JPS6169799A - Purification of interferon - Google Patents

Abstract

PURPOSE:To purify in interferon in high yield and purity, by adsorbing the interferon to a carrier having immobilized pigment, and eluting the interferon from the carrier. CONSTITUTION:An interferon-containing solution prepared by the culture of microbial strain produced by a recombinant DNA technique is passed through a column containing a pigment-immobilized carrier at a space velocity of 1-20hr<-1> or made to contact with said carrier for 1-50hr in batch process, to effect the adsorption of the interferon in said carrier. The adsorbed interferon is eluted with a salt-containing (0.5-2M) buffer solution containing 40-60vol% ethylene glycol or propylene glycol. The amount of the carrier is preferably 0.1-10l/g of the interferon.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for purifying interferon, which comprises adsorbing interferon onto a dye-immobilized carrier and then eluting the interferon.

Conventional techniques Interferon purification methods include a method using CPG (control pore glass) and zinc chelate agarose (J, W, Ierre et al, NYAS;
1980).

Blue Sepharose method (ε, Knight, g
r etal, J, B, C, 256(8) 1981
> , Can hecephas CPhenyl sephace method (A, J, !J+kulsk+e
tal, Prep, Biochem 10 (2)
1980), method using immobilized albumin (Hu
ang, J., ICet al., J.B.C.

2491974) etc. are known.

Purpose of the Invention Depending on these known methods, repeated operations are required to increase the purity, resulting in lower yields, and high purity interferon can be obtained in good yields! There is a demand for the development of an i-manufacturing method.

Means for Solving the Problems According to the method of the present invention, highly pure interferon can be obtained by adsorbing interferon onto a dye-immobilized carrier and then eluting the interferon.

Further, if necessary, highly purified interferon can be obtained by adsorbing the IIIj'J interferon onto a chelated metal-binding body and then eluting it.

Examples of the dye-immobilized carrier include those in which a dye having an anthraquinone core or a phthalo/nian core having a monochloro group or /chlor group as a reactive group and agarose having a hydroxyl group are bonded. Specifically, for example,
Madrex Gel Green A (product name, manufactured by Amicon)
, red agarose gel (product name, manufactured by Farmana)
, Madrex Gel Red A (product name, manufactured by Amicon)
etc. are commercially available and can be easily done manually.

Interferon applicable to the present invention includes natural interferon, interferon produced by culturing animal cells, and interferon produced by culturing microorganisms obtained by recombinant DNA technology. Contains interferon, as well as α and β.

Includes any type of gamma interferon.

It is sometimes useful for the purification of human interferon-β produced by culturing microorganisms obtained by recombinant DNA technology.

Interferon-containing liquids obtained from these cultures can be used as they are, but interferon-containing liquids (referred to as crude interferon solutions) that are obtained by one-time purification by known methods are preferably used. When using products produced by microorganisms, collect the microorganisms by filtering or centrifuging the culture solution, and adjust the pH to 5.
, 5-85 phosphoric acid or Tris-HCl buffer. The bacterial cell concentrate is frozen and thawed, or the bacterial cells are crushed using a mechanical crusher such as an ultrasonic homonizer or Dynomill, and interferon is extracted.

The extract is processed through a sieve or a centrifugal TS machine,
After the turbidity is removed, the supernatant is treated with a conventional purification method for interferon to obtain a crude interferon solution.

To bring the dye-immobilized carrier into contact with the interferon-containing solution, there are, for example, the Hatchi method and the column method, but the column method is preferably used, and the solution is used as it is or if necessary, phosphoric acid 1'l li solution is added. It is used after diluting with etc. In the case of a column method, the contact between the interferon-containing solution and the dye-immobilized carrier is usually carried out at a space velocity of 1 to 2 Q hr
-', and in the case of a batch method, it is maintained for 1 to 50 hours.

The amount of the dye-fixing carrier used is appropriately within the range of 0.1 to 1Oβ/g based on the amount of interferon to be treated.

After the contact is completed, the carrier is dissolved in water or phosphate buffer containing 0.5-2M sodium chloride in an amount of up to 20% propylene glycol,
By washing with a solution containing ethylene glycol or glycerin, most of the impure proteins other than the interferon can be removed.

The eluent used to elute the interferon adsorbed on the dye-immobilized carrier is a buffer containing salt (0.5M to 2M) containing 40 to 60% ethylene glycol or propylene glycol in terms of elution ratio, for example [ Phosphate buffer (pH 6-8)] is used.

The dye-immobilized carrier used is, for example, 0.0% after recovering the interferon. 1N-NaOH or 8M urine accumulation]
The column can be used repeatedly by passing 0.5 N - Na OH through +'& to remove impurity proteins other than uneluted interferon, and washing the column.

