METHOD FOR PURIFYING TGF-beta3 PROTEIN

Abstract

The invention relates to a pretreatment method for high-efficiency and high-purity separation and purification of TGF-beta3 protein from Escherichia coli, and a TGF-beta3 protein purification method comprising the pretreatment method. The pretreatment method and the purification method comprising the same according to the present invention have significantly lower process complexity than animal cell culture methods and high production efficiency due to the use of Escherichia coli, thereby enabling the separation of high-purity TGF-beta3 protein from Escherichia coli and purification with high efficiency and high purity.

Description

Technical Field

[0001] This application claims priority to Korean Patent Application No. 10-2022-0146295, filed on November 4, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a pretreatment method for efficiently and highly purely separating and purifying TGF-β3 protein from Escherichia coli (E. coli), and a method for purifying TGF-β3 protein including the pretreatment method. Background Art

[0003] TGF-β3 is a subtype of transforming growth factor-β (TGF-β) and is crucial for various biological processes, including endodermal development, organ formation, epithelial proliferation, extracellular matrix synthesis, and immune responses. Specifically, TGF-β3 stimulates mesenchymal lineage cells, inhibits epithelial or neuroectodermal lineage cells, and regulates skin wound repair, remodeling, and potential scar formation by participating in the TGF-β1 / Smad signaling pathway.

[0004] The large-scale production of bioactive human TGF-β3 faces challenges in both prokaryotic and eukaryotic expression systems. The eukaryotic expression system has low yields and complex, expensive processes, while the prokaryotic expression system produces inactive inclusion bodies. Therefore, there is an urgent need for a novel, scalable, time-saving, and economical production platform for producing highly pure TGF-β3.

[0005] [Related Technical Literature]

[0006] [Patent Literature]

[0007] (Patent Document 1) KR 10-0991203B1 (published on October 26, 2010)

[0008] (Patent Document 2) KR 10-1443257B1 (published on September 16, 2014) Summary of the Invention

[0009] Technical Problem

[0010] The inventors of the present invention have dedicated themselves to developing a novel, scalable, time-saving, and economical production platform for producing highly pure TGF-β3, and the results have confirmed that when using the pretreatment method of the present invention and the purification method including this method, highly pure TGF-β3 can be efficiently separated and purified, thus completing the present invention.

[0011] Accordingly, an object of the present invention is to provide a pretreatment method for efficiently and highly purely separating and purifying TGF-β3 protein from Escherichia coli, and a method for purifying TGF-β3 protein including the pretreatment method.

[0012] Technical solution

[0013] The present invention provides a pretreatment method for purifying TGF-β3 protein, comprising the following steps:

[0014] i) The step of recovering Escherichia coli from an Escherichia coli culture medium;

[0015] ii) The step of disrupting the recovered Escherichia coli to obtain TGF-β3 inclusion bodies;

[0016] iii) The step of washing the inclusion bodies;

[0017] iv) The step of dissolving the washed inclusion bodies; and

[0018] v) The step of refolding the dissolved inclusion bodies.

[0019] According to a preferred embodiment of the present invention, the disruption in step ii) is carried out 1 to 5 times at a pressure of 800 to 1200 bar.

[0020] According to a preferred embodiment of the present invention, the washing in step iii) is carried out 1 to 5 times.

[0021] According to a preferred embodiment of the present invention, the washing in step iii) includes first washing with a surfactant and then washing with distilled water.

[0022] According to a preferred embodiment of the present invention, the dissolution in step iv) is carried out using a buffer solution containing one or more selected from the group consisting of urea, 1,4-dithiothreitol (DTT), and tris(hydroxymethyl)aminomethane.

[0023] According to a preferred embodiment of the present invention, the dissolution in step iv) is carried out using a buffer solution with a pH of 7 to 9.

[0024] According to a preferred embodiment of the present invention, the dissolution in step iv) is carried out using a buffer solution for 1 to 24 hours.

[0025] According to a preferred embodiment of the present invention, the refolding in step v) is carried out using a buffer solution containing one or more selected from the group consisting of N-cyclohexyl-2-aminoethanesulfonic acid (CHES), NaCl, reduced glutathione, oxidized glutathione, 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate (CHAPS), L-arginine, and D-sorbitol.

