JP3004655B2 - Crystalline human granulocyte colony stimulating factor and method for producing the same - Google Patents

Description

Description: 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-CSF) is a substance that acts on human bone marrow cells to promote the differentiation and proliferation of granulocytes, and is a therapeutic and prophylactic drug and antibiotic for leukemia. It is expected to be used as a therapeutic agent for severe infectious diseases that cannot be treated by the drug alone.

Human G-CSF is a G
- So-called natural G-CSF (Japanese Patent Laid-Open No. 227526/1986), which is isolated from the culture supernatant of CSF-producing cells, and sugar chains produced using E. coli as a host by genetic recombination technology. G-CSF (JP-A-62-129298
JP-A-62-132899) and G-CSF, a glycoprotein produced using animal cells as hosts (JP-A-62-236
497) and the like are known.

Numerous attempts have been made to crystallize proteins and polypeptides, and in particular, crystallization of enzymes has been carried out as one of the effective means for actually increasing the purity. In the case of enzymes, crystallization conditions such as pH, ionic strength, temperature, and protein concentration are slightly different for each enzyme, and not all enzyme proteins can be easily crystallized.

In addition, some proteins or polypeptides with physiological activity are unstable against pH, ionic strength, temperature, etc., and may undergo structural changes and accompanying decrease in physiological activity. Therefore, proteins and polypeptides of this type are required to be crystallized under conditions that do not cause changes in their structures.

It is appropriate 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 moderately strengthen the interaction between proteins rather than the interaction between proteins and water. In molecules such as proteins, which are large and have complex surface properties, interactions between proteins naturally follow the law of free energy minimization, but they are much more complicated than ordinary organic compounds. The forces acting on the interactions between proteins can be thought of as charge, steric effect, hydrophobic effect, hydrophilic effect, and Van-der-Waals force. The proteins interact with each other so that
Therefore, proteins interact differently depending on their concentration, temperature, pH, ionic strength, etc. In some cases, they crystallize, and in other cases, they become simple aggregates. It is also known that even when crystallized, it often becomes polymorphic depending on factors such as protein concentration, temperature, pH, and ionic strength.

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

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

However, the number of proteins that have been successfully crystallized is only a small fraction of all naturally occurring proteins.
Moreover, most of the proteins that have been successfully crystallized are extracellular proteins and plant-derived proteins that are relatively stable.

The present invention relates to human G, which was thought to be difficult to crystallize.
- By crystallization of CSF, (1) purity is increased to reduce antigenicity, (2) stability is improved to facilitate handling in storage and formulation, and (3) human G-CSF Aims to improve activity.

Means for Solving the Problems The present inventors prepared and purified human G-CSF according to the inventions disclosed in the patent application publications referred to in the Background of the Invention section in a neutral or weakly acidic buffer solution. Purification is carried out by dissolving in , precipitating in the presence of a precipitating agent, and centrifuging to collect the precipitate.

It is preferable that the crystalline human G-CSF to be obtained is purified so that it contains almost no impurities and also does not contain multimers such as dimers and trimers when hydrolyzed again.

The human G-CSF that can be crystallized by the present invention does not contain natural human G-CSF, which is a glycoprotein, or human G-CSF obtained using animal cells as a host by gene recombination technology or derivatives thereof, and sugar chains. Human G-CS produced by genetic recombination technology using protein E. coli as a host
Including F or its derivatives. In general, glycoproteins are more difficult to crystallize than proteins not containing sugar chains, but according to the present invention, human G-CSF, which is a glycoprotein, was successfully crystallized.

The crystallization of human G-CSF in the present invention is carried out by the hanging drop method or depression slide method, which are gas phase equilibrium methods; the button method;
Precipitant method such as dialysis membrane method and batch method or button method; dialysis method such as dialysis membrane method.

Each method will be described below.

Gas Phase Equilibrium Method The hanging drop method and the depression slide method are representative methods.
0 buffer, such as acetate buffer, phosphate buffer, citrate buffer, citrate-phosphate buffer, etc. (buffer concentration 5 to
100 mM). The concentration of human G-CSF is 1-100mg/ml
It is preferable that it is a degree.

In addition, precipitants such _as ( _NH4 ) _2SO4 , _Na2SO4 , NaC, KC _,
Neutral or weakly acidic salts such as NH ₄ C, NaH ₂ PO ₄ , KH ₂ PO ₄ , NH ₄ H ₂ PO ₄ and LiC, or organic compound precipitants such as polyethylene glycol are added to the above human G-CSF buffer. Add to liquid.
The concentration of the precipitant used varies depending on the type and the buffer solution used, but for example, (NH ₄ ) ₂ SO ₄ is about 1 to 20%, preferably about 2 to 15% saturation concentration. For other precipitants it is about 0.1-1.5M, preferably about 0.2-1.2M.

