METHOD FOR PURIFYING DIGLYCOSYLATED INTERFERON-BETA PROTEIN
20220259260 · 2022-08-18
Inventors
Cpc classification
International classification
Abstract
The present invention relates to a method for purifying a diglycosylated interferon-beta protein and, more specifically, to a method for purifying a diglycosylated interferon-beta protein, wherein a culture solution containing interferon-beta expressed in host cells is obtained, and subjected to affinity chromatography, low-pH inactivation, hydrophobic interaction chromatography, anion-exchange chromatography, and cation-exchange chromatography, and to a diglycosylated interferon-beta protein separated by the method.
Claims
1. A method for purifying a diglycosylated interferon-beta protein comprising the following steps: (a) obtaining a culture solution comprising interferon-beta expressed in a host cell; (b) performing affinity chromatography for the culture solution obtained in step (a); (c) low pH inactivating the solution obtained in step (b); (d) performing hydrophobic interaction chromatography for the solution obtained in step (c); (e) performing anion-exchange chromatography for the solution obtained in step (d); and (f) performing cation-exchange chromatography for the solution obtained in step (e).
2. The method of claim 1, wherein the interferon-beta protein consists of a peptide sequence of SEQ ID NO: 1.
3. The method of claim 1, wherein the host cell is a chinese hamster ovary (CHO) cell.
4. The method of claim 1, wherein the affinity chromatography is performed using a blue sepharose as a resin.
5. The method of claim 1, wherein the hydrophobic interaction chromatography is performed using a butyl sepharose as a resin.
6. The method of claim 1, wherein the anion-exchange chromatography is performed using a Q resin as a resin.
7. The method of claim 1, wherein the cation-exchange chromatography is performed using SP sepharose as a resin.
8. A diglycosylated interferon-beta protein obtained by the purification method of claim 1.
9. The diglycosylated interferon-beta protein of claim 8, wherein the interferon-beta protein has a base sequence of SEQ ID NO: 1 and has sugar chains at amino acid positions 25 and 80.
10. The diglycosylated interferon-beta protein of claim 8, wherein the interferon-beta protein includes any two sugar chain structures of A2G2, FA2G2, FA2G2S1, FA2G2S2, FA2G2S*2, FA3G3S1, FA3G3S3, FA3G3S*3, FA4G4S1, FA4G4S3, FA4G4S4, FA4G4S*3, FA4G4S*4, FA4G4Lac1S3, FA4G4Lac1S*3, FA4G4Lac1S4, FA4G4Lac2S3, and FA4G4Lac1S*3.
Description
DESCRIPTION OF DRAWINGS
[0084]
[0085]
MODES FOR THE INVENTION
[0086] Hereinafter, the present invention will be described in detail.
[0087] However, the following Examples are just illustrative of the present invention, and the contents of the present invention are not limited to the following Examples.
Example 1
Expression and Obtainment of Diglycosylated Interferon-Beta in Host Cells
[0088] A culture solution expressed interferon-beta by culturing a producing CHO cell line including a corresponding diglycosylated interferon-beta gene in a 5 L scale using a medium based on optiCHO SFM of Hyclone.
[0089] The culture solution was cultured while slowly stirring at an initial temperature of 34.0° C., pH 7.0, and 50% dissolved oxygen, and cultured for 9 days, and then the culture solution recovered from an incubator was centrifuged under conditions of 4500 rpm and 4° C. for 30 minutes. After the centrifugation, a supernatant was aseptically subjected to 0.22 μm filtration, and purification was performed.
Example 2
Purification of Diglycosylated Interferon-Beta
[0090] The interferon-beta obtained in Example 1 was added to a purification process using affinity chromatography.
[0091] First, the harvested interferon-beta fractions were bound with a Blue sepharose 6 fast flow (GE) column pre-equilibrated with a solution of 10 mM sodium phosphate, 137 mM NaCl, 2.7 mM KCl, and pH 7.4, and sufficiently washed with a 700 mM sodium thiocyanate solution. Thereafter, the interferon-beta fractions were eluted with a solution of 20 mM sodium phosphate, 1.4 M NaCl, 35% propylene glycol, 250 mM L-Arginine, and pH 7.4 to be harvested.
[0092] As a result of HPLC analysis, purity of 94% to 96% was shown, and as a result of residual HCP detection, the first purification was completed with the content of 6300 to 9500 ppm.
[0093] In a second step, pH 3.5 of phosphoric acid (99%, and 10-fold diluted using distilled water) was added to adjust the pH to 3.0. Thereafter, the phosphoric acid was treated at 22° C. for 1 hour and then neutralized with 2 M Trizma base to be sampled.
[0094] In a third step, the fractions were bound to a butyl sepharose (Butyl 4 fast flow; GE) column pre-equilibrated with a solution of 20 mM Tris, 1.4 M NaCl, 300 mM Arg, and pH 7.4 and then eluted with a solution of 20 mM Tris and pH 8.0 to be harvested.
