JPH0423751B2 - - Google Patents

Description

[Detailed description of the invention]

Industrial Application Field The present invention relates to an affinity chromatography gel useful for purifying various proteins, viruses, etc.
More specifically, the present invention relates to a gel for affinity chromatography having group specificity consisting of a sulfate ester gel of solid particulate non-crosslinked cellulose, and a method for producing the same. Prior Art Conventionally, centrifugation methods, ion exchange methods, gel filtration methods, etc. have been employed to purify and obtain specific proteins, viruses, etc., and these methods are appropriately combined or used repeatedly. 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 are carried out. 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 having biological affinity. Heparin, a strongly acidic mucopolysaccharide, is a substance with such biological affinity, and it has the property of binding with proteins that have various important physiological activities in vivo, such as antithrombin and It exhibits important physiological and pharmacological effects, such as promoting blood coagulation prevention by binding to lipoprotein lipase, and exerting a blood serum clarifying effect by binding to lipoprotein lipase. Utilizing the physiological activity of heparin, it is bonded to a gel carrier for chromatography such as Sepharose CL-4B (manufactured by Pharmacia) using CNBr or the like to prepare a gel for affinite chromatography. antithrombin, using
It has been proposed to purify proteins with important physiological activities such as coagulation factors and lipoprotein lipase. However, since heparin is extracted and purified from animal lungs, kidneys, livers, etc., the process is complicated, and the properties of heparin differ depending on the raw material, so it is difficult to obtain large quantities of heparin with the same properties. It is difficult and extremely expensive. Therefore, it is currently extremely difficult to employ a method using an affinity chromatography gel bound with heparin on an industrial scale. In 1935, Jorpes et al. discovered that the anticoagulant effect of heparin is based on the presence of a sulfate group in the molecule. Since then, a high-molecular sulfate ester has been synthesized to replace heparin, and Affinitei chromatographs have been developed. Attempts have been made to prepare gels for this purpose. Examples of such substances include cellulose sulfate, dextran sulfate, and chondroitin polysulfate. Like heparin, these substances have anticoagulant effects and lipid serum clarifying effects, and also combine with basic organic substances to form complexes. Indicates the property of forming a body. Since these polymeric sulfate esters have all the major properties of heparin, they are collectively called "heparinoids." 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. (for example, U.S. Pat. No. 4,138,287) , Japanese Patent Publication No. 52-114018). However, even with this method, heparinoid itself is expensive, the method for binding heparinoid to a gel carrier is complicated, and the binding is relatively weak, so that the bound heparinoid often detaches. Purpose of the Invention As a result of various studies conducted by the present inventors in order to find an improved gel with excellent properties for use in affinity chromatography, the present inventors have discovered that solid granular particles of non-crosslinked cellulose can be maintained in their solid granular state. However, it was discovered that the gel obtained by sulfuric acid esterification and neutralization with alkali has heparin-like affinity and exhibits excellent properties as a gel for Affinity chromatography, which led to the completion of the present invention. . 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 Affinity chromatography of the present invention is obtained by sulfate-esterifying non-crosslinked cellulose, preferably crystalline cellulose or cellulose consisting of crystalline and amorphous regions. This is what I did. 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,
These include the GH-25, GC-100, GC-200 (manufactured by Chitsuso), and Avicel (manufactured by Asahi Kasei Industries). In order to produce the gel for Affinity chromatography of the present invention, the sulfuric acid esterification agent is dissolved in an amine or amide, reacted with non-crosslinked cellulose particles, and then neutralized with an alkali. . As the sulfuric acid esterification agent, sulfuric anhydride or chlorosulfonic acid is preferred, but sulfuric acid, concentrated sulfuric acid, fuming sulfuric acid, pyrosulfuric acid, sulfamic acid, and the like may also be used. However, depending on the conditions, in order to advance the hydrolysis of cellulose, 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, we do not add an excessive amount of sulfate esterification agent and use the usual raw material cellulose.
It is used in an amount of about 1 to 150 parts per 100 parts (parts by weight, same hereinafter), the reaction temperature is about -10 to 100°C, and the reaction time is about 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 granular material for column chromatography with a particle size of approximately 15 to 150 μm and a moisture content of 1%.
Use after drying to a certain extent. 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 exothermic heat, it should be sufficiently cooled with ice water or dry ice ethanol refrigerant, and care should be taken not to raise the reaction temperature above 50°C. The sulfate esterification of cellulose according to the present invention includes:
