below.

The carboxylic acid vinyl ester used in the present invention refers to a compound having one or more polymerizable carboxylic acid vinyl ester groups, such as vinyl acetate, vinyl propionate, vinyl butyrate, vinyl valerate, and vinyl pivalate. selected and used alone or in combination of two or more.

Among these, vinyl acetate and vinyl propionate are particularly preferred from the viewpoint of ease of polymerization, transesterification, saponification, and availability.

Next, the crosslinkable monomer having an isocyanurate ring used in the present invention is represented by the following structural formula.

1 B (However, R,, R2 and R8 are each independently -C11
2-CIf = C112, -C112-C=
CH also 4 - (2l+, 2- C= CIf, , not shown) Mi C1l especially R1, R2 and R3 are all -C112-C
1-Reallyl isocyanurate is preferred as a crosslinking agent because it has good copolymerizability with vinyl acetate and great stability against transesterification or saponification.

The proportion of the crosslinkable monomer having an isocyanurate ring in all the monomers is not particularly limited, but when a gel with high mechanical strength is required, it is preferably within the range of the following formula.

0.2≦3b/(a+3b)≦0.4, where a: number of moles of carboxylic acid vinyl ester b: number of moles of crosslinkable monomer having an isocyanurate ring Also, in the present invention, carboxylic acid vinyl ester When a monomer and a crosslinkable monomer having an isocyanurate ring are subjected to suspension polymerization, the monomer is dissolved in water in order to give the resulting copolymer a ξ-ras structure. A difficult organic solvent is allowed to coexist with the monomer.

The type of organic solvent used depends on the fineness of the copolymer to be produced.
In order to control the pore size and pore size distribution according to the target component to be separated, the mixture is appropriately mixed.

For example, the molecular weight of the analyte Pa1 substance is a medium-low molecule f of 10,000 or less.
In the case of silica, organic solvents that relatively easily dissolve the carboxylic acid vinyl ester polymer, such as toluene, xylene, ethyl acetate, and butyl acetate, are preferred. In addition, if the substance to be separated is a high molecular weight substance with a molecular weight of 10,000 or more, such as blood protein, etc., organic solvents that do not easily dissolve the carboxylic acid vinyl ester polymer in the above-mentioned organic solvent, such as heptano, decane, and dodecane, may be used. A chain hydrocarbon compound having 7 to 15 carbon atoms,
Alternatively, it is preferable that an ether compound having 7 to 25 carbon atoms such as dibutyl ether or di-2-ethylhexyl ether be present in an amount of 5 to 50% by weight based on the total organic solvent.

The blending of such an organic solvent not only controls the micropores necessary for the molecular sieving action, but also participates in the control of the distribution action, which is the other objective of the present invention. Although it is not necessarily clear how the organic solvent used during polymerization in the present invention is involved in the adsorption or distribution effect between the gel, eluate lfk, and the substance to be separated when separating the main components, it is not clear why. We speculate that this is because the orientation of the skeleton molecules during polymerization is affected by the organic solvent used as a porous agent.

The amount of the organic solvent used is generally 20 to 250 parts by weight, preferably 20 to 100 parts by weight, based on 100 parts by weight of the monomer.

A copolymer is obtained by suspension polymerizing the mixture of the above monomer and organic solvent in the presence of a polymerization initiator, but! ! ! The turbidity polymerization may be carried out by a general method.

The initiator used in the polymerization may be a general radical polymerization initiator used in normal suspension polymerization, such as 2
, 2'-azobisisobutyronitrile, 2,2'-azobis-(2,4-dimethylvaleronitrile), hendyl peroxide, lauroyl peroxide, di-t-butyl peroxide, etc. Peroxide-based initiators such as zide or cumene hydroperoxide can be used.

The resulting granular copolymer undergoes saponification or transesterification using an acid or alkali and water or alcohol as a solvent to generate hydroxyl groups necessary for separating body fluid components, resulting in a gel suitable for separating body fluid components. get. In the present invention, since the gel contains sufficient hydroxyl groups, the substance to be separated is not strongly adsorbed within the gel, and good separation performance is achieved without impairing the recovery rate.

