Method for purifying TGF-β3 protein

Abstract

The present invention relates to a pretreatment method for separating and purifying TGF-β3 protein from Escherichia coli with high efficiency and high purity, and a method for purifying TGF-β3 protein including the same. When using the pretreatment method of the present invention and the purification method including the same, since E. coli is used, the process is simpler and higher productivity is expected compared to animal cell culture methods, and therefore highly pure TGF-β3 protein can be separated and purified from E. coli with high efficiency.

Description

[Technical Field]

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

[0002] The present invention relates to a pretreatment method for separating and purifying TGF-β3 protein from Escherichia coli with high efficiency and high purity, and a method for purifying TGF-β3 protein including the same. [Background technology]

[0003] TGF-β3, a subtype of transforming growth factor-β (TGF-β), is essential for various biological processes, including endoderm development, organogenesis, epithelial proliferation, extracellular matrix synthesis, and immune responses. Essentially, TGF-β3 participates in the TGF-β1 / Smad signaling pathway, stimulating mesenchymal cells and suppressing epithelial or neuroectodermal cells, and regulating skin wound repair, remodeling, and potential scar formation.

[0004] Bioactive human TGF-β3 is difficult to produce on a large scale in both prokaryotic and eukaryotic expression systems. The latter have low yields, are methodologically complex, and are cost-intensive, while the former generate inactive inclusion bodies. Therefore, a new, scalable, time- and cost-saving production platform is urgently needed to produce highly pure TGF-β3. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Korean Patent No. 10-0991203 (2010-10-26) [Patent Document 2] Korean Patent No. 10-1443257 (2014-09-16) Summary of the Invention [Problem to be solved by the invention]

[0006] The present inventors have made extensive efforts to provide a new, scalable, time- and cost-saving production platform for producing highly purified TGF-β3. As a result, they have confirmed that highly pure TGF-β3 can be separated and purified with high efficiency when the pretreatment method of the present invention and the purification method including the same are used, thereby completing the present invention.

[0007] Therefore, an object of the present invention is to provide a pretreatment method for separating and purifying TGF-β3 protein from E. coli with high efficiency and purity, and a method for purifying TGF-β3 protein including the same. [Means for solving the problem]

[0008] The present invention relates to a method for producing ... ii) disrupting the collected E. coli to obtain TGF-β3 inclusion bodies; iii) washing the inclusion bodies; iv) solubilizing the washed inclusion bodies; and v) subjecting the solubilized inclusion bodies to a refolding reaction; The present invention provides a pretreatment method for purifying TGF-β3 protein, comprising:

[0009] According to a preferred embodiment of the present invention, the crushing in step ii) is performed 1 to 5 times at a pressure of 800 to 1200 bar.

[0010] According to a preferred embodiment of the present invention, the washing in step iii) is performed 1 to 5 times.

[0011] According to a preferred embodiment of the present invention, the washing in step iii) includes washing with a surfactant and then washing with distilled water.

[0012] According to a preferred embodiment of the present invention, the solubilization in step iv) is carried out using a buffer containing at least one selected from the group consisting of urea, DTT (1,4-dithiothreitol), and Tris (hydroxymethyl) aminomethane.

[0013] According to a preferred embodiment of the present invention, the solubilization in step iv) is carried out in a buffer having a pH of 7 to 9.

[0014] According to a preferred embodiment of the present invention, the solubilization in step iv) is carried out in a buffer for 1 to 24 hours.

[0015] According to a preferred embodiment of the present invention, the refolding reaction in step v) is carried out in a buffer containing at least one selected from the group consisting of CHES (N-Cyclohexyl-2-aminoethanesulfonic acid), NaCl, reduced glutathione, oxidized glutathione, CHAPS, L-arginine, and D-sorbitol.

[0016] According to a preferred embodiment of the present invention, the refolding reaction in step v) is carried out in a buffer having a pH of 8.5 to 10.5.

[0017] According to a preferred embodiment of the present invention, the refolding reaction in step v) is carried out in a buffer for 1 to 12 days.

[0018] According to a preferred embodiment of the present invention, the refolding reaction in step v) is carried out so that the final concentration is 0.1 to 2.0 g / L.