Furthermore, if necessary, stabilizers (e.g., NaC, KC, C
neutral salts such as sC, _MgC2 , _CaC2 , and organic compounds such as glycerin, dioxane, acetone), preservatives (e.g. _NaN3 ), heavy metal removers (e.g. EDTA), anti-disulfidation or antioxidants. (e.g. mercaptoethanol, cysteine, glutathione, dithiothreitol), polymerization inhibitors, surfactants (e.g. octyl-β-glucoside, hexanetriol, tween,
octaethylene glycol-n-dodecyl ether) and the like can be added.

The solution of human G-CSF prepared in this manner is deposited as a droplet on the underside of a coverslip in the hanging drop method, and is pre-treated with a buffer (usually the same buffer as used for the preparation of the human G-CSF solution). The upper opening of a reservoir containing a certain amount of liquid) is covered with a cover glass to which droplets are attached, and the container is sealed with silicon grease or the like.

In the depletion slide method, a predetermined amount of buffer solution is placed in advance in a reservoir container, a human G-CSF solution is placed on the depletion slide glass, and the container is sealed with a coverslip 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 a vibration isolator for about 1 to 21 days.

Salting-out method using a dialysis membrane (precipitant method) A 1-100 mg/ml human G-CSF buffer solution is sealed in a dialysis tube, and this is added to the same buffer with a 10-14% saturated precipitant solution (e.g. _, 10-14% saturated ( _NH4 ) _2SO4 , 0.2-1.5M
NaC, 0.2 to 1.5 M NaH ₂ PO ₄ , etc.) or polyethylene glycol aqueous solution is used as the external solution to salt out the crystals during the period of growth (usually about 1 week), and then the contents of the dialysis tube are removed. Crystals formed by centrifugation can be isolated.

Button method and dialysis method using dialysis membrane A human G-CSF buffer solution of 1 to 100 mg/ml was placed in a crystallization button, sealed with a dialysis membrane treated with EDTA in advance, and the same low-concentration buffer was used as the external solution. The resulting crystals can be isolated by dialysis for 1 day to 1 week, followed by centrifugation of the contents of the button.

Batch method Human G-CSF buffer solutions of 1 to 100 mg/ml were placed in various appropriate containers, and a precipitant solution prepared by dissolving a precipitant in the same buffer was generally poured into the container and stirred as necessary. then leave it. Colorless plate-like crystals are formed after about 1-2 weeks.

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

Confirmation of Crystallization It was confirmed by X-ray diffraction analysis that the human G-CSF crystals obtained in the present invention consisted of three kinds of crystals having the following characteristics.

I. Crystal system: orthorhombic Space group: P22 ₁ 2 ₁ Axis length (Å): a ≈ 29 b ≈ 94 c ≈ 111 Space occupied by solvent: Vs ≈ 37 (%) II. System: orthorhombic space group: P2 ₁ 2 ₁ 2 ₁ axis length (Å): a ≈ 28 b ≈ 100 c ≈ 110 Space occupied by solvent: Vs ≈ 37 (%) III. Crystal system: orthorhombic Space group: P22 ₁ 2 ₁ Axis length (Å): a ≈ 29 b ≈ 107 c ≈ 114 Space occupied by solvent: Vs ≈ 46 (%) Intermediate mixed crystals of I, II and III may appear. I,I
I's (OK) pre-session photo (μ = 10°) is the 4th
shown in the figure.

Measurement of Activity of Crystallized Human G-CSF In order to measure the activity of crystallized human G-CSF, in vitro colony forming activity (CSA) was measured by the following method.

(Measurement method) Method of Bradley TR, Metealf D. etc. (Aust.J.Exp.Bio
1. Med. Sci. 44, pp. 287-300, 1966). horse serum (Hyclone
Company) 1 ml, test sample 0.25 ml ^* , C57BL mouse bone marrow cell suspension 0.25 ml (1.5 × 10 ⁶ cells/ml), and modified McCoy's 5A culture medium 1 ml containing 0.75% agar were mixed to form a tissue culture of 35 mm in diameter. After solidifying in a plastic dish for
Incubate for 5 days under the conditions of 37°C, 5°C, 5% carbon dioxide gas/95% air, 100% humidity, and count the number of formed colonies (one colony consists of 50 or more cells). , CSA was calculated by taking the activity to form one colony as 1 unit (Unit).

