JP2001097997A - Carrier for affinity absorption from sulfated hyaluronic acid, and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new carrier for affinity adsorption, capable of fractionally adsorbing adsorbates such as proteins. SOLUTION: This carrier for affinity adsorption comprises a sulfated hyaluronic acid having a structure of the formula (n is the number capable of making the molecular weight 10,000-1,500,000, R is H or SO3-M+, with the proviso that 0.5-4 Rs in the 4 Rs in a repeating unit comprising two saccharides are each SO3-M+ as the average in the whole molecule; and M+ is independently a monovalent cation), or a derivative of the hyaluronic acid. The use thereof is also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel substance-adsorbing carrier useful for separating / separating or purifying a substance such as a protein by utilizing a difference in adsorptivity, and a use thereof.

[0002]

2. Description of the Related Art Heparin, a high molecular polysaccharide, is capable of binding a certain protein, and is used for separation or separation of proteins by utilizing the difference in the binding property to proteins or the binding power to various proteins. What you can do is well known. On the other hand, hyaluronic acid or a salt thereof, which is a high molecular polysaccharide like heparin, has no ability to bind to various proteins such as heparin. Although heparin has the ability to bind to various proteins, its binding property to various different proteins is fixed, so that there is a limit to the use of protein separation. Therefore, for substances such as various proteins,
An adsorption carrier having a binding property different from that of heparin, that is, an adsorptivity, is desired.

[0003]

SUMMARY OF THE INVENTION Accordingly, the present invention provides a substance having an adsorbing property different from that of heparin for various proteins and the like, a carrier for adsorbing proteins and the like using the substance, and an adsorbent for the protein. It is an object of the present invention to provide a method for separating or separating or purifying a substance such as a protein using a carrier for purification.

[0004]

Means for Solving the Problems As a result of various studies to solve the above-mentioned problems, the present inventor has found that by sulfating (sulfating) hyaluronic acid (salt), a protein adsorbing property different from that of heparin is obtained. It has been found that a novel effect having the following is obtained, and the present invention has been completed by using it. Accordingly, the present invention provides the following structural formula:

[0005]

Embedded image

(Wherein n has a molecular weight of 10,000 to 1.5 million)
R is hydrogen or SO_Three ^-M ⁺And where
Of the four Rs in the repeating unit consisting of two sugars, all
The average of 0.5 to 4 R in the child is SO_Three ^-M⁺In
(This number is called sulfuric acid degree (DS)); and M⁺
Are each independently monovalent ions)
Using sulfated hyaluronic acid or its salt
A carrier for adsorption is provided. M⁺Monovalent a
On is especially a monovalent metal ion, and typically Na⁺
It is.

[0007] The carrier for affinity adsorption is typically a carrier for affinity adsorption chromatography. The present invention further relates to a method for separating an adsorbed substance such as a protein, wherein the sample which is expected to contain at least one kind of adsorbed substance such as a protein which can be adsorbed on the affinity adsorbent carrier is used. When the sample contains an adsorbing substance such as a protein adsorbed on the affinity adsorbing carrier in the sample, the adsorbing substance is adsorbed, and a non-adsorbing substance such as a protein not adsorbed on the affinity adsorbing carrier is absorbed. If contained in the sample, remove or recover it from the affinity adsorption carrier, and if necessary, elute an adsorbed substance such as a protein adsorbed on the affinity adsorption carrier, such as a protein. A method for separating or purifying an adsorbed substance or a non-adsorbed substance is provided.

The present invention also relates to a method for separating or purifying a substance such as the protein of the present invention described above, and a method for separating or purifying a substance such as a protein other than the method for separating or purifying using the carrier for affinity adsorption of the present invention. And a method for separating or purifying a substance such as a protein, characterized by using For example, by using the protein separation method of the present invention in combination with a known protein separation method, adhesion proteins (such as fibronectin), blood coagulation factors (such as fibrinogen), cell growth factors (such as FGF) and the like can be obtained. It can be separated from other proteins.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION In the above-mentioned sulfated hyaluronic acid of the present invention, the number n of repeating units is a number such that the molecular weight of sulfated hyaluronic acid is 10,000 to 1.5 million, and preferably 200,000 to 800,000. Is the number In the present invention, as the hyaluronic acid for synthesizing hyaluronic acid sulfate, conventionally available hyaluronic acid can be used, and the molecular weight of hyaluronic acid is generally tens of thousands to 2,000,000. The molecular weight of sulfated hyaluronic acid is the same, or the molecular weight is slightly smaller than that of the original hyaluronic acid due to cleavage of the main chain during sulfation.