[0019] The present invention also relates to a method for producing ... ii) disrupting the collected E. coli to obtain TGF-β3 inclusion bodies; iii) washing the inclusion bodies; iv) solubilizing the washed inclusion bodies; v) subjecting the solubilized inclusion bodies to a refolding reaction; vi) purifying the solution containing the refolded TGF-β3; and vii) filtering the purified solution; The present invention provides a method for purifying TGF-β3 protein, comprising:

[0020] According to a preferred embodiment of the present invention, the purification step vi) comprises: a) performing hydrophobic interaction chromatography as a primary purification step; b) performing multimodal chromatography as a secondary purification step; and c) performing cation exchange chromatography as a tertiary purification step; It includes:

[0021] According to a preferred embodiment of the present invention, the filtration in step vii) is ultrafiltration / diafiltration.

[0022] The present invention will now be described in more detail.

[0023] The inventors have derived an optimal pretreatment step for E. coli in order to separate and purify highly pure TGF-β3, and the method for purifying TGF-β3 protein including the pretreatment step of the present invention can separate and purify highly pure TGF-β3 from E. coli with high efficiency.

[0024] In particular, the present inventors have developed a method for separating and purifying highly purified TGF-β3, and then developed an additional process that can further shorten the process time, thereby providing a more efficient method for purifying TGF-β3 protein. Specifically, when the pretreatment and purification methods of the present invention are used, 1 to 1.5 g of TGF-β3 protein can be obtained from 2.5 L of E. coli culture.

[0025] Thus, the present invention provides a method for producing a method for producing a microbial cell culture comprising the steps of: i) recovering E. coli from an E. coli culture; ii) disrupting the collected E. coli to obtain TGF-β3 inclusion bodies; iii) washing the inclusion bodies; iv) solubilizing the washed inclusion bodies; and v) subjecting the solubilized inclusion bodies to a refolding reaction; It is possible to provide a pretreatment method for purifying a TGF-β3 protein, comprising:

[0026] According to a preferred embodiment of the present invention, the crushing in step ii) may be performed 1 to 5 times at a pressure of 800 to 1200 bar, and more preferably, the crushing in step ii) may be performed 2 to 3 times at a pressure of 1000 bar.

[0027] According to a preferred embodiment of the present invention, the washing in step iii) may be performed 1 to 5 times, and more preferably, the washing in step iii) may be performed 2 to 3 times.

[0028] According to a preferred embodiment of the present invention, the washing in step iii) may include washing with a surfactant and then washing with distilled water.

[0029] The surfactant may be Tween 20.

[0030] The washing with the surfactant may be carried out for 1 to 24 hours, and more preferably for 2 to 18 hours.

[0031] The washing with distilled water may be carried out 1 to 3 times, more preferably 1 to 2 times.

[0032] The washing with distilled water may be carried out for 1 to 24 hours, more preferably for 2 to 18 hours.

[0033] According to a preferred embodiment of the present invention, the solubilization in step iv) may be performed using a buffer containing at least one selected from the group consisting of urea, 1,4-dithiothreitol (DTT), and Tris (hydroxymethyl)aminomethane (Tris).

[0034] According to a preferred embodiment of the present invention, the solubilization in step iv) may be performed in a buffer having a pH of 7 to 9. Preferably, the buffer may have a pH of 7.5 to 8.5.

[0035] According to a preferred embodiment of the present invention, the solubilization in step iv) may be performed in a buffer for 1 to 24 hours, and more preferably, the solubilization in step iv) may be performed in a buffer for 3 to 24 hours.

[0036] According to a preferred embodiment of the present invention, the refolding reaction in step (v) may be performed in a buffer containing at least one selected from the group consisting of N-cyclohexyl-2-aminoethanesulfonic acid (CHES), NaCl, reduced glutathione, oxidized glutathione, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), L-arginine, and D-sorbitol. More preferably, the refolding reaction in step (v) may be performed in a buffer containing at least one selected from the group consisting of CHES, NaCl, reduced glutathione, and oxidized glutathione.

[0037] According to a preferred embodiment of the present invention, the refolding reaction in step v) may be carried out in a buffer having a pH of 8.5 to 10.5. Preferably, the buffer has a pH of 9 to 10.