For example, the following formula Since the hydroxyl group at the 6-position of the cellobiose group has the highest reactivity as shown in , 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 the 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 only an extremely thin layer on the surface. It is thought that the cellulose is monoesterified, and it is thought that water insolubility is maintained due to the intermolecular interaction between the sulfuric acid esterified amorphous 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 affinity chromatography gel of the present invention obtained by the above method consists of water-insoluble solid particulates of non-crosslinked cellulose sulfate having a particle size of about 15 to 150 μm. The group-specific affinity chromatography gel of the present invention is extremely useful for purifying various proteins and viruses. Protein, effectively applied
For example, there are groups of viruses that have the following specificities. Protein F-HA produced by Bordetella pertussis etc.
(Filamentous Hemagglutinin) and/or
LPF-HA (Leucocytosis promoting factor)
hemagglutinin) Hepatitis B virus (HBV) and its antigens (HBcAg, HBsAg), or expressed antigens or proteins derived from transformed animal cells or transformed microorganisms Herpes simplex virus protein (HSVgB), or transformed animal cells thereof Expressed antigens or proteins derived from or transformed microorganisms Influenza virus (human, horse, pig,
avian), Japanese encephalitis virus, rabies virus, Ibaraki virus, bovine epidemic virus, porcine parvovirus, canine parvovirus, Aujeski virus, New Katsuru virus, Gambolo disease virus, etc., and/or transformed animal cells or Substances produced by transformed microorganisms Proteins with physiological activity such as antithrombin, blood coagulation factors, platelet factor 4, lipoprotein lipase, or complement components; Products derived from living organisms such as animals, plants, and microorganisms such as urokinase and invertase. Adsorption of the above various proteins, viruses, etc. using the gel for Affinity chromatography of the present invention.
For elution, operations employed in ordinary column chromatography are applied, but the conditions differ depending on the type of protein or virus to be adsorbed or eluted. However, as a general operating condition, the gel is packed in a column, equilibrated in advance with a buffer solution with a pH of 5.0 to 9.0 and a specific conductivity of 0.5 to 25.0 ms/cm, and then the gel is pre-equilibrated with a buffer solution of pH 5.0 to 9.0 and a specific conductivity of 0.5 to 25.0 ms/cm. The protein or virus to be treated is adsorbed under conditions of 0 to 60°C and a specific conductivity of 0.5 to 25.0 ms/cm, and then the same buffer as that used for the equilibration or a buffer with a higher specific conductivity is used. Finally, the ionic strength is higher than the buffer used for the above equilibration and washing, pH5.0-10.0, specific conductivity 5.0-130m.
Highly purified proteins or viruses can be obtained by elution using a buffer solution of s/cm. Incidentally, the purification operation can be carried out by either a batch method or a column method. The above purification operation will be explained 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 later is prepared in advance using a 0.01M phosphate buffer solution containing 0.2M sodium chloride with a pH value near neutrality (PH6 to 9). , and the specific conductivity is 5 to 25 m.
After equilibration using a suitable buffer solution of approximately s/cm, the sample is subjected to an F-HA adsorption operation. A series of purification operations such as adsorption of F-HA onto cellulose sulfate ester gel, washing of the gel, and elution of F-HA are carried out by commonly used industrial methods such as batch method and column method. When using the batch method, add cellulose sulfate gel to the Bordetella pertussis culture and
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 9.0°C. At this time, the specific conductivity of the B. pertussis culture was
The mixture is appropriately concentrated or diluted to a rate of about 5.0 to 25.0 ms/cm and then subjected to an adsorption operation. After the adsorption is completed, the culture-gel mixture is filled onto a strainer, and the gel and liquid are separated by suction. The separated gel has a specific conductivity of 5 to 25 ms/cm.
An appropriate buffer with a pH of about 5.0 to 10.0, such as 0.02M McIlvaine's buffer with 0.2M sodium chloride, 0.01M phosphate buffer with 0.3M sodium chloride, or
Pour 0.01M Tris-HCl buffer containing 0.3M sodium chloride and aspirate to wash. After this, the PH is about 5.0 to 10.0 and the specific conductivity is
A suitable buffer solution having a specific conductivity of about 25 to 130 ms/cm (greater than the specific conductivity of the above-mentioned washing buffer solution), e.g.
Pinekirbene buffer with 1.5M sodium chloride,
Pour phosphate buffer containing 1.5M sodium chloride 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, and the flow rate for these is 10 ml/
It is best to adjust the amount to about cm ² /Hr to 500ml/cm ² /Hr. According to the above method, cellulose sulfate ester gel has excellent specific adsorption ability for F-HA in B. pertussis culture, and the degree of purification of F-HA is several tens of times higher than that of the conventional method. -The recovery rate of HA is
90% or more, reaching close to 100%. The resulting purified F
-Specific activity of HA is 4-8×10 ⁴ HA units/mg