[0095] In a fourth step, anion-exchange chromatography filled with a Capto Q ImpRes (GE) resin using the solution of 20 mM Tris and pH 8.0 was performed using the obtained eluted fractions. The solution for eluting the solvent used 20 mM Tris, 140 mM NaCl, and pH 8.0.
[0096] In a final step, the fractions eluted in an anion-exchange resin before were added to a SP sepharose fast flow (GE) column equilibrated with 50 mM sodium acetate and pH 5.0 to perform cation-exchange chromatography by a salt concentration difference from 150 mM to 500 mM.
[0097] As a result, finally, the interferon-beta material obtained with yield of 32% exhibited purity of 95% or more, the HCP recorded 100 ppm which was a reference value or less, and as an IEF and SDS-PAGE gel analysis result, a final product with nothing significant was obtained.
Comparative Example 1
Purification According to Conventional Purification Method for Interferon-Beta Protein
[0098] Interferon-beta fractions obtained from the culture were bound with a Blue sepharose 6 fast flow (GE) column pre-equilibrated with a solution of 10 mM sodium phosphate, 137 mM NaCl, 2.7 mM KCl, and pH 7.4, and sufficiently washed with a 700 mM sodium thiocyanate solution. Thereafter, the interferon-beta fractions were eluted with a solution of 20 mM sodium phosphate, 1.4 M NaCl, 35% propylene glycol, and pH 7.0 to be harvested.
[0099] The fractions obtained in the above process were diluted 5-fold with a solution of 20 mM sodium phosphate and pH 2.7 and added to cation-exchange chromatography as a second process using a CM sepharose fast flow (GE) resin. The elution solvent used at this time was 50 mM NaPi, 0.145 M NaCl, 10% PG, and pH 7.0.
[0100] Thereafter, the eluted interferon-beta sample was eluted by using acetonitrile 30% as an organic solvent and trifluoracetic acid 0.1% as a buffer solution through an HPLC reverse-phase chromatography colum (VYDAC C4; Agilent). At this time, 20 mM sodium phosphate monobasic pH 2.9 was pre-contained in a collecting container of the eluted sample so that the sample was eluted and diluted at the same time, and the interferon-beta was obtained.
[0101] In the previous process, a 7-fold diluted interferon-beta eluting sample was concentrated about 7 times through a TFF system (Pellicon Biomax 10; Millipore) of Millipore for ultrafiltration and concentration, in order to be purified by the most efficient method while reducing the capacity. At this time, the exchanging solution was used as 20 mM sodium phosphate pH 2.9, and the concentration of the concentrate was generally based on the resin properties of size exclusion chromatography.
[0102] As a final process, unnecessary materials were removed using size exclusion chromatography (Sephacryl 100HR; GE), and at this time, the eluted sample was collected by 25 mL, and finally, the fractions including interferon-beta were obtained.
Example 3
Evaluation of Purification Method
[0103] 3-1. Evaluation of Activity, Yield, etc.
[0104] The purification methods of Example 2 and Comparative Example 1 were evaluated in terms of yield, modification of the protein, inactivity, content, and total activity.
[0105] The protein quantification used a UV measurement method. A zero point was adjusted by a solution of adding 0.5 M NaOH and a size exclusion chromatography eluting solvent at 1:1 and a product obtained in the size exclusion chromatography and 0.5 M NaOH were mixed at 1:1 and measured at UV wavelength 290 nm. The measurement was based on three times and was digitized by the following Equation.
(Average of 3 measured values÷extinction coefficient)×dilute magnification (mg/mL)
[0106] The yield was based on an elute (100%) passing through blue sepharose and a relative content thereto was measured by an enzyme-linked immunosorbent assay (ELISA) using a primary antibody specific to interferon-beta, and the ELISA used at this time was as follows. After an enzyme-conjugated antibody solution was dispensed in a measurement plate according to a concentration, a standard solution and a test solution to be measured were added two times to at least 8 dilution concentrations. Thereafter, a washing process was subjected after a reaction for a certain time, and then a substrate solution causing a coloring reaction was added to induce the reaction. After the reaction was stopped, the absorbance was measured at 450 nm.
[0107] The protein modification was measured by performing peptide mapping using HPLC equipment. An analytical sample was injected using a chromatographic system and a meter, and a protein structure and amino acid denaturation corresponding to a retention value and an integral value were confirmed with respect to values measured at a wavelength of 214 nm.
[0108] The inactivity was measured as follows by a cytopathic effect (CPE) assay. A standard material for measuring the inactivity was prepared as follows: Interferon-beta purchased by the National Institute for Biological Standards and Control (NIBSC) was mixed in an MEM culture medium to make an appropriate activity standard stock solution (2,000 IU/mL)) and then prepared by diluting STD activity (IU/mL) sequentially from 200 to 0.390625 IU/mL.
[0109] Each purified sample was diluted using an MEM culture medium so that expected activity become 50 IU/mL (volume was 1500 μl or higher) and diluted sequentially from 50 to 0.390625 IU/mL to prepare a measuring sample.