It is a ⁺ M, a result SO ₃ sulfated ^{- -} [0010] the substituents R is a hydrogen atom or SO ₃ 4 amino and M ⁺ is always present, in one repeat unit consisting of two monosaccharide components Of R, the average of all sulfated hyaluronic acid molecules (sulfation degree; De
gree of Sulfatin (DS)), 0.5 to 4 S
O ₃ ^- M ⁺ is present. The four Rs in the repeat unit are
All four theoretically is SO ₃ ^- M ⁺ and may be, but if all R attempts sulfation, and hyaluronic acid backbone is cut, there is a possibility that the molecular weight decreases.
Therefore, SO ₃ ⁻ M ⁺ in the repeating unit according to the present invention is used.
May be less than or equal to 3.5. On the other hand, SO ₃ in repeating units ^- the M ⁺ average number of is less than 0.5, an insufficient sulfation, adsorptive protein is lowered. The degree of sulfation (DS) is preferably between 1.5 and 2.5, for example about 2.

[0011] substituent SO ₃ ^- is M in M ^{+ +} is a monovalent cation, or hydrogen ions, but is preferably other than hydrogen ions ions, preferably monovalent metal ion or an ammonium ion, ( NH ₄ ⁺ ) or
It is an organic quaternary ammonium ion. Examples of the monovalent metal ion include Li ⁺ , K ⁺ , and Na ⁺ , and typically Na ⁺ .

In producing the sulfated hyaluronic acid of the present invention,
Is hyaluronic acid by the usual method for sulfating polysaccharides.
The acid may be sulfated, and the method is not limited. For example,
As a conventional technique and a method for sulfating polysaccharides, D. Sie et.al.
Method (Biochime Biophys. Acta, 966, 188 (1998)) and
The method of R.L.Whistler et.al. (Academic Press, Vol.II
 298 (1963)). As one of the methods,
Tritylamine-SO _ThreeComplex with hyaluronic acid
To cause the hyaluronic acid to be sulfated.
A specific example of this method is described in Example 1. This
Wherein the degree of sulfation (DS) is
It can be adjusted by the amount of reaction and the reaction time.

The affinity-adsorbed carrier of the present invention can be produced by binding the above-mentioned sulfated hyaluronic acid to a carrier material. As the carrier material, a common carrier material such as Sepharose or a derivative thereof, agarose, cellulose, glass, dextran and the like can be used. A conventional linker can be used for linking the carrier material and the sulfated hyaluronic acid. For example, an epoxy-activated aminohexyl (EA
H), epoxy-activated carboxylhexyl and the like. An activator, for example, carbodiimides, such as N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide hydrochloride, is used for binding the linker to sulfated hyaluronic acid and the carrier. A specific example of a method for producing a carrier for affinity adsorption is described in Example 2.

The carrier for affinity adsorption of the present invention can be used in a batch system, but is preferably packed in a column and used as a carrier for affinity chromatography. The affinity adsorption carrier of the present invention can adsorb, for example, plasma fibronectin (Fn) and fibrinogen, but cannot adsorb human serum albumin, immunoglobulin, and insulin. Therefore, fibronectin and fibrinogen can be separated from other proteins in various blood proteins.

On the other hand, heparin can adsorb fibronectin, human serum albumin and fibrinogen, but cannot adsorb immunoglobulin and insulin. That is, the sulfated hyaluronic acid of the present invention has an original adsorptive property and an adsorptive power with adsorbing substances such as various proteins. Therefore, it is possible to separate plasma fibronectin, fibrinogen and the like from other blood components by utilizing the difference in the adsorptivity and the adsorptive power. In particular, the sulfated hyaluronic acid of the present invention does not adsorb serum albumin, and thus is useful for removing serum albumin in isolating its components from blood.

Alternatively, fibronectin and fibrinogen are adsorbed by first bringing a sample containing the above blood component into contact with the sulfated hyaluronic acid of the present invention,
Leakage of unadsorbed human serum albumin, immunoglobulin and insulin, and contact of these leaked liquids with heparin allows only human serum albumin to be adsorbed and other protein components to be removed or recovered.
Next, human serum albumin adsorbed on heparin is eluted to separate human serum albumin from all other blood proteins.

