KR0184776B1 - Method of purifying recombinant human granulocyte colony stimulating factor - Google Patents

Abstract

The present invention relates to a method for purifying recombinant phosphorus granulocyte colony stimulator (rhG-CSF) expressed in yeast. G-CSF purified according to the method of the present invention, characterized in that urea is added, and cation exchange column chromatography, hydrophobic column chromatography and gel filtration chromatography are treated with the WHO NIBSS international standard. Similar cell growth was obtained and showed 2.85 × 10 ⁷ IU / ml activity.

Description

Purification method of recombinant phosphorus granulocyte colony stimulator (rhG-CSF)

1 is a result of electrophoresis (15% SDS-PAGE) of the fractions obtained in each step of the rhG-CSF purification method of the present invention,

2 is a result of reverse phase high pressure liquid chromatography after analyzing the amino terminus of rhG-CSF purified according to the purification method of the present invention through an amino acid sequence analyzer, and (A), (B) and (C) are not, respectively. No-terminal first, second and third amino acid residues,

3 is a result of measuring the activity of rhG-CSF purified according to the purification method of the present invention by an in vitro assay method, wherein (A) and (B) are proliferation curves of WHO NIBSC standards and rhG-CSF samples, respectively. .

The present invention relates to a method for purifying recombinant human granulocyte colony stimulating factor (hereinafter abbreviated to rhG-CSF) expressed in yeast, and more specifically, rhG- expressed in yeast and secreted into a culture medium. A method for purifying CSF with high activity, high purity and high yield using cation exchange chromatography, hydrophobic chromatography and gel filtration chromatography.

In the human body, hematopoiesis occurs mainly in the bone marrow, and various types of blood cells are produced through complex and diverse pathways. It is known that the formation of blood cells is regulated by the glycoprotein group, and specific glycoproteins have been found to act as regulators according to the path and stage of hematopoietic action.

The colony stimulating factor (CSF), which is a generic name for glycoproteins involved in hematopoiesis, is classified into four types according to the stage and route of hematopoiesis. That is, in the stage of hematopoiesis, granulocyte colony stimulating factor (G-CSF), which is involved in granulocyte formation, and macrophage colony stimulating factor (M-,), which is involved in macrophage formation. CSF), Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) that regulates the pathway and stage of extensive hematopoietic action. It is divided into Interleukin-3, Multilineage Colony Stimulating Factor (Multi-CSF).

Among them, G-CSF has been found to promote the growth of granulocyte blasts, in particular to activate the function of final blood cells in the hematopoietic pathway, and generally involved in the formation and activation of bone marrow leukocytes. Since G-CSF acts as a regulator of hematopoiesis, it is known to treat destroyed immune agent cells, bone marrow transplantation, severe burns and leukemia after cancer chemotherapy.

Since G-CSF is known to be involved in the growth and differentiation of granular neutrophils (Nicola, et al., J. Biol. Chem., 252, 9017 (1983)), G-CSF derived from human bladder cancer cells Purified (Welte, et al., Proc. Natl. Acad. Sci., 82, 1526 (1985)) and its cDNA sequence has been revealed (Shegekazu, et al., Nature, 319, 415 (1986); and Souza , et al., Science, 232, 61 (1986)), G-CSF is a glycoprotein of a structure having one O-glycosylation site in the polypeptide chain and has a molecular weight of 18,500 to 19,600 depending on the degree of glycosylation. The complete polypeptide of G-CSF has been found to consist of 174 amino acids, to have two disulfide bonds and to have one glycosylation site.

Natural G-CSF is a human bladder cancer cell line 5637 (Morioka, et al., Res. Exp. Med., 190, 229 (1990); Welte, et al., PNAS, 82, 1526 (1985)), endotoxin Infused mice were treated with lung tissue (Metcalf, et al., J. Cell. Physiol., 116, 198 (1983)), and human squamous cell carcinoma cell line (CHU-2) (Normura, et al., EMBO J., 5, 871 (1986)), the active fractions were separated and purified from complex extracts such as ammonium sulfate fraction, hydrophobic chromatography, ion exchange chromatography, reverse phase high pressure liquid chromatography.