[0026] According to a preferred embodiment of the present invention, the refolding in step v) is carried out using a buffer solution with a pH of 8.5 to 10.5.

[0027] According to a preferred embodiment of the present invention, the renaturation in step v) is carried out for 1 to 12 days using a buffer solution.

[0028] According to a preferred embodiment of the present invention, the renaturation in step v) is carried out such that the final concentration is 0.1 to 2.0 g / L.

[0029] In addition, the present invention provides a method for purifying TGF-β3 protein, comprising the following steps:

[0030] i) a step of recovering Escherichia coli from an Escherichia coli culture medium;

[0031] ii) a step of disrupting the recovered Escherichia coli to obtain TGF-β3 inclusion bodies;

[0032] iii) a step of washing the inclusion bodies;

[0033] iv) a step of dissolving the washed inclusion bodies;

[0034] v) a step of renaturing the dissolved inclusion bodies;

[0035] vi) a step of purifying the solution containing renatured TGF-β3; and

[0036] vii) a step of filtering the purified solution.

[0037] According to a preferred embodiment of the present invention, the purification in step vi) includes:

[0038] a) a step of performing hydrophobic interaction chromatography as primary purification;

[0039] b) a step of performing multimodal chromatography as secondary purification; and

[0040] c) a step of performing cation exchange chromatography as tertiary purification.

[0041] According to a preferred embodiment of the present invention, the filtration in step vii) is ultrafiltration / diafiltration.

[0042] The present invention will be described in detail below through specific embodiments.

[0043] The inventors of the present invention have developed an optimized pretreatment step for the isolation and purification of high-purity TGF-β3 from Escherichia coli, and the method for purifying TGF-β3 protein including the pretreatment step of the present invention can efficiently isolate and purify high-purity TGF-β3 from Escherichia coli.

[0044] Specifically, the present inventors have not only developed a method for separating and purifying high-purity TGF-β3, but also further improved the process method that can shorten the process time, thereby providing a more efficient method for purifying TGF-β3 protein. In particular, when the pretreatment method and purification method of the present invention are adopted, 1 to 1.5 g of TGF-β3 protein can be obtained from 2.5 L of Escherichia coli culture medium.

[0045] Therefore, the present invention can provide a pretreatment method for purifying TGF-β3 protein, comprising:

[0046] i) the step of recovering Escherichia coli from an Escherichia coli culture medium;

[0047] ii) the step of disrupting the recovered Escherichia coli to obtain TGF-β3 inclusion bodies;

[0048] iii) the step of washing the inclusion bodies;

[0049] iv) the step of dissolving the washed inclusion bodies; and

[0050] v) the step of renaturing the dissolved inclusion bodies.

[0051] According to a preferred embodiment of the present invention, the disruption in step ii) can be carried out 1 to 5 times at a pressure of 800 to 1200 bar. More preferably, the disruption in step ii) can be carried out 2 to 3 times at a pressure of 1000 bar.

[0052] According to a preferred embodiment of the present invention, the washing in step iii) can be carried out 1 to 5 times. More preferably, the washing in step iii) can be carried out 2 to 3 times.

[0053] According to a preferred embodiment of the present invention, the washing in step iii) may include: the step of washing with a surfactant and then washing with distilled water.

[0054] The surfactant may be Tween 20.

[0055] The washing with the surfactant can be carried out for 1 to 24 hours, more preferably 2 to 18 hours.

[0056] The number of times of washing with distilled water can be 1 to 3 times, more preferably 1 to 2 times.

[0057] The washing with distilled water can be carried out for 1 to 24 hours, more preferably 2 to 18 hours.

[0058] According to a preferred embodiment of the present invention, the dissolution in step iv) can be carried out using a buffer solution containing at least one selected from the group consisting of urea, 1,4-dithiothreitol (DTT), and tris(hydroxymethyl)aminomethane.

[0059] According to a preferred embodiment of the present invention, the dissolution in step iv) can be carried out using a buffer solution with a pH of 7 to 9. More preferably, the pH of the buffer solution can be 7.5 to 8.5.

[0060] According to a preferred embodiment of the present invention, the dissolution in step iv) can be carried out using a buffer solution for 1 to 24 hours. More preferably, the dissolution in step iv) can be carried out for 3 to 24 hours.