In particular, when the substance to be separated is a high molecular weight substance, the molar ratio of hydroxyl groups in the gel to the ester groups in the copolymer is 0.
．． Particularly good separation performance is achieved when the ratio is controlled to between 4 and 0.8. In this case, the abundance ratio of hydroxyl groups is considered to be largely involved in the balance of affinity between the gel of the substance to be separated and the eluent, contributing to the distribution effect. A suitable ratio of hydroxyl groups in such a gel can be easily set by controlling the amount of acid or alkali, reaction temperature and reaction time.

The method for producing the column of the present invention has no problems due to the generally well-known packing properties of gel.
In the method described in No. 985, when Zunawara gel slurry is introduced into a column to form a gel bed, the flow rate of gel slurry in the column is set at 0.2 to 1.5 m.
/ hr, and when increasing the packing density of the gel bed, the maximum pressure increase rate (short-time pressure increase gradient) per meter of gel bed should be 60 kg/ct or less per minute and the average pressure increase rate (long-term pressure increase gradient) If the column is prepared by increasing the pressure at 2 to 80 kg/cnl per hour, even higher separation performance can be obtained.

Next, the composition of the eluent, which is another important requirement in the present invention, will be explained in detail. As mentioned above, the object of the present invention is to combine suitable gel and eluent compositions to achieve
Although it is attempted to achieve unprecedented high separation performance by adding an appropriate distribution effect to CPC separation, it is difficult to clearly distinguish between the molecular sieving effect and the distribution effect. Therefore, in achieving the present invention, the following romatogram characteristics were utilized. That is, the elution volume VR of the substance to be separated
to the X coordinate, the logarithmically transformed value of the molecular weight of the same substance log (
A calibration curve is created with MW) as the Y coordinate, and the properties of the substance plotted on the calibration curve are assumed to be molecular sieving effects (this is a general GPC concept), and the properties of substances that elute clearly later than that curve are assumed. was used as a guideline for the distribution effect.

FIG. 1 illustrates typical examples of elution characteristics related to the molecular sieving action and distribution action that are the basis of the present invention. Figure 1 shows the elution performance of various human serum protein standards (manufactured by Midori Juji Co., Ltd.) injected into the column, starting from the high molecular weight side to IgM.

A calibration curve plotted in the order of haptoglobin, IgG, transferrin, and albumin is shown. In the figure, a is an example in which aluminum has a remarkable distribution effect, and the exclusion limit molecular weight (molecular weight) with dextran as a solute is shown.
ar weight exclusion 11m1t
) is 500,000 and the amount ratio of hydroxyl groups is 0.57.
/β sodium phosphate buffer H=7.0) is used as the eluent. In addition, b is an example showing elution characteristics close to molecular sieve action, in which a column packed with a gel in which the hydroxyl group ratio of the same gel as above was changed to 0.2.
Elute 0.1M/l sodium phosphate with 3M/1 sodium chloride ((+)H=7.0).
This is the elution characteristic when peeled. For example, when separating and analyzing serum proteins using a separation method with elution characteristics such as a, it is found that albumin is the second most abundant protein component in blood, and its molecular weight is also compared. Since IgG that is close to the target is eluted at a more distant position, the separation performance of not only albumin but also other proteins is improved. Furthermore, in order to eliminate excessive adsorption or partitioning effects, a high recovery rate (recov
ery+ 80% or more) and the elution volume had to be highly reproducible.

In the eluent used in the present invention, a buffer base consisting of at least a weak acid and a weak acid salt and a sulfate coexist, and the concentration of the buffer base is 0.01 to 0.4 M#! , preferably 0.01
~0.2M/#, more preferably 0.01~0.1M/
It is important that it be within the range of A. Specific examples of the buffer base include phosphoric acid, acetic acid, lactic acid, tartaric acid, citric acid, substituted salts of these weak acids, or combinations thereof. Further, as the sulfate, sodium sulfate, potassium sulfate, ammonium sulfate, etc. or a combination thereof can be used. According to studies conducted by the present inventors, when the gel and eluent of the present invention are used, substances that are mainly separated by molecular sieving action, such as immunoglobulin IgM, IgA, and IgG in plasma proteins, transferrin, haptoglobin, Myoglobin and the like exhibit good separation performance in a relatively low concentration range of the buffer base. However, for substances such as albumin, uric acid, creatinine, amino acids, etc., which have a remarkable distribution effect, the distribution effect becomes weak in the region where the concentration of the buffer base is low, and good separation cannot be obtained. On the other hand, in a region where the concentration of sulfate is relatively high, the separation performance of substances with a significant distribution effect is good, but the mutual separation of substances that are mainly separated by the molecular sieving effect becomes unsatisfactory. However, as mentioned above, when a buffer base consisting of a weak acid and its weak acid salt and a sulfate are properly co-existed in the eluent, the two coexisting substances, which exhibit contradictory characteristics, can be mixed together, including the substances which exhibit the same characteristics. A surprisingly good separation was achieved.