Recently, activity was reported when the recombinant G-CSF was expressed and then purified. In the case of Amgen, more than 95% of recombinant G-CSF was obtained by expressing recombinant G-CSF in Escherichia coli, dissolving with sodium laurate and purifying by reversed phase high pressure liquid chromatography (Souza, et al. , Science, 232, 61 (1986)), and in the case of Chugai, recombinant G-CSF expressed in animal cells was purified using gel filtration chromatography and hydrophobic chromatography (Tsuchiya, et al., EMBO). J., 6, 611 (1987); and Oheda, et al., J. Biochem., 103, 544 (1988).

However, when the E. coli expression system is used to produce G-CSF, since the protein is expressed as an inclusion body that was not originalized in E. coli, it is activated only when it is normalized in the purification process, and the expressed protein is glycosylated. There is a problem that the stability is not good. In addition, in the case of E. coli-derived protein, it is necessary to remove the exothermic material in order to manufacture the drug, there is also a problem that it is not easy to remove the exothermic material derived from E. coli.

In addition, the production of G-CSF using an animal cell system, in addition to the high cost of the culturing process, when the purified G-CSF is purified, the cell culture solution must be concentrated for the gel filtration chromatography, which is an initial stage of purification. It may be possible for small scale purification but it is not suitable for large scale purification.

On the other hand, when producing G-CSF using a yeast secretion expression system, since the protein is expressed in yeast and reconstituted, it is not only secreted into the culture medium in an active state, but also has a relatively low unit cost of the culture process. There is this. On the other hand, proteins secreted into yeast cultures have lower expression levels than those used with E. coli and require a completely different method from the purification of intracellular expression proteins. It has the disadvantage of being.

The present inventors continued research in consideration of the above-mentioned problems in the production and purification of G-CSF, as a result of culturing yeast to secrete G-CSF, and then concentrate the culture medium from which yeast is removed to facilitate purification. RhG-CSF purified in high activity, high purity and high yield by volume reduction, followed by cation exchange chromatography, hydrophobic chromatography and gel filtration chromatography with urea reducing the strong viscosity of the concentrated solution. By being able to obtain the present invention has been accomplished.

That is, an object of the present invention is to provide a method for purifying rhG-CSF secreted from recombinant yeast with high activity, high purity and high yield.

Purification method of the recombinant phosphorus granulocyte colony stimulating factor of the present invention for achieving the above object is, in purifying the recombinant phosphorus granulocyte colony stimulating factor (rhG-CSF) expressed and secreted in yeast, the yeast culture solution is concentrated, It is characterized by adding urea and performing cation exchange column chromatography, hydrophobic column chromatography and gel filtration column chromatography.

Hereinafter, the present invention will be described in detail.

Yeast cells transformed to produce phosphorus G-CSF, such as Sacchromyces cerevisiae, are cultured under conditions suitable for G-CSF expression in synthetic media that are maximally excluded from culture medium and color. Afterwards, the protein-expressed and secreted cultures are centrifuged to remove cell precipitates.

The cultures obtained in consideration of the activity and stability of the protein are concentrated in a low temperature room, for example using an ultrafiltration membrane, followed by diafiltration to remove salts, followed by addition of urea to reduce hydrophobicity.

The urea-added culture was adsorbed onto a cation exchange column chromatography, for example, SP sepharose cation exchange resin equilibrated with sodium acetate buffer in advance, and then washed with the same buffer to remove impurities as much as possible, and sodium acetate The pH of the buffer solution is 6.0, followed by step gradient to elute.

Hydrophobic column chromatography is then performed, preferably eluting with a phenyl sepharose resin and linear gradient of urea concentration from 0 to 4M.

Subsequently, gel filtration column chromatography is performed to remove tertiary contaminants and endotoxins that may be present in trace amounts, such as G-CSF duplex, and to remove urea to have a tertiary structure having activity. At this time, it is preferable to use Sephacryl S-100 as the resin of the gel filtration chromatography.

