JPH02209895A - Crystalline human granulocytic colony stimulation factor and production thereof - Google Patents

Abstract

NEW MATERIAL:A crystalline human granulocytic colony stimulation factor. USE:A remedy and preventive against leukemia and remedy against grave disease. PREPARATION:For example, a human granulocytic colony stimulation factor obtained by culturing a human cell having granulocytic colony stimulation factor-producing ability and isolating and purifying from the cultured cell or the human granulocytic colony stimulation factor prepared in genetic engineering manner from a main cell of procaryote is added to a buffer having 0-100mM buffer salt concentration and pH3.0-7.0 and as necessary precipitating agent such as ammonium sulfate is added thereto to dissolve the above-mentioned factor and the solution is subjected to treatment by vapor phase balance method, dialyzing membrane method, button method or batch method, etc., to produce a crystal, which is then separated by a centrifugal separation, etc., to provide the crystalline human granulocytic colony stimulation factor.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a crystalline human granulocyte colony stimulating factor useful as a pharmaceutical and a method for producing the same.

Background Art Human granulocyte colony-stimulating factor (hereinafter abbreviated as human G-C8F) is a substance that acts on human bone marrow cells to promote differentiation and proliferation into granulocytes, and is used as a therapeutic and preventive drug for leukemia and as an antibiotic. It is expected to be used as a treatment for serious infections that cannot be treated alone.

Human G-C3F is a G
- So-called natural G-C8F isolated from the culture supernatant after culturing C5F-producing cells (Japanese Patent Application Laid-Open No. 61-2275
No. 26 Publication), and G-C5F without sugar chains produced using E. coli as a host by genetic recombination technology (Japanese Patent Application Laid-open Nos. 82-129298 and 62-1328).
99), G-C3F (Japanese Unexamined Patent Publication No. 62-238497), which is a glycoprotein produced using animal cells as a host.

Many attempts have been made to crystallize proteins and polypeptides, and crystallization of enzymes in particular has been used as one of the effective means for increasing the purity. Regarding enzymes, crystallization requires slightly different conditions for each enzyme, such as pH, ionic strength, temperature, protein concentration, etc., and not all enzyme proteins can be easily crystallized.

In addition, among physiologically active proteins or polypeptides,
Some types are unstable with respect to pH, ionic strength, temperature, etc., and may cause changes in structure and associated decrease in physiological activity. Therefore, it is required that these types of proteins and polypeptides be crystallized under conditions that do not cause changes in their structure.

It is reasonable to say that protein crystallization methods currently involve many elements of trial and error, and therefore crystallization experiments often require many trials.

In order to crystallize a protein from an aqueous protein solution, it is necessary to strengthen the interaction between proteins more slowly than the interaction between proteins and water. For molecules that are large and have complex surface properties like proteins, the law of free energy minimization naturally holds true for interactions between proteins, but they are much more complex than for ordinary organic compounds. Forces that affect interactions between proteins include electric charge, steric effects, hydrophobic effects, hydrophilic effects, Van-der-Waa
The IS force may be considered, but proteins interact with each other in such a way that the system containing the protein, including the structural water of the protein, is the most stable. Therefore, the interactions between proteins vary depending on their concentration, temperature, pH, ionic strength, etc., and in some cases they crystallize, while in other cases they form mere aggregates. It is also known that even when crystallized, polymorphism often occurs depending on factors such as protein concentration, temperature, pII, and ionic strength.

It is also true that crystallization is often difficult when the purity of a protein is low.

Problems to be Solved by the Invention As mentioned above, many attempts have been made to crystallize proteins and polypeptides, and many proteins have actually been successfully crystallized.

However, only a small number of proteins have been successfully crystallized compared to all the proteins that exist in nature, and those that have been successfully crystallized are extracellular proteins with relatively excellent stability, plant proteins, etc. The majority of proteins are derived from

The present invention is based on human G-
By crystallizing CSF, we can (1) increase purity and reduce antigenicity, (2) improve stability and facilitate handling during storage and formulation, and (3) increase human G-C8F activity. The aim is to improve

Means for Solving the Problems The present inventors produced the inventions disclosed in the patent application publications mentioned in the prior art section.

