JPH06800B2 - Purification method of human interferon-β - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for purifying human interferon-β, and in particular human interferon-β using a specific adsorption carrier.
It relates to a method for purifying β.

(Conventional Technology) Interferon was synthesized by Nagano et al. (Compt.Rend.Soc.Biol., 148
Vol., 1700, 1954) and Isaacs et al. (Proc.Roy.Soc.S).
er.B, 147, 258, 1957), which is an antiviral substance, and has recently been reported to have various biological actions including an antitumor effect (Gressor, I.
Et al., BBA, vol. 516, p. 231, 1978), and its potential as a medicine has been attracting attention.

The production of interferon is generally performed by cell culture or gene recombination method, but the content of interferon produced is usually very small, and therefore, to obtain interferon that can be used as a drug,
It is necessary to establish a purification method that can obtain highly purified interferon using the crude interferon solution as a starting material.

By the way, various methods for purifying human interferon have been reported so far, for example, interferon-α.
For the zinc chelate column chromatography method (Rubinstein, M. et al., Proc. Natl. Acad. Sci. USA, 76).
Vol. 640, 1979) and the Blue Sepharose column chromatography method (Zoon, KC et al. Proc. Natl. Acad. Sci.
USA, 76, 5601, 1979) have been reported. Regarding human interferon-β, a method using ammonium sulfate salting-out method, gel filtration chromatography method and CM Sephadex column chromatography method (Knight, E. Jr., Proc. Natl. Acad. Sci. US
A., 73, p. 520, 1976) and SP Sephadex column chromatography method and zinc chelate column chromatography method (Kazuo Hosoi et al., JP-A-55).
-64799, 1980), etc. have been reported. Further, as a method for purifying interferon-γ, a porous glass chromatography method, a concanavalin A agarose column chromatography method or a combination of these methods (Yip, YK et al., Proc. Natl. Acad. Sci. USA, 78).
Vol., P. 1601, 1981).

(Problems to be Solved by the Invention) However, these conventionally reported various purification methods alone cannot sufficiently increase the degree of purification, and thus obtain interferon having a purity that can be used as a drug. For this purpose, it is unavoidable that a plurality of different purification methods are used in combination, and therefore, even if the finally obtained interferon is purified to a high purity, the yield thereof is extremely small. It wasn't.

As a result of repeated studies on a method for obtaining highly purified human interferon-β in high yield, the inventors of the present invention obtained a highly purified human interferon with only one purification operation if it was purified using a specific adsorption carrier. The present invention has been completed by finding that -β can be obtained in high yield.

(Means for Solving Problems) That is, according to the present invention, crude human interferon solution is brought into contact with an adsorption carrier, and the human interferon-β is adsorbed on the adsorption carrier.
Of human interferon-β in the eluate
In a method for purifying human interferon-β, which comprises elution of amino acids, a multimer of an amino acid having an aromatic ring as a ligand and / or an amino acid derivative having an aromatic ring as a ligand in a water-insoluble carrier made of an organic high molecular weight substance as an adsorption carrier. And a method for purifying human interferon-β using an adsorption carrier having a compound having an amino group or a carboxyl group capable of binding to a spacer molecule bound to the carrier.

Implementation of the present invention in this case is generally performed as follows. That is, the crude human interferon-β-containing crude human interferon-β solution is brought into contact with the adsorption carrier by a column method or a batch method known per se to adsorb the human interferon-β on the adsorption carrier, and then, into an appropriate buffer solution. Human interferon-β is purified by washing the adsorbent carrier with a suitable eluent and then eluting human interferon-β.

Therefore, the present invention is characterized in that a specific adsorption carrier is used in the adsorption-elution of human interferon-β.

(Operation) As described above, the adsorption carrier used in the present invention is an adsorption carrier composed of a water-insoluble carrier portion composed of an organic high molecular weight substance, a spacer portion, and a ligand portion.

In this case, examples of the water-insoluble carrier part constituting the adsorption carrier include agarose, cellulose, dextran or polyacrylamide and other organic high molecular weight compounds,
Although any of them can be used, it is particularly preferable to use agarose as the carrier part.

On the other hand, the ligand used in the present invention has a multimer of an amino acid having an aromatic ring and / or an amino acid derivative having an aromatic ring, and binds to a water-insoluble carrier through the amino group or carboxyl group possessed by the ligand. A compound capable of binding to a spacer molecule.