It is extremely high in protein and forms a single band in polyacrylamide disc electrophoresis (PH4.5) analysis, and B. pertussis endotoxin is almost completely removed. (2) Isolation and purification of LPF-HA The raw material solution, LPF-HA-containing solution, is obtained by preparing the centrifuged supernatant of a B. pertussis culture with distilled water or a buffer solution until the specific conductivity is 0.5 to 5.0 ms/cm. It is also possible to perform an adsorption operation after diluting the supernatant, but since this supernatant contains F-HA, which also has an affinity for cellulose sulfate gel, LPF-HA must be diluted in advance. Perform chromatography using cellulose sulfate gel under conditions that adsorb F-HA without adsorbing it, and adsorb the fraction that does not contain F-HA but contains a large amount of LPF-HA, which is the pass-through fraction. Subject to operation. LPF-HA to cellulose sulfate gel
A series of purification operations such as adsorption of , gel washing, and elution of LPF-HA can be performed using commonly used industrial methods such as the batch method and column method, but the column method is easier. This is convenient. In the case of the column method, the cellulose sulfate ester gel is packed into the column, and the column is pre-filled with, for example, 0.02M pine kilbene buffer (PH5.2).
PH is 5.0 to 9.0 with specific conductivity of 0.5 to 5.0 ms/cm.
After equilibrating by passing a suitable buffer solution to a certain extent, the LPF-HA adsorption operation is started. During adsorption, the pH of the solution containing LPF-HA is
5.0 to 9.0 and the specific conductivity to 0.5 to 5.0, and the solution is 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. When eluating LPF-HA, the pH is 5.0 to 9.0,
Elution is carried out by passing a suitable buffer solution having a specific conductivity of 5.0 ms/cm or more, and stepwise elution or salt concentration gradient elution is preferably carried out. That is,
When using the diluted centrifuged supernatant of B. pertussis culture as the raw material solution, under the adsorption conditions described above, LPF-HA and F-
Since HA is also adsorbed, it is necessary to elute under conditions where LPF-HA is eluted and F-HA is not eluted. For this condition, PH5-9
A suitable buffer (e.g.
A 0.02M pine kilbene buffer containing 0.7M sodium chloride) is first passed through the tube, and a fraction containing LPF-HA is collected. After this, a buffer with higher specific conductivity (100 to 300 ms/cm) than the elution buffer mentioned above is passed through to elute F-HA and other impurity components, and the cellulose sulfate ester gel is equilibrated and ready for reuse. provide Most preferably, a salt gradient elution is performed. Even when using an LPF-HA containing solution from which F-HA has been separated in advance as a raw material solution, a salt concentration gradient buffer (for example, sodium chloride 0 →
If you perform elution using a 4.0M salt concentration gradient and 0.02M pine kilbene buffer (PH5.2) and collect the LPF-HA-containing fraction, you can obtain extremely pure LPF.
- HA can be obtained. According to the above purification method, the degree of purification of LPF-HA is several tens of times higher than that of the conventional method, and
The recovery rate of HA reaches over 90% and nearly 100%.
The specific activity of the purified LPF-HA obtained is 9×
It has an extremely high protein content of 10 ⁴ LPEU/mg protein, forms a single band in polyacrylamide disc electrophoresis (PH4.5) 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 gels for affinity chromatography of the present invention and experimental examples related to the purification of various proteins or viruses using the gels. Not limited to these. Example 1 Add sulfuric anhydride to 600 ml of pyridine at a temperature below 0°C.
Drop 20g and mix. After dropping, add 30% of the mixture
Keep at ℃. Add 80g of Cellulofine GC-15 (manufactured by Chitsuso Corporation) dried to a moisture content of 1% or less to this,
React for 3 hours at 30°C with stirring. After the reaction is complete,
Cool and neutralize by adding 5% aqueous sodium hydroxide solution. The gel is over-separated and thoroughly washed with 0.01M phosphate buffered saline to obtain a cellulose sulfate gel. Elemental analysis value (measured value): C, 40.4%, O, 45.8%,
H, 6.3%, S, 0.15% and sodium content is 1400mg/Kg. Example 2 117 g of chlorosulfonic acid is added dropwise to 600 ml of pyridine at a temperature below 0° C. and mixed. After finishing dropping,
Heat the mixture to 65-70°C. Add 80 g of crystalline cellulose, Avicel for chromatography (manufactured by Asahi Kasei Industries, Ltd.), and heat the mixture at 65-70°C while stirring.
Protect for 4 hours. After the reaction is complete, cool and reduce to 10%
Neutralize by adding aqueous sodium hydroxide solution. The gel is over-separated and thoroughly washed with 0.01M phosphate buffered saline to obtain a cellulose sulfate gel. Experimental Example 1 (Purification of HBs antigen) A column (26.4 mmφ x 105 mm) filled with the cellulose sulfate gel obtained in Example 1 was
Equilibrate through 0.027M pine kilbene buffer (PH7.20) containing 0.05M sodium chloride. Add 20 ml of HBs antigen positive human serum to this column with the above buffer solution.
Pass the solution diluted to twice the volume. Then, wash thoroughly with the above buffer solution. Then, 0.027M pine kilbene buffer (PH
7.20). The results are shown in Table 1. As shown in Table 1, most of the HBs antigen was recovered in the elution fraction, and the degree of purification reached approximately 16 times.