Purification is performed by dissolving purified human G-C3F in a neutral to weakly acidic buffer solution, precipitating it in the presence of a precipitant, and collecting the precipitate by centrifugation.

The resulting crystalline human G-C8F not only contains almost no impurities, but also has two forms when hydrolyzed again.

It is preferable that it is purified to a state that does not contain multimers such as trimers.

Human G-C5F that can be crystallized by the present invention does not contain natural human G-C3F, which is a glycoprotein, or human G-C5F obtained by genetic recombination technology using animal cells as a host, or derivatives thereof, and sugar chains. E, which is a protein;
It contains human G-C8F or its derivatives produced by genetic recombination technology using eoll as a host. in general,
Glycoproteins are more difficult to crystallize than proteins that do not contain sugar chains, but according to the present invention, the glycoprotein human G-C3F was successfully crystallized.

The crystallization of human G-C3F in the present invention is carried out by hanging drop, which is a gas phase equilibrium method.
Law or depression slide
It can be carried out by a precipitant method such as a slide method; a button method; a dialysis membrane method and a batch method; or a dialysis method such as a button method; a dialysis membrane method.

Each method will be explained below.

Typical methods include the vapor phase equilibrium method, the hanging drop method, and the Depres-Notion slide method, both of which require human G-C3F to be crystallized in a weakly acidic to neutral, preferably I)11
Buffers of about 3.0 to 7.0, such as acetate buffers, phosphate buffers, citrate buffers, citric acid-phosphate buffers, etc.
(buffer concentration 5-100mM). human G-C3
The concentration of F is preferably about 1 to 100 mg/ml.

Furthermore, precipitants, such as (1'JH4)2SO4,N
a2s O4.

NaC, Q, KCD, NH4Cl1. A neutral or weakly acidic salt such as NaH2PO4°KHPO, NHHPO,a, LtC1, or an organic compound precipitant such as polyethylene glycol is added to the human G-C8F buffer described above. The concentration of the precipitant used varies depending on the type and the buffer used, but for example, for (NH) So it is about 1 to 20%, preferably about 2 to 15% saturation concentration. For other precipitants, the amount is about 0.1-1.5M, preferably about 0.2-1.2M.

Furthermore, if necessary, a stabilizer (for example, NaCl 1°KC
, 12, C5CII, MgCf1, Ca (neutral salts such as Q2, and organic compounds such as glycerin, dioxane, acetone), preservatives (e.g. Na N3)
, heavy metal removal agents (e.g. EDTA), disulfidation inhibitors or antioxidants (e.g. mercaptoethanol, cysteine, glutathione, dithiothreidol),
Polymerization inhibitors, surfactants (eg, octyl-β-glucoside, hexanetriol, Tween, octaethylene glycol-n-dodecyl ether), etc. can be added.

In the hanging drop method, the solution of human G-C8F prepared in this way is deposited as a droplet on the bottom surface of a cover glass, and is prepared in advance with a buffer solution (usually the same buffer as that used for preparing the human G-C8F solution). The upper opening of a reservoir containing a certain amount of liquid is covered with a cover glass covered with droplets, and the container is sealed using silicone grease or the like.

In the depression slide method, a buffer solution of a constant n is placed in a reservoir container in advance, a human G-C8F solution is placed on a depression slide glass, and the container is sealed with a cover glass using silicone grease. 5 to 5 sealed containers
Crystals can be obtained by standing at 30° C., preferably in a constant temperature bath equipped with an anti-vibration device, for about 1 to 210 minutes.

Salting out method using a dialysis membrane (precipitant method) A saturated or nearly saturated human G-C8F buffer solution is sealed in a dialysis tube, and this is added to the same buffer at a concentration of 10 to 14%
Precipitation side solution with saturated concentration (e.g. 10-14% saturation (N
H) 80, 0.2-1.5M NaCf
), 0.2 to 1.5 M NaH2PO4, etc.) or a polyethylene glycol aqueous solution as the external solution, salting out is carried out during the crystal growth period (usually about one week), and then the contents of the dialysis tube are drained. The resulting crystals can be isolated by centrifugation.