The amino acid having an aromatic ring is not limited to α-amino acids, but other amino acids, for example, β-amino acids, γ-amino acids, δ-amino acids and the like can be used.
Generally, α-amino acids are easily available and preferred. Among the α-amino acids having an aromatic ring, L-tryptophan, D-tryptophan, L-tyrosine, D-tyrosine, L-phenylalanine or D-phenylalanine is particularly preferable. These amino acids can be used as a multimer composed only of the same amino acid or a multimer composed of at least two kinds of amino acids. The multimer is preferably a dimer or trimer.

In these amino acid multimers, it is also possible to use a derivative of the amino acid multimer in which the free carboxyl group contained is alkyl esterified, preferably methyl esterified, ethyl esterified or propyl esterified as a ligand. is there.

Each compound used as a ligand can be purchased from, for example, Wako Pure Chemical Industries, Ltd., Kokusan Kagaku Co., Ltd. or Hanai Chemical Co., Ltd. The multimer can be purchased from Kokusan Kagaku Co., Ltd., or can be synthesized by a condensation reaction using carbodiimide. As the carbodiimide, N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide hydrochloride (Nippon Pio-Rad Laboratories) or the like can be used.

The compound that can be used as the ligand is not limited to the compounds shown above, but includes a multimer of an amino acid having an aromatic ring and an amino acid derivative having an aromatic ring as a constituent component of the compound molecule, and Any compound can be used essentially as long as it is a compound capable of binding to a water-insoluble carrier via a spacer molecule via an amino group or a carboxyl group in the compound molecule.

The adsorption carrier used in the present invention is a carrier in which a ligand is bound to a water-insoluble carrier portion through a spacer.

As a method for binding the ligand to the water-insoluble carrier via a spacer, a means for introducing by causing a condensation reaction between the amino group or carboxyl group of the ligand and the amino functional group or carboxyl functional group of the spacer molecule Is adopted.

When the ligand is introduced via the spacer molecule, the length of the spacer is preferably 2 to 15 in terms of the total number of atoms in the straight chain portion composed of carbon, nitrogen, oxygen or sulfur base paper, Spacers of length 5 to 15 are particularly preferred.

The introduction of various spacer molecules into the water-insoluble carrier can be performed by chemical synthesis, but in general, it is also possible to use commercially available products of the water-insoluble carrier already having a spacer as described below. . For example, Affi Gel 10 which is an N-hydroxysuccinimide activated ester derivative of agarose. Affi-Gel 15 (All are Japan Bio-Rad Laboratories) and activated CH-Sepharose 4B (Pharmacia Japan Co., Ltd.) and Affi-gel 202 having a free carboxyl group or amino group at the end. Affi-Gel 102 (All are Japan Bio-Rad Laboratories), AH-
Sepharose 4B CH-Sepharose 4B (Both Pharmacia Japan Co., Ltd.) and the like can be used.

The amount of the ligand introduced into the water-insoluble carrier to which the spacer molecule is bound varies depending on the water-insolubility and the type of the spacer when the ligand is introduced by, for example, a condensation reaction using carbodiimide.
5 to 20 μmole per ml is preferred. When the ligand is introduced into the N-hydroxysuccinimide activated ester derivative, the amount is preferably 5 to 15 μmole per ml of the water-insoluble carrier. A specific method for introducing a ligand into a water-insoluble carrier via a spacer molecule is usually performed as follows. For example, when a ligand is introduced into an N-hydroxysuccinimide activated ester derivative, the carrier and the ligand are mixed at room temperature under pH 5 to 10 at room temperature.
It is carried out by reacting for 4 hours to 4 hours or at 4 ° C. overnight.

As described above, the method for purifying human interferon-β of the present invention is carried out by using an adsorption carrier in which a ligand is bound to a water-insoluble carrier composed of an organic high molecular weight substance as an adsorption carrier. It is preferable to use an adsorption carrier in which a ligand is bound to a water-insoluble carrier through.

(Example) The method for purifying human interferon-β of the present invention comprises contacting a crude human interferon solution with the adsorption carrier prepared as described above, adsorbing human interferon-β on the adsorption carrier, and then eluating the solution. The method consists of eluting the adsorbed human interferon-β, and each specific step is carried out as follows, for example.