The rhG-CSF of the present invention purified through the above steps showed a cell growth similar to that of the WHO NIBSC international standard, and was estimated to be 1.72 × when the cpm value of ³ H-thymidine was absorbed by cells was estimated. 10 ⁷ IU / mg / ml showed activity.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

EXAMPLE

(Step 1) concentration of the culture solution and removal of salt

Yeast transformed to produce rhG-CSF (KCTC 0208BP, deposited on October 31, 1995) was cultured in a synthetic medium (Korea Patent Application No. 94-22289) excluding culture-derived material and color as much as possible. An ultrafiltration membrane (limit molecular weight 3000) was used to concentrate 200 liters of culture in a low temperature room. The concentrated culture solution was diafiltered to obtain 6 L of the salt-free solution, which was centrifuged to obtain a clear solution.

(Step 2) Urea Treatment and Cation Exchange Chromatography

Urea was added to the protein solution obtained in (Step 1) at a concentration of 4 to 8 M to obtain a clear solution, and adsorbed onto an SP Sepharose column (Pharmacia) previously equilibrated with 30 mM sodium acetate buffer solution (pH 4.2) containing 8 M urea. After doing this, the molecules remaining free in the column were washed with the same buffer. The protein was then eluted by step gradient using 30 mM sodium acetate buffer (pH 4.2) containing 50 mM sodium chloride and 8M urea and 60 mM sodium acetate buffer (pH 6.0) containing 8M urea. At this time, G-CSF was mostly eluted with 60 mM sodium acetate buffer (pH 6.0) containing 8M urea.

(Step 3) Hydrophobic Chromatography

The protein solution obtained in step 2 was diafiltered to remove urea, and then adsorbed onto a phenyl sepharose column (Pharmacia) previously equilibrated with 20 mM Tris buffer (pH 7.5), and the free protein in the gel with the same buffer. Washed. The protein was eluted by linear gradient of urea concentration from 0 to 4M using 20 mM Tris buffer (pH 7.5) and 20 mM Tris buffer (pH 7.5) containing 4M urea. At this time, G-CSF was mostly eluted at 2M urea concentration.

(Step 4) Gel filtration chromatography

After concentrating the protein solution obtained in (Step 3), it was injected into a Sephacryl S-100 column (Pharmacia) previously equilibrated with a buffer solution containing no endotoxin (20 mM Tris buffer, pH 7.0), and the same buffer solution. Only fractions containing G-CSF were eluted.

1 is the result of electrophoresis (15% SDS-PAGE) of the fractions obtained in each step of the rhG-CSF purification method of the present invention. Here, column M is an electrophoretic molecular weight standard sample, column 1 is a fermentation stock of yeast transformed to produce rhG-CSF, column 2 is a protein solution after condensation of the fermentation stock with an ultrafilter, and column 3 is SP sepa. Solution collected after Rose Cation Exchange Column Chromatography, column 4 shows the solution collected after phenylsepharose hydrophobic column chromatography, and column 5 shows the final purified rhG-CSF protein by Sephacryl S-100 gel filtration column chromatography. . As shown in Figure 1, the purification process of G-CSF can be confirmed in the molecular weight range of 18,500.

Reference Example 1: Amino Acid Sequence Analysis of Purified G-CSF Amino Terminals

The amino acid sequence of the rhG-CSF amino terminus purified according to the above example was analyzed using an Edman degradation reaction in an automated sequence analyzer (Model 471A, Applied Biosystem, USA) (Geoffray Zubay, Biochemistry, 2nd). Edition, 47-48 (1988). The resulting amino acid with phenylthiohydantoin was separated by reverse phase high pressure liquid chromatography column (220 mm x 2.1 mm, Model PTH-222, Applied Biosystems, USA).

