JPS6154450A - Gel for affinity chromatography having group specificity and its production - Google Patents

Abstract

PURPOSE:To obtain a gel for chromatography which maintains a solid granular state, is insoluble in an aq. solvent and has excellent physical stability by esterifying non-crosslinked cellulose by sulfuric acid. CONSTITUTION:A esterifying agent such as sulfuric acid is dissolved in amine or amide and the granular material of the non-crosslinked cellulose is brought into reaction therewith and is then neutralized with an alkali. Sulfuric anhydride or chlorosulfonic acid is preferable as the esterifying agent such as sulfuric acid and is used at about 1-50pts. (pts.wt., hereafter the same) in 100pts. the raw material cellulose and the reaction is effected at about -10-100 deg.C for about 30min-6hr. The amine or amide to be used as the solvent is exemplified by pyridine, dimethylformamide, etc. and the raw material cellulose to be added thereto so as to react therewith is used after the commercially marketed granular material having about 15-150mum grain size for chromatography is dried to about 1% moisture content. The resultant gel for affinity chromatography consists of the water-insoluble solid granular particles of the non-crosslinked cellulose sulfate having 15-150mum grain size.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to affinity chromatography gels useful for the purification of various proteins, viruses, etc., and more specifically to a group consisting of solid granular non-crosslinked cellulose sulfate ester gels. The present invention relates to a gel for affinity chromatography having specificity and a method for producing the same.

Prior Art Conventionally, centrifugation, ion exchange, gel filtration, and the like have been used to purify and obtain specific proteins, viruses, etc., and these methods are appropriately combined or repeated. By the way, when these methods are combined or repeated, each step requires complicated operations, takes a long time, and has the disadvantage that the target material is lost and reduced as the steps go through. be.

As another purification method, a so-called affinity chromatography method is also known, in which biological substances are separated and purified by chromatography using a substance that has biological affinity. Heparin, a strongly acidic mucopolysaccharide, is a substance with such biological affinity, and it has the property of binding to proteins that have various important physiological activities in vivo, such as antithrombin III. It exhibits important physiological and pharmacological effects, such as promoting blood coagulation prevention by binding to riboprotein lipase, and exhibiting lipid-serum clarifying effects by binding to riboprotein lipase.

Utilizing the physiological activity of heparin, it can be converted into Cepha-0.
A gel for affinity chromatography is prepared by binding to a chromatography gel carrier such as CL-4, 8 (manufactured by Pharmacia) using CNBr, etc., and using it, antithrombin III, coagulation factors, lipoprotein lipase, etc. It has been proposed to purify proteins with important physiological activities. Heparin is extracted and purified from animal lungs, kidneys, livers, etc., so the process is complicated, and different raw materials have different heparin properties, so it is necessary to produce large quantities of heparin with the same properties. It is difficult to obtain and extremely expensive. Therefore, the method of using affinity chromatography gel bound with heparin is currently extremely difficult to employ on an industrial scale.

1935 Jorpes et al.
Since it was revealed that the anticoagulation effect of heparin is based on the presence of sulfate groups in the molecule, attempts have been made to synthesize polymeric sulfate esters to replace heparin and prepare gels for Affinity chromatography. ing. Examples of such substances include cellulose sulfate, dekimatatrans sulfate, fundroitin boris sulfate, etc. Similar to heparin, these substances have anticoagulant effects, lipid serum clarifying effects, etc., and also bind to basic organic substances. shows the property of forming a complex. These polymeric sulfate esters have all the major properties of heparin, so they are collectively called ``heparifides.''

A method has been reported in which this heparinoid is bound to a chromatography gel using CNBr or the like to prepare an affinity chromatography gel, and this is used to purify coagulation factors, etc. (1 For example, U.S. Pat. No. 4,139 .28
No. 7, Japanese Unexamined Patent Publication No. 5'2-114018).

However, even with this method, the heparinoid itself is expensive, and the method of binding the heparinoid to the gel carrier is complicated, and the binding is relatively weak, so the bound heparinoid often detaches. be.

