JPH01100199A - Method for concentrating granulocyte-macrophage colony stimulating factor - Google Patents

Abstract

PURPOSE:To industrially and advantageously obtain the titled substance useful for phylaxis, etc., by reducing and solubilizing a granulocyte.macrophage colony stimulating factor derived from a recombinant Escherichia coli, oxidizing the solubilized factor, removing impurities by ion exchange high-speed liquid chromatography and concentrating the resultant substance. CONSTITUTION:An inert type granulocyte.macrophage colony stimulating factor derived from a recombinant Escherichia coli is treated with a reducing agent, such as dithiothreitol, to cleave intramolecular and intermolecular disulfide bonds. The obtained solubilized factor is then primarily purified by gel filtration chromatography, etc. An oxidizing agent, such as glutathione, a nonionic surfactant, such as polyoxyethylene dodecyl ether. And a chelating agent, such as ethylenediaminetetraactic acid, are added to oxidize and activate the resultant reduction product. The nonionic surfactant, oxidizing agent, reducing agent and chelating agent which are impurities are then removed by using ion exchange high-speed liquid chromatography and simultaneously concentrated to afford the aimed granulocyte.macrophage colony stimulating factor.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for concentrating granulocyte/macrophage colony stimulating factor (hereinafter abbreviated as CM-C3F). More specifically, DNA encoding GM-C3F
Inactive G produced by E. coli transformed with the sequence
In the process of concentrating the activated granulocyte/macrophage colony-stimulating factor by solubilizing, reducing, and oxidizing M-C3F, and further purifying it, ion-exchange high-performance liquid chromatography was used to remove contaminants. The present invention relates to a method for removing nonionic surfactants, oxidizing agents, reducing agents, and chelating agents while simultaneously concentrating granulocyte/macrophage colony stimulating factor.

Come ′ and. Clinical HematologV+IL
329-48 (1984); J. Biol, C.
hew,, 252.1998-2003 (1977)
;5science, 228.810-15 (198
5)).

GM-C3F can be used as a prophylactic or therapeutic drug for leukopenia and infectious diseases, and as a promoter of leukocyte recovery after bone U□ transplantation, but since it exists in extremely small amounts in vivo, it may cause genetic damage. Using recombinant technology, a technology has been established that allows for mass production using Escherichia coli. In this case, the produced protein is obtained as an inactive, insoluble aggregate, so during purification, this aggregate is solubilized by adding a reducing agent and a solubilizing agent, and then the reducing agent and solubilizing agent are added to the aggregate under appropriate conditions. After diluting or removing the agent, an operation is added to make GM-C3F into an active form by oxidizing it.

After activation, CM-C3F needs to be concentrated in order to proceed to the next purification step. Ultrafiltration has usually been used for this concentration operation, but this method is time-consuming, has a low recovery rate, and is susceptible to adsorption. It also had many problems, such as being difficult.

On the other hand, the present inventors have found that activated CM-C5F can be concentrated and purified by using reversed-phase high performance liquid chromatography. If the above impurities are included, reverse phase chromatography will detect the above impurities, 91! There was a problem that objects could not be removed.

In view of the above circumstances, as a result of intensive research, we have found that by using ion exchange high performance liquid chromatography, concentration is short, the recovery rate is high, large quantities can be easily processed, and at the same time active The inventors have discovered that it is possible to remove impurities such as nonionic surfactants, oxidizing agents, reducing agents, and chelating agents contained in the converted GM-C3F solution, and have completed the present invention.

According to the present invention, GM-CSF (Nature (London)) produced, for example, by recombinant E. coli
) 281.

544 (1979), Gene 39.247 (19
GM-C3F obtained by the known method of 85)) can be efficiently concentrated. GM-C3F accumulated in the host as insoluble aggregates is disrupted using an appropriate means, and the insoluble aggregates are removed by centrifugation.