The concentration of sulfate varies depending on the target substance to be separated, but is particularly suitable for separation of plasma proteins from 0.05 to 0.05.
A range of 0.3 M/j2 is preferred. When salts other than sulfate, such as thorium chloride and sodium nitrate, are used as the coexisting salt species, the effect of the sulfate described above does not appear as markedly, so the concentration of the buffer base should be controlled. By doing so, we will find the point of balance between substances exhibiting the above-mentioned different properties.

In this case, for example, if the concentration of the buffer base is increased in order to improve the separation performance between substances that have a remarkable distribution effect, the separation performance between substances that are mainly separated by molecular sieves will deteriorate;
On the other hand, if the concentration of the buffering base is lowered, the ability to separate substances with a significant distribution effect from each other will become insufficient. Therefore, it can be said that a separation and analysis method using an eluent having such a composition is effective for separating amounts of substances with different separation effects or for separating substances exhibiting either one of the same effects. The method of the present invention satisfies both the separation of substances with properties similar to those of molecular sieve action and substances with remarkable partitioning action, as well as the separation of substances exhibiting the same action in the coexistence of substances with both properties.

The pH of the /8 syneresis used in the present invention is preferably 5 to 8, preferably 5.5 to 7.5. A stable and good minute Pelt can be obtained within this range. In addition, the solution l1i used in the present invention
The u solution is basically an aqueous solution, but /8Iil of the method of the present invention
This does not preclude the addition of water-soluble m/8 medium, various salts, etc., as long as the characteristics or stability of the liquid II are not impaired.

The average particle size of the gel used in the present invention is usually 5 to 1OOμ
m, and a range of 5 to 20 μm is suitable for separation and analysis by high performance liquid chromatography.

The method of the present invention can be applied to the separation and analysis of various body fluids, such as blood, urine, cerebrospinal fluid, and other body fluids, preferably after removing solid substances such as blood cell components that block the flow within the column. They can be injected directly and often do not require pretreatment such as protein removal. In addition, the method of the present invention can use an eluent with the same composition throughout the process from sample injection to separation to completion of analysis, which is comparable to the separation of body fluid components by conventional ion-exchange chromatography or partitioned chromatography. There is no need to change the /88 synergic composition during such a separation step. As a result, high separation performance can be obtained with good reproducibility and simple operation.

When carrying out the method of the present invention, the separated body fluid components may be detected using commonly used detectors, such as an ultraviolet spectrophotometer, an electrochemical detector, a fluorescence spectrophotometer, or a differential refraction needle. It is also possible to selectively detect specific components by injecting an appropriate coloring reagent solution before or after the column.

The separation method of the present invention can be applied to various body fluid components, such as proteins,
Peptides, amines, amino acids, sugar IJf, etc. q+
It can be applied to analysis, but especially when used for analysis, the mutual component δ1t of various proteins with a molecular weight of 10,000 or more, and the mutual component δ11, which was conventionally thought to be difficult, can be dramatically improved.

As described above, the present invention provides an eluent that appropriately controls molecular sieving and distribution effects in a column packed with a gel that has high hydrophilicity, appropriate micropores, and has an appropriate adsorption or distribution effect. Liquid chromatography that allows liquid to flow through the liquid makes it possible to separate and analyze body fluid components that were not possible before, and also has the advantage of greatly simplifying the separation operation, which is also practically effective.

Hereinafter, the present invention will be explained in more detail with reference to Examples.
It goes without saying that the scope of the present invention is not limited to these Examples.

