JPS5843369B2 - Serum protein fractionation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a serum protein fractionation method for separating each protein component from serum.

Serum is a supernatant obtained by centrifuging clotted blood that has exited the body, and is obtained by removing fibrinogen from plasma.

The main components contained in serum or blood are albumin and globulin (α1-).

α2-, β-9γ-) and other proteins, including fibrinogen, prothrombin, antihemophilic globulin, lipoproteins, complement components, thromboplaston, isoagglutinin, cholesterol, phosphatite, and many enzymes. However, the amount is small.

That is, most of the serum proteins are albumin and globulin.

Today, serum fractionation and clinical use of separated components are commonplace.

Conventionally known methods for separating and purifying each component of serum or blood consist of one or more of the following complex operations.

(i) Precipitation with low molecular weight salts; (il) Precipitation with organic solvents; (iiD) Selective adsorption with calcium phosphate gel or barium sulfate; (iv) Isoelectric precipitation; Chromatography (VF) using adsorption or Sefferdex gel filtration method E. Amino acids such as glycine and β-alanine, water-soluble organic polymer compounds such as polyethylene glycol and polypropylene glycol, and water-insoluble electrolytes with basic amino groups. precipitation by.

However, in any case, the operation
Conditions such as pH1 ionic strength, temperature, and polymer compound concentration must be strictly controlled (e.g., ionic strength within ±0.05 and pH within ±0.1). Purifying and separating proteins with high purity required many complicated and rigorous steps.

Recently, ethylene oxide/propylene oxide block copolymers containing at least 50% ethylene oxide (polypropylene oxide unit molecular weight 900 or more)
A method using JP-A-49-108216 was disclosed, but there is still room for improvement in terms of easing operating conditions, reducing the amount of polymer compounds used, and increasing the purity of the resulting protein. There is.

This invention was made based on the above background, and it is possible to rapidly purify large quantities of each component protein with high purity from serum without causing protein denaturation even at room temperature with relatively simple and few operation steps. The object of the present invention is to provide a method for fractionating serum proteins that can be separated.

According to this invention, in a method for fractionating serum proteins by treating serum with a suitable protein separation agent, ethylene oxide and propylene oxide containing at least 50% of ethylene oxide units are used alternately or randomly as the protein separation agent. Copolymer, ketone group in the chain,
A water-soluble polymer compound selected from the group consisting of a synthetic polymer compound containing a hydroxyl group, a -grade or a secondary amine group, and a polymer electrolyte having a carboxyl group, or a water-insoluble polymer crosslinked with these polymer compounds. A method for fractionating serum proteins is provided, which is characterized by using a molecular compound.

As mentioned above, the method of the present invention can be divided into two types: one uses a water-soluble polymer compound, and the other uses a water-insoluble polymer compound obtained by crosslinking the water-soluble polymer compound with an appropriate cross-linking bond. However, the effectiveness of these macromolecular compounds in protein separation depends on the identification of the other molecule based on the strength of secondary bonding forces (especially hydrogen bonds, hydrophobic bonds, and ionic bonds) between the protein and the above-mentioned macromolecular compounds, and the accompanying This is thought to be due to the formation of cartilage aggregates.

The water-soluble polymer compounds used in this invention can be classified into the following three types.

(1) Alternating or random copolymers of ethylene oxide and propylene oxide containing at least 50% ethylene oxide units.

The ethylene oxide unit content of this copolymer is preferably 60 to 90%.

(2) A non-electrolyte synthetic polymer compound containing a polar group with hydrogen bonding ability selected from the group consisting of a ketone group, a hydroxyl group, a -class or a secondary amine group in its chain.

Specific examples include homopolymers of vinylpyrrolidone, acrylamide or vinyl alcohol, copolymers of these with each other, or copolymers of these with appropriate copolymerizable monomers such as styrene, acrylic esters, etc. be.

(3) Polymer electrolyte having a carboxyl group.

Specific examples include homopolymers of acrylic acid, methacrylic acid, itaconic acid, maleic acid, etc., copolymers of these with each other, or copolymers of these with appropriate copolymerizable monomers such as styrene, etc. be.

The separation and fractionation of proteins from serum using a water-soluble polymer compound as described above is based on precipitation due to secondary binding between the water-soluble polymer compound and serum components other than proteins, such as proteins or lipids.

According to the method of the present invention, the desired protein can be easily obtained by adjusting the pH value during the precipitation and/or regulating the concentration of the water-soluble polymer compound used, and
Adjustment of values and regulation of polymer compound concentration can also be performed over a relatively wide range, unlike conventional methods.

When using the above-mentioned ethylene oxide/propylene oxide copolymer or a synthetic polymer compound having a polar group, serum components other than proteins are generally adjusted to the pH value of serum by adding 5 to 10 weight equivalents of serum to serum. Regardless, it binds to the polymer compound used and precipitates, leaving only serum proteins in the remaining solution.