Dialysis using the button method and dialysis membrane A saturated or nearly saturated human G-C8F buffer solution is placed in a crystallization button, sealed with a dialysis membrane treated with EDTA in advance, and the same buffer at a lower concentration is used as an external solution. After dialysis for one day to one week, the contents of the button can be centrifuged to isolate the resulting crystals.

Batch method A human G-C3F buffer solution with a concentration of saturation or near saturation is placed in various appropriate containers, and a precipitate solution, which is a precipitant dissolved in the same buffer solution, is poured into the container and stirred as necessary. After that, leave it alone. Colorless plate-like crystals are formed after about 1 to 2 weeks.

Crystals can be isolated by centrifuging the buffer containing the crystals.

Confirmation of crystallization The existence of three types of crystals having the following characteristics was confirmed by X-ray diffraction analysis of the human G-C8F crystals obtained in the present invention.

■Crystal system: orthorhombic
Space group: P2212, Axis length (person): a≠29 B valve 94 C valve 111 Space occupied by solvent: vs valve valve 7%) ■, Crystal system; Orthorhombic space group: P2.2□2, Axis Length (person): A: 28 B valve 100C'':
110 Space occupied by solvent: vS and 37 (%) ■, Crystal system: Orthorhombic space Group: P22.2□ Axis length (person): a”=29 b-=107C
Valve 114 Space occupied by solvent: vs Well 46 (%) Intermediate temperature crystals of I, II and ■ may appear. I
, II (OKJ7) preset photo (μm1
0@) is shown in Figure 4.

Measurement of activity of crystallized human G-C3F In order to measure the activity of crystallized human G-C8F, in vitro colony formation promoting activity (CSA) was measured by the following method.

(Measurement method) The method of Bradley T, R, Metoalf D, etc. (Aust.

J, Exp, Blol, Mcd, Sc1.44
Volume 287-300.

(1966) using the monolayer soft agar culture method. Namely, 1 ml of horse serum (manufactured by Ilyclone),
Test sample 0.25ml *C57B L mouse bone marrow cell suspension 0.25ml (1.5X 106 cells/ml
), 1 ml of modified McCoy's 5A culture solution containing 0.75% agar was mixed, placed in a 35 mm diameter plastic tissue culture dish, allowed to solidify, and then incubated at 37°C for 50 minutes.
℃, 5% carbon dioxide/95% air, and 100% humidity for 5 days, count the number of formed colonies (a colony consisting of 50 or more cells is considered as one colony), and C8A was determined by setting the colony forming activity as 1 unit.

*The test specimen was 14 mg/ml of crystallized G-C8F (H
quantified by PLC) aqueous solution (100mM acetateb
buffer pH=4.2) 10μff 0.5% m5
PBS (phosphate buffer 5ali) containing A (7 mouse serum albumin, manufactured by Cappe1)
10 Gtlg / ml (volua+e
-1.4ml) (this will be used as the stock solution). After clearing with a Millipore filter, add 0.5% m5APB.
10.100, 500, 2,500 at s.

12.500.62,500 times was used. 0.5% m5APBS was used as a control (blank), and 1 μg/n+1 of formulated rG-C8F (non-crystalline, purified/recombinant human G-C8F) was used as a comparison control.

The results were as follows (see Figure 2).

Number of colonies formed Dilution magnification Test 1 Test 2 Average value Xi 51 55 5410 4B
47 4B, 5100 44 X
44500 30 59 44.5
2.500 18 27 22.512.5
00 8 7 13.582.500
5 8 4 Comparison 29 44
38.5 Control (blank) 5 7
6. As is clear from the experimental data, the colony formation promoting activity of the crystallized human G-C8F of the present invention is equivalent to or higher than that of amorphous, purified recombinant human G-C8F. Ta.

Hereinafter, the present invention will be explained in more detail with reference to Examples. Other human G-C8Fs that share most of the amino acid sequences can be crystallized in a similar manner.

Example 1: Depression slide method (vapor phase equilibrium method)
A solution (concentration: 1) in which human G-C8F (glycoprotein) produced by recombinant gene technology using animal cells as a host is dissolved in 10 mM citrate buffer (pH 4, 6).
5.5a+g/cnl) and 10μg of IB% saturated ammonium sulfate aqueous solution on a depression slide glass (depression 511de GL).
ass).