Example 1 200 g of hinyl acetate, ) realyl isocyanurate),
82.8 g, n-butyl acetate 158 g, decane 4
0 g and 2.2'-azobisisobutyronitrile 6.8
A homogeneous mixed solution consisting of 1.67g and water in which polyvinyl alcohol and sodium phosphate were dissolved! was placed in a 5β flask, stirred thoroughly, and then heated at 65°C for 18 hours, and then heated at 65°C for 75 hours.
Suspension polymerization was carried out by heating and stirring at °C for 5 hours to obtain a granular polymer. After filtration, water washing, and acetone extraction, NaOH94
The above copolymer was subjected to transesterification reaction at 15°C for 20 hours in a solution consisting of g and methanol 41, and then classified to obtain a gel with an average particle size of 9.5 μrn (G).The amount of hydroxyl in the gel was The ratio was 0.57.Four stainless steel columns each having an inner diameter of 7.5 m and a length of 50 am were filled with this column to prepare four similar columns.

The molecular weight limit of this column was 500,000 based on dextran. Na211PO47.2g ,,Na
112PO41.53g, Na, 230414.2
g mixture was dissolved in distilled water to make a total volume of 1z0,
Various human serum proteins (Mitri +
The following results were obtained when the recovery rate was determined by injecting each sample.

Recovery rate Immune globulin (IgM): 91% Immunoglobulin (1 g 8) = 94% Immunoglobulin (IgG): 96% Haptoglobin: 96% Transferrin: 94% Albumin: 99% Next, connect the above four columns and collect the inside of the column. Adjust the temperature to 37°C and add 1 m I of the above /8 syneresis! Wavelength 2 while passing liquid at /min.
Using a 50 nm ultraviolet spectrophotometer needle as a detector, 5 μl of Kenraito's serum was injected to separate the contained components.

Figure 2 is a chromatogram. In the figure, ■ is 1.G, 2 is transferrin, 3 and 4 are albumin,
5 is a peak whose main component is creatinine, and 6 is a peak whose main component is uric acid. Important components in serum are well separated. In addition, 2 peaks of albumin seen in peaks 3 and 4 are 81
This is also observed in commercially available high-purity human albumin, making it possible to separate albumins with different properties.

[Brief explanation of the drawing]

Figure 1 shows typical examples of elution characteristics related to molecular sieving action and distribution action. This is an example showing the characteristics. Figure 2 is an example of a chromatogram when serum of a healthy individual is separated by the method of the present invention, in which 1 is 1, G, 2 is transferrin, 3 and 4 are albumin, 5 is creatinine, and 6 is uric acid. These are the peaks whose main components are: Patent Applicant) 0 Kasei Kogyo Co., Ltd. Patent Application Agent Akira Aoki Patent Attorney Kazuyuki Nishidate Patent Attorney Ishi 1) Kei Patent Attorney Akira Yamaguchi Figure 1 1 → General Publication f4X, VR (mal No. Figure 2

Claims (1)

[Claims] 1. In separating body fluid components by liquid chromatography, at least a carboxylic acid vinyl ester monomer;
A copolymer obtained by suspension polymerizing a mixture containing a crosslinkable monomer having an insia slate ring and an organic solvent that dissolves both of the above monomers but is difficult to dissolve in water is subjected to a saponification reaction or transesterification reaction. In a liquid chromatography column packed with the granular filler obtained by the process, a buffer base consisting of at least a weak acid and a weak acid salt and a sulfate coexist, and the buffer base has a concentration of 0.01 to 0. A method for separating body fluid components, which comprises injecting and separating a body fluid sample while passing through an eluent containing a concentration of 4M/N. 2. Organic solvents that dissolve monomers but are difficult to dissolve in water,
Hydrocarbon compounds or ether compounds that are difficult to dissolve the carboxylic acid vinyl ester polymer should be added to 5 to 5% of the total organic solvent.
Contains 0% by weight, and the molar fraction of ester groups in the copolymer obtained by suspension polymerization is 0.4 to 0.8 as a filler and is converted into hydroxyl groups by saponification reaction or transesterification reaction. A method for separating body fluid components according to claim 1. 3. The concentration of sulfate in the eluent is 0.05-0.3M/l
A method for separating body fluid components according to claim 1 or 2.