When trying to obtain albumin from this residual liquid, the pH value of this residual liquid is adjusted to about 6 or more, and a smaller amount of the above-mentioned polymer compound is added, generally an amount equivalent to 2 to 5 weight φ of the residual liquid. All serum proteins other than albumin precipitate, leaving only albumin in solution.

On the other hand, if the pH value of the residual solution is kept below about 6, albumin will easily precipitate.
Globulin is obtained.

In addition, if you want to obtain only albumin from serum at once, adjust the serum to pH 6 or higher, and add the above-mentioned water-soluble polymer compound to the serum in an amount equivalent to 12 to 20 weight ★ of serum.
Only albumin will remain in solution.

When the above-mentioned polymer electrolyte is used as the water-soluble polymer compound used in this invention, serum should be made into a weakly acidic (generally p
Serum components other than albumin are precipitated by maintaining the serum at a temperature of about 5 to about 7) and adding an amount equivalent to less than 0.1 weight plate of serum, generally 0.01 weight plate.

Note that the pH of serum is approximately 7.4.

The water-soluble polymer compound used in the above operation is suitably one having a molecular weight within the range of 3,000 to 50,000.

If the molecular weight is less than 3,000, the precipitation yield will be poor, and if the molecular weight is more than 50,000, the selectivity for serum components will be poor, resulting in a decrease in the purity of each separated serum protein.

To separate the precipitate produced in the above operation, filtration, centrifugation, decantation, or similar means are used.

The separated precipitate can be completely and easily separated into the water-soluble polymer compound and the serum components secondarily bound thereto by adjusting the appropriate pH value.

Further, the pH value in the above operation can be adjusted using a physiologically acceptable weak acid such as phosphoric acid.

As already mentioned (this method of the present invention is performed by subjecting the water-soluble polymer compound described above to intermolecular crosslinking through covalent bonds such as amide bonds, ether bonds, ester bonds, secondary or tertiary amine bonds, etc. It can also be carried out using insoluble materials.

The crosslinking rate at this time varies depending on the type of polymer compound or the type of crosslinking, but is generally 5 to 60%, preferably 1%.
0 to 50%.

It will be clear to those skilled in the art what crosslinking agent to select for the polymer compound used.

For example, in the case of a polymer electrolyte having a carboxylic acid group, crosslinking through amide bonds can be achieved by using a diamine such as hexamethylene diamine.

Separation of serum components by such a crosslinked polymer compound is due to the difference in the selective adsorption power of each serum component to the crosslinked polymer compound. It can be carried out by a method or a column method.

Batch method Proteins remaining in the clear liquid and adsorbed proteins are separated.

On the other hand, when using the column method, an appropriate size such as 1
Serum is poured as it is, preferably diluted with an appropriate diluent, through a column filled with a cross-linked polymer compound arranged in a mesh size of 0 to 150, and then the desired effluent is passed through the column, and each serum protein is sequentially contained in the effluent. It's flowing out.

Physiologically acceptable media (water, saline, phosphate buffer, and other similar aqueous media) are used as diluents and effluents for the above procedures.

Note that the treated crosslinked polymer compound can be reused by washing it with the same diluent or effluent.

Considering that proteins are susceptible to thermal denaturation, it is advantageous to perform the method of this invention at a temperature below room temperature. However, since the method of this invention can rapidly process a large amount of serum, As shown in the examples of
) can also be carried out, which is convenient.

The serum proteins in the supernatant or effluent can be preserved as they are, for example, by sterile filtration using a Millipore filter or freeze-drying.

The pH in the method of this invention is generally adjusted to a pH value of 3.5 to 8.5 so that serum proteins are not denatured.
Preferably, it is within the range of 4.0 to 7.5.

As described above, the serum protein fractionation method of the present invention is easy to operate and can rapidly process a large amount of serum without requiring strict control of processing conditions such as pH value.

Moreover, the purity of the serum protein finally obtained is high, and it can be said that it is extremely useful from an industrial or clinical point of view.

Examples of this invention will be described below.

In the Examples, each protein component obtained was identified by a conventional electrophoresis method, and its purity was determined by a conventional ultracentrifugation method or column separation method.

Example 1 An ethylene oxide/propylene oxide random copolymer having a molecular weight of 8,700 and containing 80% ethylene oxide units was added to serum in an amount equivalent to 20 parts by weight of serum, and the mixture was stirred at room temperature for 30 minutes.

During this stirring a precipitate formed and the mixture became suspended. This suspension was centrifuged at 500 Or, p, m for 60 minutes, the separated precipitate was discarded, and the remaining supernatant was freeze-dried to obtain albumin.

Yield and purity were 95% and 98.5%, respectively.

Note that similar results were obtained even when the stirring time was shortened to 10 minutes.