As a result of various studies to find an improved gel with excellent properties for use in affinity chromatography, the inventors of the present invention have developed solid granular particles of non-crosslinked cellulose in a solid granular state. The present invention was completed based on the knowledge that the gel obtained by sulfuric acid esterification without retention and neutralization with alkali has heparin-like affinity and exhibits extremely excellent properties as a gel for Affinity chromatography. reached. That is, the present invention provides a novel gel for affinity chromatography that has group specificity and is useful for purifying various proteins, viruses, etc., and a method for producing the gel.

Structure and Effects of the Invention The gel for 7 Finitei chromatography of the present invention is obtained by sulfuric acid esterification of non-crosslinked cellulose, preferably crystalline cellulose or cellulose consisting of a crystalline region and an amorphous region, which is obtained by sulfuric acid esterification of crystalline cellulose or cellulose consisting of a crystalline region and an amorphous region. It has become. In this case, the cellulose sulfate ester obtained retains the shape of the raw material, is insoluble in aqueous solvents, has excellent physical stability, and is suitable as a gel for chromatography. These raw material celluloses are already commercially available, such as Cellulofine GC-15 and Cellulofine GH-25.
, GO-100, GC-200 (manufactured by Chisso Corporation), and Avicel (manufactured by Asahi Kasei Industries, Ltd.).

In order to produce the gel for 7 Finitei chromatography of the present invention, a sulfuric acid esterifying agent is dissolved in an amine or an amide, and non-crosslinked cellulose particles are reacted with this, followed by neutralization with an alkali. It will be done.

As the sulfuric acid esterifying agent, sulfuric anhydride or chlorosulfonic acid is preferable, but sulfuric acid, concentrated sulfuric acid, fuming sulfuric acid,
Pyrosulfuric acid, sulfamic acid, etc. are used. but,
Depending on the conditions, cellulose hydrolysis may proceed.
Therefore, it is necessary to carry out the sulfuric acid esterification within a limit that at least maintains the granular state of the raw material cellulose. Therefore, the sulfuric acid esterification agent is not added in excess, but is usually used in an amount of about 1 to 150 parts per 100 parts (by weight, hereinafter referred to as solid) of raw material cellulose. The duration is approximately 30 minutes to 6 hours.

Examples of the amine or amide used as a solvent include pyridine, triethylamine, and dimethylformamide. The raw material cellulose to be reacted with this is a commercially available cellulose for column chromatography with a particle size of 15 to 150μ.
In granules are dried to a moisture content of about 1% before use.

Neutralization with an alkali is carried out using an aqueous solution or a methanolic aqueous solution of sodium hydroxide, potassium hydroxide, magnesium hydroxide, etc. at an appropriate concentration. Since this neutralization reaction is usually accompanied by heat generation, it is sufficiently cooled with ice water or dry ice ethanol refrigerant, and care is taken not to raise the reaction temperature above 50°C.

In the sulfuric acid esterification of cellulose according to the present invention, for example, as shown in the following formula, the hydroxyl group at the 6-position of the cellobiose group has the highest reactivity, so it is thought that this hydroxyl group is sulfated. However, the actual proportion of sulfation is extremely small, and as is clear from the elemental analysis values in Examples described later, about 1% of the theoretical amount of S or Na is sulfated. Therefore, the cellulose sulfate ester of the present invention is not a cellulose monosulfate ester (which is water-soluble), but only the non-crystalline region of cellulose, most of which is an extremely thin layer on the surface.
It is thought that it has been monoesterified, and it is thought that it maintains its water insolubility due to the intermolecular interaction between the sulfuric acid esterified non-crystalline cellulose and the crystalline cellulose. Note that only the extremely thin surface layer of the granular cellulose is sufficient to be involved in the affinity with proteins and the like.