This aggregate is treated by using a suitable solubilizing agent (e.g., high concentration guanidine hydrochloride or urea) and a reducing agent (e.g., dithiothreitol) to cleave disulfide bonds that may exist within and between molecules. GM-C3
After solubilizing F as a reduced form, primary purification is performed by means such as gel filtration chromatography.In these operations, C, M-C3F is
Remove soluble components from the host-derived proteins, deoxidized acids, and fine and lipid membrane components contaminating the GM-CSF, and further remove contaminant components with a different molecular weight from GM-CSF by gel filtration chromatography or the like. G obtained in this way
The reduced form of M-CSF can be prepared using a suitable redox system (e.g., a mixture of oxidizing agents and reduced forms of glutathione or cysteine), surfactants (e.g., polyoxyethylene dodecyl ether or polyethylene glycol 600).
0), two sets of disulfide bonds are formed in a solution with a chelating agent (e.g. ethylenediaminetetraacetic acid), and C
Activate M-C3F. Obtained active GM-C3F
In addition to host-derived substances that could not be removed by primary purification, the solution contains impurities such as the above-mentioned redox agents, surfactants, and chelating agents. Therefore, according to the present invention, GM-C3F is concentrated while removing the impurities using ion exchange high performance liquid chromatography.

In the method of the present invention, a conventional chemically bonded filler for ion-exchange HPLC is used as the filler, for example, a porous polymer resin such as polyacrylate or silica gel with an average particle size of 5 μm or 10 μm, and diethylaminoethyl groups, An ion exchange filler to which an ion exchange group such as a diethyl-(2-hydroxypropyl) aminoethyl group, a carboxymethyl group, or a sulfopropyl group or an alkyl group such as an octadecyl group or an octyl group is chemically bonded is used. .

Examples of such fillers include TSK-gel DE
AE-5WP (manufactured by Toyo Soda), TSK-gel 5
Examples include P-5PW (manufactured by Toyo Soda).

The size of the column used in the method of the present invention is usually 4.6 to 50 m5. A length of about 150 to 300 ma+ is used, and an appropriate combination with a filler is selected depending on the objective such as the amount of sample to be loaded and the purity to be obtained.

In the method of the present invention, elution is performed by a gradient elution method. For example, by adding a low concentration of salt to the buffer solution, GM-
The column is equilibrated under pH conditions such that C3F does not elute, and a dilute aqueous sample of GM-C3F is injected into the column using an HPLC pump or other pump, and after adsorbing CM-C5F, the salt Elution is performed by a gradient elution method in which the salt concentration is increased continuously or stepwise from the initial concentration of . In the method of the present invention, the buffer used for the mobile phase has a pH of 6 to 0, for example, 0.01 to 0.
．． A 2 molar Tris-HCl or phosphate buffer is preferred. In addition, the initial equilibration of the column and the gradient elution method are performed by changing the salt concentration, and the salt used is preferably ammonium sulfate, sodium sulfate, sodium chloride, etc., and their concentration is 0.
A range of ~2M is desirable.

The flow rate of the mobile phase in the method of the present invention varies depending on the size of the column used, but is approximately 1 to 50 ad per minute, and the column temperature is constant around room temperature (25 ° C) and the column separates and elutes. The wavelength of the ultraviolet detector for detecting GM-C3F is 210-230nI11
Alternatively, a constant wavelength around 28 Onm is preferable. Also, -
The amount of sample that can be processed at one time varies depending on the size of the column used, but the amount of GM-C3F is 1 to 1000 mg.
The solution amount is preferably in the range of 10 to 2000 units.

EXAMPLES Hereinafter, the specific content of the present invention will be explained with reference to Examples, but the present invention is not limited to these Examples.

1” Horsefly 1 = Human leukemia cell line U937 (ATCC CRL1593)
Plasmid p21 containing the hGM-C3F gene derived from
GM-0” and expressed by 5G936 strain derived from E. coli strain 112 transformed by pc1857.
The concentration of 0.00465χ (W/W) obtained by performing each operation of ultrasonic disruption, centrifugation, solubilization, gel filtration chromatography, and regeneration of GM-C3F on CSF aggregates,
Purity 41.4% (W/In), 1 as GM-C3F
The regenerated solution 383 IRl containing 7.8 mg was concentrated under the conditions shown below using an ion exchange column in which diethylaminoethyl groups were introduced into a polyacrylate porous polymer resin.

This method has a recovery rate of 92.6% and a concentration of 0.0294χ(
-8), it was possible to concentrate to an aqueous solution 56H1 with a purity of 75.0% (-c).

Concentration conditions (1) are shown below.

Column: HPLC ion exchange resin with diethylaminoethyl groups introduced into polyacrylate-type porous polymer resin (Toyo Soda TSK-get DEAE-5PW, particle size 10
Mobile phase: A
Solution 20mM) Lis-hydrochloric acid buffer (pH 7,3) Solution B 20n+M) Liss-hydrochloric acid buffer with 0.5M sodium chloride added (pHL3) Gradient condition 2B
After running the solution at 5% for 10 minutes, increase solution B linearly to 45% over 60 minutes.