The chelating metal-bonded carrier used in the present invention is an insoluble polysaccharide-based carrier bound to an exchange group having chelating ability, such as a biscarboquinimino group (eg, Epokin-activated "Sepharose AB", Pharma/A!M). ) by bonding the metal to: A! ! I! I do.

This carrier is usually of the Na type with an excess of cobalt.

The interferon adsorbent is converted into the corresponding metal chelate form by contacting with a weakly acidic solution containing nickel or zinc ions, then washed with water or a weakly acidic solution, and further stabilized with a neutral buffer. can do.

When the purpose is to purify interferon to a high degree of purity, zinc is suitable as the metal to be bound, and the binding amount of zinc is preferably 500-100 Or/ml.

A commercially available product of such an adsorbent includes zinc chelate Sepharose (manufactured by Pharmania).

The metal chelate carrier and the interferon solution recovered from the dye-immobilized carrier in the above step can be brought into contact as is, but if the viscosity of the solution is high, it may be diluted with tA phosphate solution.

Contact methods include a batch method and a column method, but a column method is preferably used.

The amount of the zinc chelate carrier and the contact time are appropriately selected to provide a sufficient amount of time and time to substantially adsorb most of the interferon to be treated.

In order to recover with high purity the interferon adsorbed on the metal chelate carrier, for example, 100 mM potassium phosphate-disodium buffer at pH 7.4, 100 mM acetic acid-sodium acetate buffer at pH 5, etc. should be prepared before recovering the interferon. After selectively removing only impurities by sequentially contacting the liquids, a fraction containing highly pure interferon can be collected by changing the pH or the concentration of the collection agent continuously or stepwise.

After collecting interferon, the used metal chelate column is usually cleaned by passing an EDTA solution through it to remove zinc ions, passing it through 0.1N NaOH, and then washing it with water until it becomes neutral. It can be regenerated into the Na type, and can be repeatedly used by forming a zinc chelate by contacting with a weakly acidic solution containing zinc ions.

Examples are shown below.

Example 1 The crude human interferon-β solution obtained in Reference Example 1 was used as the crude interferon solution.

This crude solution has 4X1051 as interferon activity.
J/ml, and contains 1 mg/ml of protein.

200 Qml of the crude interferon solution was passed through 20ml of left agarose gel to adsorb interferon, and the solution was 20mM at pH 7.5! 20% ethylene glycol solution containing 20mM sodium phosphate IM at pH 7.5 (first wash), 10% ethylene glycol solution containing 20mM sodium phosphate IM, pH 7.5.
120 ml of a 50% ethylene glycol solution containing 20 mM sodium phosphate and 1 M sodium chloride at pH 7.5 was passed through the solution to elute human interferon-β.

The eluate contained 5×10 units of interferon, and the recovery rate from the crude interferon solution was 66%.
Met. The specific activity was 1 x 101 units per mg protein, and the purification ratio was 25 times.

After desalting this solution, it was treated with 2-mercaptoethanol using the method of Laemmli (Nature 227680).
-685°+q70) in the presence of sodium dodenle sulfate (SDS), electrophoretic analysis using acrylamide gel, staining with Sakumagip IJ IJ Ant Blue h250, and purity analysis using a chromato scanner (Shimadzu, C5-900). λ,=600n
m, λ==400 nm), the purity was only one band with a molecular weight of 18.000 to 19.000, which was 99.5% or more of the detection limit.

The results are shown in Table 1.

Table 1 Example 2 The same crude human interferon-β solution as in Example 1 was used.

2000 ffl+ of the crude interferon solution was added to green agarose (Madrex Gel Green A) column 21]
7 mails) & shita. 2Q at pH 7.5 on the column.
1M sodium chloride containing 20mM sodium phosphate
Qml (green color 1. 1st wash), pH
7.5 20% ethylene glycol solution containing 20mM sodium phosphate and 1M sodium chloride i o Q
ml (green column 2nd washing), and finally add 12 Q of 50% ethylene glycol solution also containing salt.
ml (green column interferon elution) i!
I used TI solution.

The exudate contained 5.8XIO' units of interferon, and the recovery rate from crude interferon was 72%. Also, its specific activity is 0.8X10' position lll1
The purification rate was 20 times for g protein.

In addition, its purity was determined by electrodynamic analysis using acrylamide in the presence of SDS, staining with Sakumazib IJ IJ Ant Blue R200, and the ratio of the human interferon band to the blue dyed band at a wavelength of 660.
When measured with a dintometer at 1 meter, it was 99.1
%Met.

From the time of passing the 50% ethylene glycol solution, the solution was passed through a 5 ml zinc chelate carrier column (inner diameter 1.5 cm) at a flow rate of 20 ml/hr to the outlet of the green agarose column.