The gel for 7 Finitei chromatography of the present invention obtained by the above method consists of water-insoluble solid particulate particles of non-crosslinked cellulose sulfate ester having a particle size of 15 to 150 μIII.

The group-specific affinity chromatography gel of the present invention is extremely useful for purifying various proteins and viruses. Proteins and viruses that can be effectively applied include:
For example, there are groups that have the following specificities.

Protein F-HA (Fila) produced by Bordetella pertussis etc.
mentous He+naHglutinin) and/or LPF HA (Leucocytos
ispro+noLingfacLor1+e+oag
glutinin) hepatitis B virus (HBV) and its antigen (HBc
AFl, HBsAg), or expressed antigens or proteins derived from these transformed animal cells or transformed microorganisms. Herpes simplex virus protein (H8VgB), expressed antigens or proteins derived from transformed animal cells or transformed microorganisms. Viruses (human, horse, pig, bird),
Japanese encephalitis virus, rabies virus, Ibaraki virus, bovine epidemic virus, porcine parvovirus, canine balbovirus, Aujesky virus, Newcastle virus, Gampolo virus, etc., and/or transformed animal cells or microorganisms thereof. Antithrombin III, blood coagulation factors, platelet factor 4, riboprotein lipase, or proteins with physiological activities such as complement components Enzymes derived from living organisms such as animals, plants, and microorganisms, such as urokinase and invertase In order to adsorb and extract the various proteins, viruses, etc. described above using the 7F chromatography column of the invention, operations employed in ordinary column chromatography are applied, and the conditions for the adsorption or Depends on the type of protein or virus to be eluted. However, as a general operation condition, the gel is packed into a column, p)
(s, o~9゜0, specific conductivity 0.5~25, 0+n
Equilibrate in advance with a buffer solution of s/c+++, and add to this a pH of 5.0 to 980, a temperature of O to 60°C, a specific conductivity of 0.
The protein or virus to be treated is adsorbed under conditions of 5 to 25.0 + Crystal Is/C111, then washed with the same buffer used for the equilibration or a buffer with a higher specific conductivity, and finally at pH 5.0, the ionic strength of which is greater than that of the buffer used for the equilibration and washing.
A highly purified protein or virus can be obtained by elution using a buffer solution having a temperature of ~10°0 and a specific conductivity of 5.0 to 130+ns/cm. Incidentally, the purification operation can be carried out by either a batch method or a column method.

The above purification operation will be described in more detail below, taking as an example the isolation and purification of F-HA and LPF-HA, which are proteins produced by Bordetella pertussis.

(1) Isolation and purification of F-HA The cellulose sulfate ester gel obtained in Example 1 described below is prepared by adding 0.2 M sodium chloride to the gel.
The pH value of a neutral buffer such as 1M'J acid buffer (p'86
~9), and after equilibration using an appropriate buffer solution with a specific conductivity of about 5 to 25 m5/cm, F-H
Used for adsorption operation of A.

A series of purification operations such as adsorption of F-HA onto the cellulose sulfate ester gel, washing of the gel, and elution of F-HA are carried out by commonly used industrial methods such as a batch method and a column method. When performing the batch method, add the cellulose sulfate gel to the Bordetella pertussis culture and adjust the pH to 6.
F-HA is adsorbed by stirring gently for about 10 to 60 minutes at a temperature of about 0 to 30'C in a range of about 0 to 9.0°C.

At this time, the specific conductivity of the B. pertussis culture is 5.0 to 25.
, 0 ms/c+o, as appropriate, and subjected to adsorption cycles.

After the adsorption is completed, the culture-gel mixture is filled onto a filter and filtered with suction to separate the gel and the filtrate. The separated gel has a specific conductivity of about 5 to 25+ns/eta and a pH of 5.
．． A suitable buffer solution having a concentration of about 0 to 10.0, for example, 0.2
M Sodium Chloride Added 0.02M Pine Kirbene (Me
I 1vaine's) buffer, 0.01M phosphate buffer with 0.3M sodium chloride, or 0.01M phosphate buffer with 0.3M sodium chloride. OIM) Pour lithium-hydrochloric acid buffer, etc. and aspirate to wash.