Alternatively, fibronectin, human serum albumin and fibrinogen are adsorbed by bringing a sample containing all of the above blood components into contact with heparin, and unadsorbed immunoglobulin and insulin are leaked out and collected. Next, the adsorbed fibronectin, human serum albumin and fibrinogen are eluted, and the eluate is contacted with sulfated hyaluronic acid to adsorb fibronectin and fibrinogen, to allow only non-adsorbed human blood albumin to leak out and to remove human serum albumin. Separate. The adsorbed fibronectin and fibrinogen can be separately eluted and recovered.

Further, according to the above-mentioned method, fibronectin and fibrinogen adsorbed to both the sulfated hyaluronic acid and heparin of the present invention, immunoglobulin and insulin not adsorbed to any of the adsorbent carriers, and only one carrier is adsorbed. It is also possible to classify into human serum albumin. The above example is an example utilizing the combination of the affinity adsorption carrier of the present invention and heparin.However, the affinity adsorption carrier of the present invention and a protein other than human serum albumin used in combination with an adsorption carrier other than heparin are used. Can also be isolated. Furthermore, isolation of a bio-related component with high purity and / or high yield by using only the method using the affinity adsorption carrier of the present invention or combining this with a method for separating other bio-related components. Is also possible.

[0019]

Next, the present invention will be described more specifically with reference to examples. Embodiment 1 FIG . Synthesis of sulfated hyaluronic acid (S-HA) Hyaluronic acid (HA150) with a molecular weight of 1.5 million dried overnight under reduced pressure
4 g were swollen in 400 ml of DMF with stirring at 30 ° C. for 48 hours, and trimethylamine-SO ₃ complex 16.6 was added.
g was added and reacted at 60 ° C. for 48 hours under a nitrogen stream. After the reaction, the reaction mixture was diluted with distilled water, neutralized with 1N NaOH, reprecipitated with acetone, dialyzed with distilled water, reprecipitated with acetone, and dried under reduced pressure to obtain S-HA. The sulfate group introduction rate per disaccharide repeating unit was defined as a degree of sulfation (DS), and DS was calculated by a chelate titration method. In this way
A sulfated heparin with a DS of 2.1 was obtained.

Embodiment 2 FIG. Preparation of Affinity Column An affinity column was prepared as follows.
Glycosaminoglycan (GAG) as a ligand, that is, heparin, hyaluronic acid (HA) or sulfated hyaluronic acid (S-HA) obtained in Example 1 is dissolved in distilled water to a concentration of 5 mM. EAH-Sepharose 4B (1 ml) was added to a mixture of 3 ml and 1 ml (10 mg / ml) of a water-soluble carbodiimide dissolved in distilled water, and pH was adjusted with 1N HCl.
After adjusting to 4.5, the mixture was incubated in a shaker at room temperature for 24 hours to prepare each GAG-carrier (GAG-Sepharose 4B). Each is packed in a glass column, and a heparin affinity column, hyaluronic acid
An affinity column and a sulfated hyaluronic acid / affinity column were used.

Embodiment 3 FIG. In vivo protein adsorption experiment The three types of affinity columns obtained in Example 2 were used for buffer A (25 mM Tris-HCl, 0.5 mM EDTA, 1.0 M Na
Cl; pH 7.6) and then buffer B (25 mM Tris-HCl,
The column was equilibrated with 0.5 mM EDTA, 50 mM NaCl; pH 7.6). 0.5 g of each protein was dissolved in 0.5 ml of buffer B to prepare a sample. This was passed through a column, and then sufficiently washed with buffer B to remove non-adsorbed components. Thereafter, buffer A and then buffer A + 4.0 urea were flowed to elute the adsorbed components. The eluate was monitored by the absorbance at 280 nm.

As for affinity chromatography showing adsorption, 10.0 ml of buffer B was further added.
10.0 ml of buffer C (25 mM Tris-HCl, 0.5 mM EDTA,
The concentration of the eluted salt was measured by performing a linear concentration gradient (0.05 to 0.5 M) of sodium chloride with two kinds of eluates (0.5 M NaCl; pH 7.6).

