KR20070091081A - Method for purifying human granulocyte microphage colony stimulating factor from the suspension culture broth of oryza sativa - Google Patents

Abstract

A method for isolating hGM-CSF(Human Granulocyte Microphage Colony Stimulating Factor) from suspension culture broth of Oryza sativa is provided to be able to purify the hGM-CSF with high purity by completely removing impurities such as contamination protein derived from plant and amylase from the suspension culture broth. A method for isolating hGM-CSF from suspension culture broth of Oryza sativa or a pre-treated solution thereof is characterized in that contamination protein having a similar molecular weight to that of the hGM-CSF and amylase generated by an Oryza sativa gene expression promoter system are removed by a combination of hydrophobic chromatography and ammonium sulfate, wherein the hydrophobic chromatography is phenyl sepharose.

Description

Method for Purifying Human Granulocyte Microphage Colony Stimulating Factor from the Suspension Culture Broth of Oryza sativa}

The present invention is a rice cell ( Oryza sativa ) hGM-CSF, a human granulocyte macrophage colony stimulating factor, is isolated from liquid suspension cultures and contains a rice cell gene expression promoter as well as a contaminant protein similar in size and size to hGM-CSF that is not easily isolated by complex sugar chain interference. The present invention relates to a method for effectively removing amylase produced by the system by a combination of hydrophobic chromatography and ammonium sulfate, and according to the present invention, rice cell-derived hGM-CSF can be isolated and purified purely from rice cell liquid suspension culture. Can be.

The present invention is a rice cell ( Oryza sativa ) The present invention relates to a method for purely separating and purifying human granulocyte macrophage colony stimulating factor (hGM-CSF), which is a human granulocyte macrophage colony stimulating factor.

Granulocyte macrophage colony stimulator GM-CSF is involved in the production of blood cells and platelets in the bone marrow, and also the separation of macrophages promotes the growth of white blood cells. As such, GM-CSF is not only a regulator of hematopoietic activity but also increases the production of immune cells (Burgess, AW et. al ., J. Biol , Chem . 252, 1988-2003 (1977); Metcalf, D. et al ., J. Cell Physiol . 111, 275-283 (1982)), which is used as a drug to be administered after bone marrow transplantation in leukemia patients, and in addition to other diseases related to blood cells such as aplastic anemia (Morstyn, G. et. al ., TIPS 10, 154-159 (1988)).

Human granulocyte macrophage colony stimulator hGM-CSF is a glycoprotein consisting of 127 amino acids and sugar chains and has various molecular weights depending on the degree of glycation (Clark, SC et al . Int . J. Cell Clon . 6, 265-377 (1988)). In the past, human cell line cultures or urine were separated and purified, but the development of genetic engineering technology has been able to obtain human granulocyte macrophage colony stimulating factor hGM-CSF from a variety of hosts, such as E. coli, yeast, animal and plant cells. Ernst et al. In the United States expressed the hGM-CSF gene and expanded the yeast transformed to secrete into the culture medium to obtain hGM-CSF, which was isolated and purified by ConA affinity chromatography and gel filtration chromatography ( Biotechnology 5, 831 ~ 834 (1987)), Lee Sang-mi of Korea, etc., were subjected to primary anion exchange chromatography, hydrophobic chromatography, secondary anion exchange chromatography, gel filtration chromatography, etc. from a yeast culture medium transformed to secrete hGM-CSF. A method of separating and purifying hGM-CSF with high purity was established (Korean Patent 10-0254829-0000). Meanwhile, Zhang et al. Expressed the hGM-CSF gene and expanded E. coli transformed to secrete it into Periplasm to obtain hGM-CSF, which was then subjected to hydrophobic chromatography, gel filtration chromatography, and anion exchange chromatography. A method of separating and purifying with purity was reported ( Process Biochemistry 34, 55-58 (1999)). As such, the methods for purely separating and purifying hGM-CSF reported so far can be seen that many use anion exchange resin, hydrophobic chromatography, gel filtration chromatography, and the like.

Human granulocyte macrophage colony stimulator hGM-CSF is a rice cell ( Oryza sativa ) has significant plant-derived sugar chains that are non-uniformly attached when expressed in sativa . These sugar chains increase the stability and persistence of hGM-CSF, but have difficulty in making purification. According to Tae-ho Kwon et al., HGM-CSF expressed in rice cells is very non-uniform, but all have titers ( Biotechnology and Bioprocess Engineering 9, 423-427 (2004)), the inventors have also been aware of the complexity of rice-derived hGM-CSF from the beginning of constructing a method for separating and purifying hGM-CSF from rice cell liquid suspension culture. In other words, the plant-derived sugar chain formed in hGM-CSF has a complicated structure and is significantly different from the sugar chain structure of animal cells or yeast cells to purify rice cell-derived hGM-CSF with high purity by applying the conditions of conventional chromatography methods. It was expected that considerable difficulties would follow.