After this, l) an appropriate buffer solution having a H of about 5.0 to 10.0 and a specific conductivity of about 25 to 130 ms/c+n (greater than the specific conductivity of the above-mentioned washing buffer), for example 1. Pour 5M sodium chloride-added pine kilbene buffer, 1.5M sodium chloride-added phosphate buffer, etc. to elute the adsorbed F-HA.

When using the column method, the conditions for the raw material solution, washing buffer, and elution buffer may be the same as for the batch method.
These liquid passing rates are 10+IIl/era2/Hr-
It is advisable to adjust the pressure to about 500+ol/cm2/Hr.

According to the above method, the cellulose sulfate ester gel has excellent specific adsorption ability for F-HA in B. pertussis culture,
The degree of purification of F-HA is several tens of times higher than that of conventional methods, and the recovery rate of F-HA is 90% or more and nearly 100%.

The specific activity of the purified F-HA obtained was extremely high, 4 to 8 x 10'HA units)/B protein, and it formed a single band in polyacrylamide disk electrophoresis (pH 4, 5) analysis, indicating that Toxins are almost completely removed.

(2) LPF-) (LPF-HA-containing solution, which is the isolated and purified raw material solution of A, is obtained by mixing the centrifuged supernatant of a B. pertussis culture with distilled water or a slow iΦj solution with a specific conductivity of 0.5 to 5. After diluting to Oms/c+o,
It can also be subjected to an adsorption operation, but this supernatant contains F, which also has an affinity for cellulose sulfate gel.
-Since HA is included, L P F
- Perform chromatography using cellulose sulfate ester gel under conditions that adsorb F-HA without adsorbing HA, and use the LP that does not contain F-HA, which is the pass-through fraction.
Both portions containing a large amount of F-HA are subjected to an adsorption operation.

Adsorption of LPF-HA onto cellulose sulfate gel,
A series of purification steps including washing the gel and elution part of LPF-HA are as follows:
This can be carried out using commonly used industrial methods such as a batch method and a column method, but the column method is easier and more convenient. In the case of the column method, a cellulose sulfate ester gel is packed in a column, and the pH is adjusted to 5.0-9 with a specific conductivity of 0.5-5.0 ms/cm, such as 0.02 M pine kilbene buffer (pH 5.2).
．． After equilibration is carried out by passing a suitable buffer solution having a concentration of approximately 0.0, the LPF-HA adsorption operation is started.

During adsorption, the solution containing LPF-HA was prepared with l) H of 5.
0 to 9.0 and the specific conductivity to 0.5 to 5.0, and passed through a column packed with cellulose sulfate gel to adsorb LPF-HA. Thereafter, a buffer similar to that used for the above-mentioned equilibration is passed through to wash the gel and wash out contaminants.

For elution of LPF-14A, either 1) H or 5. ()～
Elution is carried out by passing through an appropriate buffer solution having a specific conductivity of 9.0 and a specific conductivity of 5.0 + ns/can or more, preferably stepwise elution or salt concentration gradient elution. That is, when using the diluted centrifuged supernatant of B. pertussis culture as the raw material solution, under the above-mentioned adsorption conditions,
Since F-) (A is also adsorbed at the same time as LPF-HA, it is necessary to elute under conditions where LPF-HA elutes Jt and F-HA does not elute. These conditions include 1) H5 -9 specific conductivity 5-100 ms/
am, 'preferably 50-60 ms/can in a suitable buffer (e.g. l), 7M sodium chloride added 0
．． 02M pine kilbene buffer) was first passed through the LPF.
- Collect both parts containing HA. After this, the elution buffer with a higher specific conductivity than the above elution buffer (100-300+++s
/cm) buffer solution is passed through to elute impurity components present in the F-HA, and the cellulose sulfate ester gel is equilibrated and reused.

Most preferably, a salt gradient elution is performed.