[0061] According to a preferred embodiment of the present invention, the renaturation in step v) can be carried out using a buffer solution containing at least one selected from the group consisting of 2-(Cyclohexylamino)ethanesulfonic acid (CHES), NaCl, reduced glutathione, oxidized glutathione, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), L-arginine, and D-sorbitol. More preferably, the renaturation in step v) can be carried out using a buffer solution containing at least one selected from the group consisting of CHES, NaCl, reduced glutathione, and oxidized glutathione.

[0062] According to a preferred embodiment of the present invention, the renaturation in step v) can be carried out using a buffer solution with a pH of 8.5 to 10.5. More preferably, the pH of the buffer solution can be 9 to 10.

[0063] According to a preferred embodiment of the present invention, the renaturation in step v) can be carried out using a buffer solution for 1 to 12 days. More preferably, the renaturation in step v) can be carried out using a buffer solution for 3 to 12 days.

[0064] According to a preferred embodiment of the present invention, the renaturation in step v) can be carried out such that the final concentration is 0.1 to 2.0 g / L. Preferably, the renaturation in step v) can be carried out such that the final concentration is 0.1 to 1.5 g / L. More preferably, the renaturation in step v) can be carried out such that the final concentration is 0.1 to 0.5 g / L. Most preferably, the renaturation in step v) can be carried out such that the final concentration is 0.1 to 0.3 g / L.

[0065] In addition, the present invention provides a method for purifying TGF-β3 protein, comprising:

[0066] i) the step of recovering Escherichia coli from an Escherichia coli culture medium;

[0067] ii) the step of disrupting the recovered Escherichia coli to obtain TGF-β3 inclusion bodies;

[0068] iii) the step of washing the inclusion bodies;

[0069] iv) the step of dissolving the washed inclusion bodies;

[0070] v) the step of renaturing the dissolved inclusion bodies;

[0071] vi) Steps for purifying the solution containing refolded TGF-β3; and

[0072] vii) Steps for filtering the purified solution.

[0073] Since steps i) to v) are the same as those included in the above pretreatment method, their descriptions are incorporated by reference to the foregoing description.

[0074] According to a preferred embodiment of the present invention, the purification described in step vi) includes:

[0075] a) Performing hydrophobic interaction chromatography as a primary purification step;

[0076] b) Performing multimodal chromatography as a secondary purification step; and

[0077] c) Performing cation exchange chromatography as a tertiary purification step.

[0078] According to a preferred embodiment of the present invention, the filtration described in step vii) is ultrafiltration / diafiltration.

[0079] Advantages of the Invention

[0080] When using the pretreatment method of the present invention and the purification method including the pretreatment method, due to the use of Escherichia coli, the process complexity is significantly lower than that of animal cell culture methods, and the production efficiency is high. Therefore, high-purity TGF-β3 protein can be efficiently separated and purified from Escherichia coli. Brief Description of the Drawings

[0081] Figure 1 Showing the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis results at different dissolution times. Bands corresponding to the size of the TGF monomer were observed, and the band pattern did not change with the dissolution time.

[0082] Figure 2 Showing the SDS-PAGE analysis results at different refolding concentrations and times. Bands corresponding to the size of the TGF-dimer were observed, and the band pattern did not change with the refolding concentration or time.

[0083] Figure 3 Showing the SDS-PAGE analysis results of the TGF-β3 standard solution (lane 1) and the target protein TGF-β3 (lane 2). It was confirmed that the target protein of the present invention was purified with high purity (M: protein molecular weight marker). Detailed Description of the Embodiments

[0084] The present invention will be further described in detail below by way of examples. These examples are only intended to illustrate the present invention, and it is obvious that the scope of the present invention is not limited by these examples for those skilled in the art.

[0085] Example 1

[0086] Obtain Escherichia coli cells

[0087] Seed culture and main culture were carried out on Escherichia coli BL21(DE3) strain carrying the pTT-TGFβ3 plasmid, and the solution of the main culture process was centrifuged to recover Escherichia coli cells. The recovered cells were added to buffer A (20 mM Tris, 5 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0 ± 0.2), and then suspended.