Flow rate: 8.0 rI11/win Column temperature: Room temperature Detector: Ultraviolet absorption meter (measurement wavelength 280 nm) Sample addition: Added to the column at 8.0 m7'sin using a liquid feed pump.

The chromatogram of the GM-C3F regenerant and the chromatogram after concentration are shown in FIGS. 1 and 2.

In addition, GM-CSF in the solution! lt is the HPL shown below
Quantification was carried out under conditions C using an absolute calibration method using GM-C5F as a reference product. The analytical conditions for GM-C3F are shown below.

Column: Octadecylated silica gel for HPLC (C
osmosH5C+s made by Hanui Gyudon, spherical, particle size 5μm
1 hole diameter 12nw), inner diameter 4.6m+m, length 250
Filled in mm stainless steel tube.

Mobile phase: Solution B: 5% in 20mM sodium phosphate buffer
A solution containing mM tetra-n-butylammonium (p
H7,0) Gradient conditions: After increasing liquid B linearly from 60% to 76% over 16 minutes, increasing liquid B linearly from 60% to 76% over 50 minutes.

Flow 1: 1. Od/+win Column temperature: Room temperature Detector: Ultraviolet absorption meter (measurement wavelength 230 nm)
), purity 46.1% (K/W), and 190 d of recycled aqueous solution containing 19.0 mg of GM-C3F was concentrated by ultrafiltration under the conditions shown below.

This method has a recovery rate of 78.7% and a concentration of 0.065χ(W
/W), and was concentrated to 23 m of an aqueous solution with a purity of 54.4% (W/W).

Concentration conditions (3) are shown below.

Ultrafilter: A stirred cell with a cell capacity of 180 d and an effective filtration area of 28.7 d (manufactured by Amicon Corporation, model 8200).

Ultrafiltration membrane: Polysaccharide material with molecular weight cutoff 10,
000 (manufactured by Amicon Corporation, YMIo, 62
mm) Filtration pressure: Nitrogen, 3 kg/crM Filtration temperature 2
4°C

[Brief explanation of the drawing]

FIG. 1 shows a high performance liquid chromatogram of the regenerated liquid before i4 contraction in Example 1. FIG. 2 shows a high performance liquid chromatogram of Example 1 after concentration. Figure 1 Retention time (minutes) No. 211 Retention time (minutes) Procedure amendment (voluntary) 1982
November 13, 1988 Patent Application No. 256012 2 Name of the invention Method for concentrating granulocyte/macrophage colony stimulating factor 3 Relationship to the person making the amendment Case Patent applicant 5-15 Kitahama, Higashi-ku, Osaka ( 209) Sumitomo Chemical Co., Ltd. Representative Hideo Mori Contact information TEL (06) 220-34045,
Column 6 of Detailed Description of the Invention of the Specification Subject to Amendment, Contents of Amendment (1) Insert "r completed," after "in a short time" on page 4, line 5 of the specification. (2) On page 5, line 11, the word “oxidizing agent” has been replaced with “
``Oxidized type'' is corrected. (3) The phrase "or an alkyl group such as an octadecyl group or an octyl group" on page 6, lines 10-11 of the same document is deleted. (4) Insert "wo" after "shio" in the second line of page 7. (5) On page 7, line 7, the phrase “adsorbed” was replaced with “
I let it absorb,” he corrected. (6) From the bottom line of page 7 to the first line of page 8, the phrase ``separated by a constant temperature and a column'' is corrected to ``separated by a constant temperature and a column.'' (7) Line 2 from the bottom of No. 8, page 9, line 5,
The words "purity" on page 11, line 10 and line 14 of page 11 are corrected to "protein purity." Above-〇4

Claims (1)

[Claims] In the method of reducing and solubilizing inactive granulocyte/macrophage colony-stimulating factor derived from recombinant E. coli and further oxidizing, the activated granulocyte/macrophage colony-stimulating factor is concentrated and purified. A method that uses exchange high-performance liquid chromatography to remove impurities such as nonionic surfactants, oxidizing agents, reducing agents, and chelating agents, while simultaneously concentrating granulocyte/macrophage colony-stimulating factors.