LPF from which F7HA has been separated in advance as a raw material liquid
- Even when using an HA-containing liquid, the specific conductivity is 0.
Salt concentration gradient buffer (5 → 300 m5/cm)
For example, sodium chloride O→4. OM salt concentration gradient 0.0
If elution is performed using 2M pine kilbene buffer (pH 5.2) and a fraction containing LPF-HA is collected, LPF-HA of extremely high purity can be obtained.

According to the above purification method, the degree of purification of LPF-HA reaches several tens of times compared to the conventional method, and the recovery rate of LPF-HA reaches 90% or more and nearly 100%.

The specific activity of the purified LPF-HA obtained is 9X10'LPE.
It has an extremely high protein content of U/11g protein, forms a single band in polyacrylamide disc electrophoresis (pH 4, 5) analysis, and Bordetella pertussis endotoxin is almost completely removed.

Next, the present invention will be explained in more detail by giving examples related to the production of a gel for 7 Finitei chromatography of the present invention and experimental examples related to the purification of various proteins or viruses using the gel. is not limited to these.

Example 1 20g of sulfuric anhydride in pyridine 600- at a temperature below 0°C
Add and mix. After the addition is complete, keep the mixture at 30°C. In this, Cellulofine G dried to a moisture content of 1% or less
80 g of C-15 (manufactured by Chisso Corporation) is added, and the mixture is reacted at 30° C. for 3 hours with stirring.

After the reaction is completed, the mixture is cooled and neutralized by adding a 5% aqueous sodium hydroxide solution. The gel is separated by filtration and thoroughly washed with 0.01M phosphate buffered saline to obtain a cellulose sulfate gel.

Elemental analysis value (measured value): C, 40.4%, 0.45°8
%, H, 6.3%, S, 0.15% and the sodium content is 1 + 400 mg/kg.

Example 2 117 g of chlorosulfonic acid is added dropwise to pyridine 60 (h, Q) at a temperature below 0°C and mixed. After the addition is complete,
Heat the mixture to 65-70°C.

Add 80 g of crystalline cellulose, Avicel for chromatography (manufactured by Asahi Kasei Industries, Ltd.), and add 65 to 70 g of crystalline cellulose while stirring.
Hold at 0'C for 4 hours. After the reaction is completed, cool 1
Neutralize by adding +7um aqueous solution (0% hydroxide). The gel was separated by filtration and 0. Wash thoroughly with OIM phosphate buffered saline solution to obtain cellulose sulfate ester gel.

Experimental Example 1 (Purification of HBs antigen) A column packed with the cellulose sulfate gel obtained in Example 1 (26,4+face φX 1 (') 5
0.027 containing 0.05M sodium chloride in m+J
Equilibrate through M manokilbene buffer (p) (7,20). 20 ml of HBs antigen positive human serum diluted 3 times with the above buffer solution is passed through this column.

Then, wash thoroughly with the above buffer solution. Then, 0.6
Elute with 0.027M pine kilbene buffer (pH 7.20) containing M sodium chloride. The results are shown in Table 1. As shown in Table 1, ) (Bs antigen was almost completely recovered in the elution fraction, and the degree of purification reached approximately 16 times.

Table 1 *1) Lanoium/assay (manufactured by Guinabot,
Measured with Auxeria ll-125).

(Kuroyo Seishin, Saito Haru: Japan-J, Med.

Sci, Biol, 32:47-52 (1979)) Wood 2) Contains effluent and wash liquor.

Experimental Example 2 (Purification of F-HA) Cellulose sulfate gel prepared in the same manner as in Example 1 was applied to a column (16+n+oφX100m+Il).
to which 0.2 M sodium chloride was added 0.0
1M phosphate buffer (pH 8.0, specific conductivity 17.5
ms/cm) to equilibrate. 800 J of pertussis I phase fungus Higashihama strain fermentor culture supernatant was diluted into this column, and the specific conductivity was 17.5 Qlξ/am, l.
)88, Pour the solution adjusted to 0. After passing through the solution, it is washed with the above buffer solution to wash out contaminants.