[0088] Example 2

[0089] Cell disruption

[0090] <2-1>Set the number of cell disruptions

[0091] The cells in the cell recovery solution of [Example 1] were disrupted a total of three times using a high-pressure crusher at a pressure of 1000 bar. During the disruption process, 1 mL samples of the culture solution obtained before disruption, the culture solution after the first disruption, the culture solution after the second disruption, and the culture solution after the third disruption were taken respectively, and the optical density (OD) at a wavelength of 600 nm was measured. In addition, when obtaining the disrupted culture solution, the culture solution obtained in each step was weighed. As shown in the following [Mathematical Formula 1], the disruption rate was calculated using the culture solution weight and the OD value measured at a wavelength of 600 nm.

[0092] Mathematical Formula 1:

[0093] Cell disruption rate (%) = 100 - { (cell concentration in the cell lysate (OD 600 )) / (cell concentration before cell disruption (in the cell recovery solution) (OD 600 ))} × 100

[0094] As a result, as shown in the following [Table 1], it was confirmed that the disruption rate increased by about 13% in the second disruption and about 4% in the third disruption.

[0095] Table 1

[0096] <2-2>Cell disruption process

[0097]

[0098] Inject the cell recovery solution described in [Example 1] into a cell crusher, and perform the first cell disruption at a pressure of 1000 ± 200 bar. Collect the first cell lysate into a 5 L beaker, stir the collected cell lysate and store it. Measure the cell concentration using the first cell lysate.

[0099] Calculate the cell disruption rate according to [Mathematical Formula 1] and confirm its value.

[0100] Inject the first cell lysate into a cell disruptor and perform a second cell disruption at a pressure of 1000 ± 200 bar. Collect the second cell lysate in a 10 L beaker, stir the collected cell lysate, and store it. Use the second cell lysate to measure the cell concentration. Calculate the cell disruption rate according to [Mathematical Formula 1] and confirm its value.

[0101] Example 3

[0102] Inclusion body washing

[0103] <3-1>Washing condition setting

[0104] As a washing step for removing host cell proteins (HCP) and host cell DNA (HCD), a comparative experiment was conducted according to the washing buffer, washing steps, and washing time.

[0105] The washing buffers are as follows: i) 20 mM Tris, 5 mM EDTA, pH 8.0; ii) 20 mM Tris, 5 mM EDTA, 1% Tween 20 (v / v), pH 8.0; iii) 20 mM Tris, 5 mM EDTA, 1.5% Tween 20 (v / v), pH 8.0; and iv) 20 mM Tris, 5 mM EDTA, 1% Triton X-100 (v / v), pH 8.0.

[0106] Centrifuge the cell lysate described in [Example 2] at 8000 rpm for 30 minutes to obtain inclusion bodies (IB). Weigh the obtained IB and divide it into 12 equal parts, and suspend them in 400 mL of washing buffer prepared according to the conditions in [Table 2] below. After washing for an appropriate time (16 hours or 2 hours) under each condition, centrifuge at 8000 rpm for 30 minutes to obtain the first washed IB. The supernatant obtained at this time is stored below -20 °C for analysis and detection. Subsequently, suspend the first washed IB in 400 mL of distilled water (DW) and perform a second wash for an appropriate time according to the conditions in [Table 2]. After the second wash is completed, centrifuge at 8000 rpm for 30 minutes to obtain the washed IB. The supernatant obtained at this time is also stored below -20 °C for analysis and detection.

[0107] Table 2

[0108]

[0109]

[0110] The supernatant obtained after the first wash was analyzed for residual host cell protein (HCP) and host cell DNA (HCD) to evaluate the impurity removal ability of the first washing process. In addition, since the second washing process using distilled water (DW) was designed to remove the detergent used in the first wash, residual Tween 20 was detected for other analysis samples except for samples in conditions 9, 10, and 11 without Tween 20.

[0111] As a result, as shown in [Table 3] below, it was confirmed that after the first wash with a detergent containing Tween 20, the HCD removal rate exceeded 99% under all conditions regardless of the detergent concentration or washing time, and the HCD removal amount was higher compared to the conditions using Triton X-100 as the detergent. It was also confirmed that the HCP removal amount was higher when using Tween 20 as the detergent than in the conditions using Triton X-100 regardless of the concentration and time. When the wash buffer contained no detergent (conditions 9, 10), the HCP removal ability was better than the existing conditions, but the HCD removal ability was comparable to the control conditions and the removal ability decreased compared to the washing conditions with Tween 20.