12% dissolved in 1100I citric acid 7F (pH 4,8) in a reservoir.
Pour 1 ml of saturated ammonium sulfate solution into the container, seal it with a cover glass using silicone grease, and let it cool at room temperature (20~20°C).
4° C.) for about 3 weeks to obtain a human G-C3F single crystal (colorless transparent plate substitute) (Fig. 1).

In order to identify the characteristics of the crystal, the obtained single crystal was sealed in a quartz capillary together with the mother liquor and subjected to X-ray diffraction analysis.

As a result of this analysis, three types of crystals with the following characteristics were confirmed.

■, Crystal system: Orthorhombic space group: P22□21 Dimensions of unit crystal lattice (people) = a cow 29 b well 94 c to 10 Number of molecules per unit cell = 8 Volume per unit cell: 303,900 (Person 3) Space occupied by solvent: vS well 37 (%) ■, Crystal system: Orthorhombic space group: P2, 2□2, Dimensions of unit crystal lattice: A valve 28 (person) B valve 100 c: 110 Number of molecules per unit cell: 8 Volume per unit cell: 303,600 (3 people) Space occupied by solvent: VSS Milt 7%) ■, Crystal system; Orthorhombic space group: P22□2, Unit crystal Dimensions of lattice: a valve 29 (person) b: 107 c: 114 Number of molecules per unit cell = 8 Volume per unit cell: 353.742 (person 3) Space occupied by solvent: Vs' = 46 (%) or The obtained crystals are 2
After washing twice with buff γ-containing 5% saturated ammonium sulfate, HPLC using a GPC column (TSK G3o00) (solvent: 101 phosphate buffer (pH
6,8) +100mM NaCρ; flow rate 1ml
/5in) in 19.75 minutes. Results in Part 3a
As shown in Figure (control) and Figure 3b, this crystal is G-C8F
It was confirmed that

Example 2: Hanging drop method (vapor phase equilibrium method) Human G-C8F, the glycoprotein used in Example 1, was mixed with a precipitant and/or
Alternatively, a solution (18.3 mg/ml) dissolved in 100 mM acetate buffer (pH 4.2) to which a stabilizer was added in advance
) 2ttQ and 0, 8 to 1 with varying concentrations in 0.1M increments.
3 M NaH2PO4 aqueous solution 2μρ was mixed on a silylated cover glass, and 0.7~
Na concentration was changed in steps of 0.1M over a range of 1.2M.
8n+I of H2PO4 aqueous solution was added, covered with a cover glass, sealed with silicone grease, and left to stand. Among the samples of each combination, the early ones precipitated crystals as colorless plate-like crystals after about 24 hours.

After about 2 weeks, the precipitated crystals were subjected to HPLC and G-C8F
It was confirmed that

The conditions and crystallization status of each test sample are summarized in the following table.

Example 3: Dialysis membrane method A solution (16,3
a+g/ml) 1050 μQ was sealed in a dialysis tube (bore size: MW 7.000, impermeable, EDTA treated). Transfer this tube to IM KC, mother liquor (1
0mM acetate buffer - (p114.2)) 200ml
The solution was slowly stirred and salted out for about one week. Afterwards, the contents were transferred to a V-vial and centrifuged (5°C,
15.00 rpm, 10 minutes) to obtain crystals (colorless plate-like crystals).

Example 4: Batch method (1) Human G-C3F used in Example 1 was added to 100 mM
A solution (15,
58mg/ml) was placed in a container and added with 100mM containing 1.2M NaH2PO4.
Acetate buffer was added and left at room temperature for 2 weeks.

The precipitated crystals were centrifuged (5°C, 15,000 rpm)
.

10 minutes). The crystals were colorless plate-like crystals.

(2) Similarly to (1) above, human G-CSF/l
oOmM 100mM acetate buffer 4.2) solution (13,
After standing for 3 weeks, colorless plate crystals were obtained by centrifugation using 300 μQ (08 mg/ml) and 300 μg of 100 mM acetate buffer containing (NH) SO2 at a saturation concentration of 16%.

Example 5: Dialysis membrane method 15% of human G-C3F used in Example 1 was added in advance.
Salting out with saturated ammonium sulfate, pH 4, 6, 5
Dissolve in mM citrate buffer and make 200μ of its saturated solution.
g into a dialysis tube and dialyze against 80 ml of water. Very long needle-like crystals as shown in the photograph (Fig. 4) were obtained.