Then, 1.5M sodium chloride-added phosphate buffer (p
Elute with H7,6) to obtain 30 mj2 of both fractions containing F-HA.

The analysis results of the culture supernatant, flow-through fraction, purified F-) (A fraction) are shown in Table 2.

The recovery rate of F-HA was 93.8%, and the purity (purified F-H
The specific activity of fraction A/specific activity of culture supernatant) reached 34 times. LPF-HA activity of purified F-HA fraction was determined by HUB)EL
ISA method [Tsumi Sato Proceedings of the 28th Toxin Symposium 14
1 (1981)], 10ELISA Units)
/mj2 or less.

Table 2 J) F-HA hemagglutination test [5ato, Y, eLa
l+ I revised ecL.

Immun, 7, 929-999 (1973)]2
) Displayed as protein content using Kirbourg method protein nitrogen measurement value
The test was carried out in accordance with the Biological Products Standards (Yakuhatsu No. 287, 1981).

Experimental Example 3 (Purification of LPF-HA) A cellulose sulfate ester gel prepared in the same manner as in Example 1 was filled, and 1. Pass the Ol solution through. In this column, 50% of the centrifuged supernatant of a standing culture of Pertussis
0J diluted 10 times with distilled water (specific conductivity 1,
5 ms/cm). 0,0 of about 500J
After passing 2M pine kilbene buffer (pH 5.2) through the column and washing the gel, pine kirbene buffer containing 0.02 M sodium chloride (specific conductivity 2, 0+ns/c++
+. pH5.2) 2000- ν1, sodium chloride O→4. Elution was performed using a salt concentration gradient of OM, approximately 20 m
After fractionation and fractionation, 130 J of both fractions containing LPF-HA are pooled.

Table 3 shows the analysis results and experimental results of the raw material solution and purified LPF-HA fraction.

Table 3 1) In viLro test of LPF-HA: Hapto-
LPF-H by ELISA method [see Tsutomu Sato 28th Toxin Symposium Proceedings 141-144 (1981)]
Unit of A2) Displayed as protein content based on the protein nitrogen measurement value X6.25 using the Kirtar method.3) After dilution to a content of 6.25 μg protein/ml, it was determined to be non-defective in accordance with the Biological Product Standards (Yakuhatsu No. 287, 1981). It was revitalized and implemented.

1 Example 4 (Purification of Influenza Virus) Above Example 1
The cellulose sulfate gel obtained in
mφX17c+n), and o. 0.010M phosphate buffer containing 14M sodium chloride (pH 7.4)
) to equilibrate. This column was inoculated and cultured with 11-day-old hatched chicken eggs (34℃, 48 hours) A/Philippines (
83N2) Type influenza virus infected allantoic vaginal fluid [viral amount 77CCA, protein nitrogen amount 377 μg/m
ffi (o-saw method), specific activity (OCA/TCA-
N) 0.2014, 200Inf! ．． Pass the liquid through. This is followed by washing with 2500+J of 0.01M phosphate buffer containing 0.19M sodium chloride. Then, 1.4
Elute with 0.0-10M phosphate buffer containing 9M sodium chloride to obtain elution fraction 17 (h, 9. Then, 4.

0, OIM phosphoric acid mild +11 containing 99M sodium chloride
Elute and wash the gel with J solution.

The results are shown in Table 4.

Table 4*) CCA (cl+ick cell a
gglutination) = unit of virus amount measured by chicken hemagglutination reaction, specific activity is determined by colorimetrically quantifying the amount of protein nitrogen precipitated using trichloroacetic acid.
The ratio of the value (TCA-N) to the amount of virus (CCA) indicates the amount of virus (antigen) per amount of protein nitrogen.

As shown in Table 4, influenza virus. was recovered in the elution fraction, and the degree of purification reached approximately 20 times.