[0112] Table 3

[0113]

[0114] In addition, it was confirmed that when using distilled water (DW), the Tween 20 removal rate exceeded 99.8% under all conditions regardless of the washing time and number of washes.

[0115] Therefore, the final washing process was set as follows: the first wash was carried out with 1% to 1.5% Tween 20 for 2 to 16 hours, and the second wash was carried out for 2 to 16 hours.

[0116] <3-2>Washing process

[0117] When performing the first inclusion body wash, the cell lysate described in [Example 2] was centrifuged at 4°C and 8000 rpm for 30 minutes. The inclusion bodies recovered by centrifugation were added to buffer B (20 mM Tris, 5 mM EDTA, 1% (v / v) Tween 20, pH 8.0 ± 0.2) and stirred at room temperature for 1 day.

[0118] When performing the second inclusion body wash, the solution from the first inclusion body wash process was centrifuged at 4°C and 8000 rpm for 30 minutes. The inclusion bodies recovered by centrifugation were added to distilled water (DW), and the second inclusion body wash was carried out with stirring at 300 rpm at room temperature for 2 ± 1 hour.

[0119] The solution from the second inclusion body wash process was centrifuged at 4°C and 8000 rpm for 30 minutes, and finally the inclusion bodies were recovered and weighed.

[0120] Example 4

[0121] Dissolution treatment

[0122] <4-1>Dissolution time setting

[0123] The washed inclusion bodies (IB) obtained from [Example 3] were added to a buffer solution (6 M urea, 100 mM dithiothreitol (DTT), 50 mM Tris, pH 8.0), and dissolution treatment was carried out for 24 hours. At 3 hours, 6 hours, 9 hours, and 24 hours after the start of dissolution, 1 mL of the dissolution solution sample was taken for analysis and detection respectively. The sample analysis was carried out using a 660 nm detection kit for concentration determination (twice), and whether the measured sample concentration showed a statistical trend over time was verified by SDS-PAGE.

[0124] As a result, the p-values of the regression analysis results of the concentrations measured twice were 0.2674 and 0.6116, which confirmed that there was no statistical significance in the change of the dissolution solution concentration over time. Based on this result, it was determined that the sample concentration was not affected when the dissolution treatment time was within the range of 3 to 24 hours. In addition, the SDS-PAGE analysis results confirmed that the bands shown due to dissolution corresponded to the molecular weight size of the TGF-β3 monomer, and no change in the SDS-PAGE pattern was observed over time, thereby confirming that the dissolution time had no effect ( Figure 1 ).

[0125] <4-2>Dissolution process

[0126] All the inclusion bodies obtained from 2.5 L of the culture medium were added to 1.2 L of buffer C (6 M urea, 100 mM dithiothreitol (DTT), 50 mM Tris, pH 8.0 ± 0.2). The dissolution solution was stirred at 200 rpm for 16 ± 4 hours for dissolution.

[0127] After the reaction was completed, 5 mL of the dissolution solution was aliquoted into two 15 mL conical tubes, and one of them was stored frozen (-20 ± 5 °C). The 5 mL of the dissolution solution taken was used for protein concentration analysis (660 nm protein assay method).