The present inventors have applied rice cell-derived hGM by combining anion exchange chromatography, gel filtration chromatography, and hydrophobic chromatography, which several researchers used to separate and purify human granulocyte macrophage colony stimulator hGM-CSF derived from yeast and Escherichia coli in high purity. To find a method to separate and purify -CSF with high purity.

However, under conventional chromatographic conditions, amylase produced by the rice cell gene expression promoter system, as well as plant-derived contaminating proteins similar to the molecular weight of rice cell-derived hGM-CSF, was added to hGM-CSF-containing rice cell liquid suspension cultures. It was not easily removed from. Therefore, it is a major technical task of the present invention to establish a method for separating and purifying rice cell-derived hGM-CSF with high purity by completely removing these impurities from rice cell liquid suspension culture.

In order to solve these technical problems, the present inventors have repeated numerous experiments and found that contaminating proteins, which are difficult to remove by the conventional method, can be effectively removed from the hGM-CSF solution by using a combination of hydrophobic chromatography and ammonium sulfate. The present invention has been completed.

The purely purified and purified rice cell-derived hGM-CSF showed different reaction patterns when compared to the hGM-CSF international standard, and other hGM-CSF derived from E. coli and yeast, but the biological activity was excellent.

The present invention is a rice cell ( Oryza sativa ) hGM-CSF, a human granulocyte macrophage colony stimulating factor, is isolated from liquid suspension cultures and contains a rice cell gene expression promoter as well as a contaminant protein similar in size and size to hGM-CSF that is not easily isolated by complex sugar chain interference. The present invention relates to a method for effectively removing amylase produced by the system by hydrophobic chromatography and ammonium sulfate elution combination, wherein the rice cell-derived hGM-CSF is isolated and purified purely from rice cell liquid suspension culture according to the present invention. can do.

Rice cell-derived hGM-CSF is expressed by acting on the alpha amylase promoter by sugar depletion in rice cell liquid suspension culture and secreted into the culture. At the end of the incubation, the rice cells in the liquid suspension culture are removed using a filter. Since rice cell-derived hGM-CSF is non-uniform but has an average molecular weight of about 25 kDa, a 10 KDa UF membrane is used to concentrate the hGM-CSF. Filter the cultures with pre-filter, concentrate the solution volume to 1/10 using a 10 KDa UF membrane, desalting and load into DEAE anion exchange chromatography previously equilibrated with buffer.

Since rice cell-derived hGM-CSF has an isoelectric point at an acidic pH, concentrated desalted hGM-CSF is well adsorbed on DEAE anion exchange chromatography at neutral or higher pH conditions. The rice-derived hGM-CSF was best adsorbed in pH 8.5 buffer, the adsorption rate was slightly lowered at pH 6.5, while the separation of hGM-CSF and impure protein was the best at pH 7.5. At 7.5. Rice cell-derived hGM-CSF adsorbed on DEAE anion exchange chromatography was eluted by adding gradients of 50, 100, 150, 200, and 250 mM NaCl. SDS-PAGE confirmed the elution of rice cell-derived hGM-CSF in 20 mM phosphate buffer (pH 7.5) and 100 mM NaCl concentration gradient, many impurities in the rice cell liquid suspension culture was removed and removed (Fig. 1).

However, amylase produced by the rice cell gene expression promoter system was not well removed by DEAE anion exchange chromatography method under these conditions. Thus, gel filtration chromatography was applied to effectively remove amylase from rice cell-derived hGM-CSF, but amylase was not isolated at all despite its significantly higher molecular weight than rice cell-derived hGM-CSF. This was judged to be due to amylase adsorbed to sugar chains of hGM-CSF or dissolved physically with hGM-CSF.

To effectively remove amylase, which is difficult to separate in gel filtration chromatography, the pH is gradually adjusted to 5.0 by adding 1 M HCl in a magnetic stirrer to precipitate. In this process, amylase is substantially removed from hGM-CSF (Fig. 2). In addition, hydrophobic chromatography was applied to effectively remove impurity proteins having a molecular weight around hGM-CSF which is not easy to remove. The pH of the DEAE anion exchange chromatography eluate containing hGM-CSF derived from rice cells was adjusted to 7.0 and the salt concentration was adjusted to 2 M NaCl and 2.5 M NaCl by addition of NaCl, followed by hGM on hydrophobic phenyl sepharose chromography. -CSF was adsorbed. Thereafter, the concentration of NaCl was lowered to elute the hGM-CSF, but the adsorption of the hydrophobic phenyl sepharose hGM-CSF was not good when the NaCl salt was used, and the separation of the hGM-CSF was not so good even when the elution was performed. 6 and FIG. 7).