As various in vivo proteins, plasma fibronectin was used as an adhesive protein, human serum albumin was used as a blood protein, immunoglobulin was used as an antibody, fibrinogen was used as a blood coagulation factor, and insulin was used as a hormone. The results for fibronectin are shown in FIG. 1 (hyaluronic acid column), FIG. 2 (sulfated hyaluronic acid column) and FIG. 3 (heparin column), and the results for human serum albumin are shown in FIG. 4 (hyaluronic acid column) and FIG. The results for immunoglobulins are shown in FIGS. 7 (hyaluronic acid column), FIG. 8 (sulfated hyaluronic acid column) and FIG. 9 (heparin column).
The results for fibrinogen are shown in FIG. 10 (hyaluronic acid column), FIG. 11 (sulfated hyaluronic acid column) and FIG. 12 (heparin column), and the results for insulin are shown in FIG. 13 (hyaluronic acid column). Fig. 14 (sulfated hyaluronic acid column) and Fig. 15
(Heparin column).

The results are summarized in Table 1. The hyaluronic acid column did not adsorb any protein, whereas the sulfated hyaluronic acid column adsorbed fibronectin and fibronogen and eluted at different salt concentrations. The heparin column showed the same results as the S-HA affinity column, but also adsorbed albumin and eluted at the same salt concentration. Note that albumin is one of the most abundant proteins in blood, and may interfere with analysis, isolation and purification of in vivo components (eg, proteins).

[0025]

[Table 1]

Embodiment 4 FIG. Experimental separation of biological protein mixture The heparin column and the sulfated hyaluronic acid column obtained in Example 2 were buffer A (25 mM Tris-HCl, 0.5 mM
M EDTA, 1.0M NaCl; pH7.6)
(25 mM Tris-HCl, 0.5 mM EDTA, 50 mM NaCl; pH 7.6
) To equilibrate the column. As a biological protein mixture, 0.5 g of a mixture of plasma fibronectin and human serum albumin mixed at a weight ratio of 1: 1 was dissolved in 0.5 ml of buffer B to prepare a sample. This is passed through the column, and then buffer B
After washing thoroughly with 7.0 ml, non-adsorbed components were removed. 7.0m after this
l of buffer B and 7.0 ml of buffer C (25 mM Tris-HCl
, 0.5 mM EDTA, 0.5 M NaCl; pH 7.6), and a linear gradient of sodium chloride (0.05 to 0.5).
M) was performed, and then the buffer A was flowed to elute the adsorbed components. The eluate was monitored by the absorbance at 280 nm.

The elution results are shown in FIG. 16 and FIG. Elution was overlapped with the heparin column, and the two types of proteins derived from blood could not be separated, whereas the sulfated hyaluronic acid column could. In FIG. 16, the first peak from the heparin column is a peak from which the excess mixed sample has leaked, and the second peak and the shoulder are peaks from which fibronectin and human serum albumin once adsorbed are eluted together.

Embodiment 5 FIG. Experimental separation of biologically purified crude product The sulfated hyaluronic acid column obtained in Example 2 was used for buffer A (25 mM Tris-HCl, 0.5 mM EDTA, 1.0 M NaC).
l; pH7.6), and then washed with buffer B (25 mM Tris-HCl,
The column was equilibrated with 0.5 mM EDTA, 50 mM NaCl; pH 7.6). 0.5 g of bovine-derived pituitary extract (BPE) containing a growth factor was dissolved in 0.5 ml of buffer B as a biologically purified product to obtain a sample. This is applied to the column and then 7.0 ml of buffer B
And the non-adsorbed portion was removed. After this, buffer C
(25 mM Tris-HCl, 0.5 mM EDTA) +100 mM NaCl, then buffer C + 300 mM NaCl, then buffer C + 500 mM Na
The adsorbed components were eluted by flowing Cl followed by buffer C + 2M NaCl. The eluate was monitored by the absorbance at 280 nm. Further, the eluted fraction was examined for adsorbed substances by polyacrylamide electrophoresis.

FIG. 18 shows the elution results, and FIG. 19 shows the results of the electrophoresis. Specific proteins in the sulfated hyaluronic acid column BPE were specifically adsorbed and separated. According to the results of the electrophoresis, several types of proteins were adsorbed on the sulfated hyaluronic acid column. Especially about 14,000 molecular weight
Was consistent with fibroblast growth factor (FGF). Examples 1 to 5 show that the affinity adsorbent using sulfated hyaluronic acid can analyze, separate and purify proteins and biological components from raw materials or crude products containing them.

[Brief description of the drawings]

FIG. 1 is a diagram showing an elution profile when fibronectin is applied to a hyaluronic acid column and eluted.

FIG. 2 is a diagram showing an elution profile when fibronectin is applied to a sulfated hyaluronic acid column and eluted.

FIG. 3 is a view showing an elution profile when fibronectin is applied to a heparin column and eluted.