[0038] According to a preferred embodiment of the present invention, the refolding reaction in step v) may be carried out in a buffer for 1 to 12 days, and more preferably, the refolding reaction in step v) may be carried out in a buffer for 3 to 12 days.

[0039] According to a preferred embodiment of the present invention, the refolding reaction in step v) may be carried out to a final concentration of 0.1 to 2.0 g / L. Preferably, the refolding reaction in step v) may be carried out to a final concentration of 0.1 to 1.5 g / L, more preferably, the refolding reaction in step v) may be carried out to a final concentration of 0.1 to 0.5 g / L, and most preferably, the refolding reaction in step v) may be carried out to a final concentration of 0.1 to 0.3 g / L.

[0040] The present invention also provides a method for producing a method for producing a microbial cell culture comprising the steps of: i) recovering E. coli from an E. coli culture; ii) disrupting the collected E. coli to obtain TGF-β3 inclusion bodies; iii) washing the inclusion bodies; iv) solubilizing the washed inclusion bodies; v) subjecting the solubilized inclusion bodies to a refolding reaction; vi) purifying the solution containing the refolded TGF-β3; and vii) filtering the purified solution; A method for purifying TGF-β3 protein comprising the steps of:

[0041] The steps i) to v) are the same as those included in the pretreatment method, and therefore the description thereof will be substituted.

[0042] According to a preferred embodiment of the present invention, the purification step vi) comprises: a) performing hydrophobic interaction chromatography as a primary purification step; b) performing multimodal chromatography as a secondary purification step; and c) performing cation exchange chromatography as a tertiary purification step; It may also include.

[0043] According to a preferred embodiment of the present invention, the filtration in step vii) may be ultrafiltration / diafiltration. [Effects of the Invention]

[0044] When using the pretreatment method of the present invention and the purification method including the same, since E. coli is used, the process is simpler and higher productivity is expected compared to animal cell culture methods, and therefore highly pure TGF-β3 protein can be separated and purified from E. coli with high efficiency. [Brief explanation of the drawings]

[0045] [Figure 1] The results of SDS-PAGE analysis according to solubilization time are shown. Bands were observed at the size of TGF-monomers, and no change in pattern was observed depending on the solubilization time. [Figure 2] The results of SDS-PAGE analysis of the refolding concentration and time are shown. Bands were observed at the TGF-dimer size, and no change in the pattern was observed depending on the refolding concentration and time. [Figure 3] The results of SDS-PAGE analysis of a TGF-β3 standard solution (lane 1) and the target protein TGF-β3 (lane 2) are shown. It was confirmed that the target protein of the present invention was highly purified (M: protein marker). DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention will be described in more detail below through examples. These examples are provided solely for the purpose of illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

[0047] [Example 1] Escherichia coli cell harvest A strain of Escherichia coli (E. coli BL21 (DE3)) transformed with the pTT-TGFβ3 plasmid was subjected to seed culture and main culture, and the main culture solution was centrifuged to recover the E. coli cells. The recovered cells were added to Buffer A (20 mM Tris, 5 mM EDTA, pH 8.0 ± 0.2) and suspended.

[0048] [Example 2] Cell disruption <2-1> Setting the number of cell disruption times The cell collection solution from Example 1 was disrupted three times using a high-pressure disrupter at a pressure of 1000 bar. During the disruption process, 1 mL of culture medium was sampled from the culture medium harvested before disruption, after the first disruption, after the second disruption, and after the third disruption, and the OD at 600 nm was measured. In addition, when harvesting the culture medium after disruption, the weight of the culture medium harvested at each stage was measured. The disruption rate was calculated using the weight of the culture medium and the measured OD at 600 nm according to the following Equation 1.

[0049] [Formula 1] Cell disruption rate (%) = 100 - {(cell concentration of the cell lysate (OD 600 )) / (cell concentration (OD) before cell lysis (cell recovery solution) 600 ))}×100 As a result, as shown in Table 1 below, it was confirmed that the crushing rate during secondary crushing increased by approximately 13%, and the crushing rate during tertiary crushing increased by approximately 4%.