[0128] Example 5

[0129] Renaturation treatment

[0130] <5-1>Renaturation condition setting

[0131] Prepare a refolding buffer with a total volume of 4 L (containing 0.7 M CHES (cyclohexylaminoethanesulfonic acid), 1 M NaCl, 2 mM reduced glutathione (GSH), 0.4 mM oxidized glutathione (GSSG), pH 9.5). When preparing the buffer, first adjust the pH to 9.2, let it stand overnight (O / N) in a low-temperature chamber, and then readjust the pH to 9.5. Divide the prepared refolding buffer into six equal parts, each part being 500 mL. Based on the concentration of the solution dissolved for 24 hours, add the dissolution solution at final refolding concentrations of 0.1, 0.3, 0.5, 1.0, 1.5, and 2.0 g / L. Before adding the dissolution solution, first pipette out a volume of liquid equal to the volume of the dissolution solution to be added from the aliquoted refolding solution, and then add the dissolution solution to keep the final volume at 500 mL. At 0, 1, 3, 5, 7, and 12 days after the start of refolding, take 1 mL of refolding solution samples, and add 37% hydrochloric acid (HCl) approximately 5% of its volume to the samples to terminate the refolding reaction. After terminating the refolding reaction, centrifuge at 13,000 rpm for 30 minutes, and store the supernatant at -20 °C or below for analysis. Analyze the refolding rate by calculating the peak area of the main peak of the reverse-phase high-performance liquid chromatography (RP-HPLC) of the samples undergoing each refolding cycle reaction. In addition, verify the change of monomer and dimer band patterns over time by SDS-PAGE.

[0132] Perform RP-HPLC analysis on the refolding process solutions sampled at different concentrations and times. The percentage of the dimer peak area (% area) in RP-HPLC under each condition is shown in the following [Table 4].

[0133] Table 4

[0134]

[0135] The results show that when the concentration ≥ 1.0 g / L, a large number of aggregates were formed during the refolding reaction, so the process solution became very turbid. It was confirmed that when refolding was carried out at a concentration ≤ 0.5 g / L, a dimer efficiency of about 55% to 60% was observed, and when additional refolding was carried out from the 3rd day to the 12th day, the dimer efficiency increased by about 3%p at most.

[0136] In addition, the dimer efficiency of each sample was confirmed using SDS-PAGE. No change in the SDS-PAGE pattern was observed, confirming that the refolding concentration and time had no effect ( Figure 2 ).

[0137] <5-2>Renaturation process

[0138] For the refolding process with a protein concentration of 0.1 g / L, the amount of the dissolution solution to be added is calculated according to the following [Mathematical Formula 2].

[0139] [Mathematical Formula 2]

[0140] Volume of dissolution solution added (L) = (Volume of solution during refolding process (L) × 0.1 g / L) / Protein concentration of dissolution solution (g / L)

[0141] After taking out buffer D equivalent to the input volume of the dissolution solution calculated according to [Equation 2] from a 50 L mobile tank, the same volume of the dissolution solution was added. The refolding process was completed after refolding for about 72 hours. The solution after three days of refolding was pretreated by adjusting the pH to 2.0 ± 0.2 with hydrochloric acid solution, and then filtered with a capsule filter.

[0142] The final filtrate was divided into two 5 mL aliquots in 15 mL conical tubes for subsequent process solution analysis (total protein concentration, protein determination at 660 nm). The samples remaining after the process solution analysis were stored frozen (-20 ± 5 °C). The final filtrate was stored at low temperature until used as the injection sample for Purification 1.

[0143] Example 6

[0144] Purification 1 - Hydrophobic Chromatography

[0145] The chromatographic column was equilibrated by injecting more than 2 column volumes (CV) of buffer E (4 M urea, 20 mM sodium acetate (Na-AcOH), 0.1 M NaCl, pH 4.0 ± 0.2). All the pretreated refolding process solution was injected into the chromatographic column. Unbound elution was performed by injecting 10 CV of buffer E into the column.

[0146] Washing was performed by injecting 4 CV of buffer F (20 mM Tris, pH 8.0 ± 0.2) into the column.

[0147] Elution was performed by injecting 3 CV of buffer G (4 M urea, 20 mM Tris, pH 9.5 ± 0.2) into the column. All 3 CV volumes of the eluate were collected starting from the injection of buffer G.

[0148] At the end of the elution, 5 mL of the elution fraction was aliquoted into 15 mL conical tubes for the following process solution analysis: purity (SDS-PAGE electrophoresis), purity (reverse phase high performance liquid chromatography, RP-HPLC) (ion pair chromatography, IPC), and concentration (protein determination at 660 nm).

[0149] Example 7

[0150] Purification 2 - Multimode Chromatography

[0151] The column was equilibrated by injecting more than 2 CV of buffer G (4 M urea, 20 mM Tris-HCl, pH 9.5 ± 0.2). All the eluate of Purification 1 was injected into the column. The unbound substances were eluted by injecting 5 CV of buffer G into the column.