Next, the zinc chelate support column was treated with 20 mM potassium phosphate-disodium buffer at pH 7.4 containing IM sodium chloride (first wash of zinc column), and then 0.5
100mM acetic acid at pH 5 containing IJM chloride
(acetic acid solution) IJium buffer (2nd zinc column wash) and 0.5M chloride) pH 3.7 containing IJum
5 of 100mM acetic acid-sodium acetate buffer, pH 3,
116 in 100mM acetic acid-sodium acetate buffer
The liquid was passed through.

This acetic acid-sodium acetate solution at pH 3.75 and pH 3.3 contains about 3.5 x 108 m of interferon, and the recovery rate from the crude interferon solution is 4.
It was 3%. Also, its specific activity is 1,2 x 10°
Purification magnification was 300 times in units/mg protein.

Purity was determined by electrophoresis under the same conditions as above.
It was 99.9%.

The results are shown in Table 2.

Table 2 Reference Example 1゜Culture 10β of human interferon-β producing bacteria was collected, and the bacterial cells were dissolved in 50 mM IJ acid buffer (pH 7).
, 5) On 2F! ! ! After turbidity, the bacterial cells were crushed using Manton Galarin at a pressure of 400 kg/cd.The resulting crushed bacterial cells were centrifuged, and the extract 161 was obtained.
Doozoni.

This extract was passed through a carat filled with 50 Qml of CPG (control pore glass with a pore size of 300) to adsorb human interferon-β, and then passed through a 100 m
Wash with 1M phosphate buffer (pH 7,5) and 1M chloride) 100mM borate buffer containing IJum (pH 9)
, 0) 2β to elute all of the human interferon-β, and the crude human interferon-β solution was 1 grade.

Effects of the Invention Highly purified interferon can be obtained by the method of the present invention.

Patent Applicant (102>Kyowa Hakko Kogyo Co., Ltd. Representative Mikio Kato, ``:', 1+°, □ Pano ~ - Nishi'' Proceedings Rectification 1, Indication of Case 1982 Patent Application No. 192788 No. 2, Name of the invention Method for purifying interferon 3, Relationship with the amended case Patent applicant postal code 100 Address 6-1 Otemachi-chome, Chiyoda-ku, Tokyo Name Ya (102) Kyowa Hakko Kogyo Co., Ltd. (Placement: 03-20
1-7211 Extension 3391> Representative Kato
Mikio 4. In the detailed explanation of the invention in the specification subject to amendment, the following sentence is added between the 5th and 6th lines of page 11 of the specification of contents of the amendment.

Example 3 In Example 1, 120 ml (500 x 10' units) of the 50% ethyl L/7 glycol solution containing interferon obtained from the red agarose gel was adjusted to pH 7.5.
diluted 3 times with Enoki's solution containing 20mM sodium phosphate, and transferred 23ml onto a 5ml zinc chelate carrier column.
The liquid was passed at a flow rate of /hr. The zinc chelate support column was then treated with 20 mM potassium phosphate, disodium lli solution, pH 7.4, containing 1 M sodium chloride, 0
5M chloride) 100mM of pf-15 containing IJium
Pour 1 ￥￥ acid-sulfur acetate-IJum buffer solution in this order, and fill it with 1 ￥￥ acid, pH 3.75 containing 0.5 M sodium chloride.
When 00mM acetic acid-acetic acid solution was added to blood, Interferon 300 was added to the collected solution (5Qml).
It contained X l 06 units and the recovery rate was 60%. The specific activity was 1X10' units/11g protein, and the purification ratio was 250 times.

Example 4 A 5 m
1 of red agarose carrier (this column was passed through the column at a flow rate of 30 ml/hr. Next, the left agarose carrier column was injected with 20 mVI sodium phosphate at pH 7.5 and 1
50 ml of a solution containing M sodium chloride was passed through. In addition, the same product containing 2096 ethylene glycol, night 2
To elute human ink feron-β, add 30 ml of the same solution containing 50% ethylene glycol.
1 was passed through.

The eluate contained 280×10′ units of interferon, and the recovery rate was 80%.

In addition, the specific activity was 1.4 x 10"jlt/mg protein, the purification ratio increased to 360 times, and the protein concentration of the eluate was 1.6
0 or/ml, and the concentration ratio increased to 30 times.

Example 5 In Example 3, 60 ml of the recovered solution containing interferon recovered from the zinc chelate, g interferon activity 300 x 10 units, protein concentration 50 γ/ml) was added to a 5 ml green agarose carrier column at 30 ml/h.
The liquid was passed through at a flow rate of r.

Thereafter, elution was carried out in the same manner as in Example 4.

The eluate contained 265×10 6 units of interferon, and the recovery rate was 85%.

In addition, the specific activity was 1.4 x 10° units/mg protein, the purification ratio increased to 350 times, and the protein concentration of the eluate was 200
At 0 γ/ml, the concentration factor increased to 40 times. ”

Claims (1)

[Claims] A method for purifying interferon, which comprises adsorbing interferon to a dye-immobilized carrier and then eluting the interferon.