[0050] [Table 1]

[0051] <2-2> Cell disruption process The cell recovery solution from Example 1 was injected into a cell disrupter, and primary cell disruption was carried out at a pressure of 1000±200 bar. The primary cell disruption solution was collected in a 5 L beaker, stirred, and stored. The primary cell disruption solution was used to measure cell concentration.

[0052] The cell disruption rate was calculated according to the above [Equation 1] and the value was confirmed.

[0053] The primary cell lysate was injected into a cell lysator, and secondary cell lysis was performed at a pressure of 1000 ± 200 bar. The secondary cell lysate was collected in a 10 L beaker, stirred, and stored. The secondary cell lysate was used to measure cell concentration. The cell lysis rate was calculated according to the above [Equation 1], and the value was confirmed.

[0054] [Example 3] Inclusion body washing <3-1> Setting cleaning conditions As a washing step for removing HCP (Host Cell Protein) and HCD (Host Cell DNA), a comparative experiment was carried out on washing buffers, washing steps, and washing times.

[0055] The wash buffers were 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.

[0056] The cell lysate from Example 2 was centrifuged at 8000 rpm for 30 minutes to obtain inclusion bodies (IBs). The obtained IBs were weighed, divided into 12 equal parts, and suspended in 400 mL of wash buffer prepared according to the conditions listed in Table 2 below. After washing for either 16 or 2 hours, the IBs were centrifuged at 8000 rpm for 30 minutes to obtain the primary washed IBs. The resulting supernatant was stored at -20°C or below for analytical testing. The primary washed IBs were then suspended in 400 mL of distilled water (DW) and subjected to a secondary wash according to the conditions listed in Table 2. After the secondary wash, the IBs were centrifuged at 8000 rpm for 30 minutes to obtain the washed IBs. The resulting supernatant was also stored at -20°C or below for analytical testing.

[0057] [Table 2]

[0058] The supernatant obtained after the first wash was analyzed for residual HCP and HCD to evaluate the ability to remove impurities during the first wash. In addition, since the second wash using DW is a step to remove the detergent used in the first wash, the analytical samples were analyzed for residual Tween 20 except for conditions 9, 10, and 11, which do not contain Tween 20.

[0059] As a result, as shown in Table 3 below, when a detergent containing Tween 20 was used, more than 99% of HCD was removed under all conditions, regardless of detergent concentration or washing time, demonstrating greater removal of HCD than when Triton X-100 was used as the conventional detergent. For HCP, when Tween 20 was used as the detergent, greater removal of HCP was observed than when Triton X-100 was used, regardless of concentration or time. When detergent-free washing buffers (conditions 9 and 10) were used, HCP removal was superior to the conventional conditions, while HCD removal was similar to the control and reduced compared to the washing conditions containing Tween 20.

[0060] [Table 3]

[0061] It was also confirmed that when washing with DW, 99.8% or more of Tween 20 was removed under all conditions, regardless of the time or number of washes.

[0062] Therefore, the final washing step was set to use 1% to 1.5% Tween 20 for the first washing step over 2 to 16 hours, and for the second washing step over 2 to 16 hours.

[0063] <3-2> Cleaning process For primary inclusion body washing, the cell lysate from Example 2 was centrifuged at 8,000 rpm for 30 minutes at 4°C. 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 washed with stirring at room temperature for 1 day.

[0064] For the secondary inclusion body washing, the solution from the primary inclusion body washing step was centrifuged at 8,000 rpm at 4°C for 30 minutes. The inclusion bodies recovered by centrifugation were added to distilled water (DW) and stirred at 300 rpm at room temperature for 2±1 hours to perform the secondary inclusion body washing.

[0065] The secondary inclusion body washing solution was centrifuged at 4° C. and 8000 rpm for 30 minutes, and the inclusion bodies were finally collected and weighed.

[0066] [Example 4] solubilization <4-1> Solubilization time setting The washed inclusion bodies (IBs) obtained in Example 3 were solubilized for 24 hours in 6 M urea, 100 mM DTT, and 50 mM Tris (pH 8.0). After solubilization, 1 mL of the solubilized solution was sampled at 3, 6, 9, and 24 hours for analytical testing. Sample analysis consisted of concentration analysis (in duplicate) using a 660 nm assay kit and SDS-PAGE. The statistical trends over time in the measured sample concentrations were examined.