[0152] The column was washed by injecting 10 CV of buffer H (4 M urea, 20 mM Tris-HCl, 0.2 M sodium chloride, pH 9.5 ± 0.2).

[0153] Elution was carried out by injecting 3 CV of buffer I (4 M urea, 20 mM sodium acetate, pH 4.0 ± 0.2) into the column. The eluate was collected starting from the injection of 0.5 CV of buffer I until 3 CV had flowed out.

[0154] At the end of elution, 5 mL of the elution fraction was aliquoted into 15 mL conical tubes for the following process solution analysis: purity (SDS-PAGE), purity (reverse-phase high performance liquid chromatography, RP-HPLC) (ion pair chromatography, IPC), and concentration (protein assay at 660 nm).

[0155] Example 8

[0156] Purification 3 - Ion Exchange Chromatography

[0157] <8-1>Ion exchange chromatography

[0158] The column was equilibrated by injecting more than 2 CV of buffer I (4 M urea, 20 mM sodium acetate, pH 4.0 ± 0.2). All the eluate of Purification 2 was injected into the column. The unbound substances were eluted by injecting 5 CV of buffer I into the column.

[0159] The column was washed by injecting 7 CV of buffer J (4 M urea, 20 mM sodium acetate, 0.2 M sodium chloride, pH 4.0 ± 0.2).

[0160] Elution was carried out by injecting 5 CV of buffer K (4 M urea, 20 mM sodium acetate, 0.5 M sodium chloride, pH 4.0 ± 0.2) into the column. The entire 5 CV volume was collected as the eluate starting from the injection of buffer K. When the collection during elution was completed, the column regeneration and preservation steps could be directly entered. At the end of elution, 5 mL of the elution fraction was aliquoted into 15 mL conical tubes for the following process solution analysis: purity (SDS-PAGE method), purity (RP-HPLC) (IPC), and concentration (protein assay at 660 nm).

[0161] <8-2>Final yield after final purification

[0162] The final yield of transforming growth factor-β3 (TGF-β3) obtained after the third and final purification process is as follows: when the volume of the process solution is 0.732 liters, the TGF-β3 protein concentration is 0.977 g / L, and the amount of TGF-β3 protein is 715.2 mg.

[0163] Example 9

[0164] Filtration

[0165] Install the membrane (10 kDa, 0.1 m 2 *1 piece) in an ultrafiltration / diafiltration system (UF / DF system).

[0166] Inject buffer L (20 mM sodium acetate, pH 3.8 ± 0.2) into the membrane for equilibration. The equilibration is completed when the pH value of the permeate / filtrate reaches 3.8 ± 0.3 and the conductivity is 2 bar or lower.

[0167] Perform concentration filtration (ultrafiltration) under the condition that the transmembrane pressure (TMP = [(feed pressure (P Feed ) + retentate pressure (P Ret )] / 2) is 2 bar or lower.

[0168] The concentration volume is calculated by the following [Equation 3]. The set concentration of the diafiltration substance (DS) is 1.0 g / L. However, considering the difference between the eluate of Purification 3 and the DS concentration analysis method, it is first concentrated to twice the set concentration and then diluted.

[0169] [Equation 3]

[0170] Concentration volume = content of rhTGF-β3 protein in the eluate of Purification 3 (g) / 2.0 g / L

[0171] Inject buffer L equivalent to the concentrated volume for buffer exchange (diafiltration). After six diafiltration cycles, when the pH value of the permeate / filtrate is 3.8 ± 0.2, the filtration process terminates. If not meeting the requirements, perform another cycle of diafiltration and confirm whether the permeate / filtrate meets the pH standard at the end of each cycle.

[0172] To recover the liquid in the pipeline, perform additional concentration. After closing the permeate valve, use buffer L for pipeline recovery.

[0173] The concentration of the recovered solution was measured using the A280 method. An appropriate amount of buffer L was calculated and added to bring the final concentration of the stock solution to 1.0 g / L. When measuring the A280 protein concentration, 500 μL of DS was added to the cuvette, and the absorbance was measured at a wavelength of 280 nm using a multifunctional spectrophotometer (microplate reader). When the measured absorbance was not within the range of 0.3 to 0.7, the dilution factor was adjusted to make the absorbance fall within this range. The measured absorbance was multiplied by the dilution factor and then divided by the extinction coefficient of 1.841 to calculate the protein concentration. The recovered solution with the calculated volume of buffer L was diluted, and after confirming the final concentration using the A280 method, it was filtered through a 0.2-μm bottle-top filter.