*Test sample is 14 mg/ml of crystallized G-CSF (quantified by HPLC)
Aqueous solution (100mM acetatebuffer pH = 4.2) 10μ 0.5%
mSA (mouse serum albumin, manufactured by Cappel) in PBS (pho
100 μg / ml (volume = 1.4 m
l) Prepare (this is the stock solution). After passing through a Millipore filter, this was filtered with 0.5% mSAPBS for 10, 100, 500, 2, 50
0, 12, 500, 62, 500 times were used. 0.5% mSAPBS as a control (blank) and formulated r
G-CSF (amorphous, purified recombinant human G-CSF)
1 μg/ml was used.

The results were as follows (see Figure 2).

As is clear from the experimental data, the colony formation-promoting activity of the crystallized human G-CSF of the present invention was confirmed to be equal to or higher than that of non-crystallized, purified recombinant human G-CSF.

EXAMPLES The present invention will be described in more detail below with reference to examples. Other human G-CSFs sharing most amino acid sequences can be crystallized in a similar manner.

Example 1: Depression Slide Method (Gas Phase Equilibrium Method) Human G-CSF (glycoprotein) produced by recombinant gene technology using animal cells as hosts was dissolved in 10 mM citrate buffer (pH 4.6). (Concentration: 15.5mg/ml) 10μ
and 10 μl of 16% saturated aqueous ammonium sulfate solution were mixed on a depression slide glass. Put 1 ml of 12% saturated ammonium sulfate solution dissolved in 10 mM citrate buffer (pH 4.6) into a reservoir, seal the container with a cover glass using silicon grease, and store at room temperature (20-24°C) for about 3 hours. After standing for a week, human G-CSF single crystals (colorless transparent plates) were obtained (Fig. 1).

In order to identify the characteristics of the crystal, the resulting 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 having the following characteristics were confirmed.

I. Crystal System: Orthorhombic Space Group: P22 ₁ 2 ₁ Unit Crystal Lattice Dimensions (Å): a ≈ 29 b ≈ 94 C ≈ 111 Number of Molecules per Unit Cell: 8 Volume per Unit Cell: 303,900 (Å ³ ) Space occupied by solvent: Vs ≈ 37 (%) II. Crystal system: orthorhombic Space group: P2 ₁ 2 ₁ 2 ₁ Unit crystal lattice dimensions: a ≈ 28 (Å) b ≈ 100 c ≈ 110 Number of molecules per unit cell: 8 Volume per unit cell: 303,600 (Å ³ ) Space occupied by solvent: Vs ≈ 37 (%) III. Crystal system: orthorhombic Space group: P22 ₁ 2 ₁ Unit crystal lattice dimensions : a ≈ 29 (Å) b ≈ 107 c ≈ 114 Number of molecules per unit cell: 8 Volume per unit cell: 353,742 (Å ³ ) Space occupied by solvent: Vs ≈ 46 (%) The number of obtained crystals is 25 After washing twice with a buffer containing % saturated ammonium sulfate, HPLC using a GPC column (TSK G3000) (solvent: 10 mM phosphate buffer (pH 6.8) + 10
19.75 min at 0 mM NaC; flow rate 1 ml/min). The results are shown in Figures 3a (control) and 3b.
It was confirmed that

Example 2: Hanging Drop Method (Gas Phase Equilibrium Method) Human G-CSF, which is the glycoprotein used in Example 1, was dissolved in 100 mM acetate buffer (pH 4.2) to which a precipitant and/or stabilizer had been previously added. 2 μl of the dissolved solution (18.3 mg/ml) and 2 μm of 0.8-1.3 M NaH ₂ PO ₄ aqueous solution with a concentration of 0.1 M steps were mixed on a silylated coverslip, and 0.7 μl was added to the reservoir container. NaH with concentration changed every 0.1M in the range of ~1.2M
8 ml of a ₂ PO ₄ aqueous solution was added, covered with a cover glass, sealed with silicone grease, and allowed to stand. Among the samples of each combination, crystals precipitated as colorless platelets after about 24 hours.

After about 2 weeks, the precipitated crystals were subjected to HPLC and confirmed to be G-CSF.