[0067] As a result, regression analysis of duplicated concentrations yielded p-values ​​of 0.2674 and 0.6116, confirming that there was no statistical significance between the solubilization time and the solubilization concentration. Based on these results, it was determined that the solubilization time, from 3 to 24 hours, had no effect on sample concentration. Furthermore, SDS-PAGE analysis confirmed a solubilized band of TGF-β3 monomer size, and no change in the SDS-PAGE pattern was observed over time, confirming that the solubilization time had no effect (Figure 1).

[0068] <4-2>Solubilization process The entire amount of inclusion bodies collected from 2.5 L of culture medium was added to 1.2 L of Buffer C (6 M urea, 100 mM DTT, 50 mM Tris pH 8.0 ± 0.2), and the solubilized solution was stirred at 200 rpm for 16 ± 4 hours to solubilize the inclusion bodies.

[0069] After the reaction, 5 mL of the solubilized solution was sampled in two 15 mL conical tubes, one of which was frozen and stored at -20 ± 5°C. The 5 mL sampled solubilized solution was used for protein concentration analysis (660 nm protein assay).

[0070] [Example 5] Refolding <5-1> Setting refolding conditions A total of 4 L of refolding buffer containing 0.7 M CHES (N-cyclohexyl-2-aminoethanesulfonic acid), 1 M NaCl, 2 mM glutathione reduced (GSH), and 0.4 mM oxidized glutathione (GSSG), pH 9.5, was prepared. The buffer was first titrated to pH 9.2, then left overnight in a cold room, and then titrated to pH 9.5. The prepared refolding buffer was divided into six 500 mL aliquots. Based on the concentration of the solution after 24 hours of solubilization, solubilization solution was added to achieve final refolding concentrations of 0.1, 0.3, 0.5, 1.0, 1.5, and 2.0 g / L. Before adding the solubilization solution, an aliquot of the refolding solution was removed from the dispensed solution, and then the solubilization solution was added to maintain a final volume of 500 mL. After the initiation of refolding, 1 mL of the refolding solution was sampled at 0, 1, 3, 5, 7, and 12 days, and approximately 5% by volume of 37% HCl was added to the sampled refolding solution to terminate the refolding reaction. After the refolding reaction was completed, the sample was centrifuged at 13,000 rpm for 30 minutes, and the supernatant was stored at -20°C or below for analytical testing. The refolding rate of the samples reacted at each refolding period was analyzed by calculating the main peak area of ​​RP-HPLC. Furthermore, SDS-PAGE was used to confirm whether the band patterns of the monomer and dimer changed over time.

[0071] The refolding process solution sampled at each concentration and time was analyzed by RP-HPLC. The % area of ​​the dimer peak on RP-HPLC for each condition is shown in Table 4 below.

[0072] [Table 4]

[0073] As a result, at concentrations above 1.0 g / L, a large amount of aggregation was formed during the refolding reaction, causing the process solution to become very turbid. When refolding was performed at concentrations below 0.5 g / L, the dimer efficiency was approximately 55-60%, and further refolding from days 3 to 12 increased the dimer efficiency by up to approximately 3%.

[0074] Furthermore, dimer efficiency for each sample was confirmed by SDS-PAGE. No changes in the SDS-PAGE pattern were observed, confirming that the refolding concentration and time had no effect (Figure 2).

[0075] <5-2> Refolding process The amount of solubilization solution to be added for the refolding step at a protein concentration of 0.1 g / L was calculated according to the following [Equation 2].

[0076] [Formula 2] Input volume of solubilization solution (L) = (volume of refolding step (L) × 0.1 g / L) / (protein concentration of solubilization solution (g / L)) A volume of Buffer D equal to the amount of the solubilization process solution calculated in [Equation 2] was removed from the 50 L mobile tank, and the same amount of solubilization process solution was then added. The refolding process was terminated after approximately 72 hours of refolding. The process solution that had been refolded for 3 days was pretreated by adjusting the pH to 2.0±0.2 with hydrochloric acid solution, and then filtered using a capsule filter.