Example '6: Hanging drop method (vapor phase equilibrium method) The human G-C3F used in Example 1 was salted out in advance with 15% saturated ammonium sulfate, and this was salted out at pH 4, 6, and 5mM.
Dissolve in citrate buffer (containing 3mM octyl-β-glucoside) and add 5ml of 15~
22% saturated ammonium sulfate or 5mM sodium citrate was mixed, and 8 ml of 11-13% saturated ammonium sulfate or 5+nM sodium citrate was used as an external solution to crystallize. Plate-shaped crystals were obtained in several days to two weeks.

Example 7: Dialysis membrane method A solution (11,45
mg/ml) was sealed in a dialysis tube. This tube was immersed in 200 ml of IM KCΩ solution (10mM acetate buffer - (1)H4,2)), and the KCΩ concentration was increased at a rate of 0.1M per day from the next day until the final concentration was 1.4MKCΩ. And so. Thereafter, the contents were transferred to a V-vial and centrifuged (10°C, 15,000
rpI5. 5 minutes) and then the mother liquor was removed to obtain crystals (colorless plate-like crystals). The obtained crystals were added to 10mM acetate buffer (p
114.2) Dissolve in 400μg and centrifuge (10°
C, 12,000 rpm, 5 minutes) supernatant liquid 42
The G-C8F concentration of 0uQ was 15.05 mg/ml. Concentration was measured by HPLC method (yield 90.7%)
.

Example 8: Dialysis membrane method A solution of human G-C8F used in Example 1 dissolved in 10mM acetate buffer (pH 4,2)
g/mf) 680 μQ was sealed in a dialysis tube.

This tube was immersed in 200 μg of 5% saturated ammonium sulfate solution (10mM acetate buffer - pH 4,2), and from the next day, the ammonium sulfate concentration was increased at a rate of 1% saturation per day, until the final concentration was 13% saturated ammonium sulfate. . After that, the contents were transferred to a V vial and centrifuged (10°C, 15,000 rpm,
5 minutes) and then the mother liquor was removed to obtain crystals (colorless plate-like crystals).

The obtained crystals were added to 10mM acetate buffer (pH 4,2
) Dissolved in 1200 μl and centrifuged (10'c, 1
2.00Orpm, 5 minutes) supernatant liquid 1290μ
The G-C3F concentration in Ω is 18.32mg/m! Met.

The concentration was determined by HPLC method (yield 96.4%).

Example 9: Batch method Human G-C8F used in Example 1 was dissolved in 10mM acetate buffer (pH 4,2) (19.84m
g/ml) 1 in 200 μg, 5M Na
Add 250 μo of H2P O4 solution, leave for 1 week, transfer the contents to a V vial, and centrifuge (10℃, 15.0 μo
Orpm for 5 minutes) and the mother liquor was removed to obtain crystals (colorless plate crystals). The obtained crystals were dissolved in 400 μg of 10 mM acetate buffer (pold, 2) and centrifuged (10°
C, 12,00 Orpm, 5 minutes) supernatant liquid 45
0. The G-C8F concentration of czR was 12.51 mg/ml. The concentration was determined by HPLC method (yield 78.8%).
).

Example 1O: Batch method A solution (28.91 g) of human G-C5F used in Example 1 dissolved in 10 mM acetate buffer (pH 4.2)
g/ml) 34% saturated ammonium sulfate solution 80% under water cooling to 1200μg
0 μg was added and left at 14°C, and then the temperature was raised at a rate of 1°C per day to 24°C. Transfer the contents to a V-vial and centrifuge (10°C, 15,000 rpm).
m, 5 minutes), remove the mother liquor, and collect the crystals (colorless plate-like crystals).
I got it. The obtained crystals were added to 10mM acetate buffer (pl
! 4.2) and centrifugation (10°C, 12.00
Orpm, 5 min) supernatant liquid 1380u6
C8F concentration was 24.05 mg/ml.

The concentration was determined by HPLC method (yield 95.7%).