That is, in the case of contacting a solution containing human interferon-β (crude human interferon solution) with the adsorption carrier, the pH at the time of contact is preferably 4 to 9, and for example, 0.01M phosphate buffer of pH 7 is used. Can be used as a solvent. Further, the ionic strength of the human interferon-β-containing solution when contacting with the adsorption carrier is not particularly limited, and for example, when the ionic strength is adjusted with sodium chloride, the final concentration thereof is 0.14M. Human interferon-β can be adsorbed on an adsorption carrier. Further, when the ionic strength is further increased, for example, when sodium chloride is added to a final concentration of 3M, human interferon-β can be sufficiently adsorbed on the adsorption carrier. The contact between the adsorption carrier and the human interferon-β-containing liquid can be carried out by a batch method or a column method, but the column method is preferable because of high purification efficiency. The adsorption carrier that adsorbs human interferon-β is
For the purpose of removing non-adsorbed mixed proteins and the like, before the operation of eluting human interferon-β, a sufficient amount of a buffer solution, for example, 0.14 M sodium chloride at pH 7 of 0.
It is preferable to wash with 01M phosphate buffer.

Next, as a method for eluting human interferon-β from the adsorption carrier on which human interferon-β has been adsorbed, a liquid polyhydric alcohol such as ethylene glycol or propylene glycol in 30 to 80% (w / v) of pH 4 to 8 is used. It can be carried out using a buffer.

Although highly purified human interferon-β is obtained as described above, human interferon-β that can be purified by the above method of the present invention is not only human interferon-β produced by the cell culture method, but also the gene Human interferon produced using recombinant technology-
It is also possible to purify β, for example, human interferon-β produced by incorporating the human interferon-β gene into E. coli, yeast or mammalian cells.

Since the adsorption carrier used in the purification method of the present invention is capable of adsorbing sugar-free human interferon-β produced by Escherichia coli, the binding site of the adsorption carrier in the human interferon-β molecule is sugar. Inferred to be the peptide part, not the part. Therefore, the adsorption carrier of the present invention can be used not only for the purification of natural human interferon-β, but also for the purification of human interferon-β whose amino acid sequence is slightly changed or human interferon-β whose sugar moiety is changed. Is expected.

Thus, the adsorption carrier used in the present invention is not only for purifying a crude human interferon solution, but also for the purpose of further increasing the degree of purification of human interferon-β partially purified by other known purification means. It is possible to use. It is also possible to completely remove the mixed protein other than human interferon-β by using various adsorption carriers used in the present invention in combination.

Hereinafter, the present invention will be described more specifically with reference to Experimental Examples and Examples, but the present invention is not limited to these Experimental Examples and Examples. The titers of human interferon shown in Examples and Examples are CPE (Cytopathic Eff) using FL cells and Sindbis virus.
ect) method. The titer of human interferon-α is the international standard of human interferon-α (GO23
-901-527) was used as a reference, and the titer of human interferon-β was shown using the international standard of human interferon-β (GO23-902-527) as a reference.

Experimental Example 1 After washing 40 ml of Affi-Gel 15 with cold distilled water 4, 0.05
Further wash with M phosphate buffer (pH 6) and use the same buffer
Suspended to 40 ml. This suspension was divided into 10 ml aliquots, and each was adjusted to 1 mM, 5 mM, 20 mM or 40 mM D-
10 ml of an aqueous phenylalanyl-D-phenylalanine solution was added, and the mixture was stirred at 4 ° C. for 18 hours to prepare adsorbent carriers having different amounts of introduced D-phenylalanyl-D-phenylalanine as a ligand. did. The amount of ligand bound to the carrier was calculated by subtracting the amount of unadsorbed ligand obtained by measuring the absorbance of the solution and the washing solution after the completion of the ligand binding reaction from the total amount of ligand used in the binding reaction. Each adsorption carrier prepared as described above contained 0.14 M sodium chloride.
After washing with 01M phosphate buffer (pH 7), each was packed in a column having a diameter of 1.5 cm. Then Vilcek et al. (Antimicrob.Ag.Ch
Emother., Vol. 2, p. 476, 1972) Produced normal human fibroblast MRC-5 cell crude interferon-β solution 500 ml (specific activity 6.3 × 10 ⁴ units / mg, total interferon titer 7.2) × 10 ⁶ units). Next, after washing the column having adsorbed interferon with the above-mentioned pH 7 buffer solution, the adsorbed interferon is removed by passing the above-mentioned pH 7 buffer solution containing 50% (w / v) ethylene glycol. It eluted.

As a result of purification using each adsorption carrier having a different amount of bound ligand, the recovery rate and specific activity of the purified interferon in the eluate obtained are shown in Table 1.