[0077] The final filtrate was dispensed into two 15 mL conical tubes at 5 mL each and used for the following process liquid analysis (total protein concentration, 660 nm protein assay). The sample remaining after process liquid analysis was stored frozen (-20 ± 5°C). The final filtrate was stored at a low temperature until it was used as the injection sample for purification 1.

[0078] [Example 6] Purification 1_Hydrophobic Chromatography The column was equilibrated by injecting Buffer E (4 M urea, 20 mM Na-AcOH, 0.1 M NaCl, pH 4.0 ± 0.2) for 2 CV or more. The entire pretreated refolding solution was then injected onto the column. Buffer E was then injected for 10 CV to perform an unbound wash.

[0079] The column was washed with 4 CV of Buffer F (20 mM Tris, pH 8.0±0.2).

[0080] Elution was performed by injecting 3 CV of Buffer G (4 M urea, 20 mM Tris, pH 9.5±0.2) into the column. From the point of injection of Buffer G, a total of 3 CV was collected as the eluate.

[0081] At the end of the elution, 5 mL of the elution fraction was dispensed into a 15 mL conical tube and used for the following analyses of the process liquid: purity (SDS-PAGE), purity (RP-HPLC) (IPC), and concentration (660 nm protein assay).

[0082] [Example 7] Purification 2: Multimodal Chromatography The column was equilibrated by injecting 2 CV or more of Buffer G (4 M urea, 20 mM Tris-HCl, pH 9.5±0.2). The entire volume of the Purification 1 eluate was then injected into the column. Five CV of Buffer G was then injected into the column to perform an unbound wash.

[0083] The column was washed with 10 CV of Buffer H (4 M urea, 20 mM Tris-HCl, 0.2 M NaCl, pH 9.5±0.2).

[0084] Elution was performed by injecting 3 CV of Buffer I (4 M urea, 20 mM Na-AcOH, pH 4.0 ± 0.2) into the column. Collection of the eluate began when 0.5 CV of Buffer I had been injected into the column, and continued until 3 CV had flowed through.

[0085] At the end of the elution, 5 mL of the eluted fraction was dispensed into a 15 mL conical tube and used for the following in-process analysis: purity (SDS-PAGE), purity (RP-HPLC) (IPC), and concentration (660 nm protein assay).

[0086] [Example 8] Purification 3: Ion exchange chromatography <8-1> Ion exchange chromatography The column was equilibrated by injecting 2 CV or more of Buffer I (4 M urea, 20 mM Na-AcOH, pH 4.0 ± 0.2). The entire volume of the purified 2 eluate was then injected onto the column. Five CV of Buffer I was then injected onto the column for unbound wash.

[0087] The column was washed with 7 CV of Buffer J (4 M urea, 20 mM Na-AcOH, 0.2 M NaCl pH 4.0±0.2).

[0088] Elution was performed by injecting 5 CV of Buffer K (4 M urea, 20 mM Na-AcOH, 0.5 M NaCl pH 4.0 ± 0.2) into the column. From the point of injection of Buffer K, the entire 5 CV volume was collected as eluate. Once elution collection was complete, the column could be immediately regenerated and stored. At the end of the elution, 5 mL of the eluted fraction was dispensed into a 15 mL conical tube and used for the following in-process analyses: purity (SDS-PAGE), purity (RP-HPLC) (IPC), and concentration (660 nm protein assay).

[0089] <8-2> Final yield after final purification The final yield of TGF-β3 obtained after the final tertiary purification process was 0.732 L, the process liquid volume was 0.977 g / L, and the amount of TGF-β3 protein obtained was 715.2 mg.

[0090] [Example 9] Filtration The ultrafiltration / diafiltration system (UF / DF system) was equipped with a membrane (10 kDa, 0.1 m 2 * 1 piece) was attached.

[0091] Equilibration was performed by injecting Buffer L (20 mM Na-OAc, pH 3.8±0.2) into the membrane. Equilibration was completed when the pH of the permeate filtrate was 3.8±0.3 and the electrical conductivity was 2 bar or less.

[0092] Filtration for concentration (Ultrafiltration) was performed using TMP ([P Feed +P Ret ] / 2)The test was carried out under conditions of 2 bar or less.