2 is a result of reverse phase high-pressure liquid chromatography after analyzing the amino terminus of rhG-CSF purified according to the purification method of the present invention through an amino acid sequence analyzer, wherein (A), (B) and (C) are respectively amino Terminal first, second and third amino acid residues. As a result of comparing the retention time of standard amino acids in the same column as the results of (A), (B) and (C), it was confirmed that they were threonine (Thr), proline (Pro) and leucine (Leu), respectively. Therefore, it can be seen that the amino acid sequence of the rhG-CSF amino terminus purified by the method of the present invention is Thr, Pro, and Leu in turn. Purity of the purified rhG-CSF was 95%.

Reference Example 2: Determination of titer of secreted G-CSF protein through in vitro analysis

The titer of G-CSF in in vitro assays can be determined to the extent of the proliferation of Granulocyte Progenitor Cells (GPC, NFS-60) by G-CSF. In other words, a titer determination method ( PNAS USA, 83, 7633 (1986)), which quantifies and measures the growth rate of cells by treating GPC with G-CSF and adding ³ H-thymidine (radioactivity-labeled substance, Amersham) It can be modified and used as follows.

50 µl each in columns A-D with serial dilution of WHO NIBSC International Standard from 12.5 IU / ml in RPMI medium (GibcoBRL # 23400-062) containing 5% fetal bovine serum (GibcoBRL # 16000-044) in a 96-well container G-CSF protein purified in the same manner was serially diluted twice and 50 µl was added to each corresponding hole in the E-H rows. Cell line M-NFS-60 (ATCC, CRL-1838) is used as an analytical cell.The cell suspension, centrifuged three times or more with G-CSF-free medium, at 5 × 10 ⁵ cells / ml 50 μl each was added to the empty container and shaken, followed by incubation for 24 hours in a 5% CO ₂ incubator. ^After adding ³ H-thymidine to 0.25 μCi per cavity and incubating for 4 hours at 37 ° C., 5% CO ₂ incubator, the contents of each ball were harvested on a glass fiber filter using a microharvester. The cell-harvested glass fiber filter was placed in a scintillation bottle, and 5 ml of scintillation solution (Packard # 6013329) was added to measure radioactivity with a liquid scintillation counter (Packard Tri-Carb 2500 TR).

3 is a result of measuring the activity of rhG-CSF purified according to the purification method of the present invention by an in vitro assay method, wherein (A) and (B) are proliferation curves of WHO NIBSC standards and rhG-CSF samples, respectively. The following table shows the calculated activity of G-CSF.

G-CSF activity 2.85 × 10 U / ml

1 is the cpm value after 2-fold diluted G-CSF treatment;

2 is a value obtained by substituting the cpm value of the G-CSF sample into an international standard curve in terms of logarithmic index; log'A '= {sample cpm-standard Y-intercept cpm} / standard slope

3 is the dilution factor (D.F); And

4 is the product of the logarithm of the second row, multiplied by the dilution factor.

'B'U / ml = 2 logA × D.F / 1000

As shown in FIG. 3, even when treated with G-CSF purified according to the method of the present invention, it can be seen that similar cell growth can be obtained when treated with WHO NIBSC International Standard. Also 2.85 × 10 when the titer is estimated by the cpm value that cells receive when H-thymidine is taken up It showed IU / mg / ml activity.

Claims (7)

In purifying recombinant phosphorus granulocyte colony stimulator (rhG-CSF) expressed and secreted in yeast, the yeast culture is concentrated, urea is added to the concentrate, cation exchange column chromatography, hydrophobic column chromatography and gel filtration column chromatography. Purification method of rhG-CSF, characterized in that to perform the chromatography. The method of claim 1, wherein urea is added to the concentrate at a concentration of 4 to 8 M. The purification method according to claim 1, wherein the cation exchange column chromatography is performed on an SP Sepharose resin. 4. The sodium acetate buffer of claim 3 wherein in the cation exchange column chromatography Purification method characterized by eluting with a step gradient of solution (pH 6.0). The method of claim 1, wherein the hydrophobic column chromatography is performed on a phenyl sepharose resin. The purification method according to claim 5, wherein in the hydrophobic column chromatography, the concentration of urea is eluted by linear gradient from 0 to 4M. 8. The method of claim 7, wherein said gel filtration column chromatography is performed on Sephacryl S-100 resin.