Experimental Example 2 For the purpose of investigating the length of the spacer portion in the adsorption carrier that affects the degree of purification and recovery of interferon, L-tryptophyll-L-tryptophan methyl ester was used as a ligand, and the length of the spacer bound with this ligand was determined. Different adsorption carriers were made.

An adsorption carrier containing no spacer was prepared as follows. That is, a commercially available bromocyan-activated Sepharose 4B (Pharmacia Japan Co., Ltd.) was swollen in a 1 mM hydrochloric acid solution, washed with the same solution, and then a cup made of a 0.1 M carbonate buffer solution (pH 8.3) containing 0.5 M sodium chloride. The ligand dissolved in the ring buffer was added to 10 μmole per ml of the carrier. After stirring overnight at 4 ° C., unreacted groups were blocked with glycine and then subjected to an experiment.

An adsorption carrier having a length of 6 carbon atoms as a spacer was prepared by binding a ligand to commercially available CH-Sepharose 4B (Pharmacia Japan Co., Ltd.) by a condensation reaction using carbodiimide. That is, a dry powder of CH-Sepharose 4B was swollen in a 0.5 M sodium chloride solution and further washed with the same solution. After dissolving the ligand in distilled water, adjust the pH to 4.5 and
A 0 mM solution was prepared. 20 ml of this ligand solution was mixed with 20 ml of the washed CH-Sepharose 4B, and the pH was adjusted.
5 ml of 0.2 M carbodiimide aqueous solution adjusted to 4.5 was immediately added, and after mixing, the mixture was stirred overnight at room temperature.

An adsorption carrier having a spacer having a length of 10 and 15 atoms was prepared by introducing a ligand into Affi-gel 10 and Affi-gel 15 (both Japan Bio-Rad Laboratories) having an N-hydroxysuccinimide derivative. did. That is, 30 ml of each resin was washed with cold distilled water, and then the pH of Affi-Gel 10 was adjusted to 0.7.
05M phosphate buffer and pH for Affi-Gel 15
6. Washed with cold coupling buffer consisting of 0.05M phosphate buffer of 6. 20 ml of each carrier after washing, 20 ml of 20 mM ligand solution prepared separately with each cold coupling buffer
Were mixed with each other and stirred overnight at 4 ° C.

10 ml of each adsorption carrier prepared as described above was packed in a column having a diameter of 1.5 cm, and equilibrated with a 0.01 M phosphate buffer solution (pH 7.5) containing 0.14 M sodium chloride. Zoon et al. (J. Clin. Mi
crobiol., 7, p. 44, 1979) Namalwa
Crude human interferon-α solution produced by cells 500m
l (specific activity 8.3 × 10 ⁴ units / mg, total interferon titer
2.4 x 10 ⁶ units) through each column, and then add the above pH 7.
After washing with buffer solution 5
The adsorbed interferon was eluted with the above pH 7.5 buffer solution contained in 10% (w / v).

Table 2 shows the purification effect of crude interferon-α by each adsorption carrier.

Experimental Example 3 Affi-Gel 10 was washed with cold distilled water and cooled 0.05M phosphate buffer (pH 7), and divided into 10 ml each, and various L-type amino acids, multimers of L-type amino acids or their methyl groups. An adsorption carrier in which ethyl ester was introduced as a ligand was prepared in the same manner as in Experimental Example 2. The amount of ligand added to the ligand binding reaction system is 1 ml of the carrier.
It was 20 μmole. Each adsorption carrier produced in this way
10 ml of each was packed in a column having a diameter of 1.5 cm, equilibrated with 0.01 M phosphate buffer (pH 7) containing 1 M sodium chloride,
500 ml of crude human interferon-β solution for MRC-5 cell production with a final sodium chloride concentration adjusted to 1 M (specific activity
8.5 × 10 ⁴ units / mg, total interferon titer 9.6 × 10 ⁶ units) were passed. After washing with the above-mentioned buffer solution containing sodium chloride, further washing with the same solution to which 3% (w / v) propylene glycol was added, and then 80% (w /
The adsorbed interferon was eluted with 0.01M phosphate buffer (pH 7) containing 1M sodium chloride containing v) of propylene glycol. Table 3 shows the purification effect of crude interferon-β by each adsorption carrier.