[0093] The concentrated volume was calculated using the following formula 3. The set DS concentration was 1.0 g / L, but considering the differences between the purified 3 eluate and the DS concentration analysis method, the solution was concentrated to twice the set concentration and then diluted.

[0094] [Formula 3] Concentrated volume = amount of rhTGF-β3 protein in the purified 3 eluate (g) / 2.0 g / L Buffer exchange (diafiltration) was performed by adding 1 L of Buffer corresponding to the volume after concentration. The filtration process was terminated when the pH of the permeate filtrate was 3.8 ± 0.2 after six diafiltration cycles. If the pH was incompatible, one additional diafiltration cycle was performed to confirm whether the permeate filtrate at the end of each cycle met the pH standard.

[0095] Additional concentration was performed for line recovery. After closing the permeate valve, line recovery was performed using Buffer L.

[0096] The concentration of the recovered solution was measured using the A280 method, and the volume of Buffer L to be added was calculated to achieve a final concentration of 1.0 g / L. To measure the A280 protein concentration, 500 μL of DS was placed in a cuvette and the absorbance was measured at 280 nm using a multispectrophotometer (ELISA reader). If the measured absorbance was outside the 0.3–0.7 range, the dilution factor was adjusted to bring it within the range. The protein concentration was calculated by multiplying the measured absorbance by the dilution factor and dividing by the extinction coefficient of 1.841. The recovered solution was diluted with the calculated volume of Buffer L, and the final concentration was confirmed using the A280 method. The recovered solution was then filtered through a 0.2 μm bottle top filter.

[0097] The filtrate whose concentration was confirmed was filtered through a 0.2 μm bottle top filter (PES) into a sterilized glass bottle, and the final stock solution was dispensed into 50 1.5 mL EP tubes, each containing 1 mL, for use in the lot release test.

[0098] [Example 10] Standards evaluation The final TGF-β3 separated and purified through the processes of Examples 1 to 9 was subjected to a standard evaluation, and the results are shown in Table 5. In particular, the electrophoresis results were consistent with the TGF-β3 standard solution (lane 1), and no additional impurity proteins were detected, confirming that the target protein, TGF-β3 (lane 2), was purified with high purity (Figure 3).

[0099] [Table 5] [Industrial Applicability]

[0100] When using the pretreatment method of the present invention and the purification method including the same, since E. coli is used, the process is simpler and higher productivity is expected compared to animal cell culture methods, and therefore high-purity TGF-β3 protein can be separated and purified from E. coli with high efficiency, making it industrially applicable.

Claims

1. i) recovering E. coli from the E. coli culture; ii) disrupting the collected E. coli to obtain TGF-β3 inclusion bodies; iii) washing the inclusion bodies; iv) solubilizing the washed inclusion bodies; and v) subjecting the solubilized inclusion bodies to a refolding reaction; A pretreatment method for purifying TGF-β3 protein, comprising:

2. 2. The pretreatment method according to claim 1, wherein the crushing in step ii) is performed 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 performed 1 to 5 times.

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

5. 2. The pretreatment method according to claim 1, wherein the solubilization in step iv) is performed using a buffer containing at least one 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 solubilization in step iv) is carried out in a buffer having a pH of 7 to 9.

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

8. 2. The method of claim 1, wherein the refolding reaction in step v) is carried out in a buffer containing at least one selected from the group consisting of N-cyclohexyl-2-aminoethanesulfonic acid (CHES), NaCl, reduced glutathione, oxidized glutathione, CHAPS, L-arginine, and D-sorbitol.

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

5.

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

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

12. i) recovering E. coli from the E. coli culture; ii) disrupting the collected E. coli to obtain TGF-β3 inclusion bodies; iii) washing the inclusion bodies; iv) solubilizing the washed inclusion bodies; v) subjecting the solubilized inclusion bodies to a refolding reaction; vi) purifying the solution containing the refolded TGF-β3; and vii) filtering the purified solution; A method for purifying a TGF-β3 protein, comprising:

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

14. 13. The method of claim 12, wherein the filtration in step vii) is ultrafiltration / diafiltration.

Images