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

Example 3: Dialysis Membrane Method 1050 μl of a solution (16.3 mg/ml) of human G-CSF dissolved in 100 mM acetate buffer (pH 4.2) used in Example 1 was passed through a dialysis tube (pore size: MW 7,000 impermeable). Thing, EDTA
treated). 1M KC solution (10mM
It was immersed in 200 ml of acetate buffer (pH 4.2) and slowly stirred to salt out for about one week. After that, the contents are V
It was transferred to a vial and centrifuged (5°C, 15,000 rpm, 10 minutes) to obtain crystals (colorless platelets).

Example 4: Batch method (1) 200 μl of a solution (15.58 mg/ml) of human G-CSF used in Example 1 dissolved in 100 mM acetate buffer (pH 4.2)
into _a container, to which ₁₀₀
mM acetate buffer was added and allowed to stand at room temperature for 2 weeks.

The precipitated crystals were isolated by centrifugation (5°C, 15,000 rpm, 10 minutes). The crystals were colorless plate crystals.

(2) As in (1) above, human G-CSF/100 mM acetate buffer (pH 4.2) solution (13.08 mg/ml) 300 µ and 18%
100 _mM acetate buffer containing saturating concentration of ( _NH4 ) _2SO4
Colorless plate crystals were obtained by centrifugation after standing for 3 weeks.

Example 5: Dialysis Membrane Method The human G-CSF used in Example 1 was salted out in advance with 15% saturated ammonium sulfate, dissolved in pH 4.6, 5 mM citrate buffer, and dialyzed about 200 μl of the saturated solution. Place in a tube and dialyze against 80 ml of water. Very long needle crystals were obtained as shown in the photograph (Fig. 4).

Example 6: Hanging Drop Method (Gas Phase Equilibrium Method) The human G-CSF used in Example 1 was salted out in advance with 15% saturated ammonium sulfate, and this was added to pH 4.6, 5 mM citrate buffer (containing 3 mM octyl- β-glucoside), 5 ml of which is mixed with the same amount (5 ml) of 15-22% saturated ammonium sulfate or 5 mM sodium citrate, and 11-13%
8 ml of saturated ammonium sulfate or 5 mM sodium citrate
was crystallized as the external solution. Plate-like crystals were obtained in several days to two weeks.

Example 7 Dialysis Membrane Method 610 μl of a solution (11.45 mg/ml) of human G-CSF dissolved in 10 mM acetate buffer (pH 4.2) used in Example 1 was enclosed in a dialysis tube. This tube is 1M KC solution (10
The membrane was immersed in 200 ml of mM acetate buffer (pH 4.2), and from the next day, the KC concentration was increased at a rate of 0.1 M per day to a final KC concentration of 1.4 M. Thereafter, the content was transferred to a V vial and centrifuged (10°C, 15,000 rpm, 5 minutes) to remove the mother liquor and obtain crystals (colorless platelets). The obtained crystals were dissolved in 400 μl of 10 mM acetate buffer (pH 4.2) and centrifuged (10
℃, 12,000 rpm, 5 minutes), the G-CSF concentration of 420 μl of the supernatant is
It was 15.05 mg/ml. Concentration was measured by HPLC method (yield
90.7%).

Example 8 Dialysis Membrane Method 680 μl of a solution (32.09 mg/ml) obtained by dissolving human G-CSF in 10 mM acetate buffer (pH 4.2) used in Example 1 was enclosed in a dialysis tube. This tube was immersed in 200 μm of a 5% saturated ammonium sulfate solution (10 mM acetate buffer pH 4.2), and the ammonium sulfate concentration was increased at a rate of 1% saturation per day from the next day,
The final concentration was 13% saturated ammonium sulfate. After that, the content was transferred to a V vial and centrifuged (10°C, 15,000 rpm, 5 minutes) to remove the mother liquor and obtain crystals (colorless platelets). 10 crystals obtained
G-CS of 1290 μl of supernatant dissolved in 1200 μm of mM acetate buffer (pH 4.2) and centrifuged (10°C, 12,000 rpm, 5 minutes)
The F concentration was 16.32 mg/ml. Concentration was measured by HPLC method (yield 96.4%).

Example 9: Batch method 1.1.
Add 250 μl of 5M NaH ₂ PO ₄ solution, leave for 1 week, and remove the contents.
The mixture was transferred to a vial and centrifuged (10°C, 15,000 rpm, 5 minutes) to remove the mother liquor to obtain crystals (colorless platelets). The resulting crystals were dissolved in 400 μl of 10 mM acetate buffer (pH 4.2) and centrifuged (10°C, 12,000 rpm, 5 minutes).
The SF concentration was 12.51 mg/ml. Concentration was determined by HPLC method (yield 78.8%).