From the above, the crystalline human G-C3F of the present invention was obtained in Examples 7 to 1.
0, it was efficiently produced with a high yield of 78.8% to 96.4%. In addition, as shown in Figure 2, the crystalline human G-C3F of the present invention has a colony forming ability comparable to or higher than that of the non-crystalline G-C3F of the comparison control, and from the viewpoint of activity, it is not suitable as a drug. Very useful. Furthermore, since it is a crystal, it has the advantage of good stability and easy formulation.

Example 11: Button method Approximately 10 m
G-C3 obtained by salting out method so that g/n+IG-C5F
F was dissolved, and the solution was poured into a 30 μg crystallization button, which was sealed with a dialysis membrane previously treated with EDTA to prevent air bubbles, and firmly secured with a rubber ring. By dialyzing this button into 2mM citrate buffer, 1
Needle crystals were obtained in 1 to 3 days.

Similarly, crystals were obtained under the following conditions.

[Brief explanation of the drawing]

Figure 1 shows human G-CSF crystallized according to Example 1.
This is a micrograph (X 100). Figure 2 shows human G-C8F crystallized according to Example 1.
1 is a graph showing the colony formation promoting activity of . Figures 3a and 3b show HPLC analysis of human G-C3F stock solution and human G-C3F crystallized according to Example 1, respectively.
This is a chart analyzed by (measurement wavelength 220 nm
; Elution solvent buffer 110ff1 phosphate buffer
-(pJ(6,8)+10h+MNaCρ; Column GPC (TSK G3000); Flow rate, 1ml
/win). Figure 4 shows human G-C3F crystallized according to Example 1.
These are preset photos of I and II (OK, Q). Figure 5 shows human G-C8F crystallized according to Example 5.
This is a micrograph (x84). Figure 7 Ground 3α Concave tail 3b Figure 1τ-F Figure tail 4 Leap procedure amendment 1, Description of the case 1999 Patent Application No. 1 392 2, Title of the invention Crystalline human granulocyte colony stimulating factor and method for producing the same 3. Person making the amendment 6゜ Contents of the amendment (1) The “saturated or nearly saturated state” described in page 10, line 11 of the specification and page f511, line 2 is changed to “1 to 110Ox
Corrected to "/waste". (2) The statement “Saturation or near-saturation concentration” on page 10, item 11 of the specification is corrected to “1 to 100 pg/zlJ.”

Claims (1)

[Claims] 1) Crystalline human granulocyte colony stimulating factor. 2) The crystalline human granulocyte colony-stimulating factor according to claim 1, wherein the human granulocyte colony-stimulating factor is obtained by genetic engineering from animal cells and other eukaryotic cells. 3) The crystalline human granulocyte colony-stimulating factor according to claim 1, wherein the human granulocyte colony-stimulating factor is obtained by culturing human cells capable of producing granulocyte colony-stimulating factor and is isolated and purified from the cells. 4) The crystalline human granulocyte colony-stimulating factor according to claim 1, wherein the human granulocyte colony-stimulating factor is obtained by genetic engineering from a prokaryotic microbial host cell. 5) From a solution prepared by dissolving human granulocyte colony stimulating factor in a buffer solution with a buffer salt concentration of 0 to 100 mM and a pH of 3.0 to 7.0, and a precipitant if necessary, a gas phase equilibrium method, a dialysis membrane method,
A method for producing crystalline human granulocyte colony-stimulating factor, which comprises crystallizing human granulocyte colony-stimulating factor by a button method or a batch method. 6) Human granulocyte colony stimulating factor 1-100mg/m
Claim 5 characterized in that it is dissolved in a buffer solution at a concentration of 1.
Method described. 7) If a precipitant is used, it is (NH_4)_2S
O_4, Na_2SO_4, NaCl, KCl, NH_
4Cl, NaH_2PO_4, KH_2PO_4, NH
_4H_2PO_4, LiCl, CsCl, CaCl_
6. The method according to claim 5, characterized in that it is selected from one of the following: 2, MgCl_2, MgSO_4, polyethylene glycol, sodium citrate, sodium oxalate, or a mixture thereof. 8) The method according to claim 5, wherein when a precipitant is used, its concentration is 0.1 to 1.5 M of the human granulocyte colony stimulating factor-containing solution.