Example 1 50 ml of Affi-Gel 10 was cold distilled water and chilled 0.05 M
After washing with phosphate buffer (pH 7), use the above buffer
The mixture was mixed with 50 ml of D-tryptophyll-D-tryptophan solution adjusted to 20 mM, and the mixture was stirred overnight at 4 ° C. under pH 7 conditions. The adsorption carrier having the ligand bound thereto was packed in a column having a diameter of 2.5 cm and equilibrated with a 0.01 M phosphate buffer solution (pH 8) containing 1 M sodium chloride. Taniguchi et al. (Proc.Natl.Acad.S
Ci. USA, 77, 5320, 1980), crude human interferon-β solution 2 (specific activity 4.8 × 10 ⁴ units / mg, total interferon titer 3.3 × 1) produced in E. coli according to the method of
( ⁷ units) to which sodium chloride was added to a final concentration of 1M,
Passed through the column. PH 8 buffer and 10% (w
60% (w / v) after continuously washing the column with the above-mentioned pH 8 buffer solution containing propylene glycol.
The human interferon-β adsorbed was eluted with 0.01M phosphate buffer (pH 8) containing propylene glycol and 1M sodium chloride. The recovery of eluted human interferon-β is 70% and the specific activity is 5.
It was 1 × 10 ⁷ units / mg.

Example 2 50 ml of Affi-Gel 10 was cold distilled water and chilled 0.05 M
After washing with phosphate buffer (pH 7), use the above buffer
Mixed with 50 ml of L-phenylalanyl-L-phenylalanine methyl ester solution adjusted to 20 mM, under the condition of pH 7,
Stir overnight at 4 ° C. After the reaction was completed, the adsorption carrier having the ligand bound thereto was packed in a column having a diameter of 2.5 cm, and then equilibrated with a 0.01 M phosphate buffer solution (pH 7) containing 1 M sodium chloride. Crude human interferon-β solution 2.5 (specific activity 8.0 × 10 ⁴ units / mg, total interferon titer 4.2 × 10 ⁷ units) produced by MRC-5 cells was added with sodium chloride to a final concentration of 1 M, and then passed through a column. Let After washing with the above buffer containing sodium chloride, 3% (w
/ V) The adsorbent carrier was further washed with a solution to which propylene glycol was added, and then adsorbed with 0.01M phosphate buffer (pH 7) containing 80% (w / v) propylene glycol and 1M sodium chloride. Human interferon-β was eluted. The recovery rate of the eluted interferon was 89%, and its specific activity was 2.3 × 10 ⁸ units / mg.

(Effect) As described above, the human interferon-β of the present invention
In the purification method by adsorption-elution, an adsorbent carrier composed of a water-insoluble carrier part composed of an organic high molecular weight substance, a spacer part and a ligand part is used as an adsorbent carrier, and in particular, an amino acid having an aromatic ring as a ligand part. Alternatively, a compound having at least one amino acid derivative having an aromatic ring and capable of binding to a spacer molecule bound to a water-insoluble carrier via an amino group or a carboxyl group possessed by the compound is selected. By using the adsorption carrier, highly purified human interferon-β can be obtained in a high yield by one purification operation, and it can be said that the method is particularly excellent as compared with the conventional method.

Claims (6)

[Claims] 1. A method for purifying human interferon-β, which comprises contacting crude human interferon with an adsorption carrier, adsorbing human interferon-β on the adsorption carrier, and then eluting human interferon-β with an eluent. A spacer molecule, which is a water-insoluble carrier composed of an organic high molecular weight substance as an adsorption carrier, has a multimer of an amino acid having an aromatic ring and / or an amino acid derivative having an aromatic ring as a ligand, and is bound to the carrier. The method for purifying human interferon-β, which comprises using an adsorption carrier to which a compound having an amino group or a carboxyl group capable of binding to is used. 2. The purification method according to claim 1, wherein the multimer is a dimer or trimer. 3. The purification method according to claim 1, wherein the amino acid derivative having an aromatic ring is an amino acid having a carboxyl group alkylated. 4. The purification method according to claim 3, wherein the alkyl ester is methyl ester, ethyl ester or propyl ester. 5. The method according to any one of claims 1 to 3, wherein the amino acid having an aromatic ring is L-tryptophan, D-tryptophan, L-tyrosine, D-tyrosine, L-phenylalanine or D-phenylalanine. The purification method according to Item 1. 6. An amino acid derivative having an aromatic ring is L-tryptophan, D-tryptophan, L-tyrosine or D.
-The tyrosine, L-phenylalanine or D-phenylalanine derivative as claimed in any one of claims 1 to 3.