[Table] Experimental Example 2 (Purification of F-HA) Cellulose sulfate ester gel prepared in the same manner as in Example 1 was packed into a column (16 mmφ x 100 mm), and 0.01 M sodium chloride was added to the column (16 mmφ x 100 mm).
Phosphate buffer (PH8.0, specific conductivity approximately 17.5ms/cm)
Equilibrate by passing the solution through. Through this column, dilute 800 ml of culture supernatant of Pertussis faecium Higashihama strain fermenter and adjust the specific conductivity to approximately 17.5 ms/cm and pH 8.0. After passing through the solution, it is washed with the above buffer solution to wash out contaminants. Then, elute with phosphate buffer (PH7.6) containing 1.5M sodium chloride,
Obtain 30 ml of fraction containing F-HA. Culture supernatant, flow-through fraction, purified F-HA
The analysis results of the fractions are shown in Table 2. The recovery rate of F-HA was 93.8%, and the purity (purified F-HA) was 93.8%.
- specific activity of HA fraction/specific activity of culture supernatant) reached 34 times. The LPF-HA activity of purified F-HA fraction is
Analysis using the Hapto ELISA method ``Sato et al., Proceedings of the 28th Toxin Symposium 141 (1981)'' showed that the amount was 10 ELISA units/ml or less.