Experimental Example 5 (Purification of Japanese Encephalitis Virus) 37.5J of cellulose sulfate gel prepared in the same manner as in Example 1 was placed in a column (25mmφX 400m
n+), and 375 J of distilled water was passed through it. Next, after equilibrating the gel with 375 J of 0.14 M NaC (additional 0.01 M phosphate buffer), 50+ J of an inactivated Japanese encephalitis virus suspension obtained by protamine-end treatment was passed through this column. 0.14M NaCl addition 0
After thoroughly washing the gel with 01M phosphate buffer,
．． Elution was performed using 100 J of 100 MIJ acid buffer (specific conductivity 120 m5/etQ, pH 7.2) supplemented with 5 M NaCl, and after fractionation into approximately 5-fold fractions, both fractions containing Japanese encephalitis virus were separated. Pool 10J.

Table 5 shows the analysis results and experimental results of the raw material liquid and purified Japanese encephalitis virus fraction.

Table 5 Experimental Example 6 (Purification of Rabies Virus) Cellulose sulfate ester gel 0,141 prepared in the same manner as in Example 1 was applied to a column (2,5n++nφX 4
00 + nm), and 1400 J of distilled water was passed through it. Next, 0.14M NaCf was added. OIMIJ
After equilibrating the gel with 1,400+ njg of phosphate buffer, the inactive rabies virus suspension obtained by inoculating and proliferating primary cultured chicken embryo cells into this column was 2.9! The flow rate 5
The liquid is passed at a rate of 00ml/hour.

After thoroughly washing the gel with 0.01M phosphate buffer containing 0.14M NaCl, add 0.01 g of IMNaC.
M phosphate buffer (specific conductivity 87, 6 ms/can
, pH 7,26) using 500 mj2 and a flow rate of 1 to
After elution is carried out at 1/min and fractionated into approximately 10 J fractions, 30 J of both fractions containing rabies virus are pooled.

Table 6 shows the analysis results and experimental results of the raw material liquid and purified rabies virus fraction.

Table 6 Experimental Example 7 (Purification of Antithrombin III) Fresh frozen plasma was used as a raw material for treatment, thawed and heated at 8000 rpm.
Centrifuged for 30 minutes at m, 10% polyethylene glyfur with 1400 molecules was added to the supernatant, and stirred for 1 hour.
Centrifuge at 8000 rpm for 60 minutes. Next, this supernatant was dissolved in 0.02M phosphorus era buffer (Na2HPO4.12H
20 (0.2M, 76%) and NaH2PO4・2H20
(0.2M, 24% mixed solution).

A column packed with 0.521 cellulose sulfate gel prepared in the same manner as in Example 1 (1°5 canφ)
After equilibrating the gel by passing the same 0.02 M phosphate buffer as above into the gel, 20 mff1 of the dialyzed plasma supernatant (pH 5.0) was passed through the gel. Next, the column was washed with the same buffer as above until the absorbance of the effluent decreased sufficiently, and then the elution buffer (0.02
Elute with M phosphate buffer +2, OM sodium chloride). The absorbance was measured at 280 nm (OD
2. . ) are obtained, showing two 7-raction peaks at ). Table 7 shows the results of analysis of this 7-luxion.

Table 7

Claims (4)

[Claims] (1) A gel for affinity chromatography having group specificity obtained by converting solid particles of non-crosslinked cellulose into a sulfuric acid ester while maintaining its solid particle state and then neutralizing it with an alkali. (2) The gel according to item (1) above, wherein the non-crosslinked cellulose is crystalline cellulose or cellulose consisting of a crystalline region and an amorphous region. (3) dissolving a sulfate esterification agent in an amine or amide and reacting it with solid particles of non-crosslinked cellulose;
A method for producing a gel for affinity chromatography having group specificity, which comprises then neutralizing with an alkali. (4) The method according to item (3) above, wherein the sulfuric acid esterifying agent is sulfuric anhydride or chlorosulfonic acid.