[0110] The measuring sample and the standard material were dispensed in a 96-well culture plate, respectively, and A549 cells were added to wells at 3×10.sup.4 cells/well and then incubated for 22 hours under 37° C. and 5% CO.sub.2 conditions. The medium was removed from the plate, and a virus solution (1000 TCID50/ml) was dispensed in each well, and incubated for 22 hours under 37° C. and 5% CO.sub.2 conditions. The solution was removed from the plate and then stained with a Crystal Violet solution at room temperature, and bleached with 2-Methoxyethanol, and then the absorbance was measured at 570 nm to calculate the inactivity.
[0111] The results of respective analysis items were shown in the following table.
TABLE-US-00002 TABLE 2 Analysis item Comparative Example 1 Example 2 Final product Total quantity (UV) 16.5 mg 26.4 mg Yield (ELISA, First) 10.9% 19% Yield (ELISA, Second) 11.5% 20.1% N’ -Met deletion form 3.6% 2.8% (Peptide mapping) Specific activity (CPE/UV) 327 MIU/mg 247 MIU/mg Total activity 5395 MIU 6520 MIU
[0112] As the measuring result, in the method of Comparative Example 1, the total content was 16.5 mg, the yield was about 11%, and a Met deletion form was shown as 3.6%. The inactivity was higher than the method of the present invention as 327 MIU/mg, and the purified content was not only about 16.5 mg, and the total activity was only 5395 MIU.
[0113] On the contrary, in the method of the present invention, the total content was 26.4 mg, the yield was about 20%, the Met deletion form was 2.8%, and the total activity was high as 6520 MIU.
[0114] Therefore, it could be seen that the diglycosylated interferon-beta may be separated/purified by the method of the present invention to have excellent quality and yield and higher activity.
[0115] 3-2. Evaluation of Impurities
[0116] In the purification of Comparative Example 1 and Example 2, the impurities were confirmed in terms of a cell-derived protein (CHO-HCP) content of CHO cells, an endotoxin content, and patterns in IEF.
[0117] A CHO cell-derived protein content to be separated from the interferon-beta as the target protein was measured by using the ELISA. A dilution solution was prepared and dispensed in the plate and the analytical sample was diluted to the final half. In addition to the diluted sample, the standard solution was transferred and dispensed to a plate included in an analytical kit (wherein the enzyme-conjugated antibody was applied to a kit-attached plate), and a substrate solution representing the coloring reaction was added to induce the reaction. Thereafter, in the plate, the CHO cell-derived protein amount to a standard calibration curve was analyzed by measuring the absorbance at a wavelength of 450 nm.
[0118] Limulus amebocyte lysate (LAL) assay was used to measure a remaining endotoxin level. An endotoxin standard sample was mixed well with LAL analysis-dedicated distilled water, and then prepared at a concentration to draw the standard calibration curve in a test tube for analysis. A standard sample, a zero point solution, and an analysis sample for each concentration prepared were dispensed to the plate at the same amount. An LAL reagent was well dissolved in the analysis-dedicated distilled water, and then uniformly dispensed to the above prepared plate to induce the reaction. In the plate after the reaction ended, an endotoxin level present in a target material was measured by measuring the absorbance at a wavelength of 405 nm.
[0119] Positive and negative buffers to be used for IEF gel analysis were prepared and mixed with an IEF analysis buffer (pH 3 to 10) by calculating the amount of the analystic sample and the standard sample to be the same as each other to make a development sample for analysis. An IEG gel of pH 3 to 10 was attached to a development container and then the positive buffer was filled in the development container and the negative buffer was filled in an external space, and the prepared samples were injected to the gel. The samples were developed in the prepared development container in the order of 200 V for 1 hour and 500 V for 30 minutes and then the completed gel was separated and subjected to coomassie dyeing and bleaching, and isoelectric points of the protein was determined through a chemiluminescence analyzer.
TABLE-US-00003 TABLE 3 Analysis item Comparative Example 1 Example 2 CHO-HCP 65 ppm 42 ppm Endotoxin 0.00 EU/ug 0.00 EU/ug IEF Result (Fig 1) Confirm basic variant Non-confirm basic variant
[0120] As a result, the protein content of CHO cells was 65 ppm in Comparative Example 1 and 42 ppm according to the method of the present invention. Although the protein contents of Comparative Example 1 and the present invention were presented to 100 ppm or less as a general reference value, in the present invention, it was confirmed that the protein content was slightly low by performing cation-exchange chromatography following anion-exchange chromatography.
[0121] The endotoxin was not contained in both cases, and as a result of confirming the IEF result (
INDUSTRIAL APPLICABILITY
[0122] As described above, the purification method of the interferon-beta protein provided in the present invention is significantly excellent in purification yield of the diglycosylated interferon-beta protein, easy in manufacturing management and quality management in mass culture, and may purify the diglycosylated interferon-beta protein with excellent quality suitable for regulations to be used as medicines, and thus has large industrial applicability.