[Table] Experimental Example 3 (Purification of LPF-HA) Cellulose sulfate ester gel prepared in the same manner as in Example 1 was packed into a column (40 mmφ x 200 mm), and 1.0 g of distilled water was passed through it. In this column, 500 ml of the centrifuged supernatant of a static culture of Pertussis faecium Higashihama strain was diluted 10 times with distilled water (specific conductivity: approx.
1.5ms/cm). Approximately 500 ml of 0.02 M pine kilbene buffer (PH5.2) was passed through the column to wash the gel, and then 0.02 M pine kirbene buffer (specific conductivity approximately 2.0 ms/cm, PH
5.2) Using 2000 ml, elute with a salt concentration gradient from 0 to 4.0 M sodium chloride, fractionate approximately 20 ml each, and collect approximately 130 fractions containing LPF-HA.
Pool ml. Table 3 shows the analysis results and experimental results of the raw material solution and purified LPF-HA fraction.

[Table] Experimental Example 4 (Purification of Influenza Virus) The cellulose sulfate ester gel obtained in Example 1 was packed into a column (5 cmφ x 17 cm), and 0.010 M phosphate buffer containing 0.14 M sodium chloride ( PH7.4) and equilibrate. This column was inoculated with 11-day-old hatched chicken eggs and cultured (34℃, 48 hours).
A/Philippine ( _H3N2 ) _type influenza virus infection allantoic fluid [viral load 77CCA,
4,200 ml of protein nitrogen content (377 μg/ml (Lowry method), specific activity (CCA/TCA-N) 0.20) was passed through. This is followed by washing through 2500 ml of 0.01M phosphate buffer containing 0.19M sodium chloride. Then, 1.49M
Elute with 0.010M phosphate buffer containing sodium chloride to obtain 170 ml of eluate fraction. Then, elute and wash the gel with 0.01M phosphate buffer containing 4.99M sodium chloride. The results are shown in Table 4.

[Table] As shown in Table 4, most of the 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) 37.5 ml of cellulose sulfate gel prepared in the same manner as in Example 1 was added to a column (25 mmφ x 400
mm) and pour 375 ml of distilled water through it.
Next, 375 ml of 0.01 M phosphate buffer with 0.14 M NaCl added.
After equilibrating the gel, 50 ml of an inactivated Japanese encephalitis virus suspension obtained by treatment with protamine and charcoal powder was passed through the column. 0.14M NaCl addition 0.01M
After washing the gel thoroughly with phosphate buffer,
100M phosphate buffer with 1.5M NaCl (specific conductivity approx.
Elution is performed using 100 ml (120 ms/cm, PH7.2), fractionated into approximately 5 ml portions, and approximately 10 ml of fractions containing Japanese encephalitis virus are pooled. Table 5 shows the analysis results and experimental results of the raw material liquid and purified Japanese encephalitis virus fraction.

[Table] Experimental Example 6 (Purification of Rabies Virus) Cellulose sulfate gel 0.14 prepared in the same manner as in Example 1 was applied to a column (2.5 mmφ x 400
mm) and pour 1400ml of distilled water through it.
Next, 1400 ml of 0.01 M phosphate buffer with 0.14 M NaCl added.
After equilibrating the gel, 2.9 g of an activated rabies virus suspension obtained by inoculating and proliferating primary cultured chicken embryo cells was passed through the column at a flow rate of 500 ml/hour. After thoroughly washing the gel with 0.01M phosphate buffer containing 0.14M NaCl, add 0.01M 1M NaCl.
Phosphate buffer (specific conductivity 87.6ms/cm, PH7.26)
Elution was performed using 500 ml at a flow rate of 1 ml/min.
After fractionating into 10 ml portions, approximately 30 ml of 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] Experimental example 7 (Purification of antithrombin) Fresh frozen plasma was used as a processing raw material. It was thawed and centrifuged at 8000 rpm for 30 minutes. To the supernatant, 10% polyethylene glycol with a molecular weight of 4000 was added and incubated for 1 hour. Stir and centrifuge at 8000 rpm for 60 minutes. This supernatant _was then mixed with 0.02M phosphate buffer ( _{Na2HPO4.12H2O} (0.2M, 76%)) _.
Dialyze against a mixture of NaH ₂ PO ₄ and 2H ₂ O (0.2M, 24%). A column packed with cellulose sulfate gel 0.52 (1.5
After equilibrating the gel by passing the same 0.02 M phosphate buffer as above into the gel (cmφ x 30 cm), 20 ml of the dialyzed plasma supernatant (PH 5.0) is passed through it. Then,
After washing the column with the same buffer as above until the absorbance of the effluent drops sufficiently, it is eluted with an elution buffer (0.02M phosphate buffer + 2.0M sodium chloride). Fractionate into approximately 10ml portions and absorbance at 280nm
Two fractions are obtained showing an absorption peak at (OD ₂₈₀ ). Table 7 shows the results of analysis of this fraction.

【table】

Claims (1)

[Scope of Claims] 1. A gel for Affinity chromatography having group specificity obtained by converting solid particles of non-crosslinked cellulose into sulfuric acid ester while maintaining the solid particle state and then neutralizing with 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. A gel for Affinity chromatography having group specificity characterized by dissolving a sulfuric acid esterification agent in an amine or amide, reacting it with solid particles of non-crosslinked cellulose, and then neutralizing with an alkali. Manufacturing method. 4. The method according to item 3 above, wherein the sulfuric acid esterifying agent is sulfuric anhydride or chlorosulfonic acid.