FIG. 4 is a view showing an elution profile when human serum albumin is applied to a hyaluronic acid column and eluted.

FIG. 5 is a view showing an elution profile when human serum albumin is applied to a sulfated hyaluronic acid column and eluted.

FIG. 6 is a diagram showing an elution profile when human serum albumin is applied to a heparin column and eluted.

FIG. 7 is a diagram showing an elution profile when immunoglobulin is applied to a hyaluronic acid column and eluted.

FIG. 8 is a diagram showing an elution profile when immunoglobulin is applied to a sulfated hyaluronic acid column and eluted.

FIG. 9 is a diagram showing an elution profile when immunoglobulin is applied to a heparin column and eluted.

FIG. 10 is a view showing an elution profile when fibrinogen is applied to a hyaluronic acid column and eluted.

FIG. 11 is a view showing an elution profile when fibrinogen is applied to a hyaluronic acid sulfate column and eluted.

FIG. 12 is a diagram showing an elution profile when fibrinogen is applied to a heparin column and eluted.

FIG. 13 is a diagram showing an elution profile when insulin is applied to a hyaluronic acid column and eluted.

FIG. 14 is a view showing an elution profile when insulin is applied to a hyaluronic acid sulfate column and eluted.

FIG. 15 is a graph showing an elution profile when insulin is applied to a heparin column and eluted.

FIG. 16 is a diagram showing an elution profile when fibronectin and human serum albumin were applied to a hyaluronic acid sulfate column (black triangles) and a heparin column (white circles) and eluted. The first peak in the hyaluronic acid sulfate column shows a leakage peak of unbound serum albumin, and the second peak shows an elution peak of fibronectin adsorbed on the column. In the heparin column, the first peak indicates a leak peak of the mixed sample that could not be completely absorbed, and the second peak (with a shoulder) indicates an elution peak of the adsorbed human serum albumin and fibronectin.

FIG. 17 is a drawing-substituting photograph showing the results of analyzing the components of each peak in FIG. 16 by SDS-polyacryl gel electrophoresis.

FIG. 18 shows an elution profile when bovine pituitary extract was applied to a hyaluronic acid sulfate column (closed triangles) and a heparin column (open circles) and eluted.

FIG. 19 is a drawing substitute photograph showing the result of analyzing each peak component in FIG. 18 by SDS-polyacrylamide gel electrophoresis.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/75 C07K 14/75 14/76 14/76 14/78 14/78 C08B 37/08 C08B 37 / 08 A G01N 30/48 G01N 30/48 RN 30/88 30/88 J F term (reference) 4C090 AA08 BA67 BD36 BD37 CA38 DA05 4D017 AA09 BA07 CA11 CB01 DA03 4G066 AC01B AD01B CA54 DA11 EA02 4H045 AA20 DA37 DA65 DA70 DA75

Claims (8)

[Claims] (1) The following structural formula:(Wherein, n is a number such that the molecular weight is 10,000 to 1.5 million;
R is hydrogen or SO_Three ^-M ⁺Where two sugars
Of the four Rs in the repeating unit consisting of
0.5 to 4 R are SO_Three ^-M⁺And
M⁺Are independently monovalent cations)
A) comprising sulfated hyaluronic acid or a salt thereof
A carrier for affinity adsorption. 2. The hyaluronic acid or a salt thereof according to claim 1, wherein M ⁺ is Na ⁺ . 3. The carrier for affinity adsorption according to claim 1, wherein the carrier for affinity adsorption is a carrier for affinity adsorption chromatography. 4. A sample which is expected to contain at least one kind of adsorbent which can be adsorbed on the carrier for affinity adsorption according to any one of claims 1 to 3, is brought into contact with the carrier for affinity adsorption. At the very least, the adsorbed substance is adsorbed, and if there is a non-adsorbed substance that is not adsorbed on the affinity adsorption carrier, it is removed or recovered from the affinity adsorption carrier, and if desired, the adsorption adsorbed on the affinity adsorption carrier. A method for separating or purifying an adsorbed substance or a non-adsorbed substance, comprising eluting the substance. 5. The method according to claim 4, wherein the adsorbed substance is a protein.
The separation or purification method described in 1. 6. The method according to claim 4, wherein the adhesion protein such as fibronectin is separated / fractionated or purified. 7. The method according to claim 4, wherein blood components such as fibrinogen and albumin are separated, separated or purified. 8. The method according to claim 4, wherein a cell growth factor such as fibroblast growth factor (FGF) is separated, fractionated or purified.
The method described in.