[0174] The filtrate with the confirmed concentration was filtered through a 0.2-μm bottle-top filter (polyethersulfone (PES)) into a sterilized glass bottle, and the final solution was dispensed into 50 1.5-mL EP tubes, 1 mL per tube, for batch release testing.

[0175] Example 10

[0176] Specification evaluation

[0177] Specification evaluation was performed on the TGF-β3 finally separated and purified through the processes of Examples 1 to 9, and the results are shown in Table 5 below. Specifically, the electrophoresis results were consistent with the TGF-β3 standard solution (lane 1), and no additional impurity proteins were detected, which confirmed that the target protein TGF-β3 (lane 2) had been purified with high purity ( Figure 3 ).

[0178] Table 5

[0179]

[0180]

[0181] Industrial applicability

[0182] Since the pretreatment method of the present invention and the purification method including the pretreatment method use Escherichia coli, compared with the animal cell culture method, the process is simple and is expected to achieve a high production efficiency. Therefore, high-purity TGF-β3 protein can be efficiently separated and purified from Escherichia coli. Thus, the pretreatment method and the purification method have industrial applicability.

Claims

1. A pretreatment method for purifying TGF-β3 protein, comprising the following steps: i) The step of recovering Escherichia coli from an Escherichia coli culture medium; ii) The step of disrupting the recovered Escherichia coli to obtain TGF-β3 inclusion bodies; iii) The step of washing the inclusion bodies; iv) The step of dissolving the washed inclusion bodies; and v) The step of renaturing the dissolved inclusion bodies.

2. The pretreatment method according to claim 1, wherein the disruption in step ii) is carried out 1 to 5 times at a pressure of 800 to 1200 bar.

3. The pretreatment method according to claim 1, wherein the washing in step iii) is carried out 1 to 5 times.

4. The pretreatment method according to claim 1, wherein the washing in step iii) includes first washing with a surfactant and then washing with distilled water.

5. The pretreatment method according to claim 1, wherein the dissolution in step iv) is carried out using a buffer containing one or more selected from the group consisting of urea, 1,4-dithiothreitol (DTT), and tris(hydroxymethyl)aminomethane.

6. The pretreatment method according to claim 1, wherein the dissolution in step iv) is carried out using a buffer with a pH of 7 to 9.

7. The pretreatment method according to claim 1, wherein the dissolution in step iv) is carried out using a buffer for 1 to 24 hours.

8. The pretreatment method according to claim 1, wherein the renaturation in step v) is carried out using a buffer containing one or more selected from the group consisting of N-cyclohexyl-2-aminoethanesulfonic acid (CHES), NaCl, reduced glutathione, oxidized glutathione, 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate (CHAPS), L-arginine, and D-sorbitol.

9. The pretreatment method according to claim 1, wherein the renaturation in step v) is carried out using a buffer with a pH of 8.5 to 10.

5.

10. The pretreatment method according to claim 1, wherein the renaturation in step v) is carried out using a buffer for 1 to 12 days.

11. The pretreatment method according to claim 1, wherein the renaturation in step v) is carried out such that the final concentration is 0.1 to 2.0 g / L.

12. A method for purifying TGF-β3 protein, comprising the following steps: i) The step of recovering Escherichia coli from an Escherichia coli culture medium; ii) The step of disrupting the recovered Escherichia coli to obtain TGF-β3 inclusion bodies; iii) The step of washing the inclusion bodies; iv) The step of dissolving the washed inclusion bodies; v) The step of renaturing the dissolved inclusion bodies; vi) The step of purifying the solution containing the renatured TGF-β3; and vii) The step of filtering the purified solution.

13. The purification method according to claim 12, wherein the purification in step vi) includes: a) The step of performing hydrophobic interaction chromatography as primary purification; b) The step of performing multimodal chromatography as secondary purification; and c) The step of performing cation exchange chromatography as tertiary purification.

14. The purification method according to claim 12, wherein the filtration in step vii) is ultrafiltration / diafiltration.

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