Further, when the same operation was performed using a block copolymer having the same composition ratio and the same molecular weight as the above copolymer, the yield of albumin was 92[y] and the purity was 95%.

Example 2 Molecule 1 containing 80% ethylene oxide units in serum
x1ooo of ethylene oxide/propylene oxide random copolymer was added in an amount equivalent to 5 weight φ of serum, and the mixture was stirred at room temperature for 30 minutes.

The resulting suspension was centrifuged at 500 Or, p, m for 60 minutes, the separated precipitate was discarded, and sodium phosphate was added to the remaining supernatant to adjust the pH to 4.6.

The above-mentioned copolymer was added to this supernatant liquid in an amount equivalent to 16 weight □□□, and the mixture was stirred at room temperature for 30 minutes.

The resulting suspension was centrifuged at 500 Or, p, m for 60 minutes.

Discard the separated precipitate 1. Filter the supernatant through a Millipore filter (
γ-globulin was obtained by sterile filtration (0.3 micron) or lyophilization.

The yield was 82% and the purity was 98.2%.

Note that similar results were obtained even when the stirring time was shortened to 10 minutes.

Furthermore, when the same operation was carried out using a block copolymer having the same composition ratio and the same molecular weight as the above copolymer, the yield of globulin was only 75□□□, and the purity was 88%.

Example 3 Styrene/maleic anhydride copolymer 1 with a molecular weight of 4000
Dissolve 0.1 grams in tetrahydrofuran 750a,
Add 0.5 hexamethylene diamine as a crosslinking agent to this solution.
A solution of 5 grams dissolved in 250 ml of tetrahydrofuran was slowly added.

After the addition was completed, boiling point reflux was carried out for 20 hours.

The solution became heterogeneous.

After the reaction was completed, the reaction solution was evaporated to dryness, the residue was washed with water all day and night, and after confirming that there was no elution valve, it was dried to obtain the desired water-insoluble crosslinked copolymer.

The above-mentioned crosslinked copolymer was made into a size of 100 to 150 meshes, and packed into a column having a diameter of 3 to 5 crrL.

This column was filled with 3 volumes of phosphate buffer at pH 5.6.
I poured more than double the amount.

Next, this column was soaked with a phosphate buffer solution of pH 5 or 6.
Serum diluted 0 times was run.

Thereafter, when a phosphate buffer solution of pH 7.4 was flowed as an effluent, each protein was separated in the effluent in the order of albumin, γ-globulin, macroglobulin, etc.

Collect each effluent and filter it through a Millipore filter (0,3
Each protein was purified by sterile filtration (micron) or lyophilization.

When the same operation as above was carried out using a crosslinked polymer produced in the same manner except that methyliminobispropylamine was used as a crosslinking agent, heating at 70° C. or higher was required for protein separation.

Claims (1)

[Scope of Claims] 1. A method for fractionating serum proteins by treating serum with a protein separating agent, wherein the protein separating agent is an alternating or random combination of ethylene oxide and propylene oxide containing at least 50% ethylene oxide units. A method for fractionating serum proteins, characterized in that a water-soluble polymer compound made of a polymer is used at a temperature below room temperature. 2 The molecular weight of the water-soluble polymer compound is 3000 to 50
000. The method for fractionating serum proteins according to claim 1. 3. Serum components other than albumin are precipitated and separated by maintaining the serum at a pH of approximately 6.0 or higher and adding a water-soluble polymer compound to the serum in an amount equivalent to 12 to 20 weight of the serum to obtain only albumin. A method for fractionating serum proteins according to claim 1 or 2, characterized in that: 4. By adding a water-soluble polymer compound to the serum in an amount equivalent to 5 to 10 weight φ of the serum, the serum components of the protein salt series are precipitated and separated, and the remaining liquid is maintained at a pH of less than about 6.0, and the water-soluble polymer compound is added to the serum. The serum protein preparation according to claim 1 or 2, characterized in that serum proteins other than globulin are precipitated and separated by adding a compound in an amount equivalent to 2 to 5 weight φ of the remaining liquid to obtain only globulin. Fractionation method. 5. Any one of claims 1 to 4, wherein the content of ethylene oxide units in the alternating or random copolymer is 60 to 90% (method for fractionating serum proteins described herein). In the method of fractionating serum proteins by treatment, a water-insoluble polymer compound obtained by crosslinking a polymer electrolyte having a carboxyl group with an amide bond using hexamethylene diamine is used as a protein separation agent at a temperature below room temperature. 7. A method for fractionating serum proteins, characterized in that the serum proteins are adsorbed on the adsorbent by flowing a diluted serum solution through a column packed with an adsorbent made of a water-insoluble polymer compound of a predetermined size. 7. The method for fractionating serum proteins according to claim 6, wherein a physiologically acceptable medium is then passed through the column as an effluent, and each serum protein component is sequentially eluted.