Therefore, the present inventors have tried different salts. As a result, when using ammonium sulfate salt ((NH ₄ ) ₂ SO ₄ ), hGM-CSF having an excess sugar chain derived from rice cells was well adsorbed, and the hGM-CSF was released even when eluted. The present invention was completed by discovering high eluting. If this is explained in more detail as follows. Adjust the salt concentration of the DEAE anion exchange chromatography eluate with ammonium sulphate to 1.5 M, and then adsorb the hGM-CSF to the hydrophobic phenyl sepharose chromatography and lower the concentration of ammonium sulphate (1.2 M, 0.9 M, 0.6 M, 0.3 M, gradient to 0 M sequence) to elute hGM-CSF. Analysis of the elution fraction by SDS-PAGE confirms that the hGM-CSF is eluted in 1.2 M, 0.9 M, and 0.6 M ammonium sulfate. The highest purity of the hGM-CSF is eluted in 0.9 M ammonium sulfate (FIG. 3). Ammonium sulfate of 0.9 M can be easily removed by Diafiltration using a 10 KDa Molecular Cut-off UF concentrator. The salt-removed solution is finally substituted with PBS buffer.

As described above, it can be seen that the combined use of hydrophobic chromatography and ammonium sulfate salt is very effective for the separation and purification of hGM-CSF to which the sugar cell-derived sugar chains are non-uniformly attached. Through this core process, a pattern of a typical hGM-CSF by sugar chains derived from rice cells can be obtained, and the impurity bands around the hGM-CSF or the Amylase impurity bands above can be removed very effectively. The purely purified rice cell-derived hGM-CSF showed excellent biological activity when compared to the titer of hGM-CSF international standard.

The use of a combination of hydrophobic chromatography and ammonium sulphate found by the inventors of the present invention is very specific when the hydrophobic chromatography does not show such resolution when NaCl salt is used. It is expected to be very effective.

While the invention has been described in detail with reference to the described embodiments, it will be apparent to those skilled in the art that various modifications and variations are possible within the scope and spirit of the invention, and such variations and modifications are within the scope of the appended claims. will be.

Example 1 Pretreatment of Cell Suspension Culture Solution Containing Rice Cell-derived hGM-CSF

20 L of rice cell suspension culture was used to filter the cells and the culture. The culture medium filtered by the pre-filter was secondary filter using a 0.45 microcapsule filter, and the culture medium passed through the filtered culture medium using a Millipore 10 KDa Molecular Cut-off UF concentrator (membrane area 0.5M ² ) was discarded. The solution was concentrated to 1 L of volume. After diluting the media components and salts and adsorbing the DEAE ion exchange resin to a total volume of 2 L with 20 mM Phosphate buffer (pH7.5), UF concentration dilution was repeated about 10 times. Thereafter, 1 L of the concentrated solution was transferred to a beaker, and the UF membrane was washed with Phosphate buffer to recover a total of 1.5 L solution.

The collected solution was placed in a 500 mL centrifuge container, about 300 mL, centrifuged at 15,000 g for 30 minutes, and the solution was centrifuged in Whatman filter paper 4, collected in a beaker, and refrigerated until use in the next step. .

Example 2 Recovery and Purification of Rice Cell-derived hGM-CSF Using Anion Exchange Resin

500 mL of DEAE Sepharose anion exchange resin was packed into B & B BPG100 / 500 column, and 20 mM Phosphate buffer (pH7.5) was equilibrated by flowing the flow at 100 mL / min, and the refrigerated solution containing hGM-CSF was 100 Adsorption was carried out on the anion exchange resin at a mL / min flow rate, and the equilibrium left buffer solution was loaded. Solutions were prepared in equilibration buffers with NaCl concentrations of 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, eluted hGM-CSF from low salt concentration to high salt concentration and fractionated into several 1 L vessels. . The fractionated eluate was checked for the presence and purity of hGM-CSF by 15% SDS-PAGE (Fig. 1). HGM-CSF was identified in the 100 mM NaCl fraction.