Example 10: Batch method To 1200 µl of the solution (28.91 mg/ml) of human G-CSF dissolved in 10 mM acetate buffer (pH 4.2) used in Example 1, 800 µl of 34% saturated ammonium sulfate solution was added under ice-cooling. After standing at 14°C, the temperature was raised at a rate of 1°C per day until it reached 24°C. Transfer the contents to V vials and centrifuge (10°C, 15,
000 rpm, 5 minutes), the mother liquor was removed, and crystals (colorless platelets) were obtained. The obtained crystals were dissolved in 10 mM acetate buffer (pH 4.2), centrifuged (10°C, 12,000 rpm, 5 minutes), and the supernatant was 1380 ml.
The μ G-CSF concentration was 24.05 mg/ml. Concentration is HPLC
(Yield 95.7%).

From the above, the crystalline human G-CSF of the present invention can be obtained from Examples 7 to 10.
was efficiently produced in high yields of 78.8% to 96.4%. Also, as shown in FIG. 2, the crystalline human G-
CSF has a colony-forming ability comparable to or higher than that of amorphous human G-CSF as a control, and is very useful as a pharmaceutical in terms of activity. Furthermore, since it is a crystal, it has the advantage of being stable and easy to formulate.

Example 11: Button Method Approximately 10 mg/ml G-CSF in 5 mM citrate buffer (pH 4.6)
Dissolve the G-CSF obtained by the salting-out method so that
The solution was put into the crystallization button of , and sealed with a salting-out film treated with EDTA in advance so as to prevent air bubbles from entering, and then tightly secured with a rubber ring. By dialysis of this button against 2 mM citrate buffer, needle-like crystals were obtained in 1 to 3 days.

Similarly, crystals were obtained under the following conditions.

[Brief description of the drawing]

1 is a photomicrograph (×100) showing the structure of human G-CSF crystals crystallized according to Example 1. FIG. 2 is a graph showing the colony formation promoting activity of human G-CSF crystallized according to Example 1. FIG. Figures 3a and 3b are charts of HPLC analysis of human G-CSF undiluted solution and human G-CSF crystallized according to Example 1, respectively (measurement wavelength 220 nm; elution solvent buffer 10 mM phosphate buffer (pH 6.8); ) + 100 mM NaC; column GPC (TSK G3000); flow rate, 1 ml/min). 4 is a photomicrograph (×84) showing the structure of human G-CSF crystals crystallized according to Example 5. FIG.

Continuing from the front page (56) References JP-A-62-236497 (JP, A) JP-A-62-132899 (JP, A) JP-A-62-129298 (JP, A) JP-A-61-227526 (JP) , A) J. Biol. Chem. , 262 [25] (1987) p. 12306-12308J. Biol. Chem. , 262 [25] (1987) p. 12323-12324J. Biol. Chem. , 257 [17] (1982) p. 9883−9886 (58) Researched field (Int.Cl. ⁷ , DB name) C12N 15/00 - 15/90 BIOSIS (DIALOG) WPI (DIALOG) JICST file (JOIS)

Claims (7)

(57) [Claims] 1. Human granulocyte colony-stimulating factor having a crystalline sugar chain. 2. The crystalline human granulocyte colony-stimulating factor according to claim 1, which is genetically engineered 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 isolated and purified from cultured human cells having granulocyte colony stimulating factor-producing ability. factor. 4. A solution prepared by dissolving human granulocyte colony-stimulating factor having a sugar chain in a buffer solution having a buffer salt concentration of 0 to 100 mM and a pH of 3.0 to 7.0 and, if necessary, a precipitant, is prepared by gas phase equilibrium method and dialysis. A method for producing crystalline human granulocyte colony-stimulating factor, which comprises crystallizing human granulocyte colony-stimulating factor by a membrane method, a button method or a batch method. 5. human granulocyte colony stimulating factor at 1 to 100 mg/
Claim 4, characterized in that it dissolves in a buffer at a concentration of ml.
described method. 6. When a precipitant is used, it is (NH ₄ ) ₂ SO
₄ , _{Na2SO4 , NaC ,} KC, _{NH4C , NaH2PO4} , _KH2PO4 , _NH4H2P
5. Process according to claim 4, characterized in that it is selected from one of O4, LiC, CsC, _CaC2 , _MgC2 , _MgSO4 , polyethylene glycol, sodium citrate, sodium oxalate or mixtures thereof. . 7. The method according to claim 4, wherein the concentration of the precipitating agent is 0.1 to 1.5M of the solution containing human granulocyte colony stimulating factor.