Example 3 Impurity Removal by Acid Treatment

The 100 mM NaCl fraction solution eluted from the DEAE Sepharose anion exchange resin of Example 2 was recovered and concentrated by a 10 KDa Molecular Cut-off UF concentrator (Millipore Lab Scale TFF System) to reduce the volume to 200 mL and recovered in a beaker. The pH is then slowly adjusted to 5.0 by adding 1 M HCl in a magnetic stirrer. At this time, precipitates were generated. In order to remove them, 200 mL was placed in a 500 mL centrifuge container and centrifuged at 15,000 g for 30 minutes, and filtered with Whatman filter paper 4. The filtered solution was slowly raised to 7.0 with 1 M NaOH while stirring in a magnetic stirrer and then adjusted to 1.5 M ammonium sulfate (pH 7.0). In this process, amylase impurities can be significantly reduced (FIG. 2).

Example 4 Purity of Rice Cell-derived hGM-CSF by Hydrophobic Chromatography and Ammonium Sulfate Combination

Hydrophobic phenyl sepharose was packed into 50 mL of B & B's XK50 column and equilibrated at 20 mL / min flow rate using 20 mM Phosphate buffer (pH 7.0) containing 1.5 M ammonium sulfate. The filtrate of Example 3 was then passed through hydrophobic phenyl sepharose to adsorb hGM-CSF and washed with hydrophobic phenyl sepharose with 20 mM Phosphate buffer (pH 7.0) containing 1.5 M ammonium sulfate, followed by 1.2 M, Ammonium sulfate solution was added to hydrophobic phenyl sepharose in a 0.9 M, 0.6 M, 0.3 M, 0 M gradient to elute hGM-CSF. The elution fractions were divided into 10 mL for each ammonium sulfate concentration in 50 mL plastic containers by concentration. The fractionated eluate was refrigerated until analysis, and the content and purity of hGM-CSF were confirmed by 15% SDS-PAGE (FIG. 4).

Example 5 Removal of Ammonium Sulfate

15% SDS-PAGE confirmed that the highest purity of 0.9 M ammonium sulphate eluting solution 1 to 10 and 0.6 M ammonium sulphate eluting solution 1 to 3 was obtained in 50 mL fractions eluted from hydrophobic phenyl sepharose (FIG. 4). ). After collecting the ammonium sulfate by dialfiltration method using a 10 KDa Molecular Cut-off UF concentrator, the resultant solution was converted into PBS buffer solution and finally sterilized by a 0.2 um filter to prepare a final concentrated stock solution that can be used as a biopharmaceutical raw material.

To confirm the purity of the concentrated stock solution, 10-fold and 20-fold dilutions were separated by 15% SDS-PAGE and stained with Silver Staining. As a result, a typical pattern of hGM-CSF due to sugar chains derived from rice cells was observed. No band or upper Amylase impurity band could be observed at all (FIG. 5). Therefore, it can be seen that the combined use of hydrophobic chromatography and ammonium sulfate salt is very effective for the separation and purification of hGM-CSF to which the sugar cell-derived sugar chains are attached in a large amount.

1 is a view showing the results of SDS-PAGE verification of the DEAE ion exchange chromatography separation purification process.

2 is a SDS-PAGE confirming result of the effect of removing Amylase impurities by acid treatment.

FIG. 3 is an SDS-PAGE confirming diagram of hGM-CSF separation and purification effect by a combination of hydrophobic phenyl sepharose chromatography and ammonium sulfate. FIG.

4 is a SDS-PAGE confirming result of ammonium sulfate concentration gradient (0.9 M-0.6 M) during hydrophobic phenyl sepharose chromatography separation and purification process.

5 is a diagram showing the purity of the final concentration of hGM-CSF after removal of ammonium sulfate by Diafiltration.

FIG. 6 is a diagram showing the results of SDS-PAGE identification of hGM-CSF separation and purification by hydrophobic phenyl sepharose chromatography and 2M NaCl.

7 is a SDS-PAGE confirming result of the purification process of hGM-CSF separation by hydrophobic phenyl sepharose chromatography and 2.5 M NaCl combination.

Claims (3)

A method for purely separating and purifying recombinant human granulocyte macrophage colony stimulator hGM-CSF from rice cell liquid suspension culture or pretreated solution using a combination of hydrophobic chromatography and ammonium sulfate. The method of claim 1, wherein the hydrophobic chromatography is phenyl Sepharose (Phenyl Sepharose). The method according to claim 1, wherein the applied salt concentration condition of hydrophobic chromatography is at least 1.2 M ammonium sulfate, and the eluted salt concentration condition of hGM-CSF is between 1.2 M and 0.6 M ammonium sulfate salt.

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