Method for manufacturing recombinant insulin-like growth factor I

Abstract

The present invention provides a manufacturing method as an improved method for obtaining correctly folded rIGF-1 with high purity from recombinant insulin-like growth factor I (rIGF-1) with relative misfolding. The manufacturing method of recombinant insulin-like growth factor I (rIGF-1) includes: (1) obtaining rIGF-1 as a fusion protein (SUMO-IGF-1) with ubiquitin-like protein SUMO (small ubiquitin-related modifier); (2) treating SUMO-IGF-1 with SUMO protease; and (3) applying the treatment solution of step (2) to cation exchange chromatography by gradient dissolution to obtain a dissolution solution containing rIGF-1.

Description

[Technical Field]

[0001] This invention relates to a method for manufacturing a recombinant insulin-like growth factor I (rIGF-1). [Previous Technology]

[0002] In the production of recombinant proteins using E. coli, there are problems with the expression of protein insolubilization and misfolding. Regarding recombinant insulin-like growth factor I (rIGF-1), in order to combat the insolubilization problem, a technique has been reported to express rIGF-1 as a fusion protein (SUMO-rIGF-1) with the ubiquitin-like protein SUMO (small ubiquitin-related modifier) ​​(see Non-Patent Literature 1). Furthermore, in order to separate correctly folded rIGF-1 from misfolded rIGF-1, reverse-phase high-performance liquid chromatography (HPLC) can be used (see Patent Literature 1).

[0003] The production of rIGF-1 by means of SUMO-rIGF-1, as described in Non-Patent Document 1, involves the purification of rIGF-1 using a nickel column (Ni-NTA resin column). Specifically, a solution of expressed SUMO-rIGF-1 treated with SUMO protease (containing SUMO-rIGF-1, SUMO protease, SUMO, and rIGF-1) is applied to Ni-NTA (nickel nitrilotriacetic acid) affinity chromatography. By adsorbing the histidine-tagged SUMO-rIGF-1, SUMO protease, and SUMO onto the column, only rIGF-1 is precipitated, thus purifying rIGF-1. [Prior Art Documents] [Patent Documents]

[0004] Patent Document 1: Japanese Patent Publication No. 07-501704 [Non-Patent Document]

[0005] Non-patent document 1: "Soluble expression of human insulin-like growth factor-1 with the assistance of SUMO fusion partner", Journal of Chemical and Pharmaceutical Research, 2014, 6(4):1059-1066 [Summary of the Invention]

[0006] [The problem the invention aims to solve]

[0007] The main objective of this invention is to provide an improved method for obtaining rIGF-1 that corrects folded rIGF-1 with high purity. [Means for solving the problem]

[0008] In order to solve the above-mentioned problems, the present invention provides the following [1]-

[11] . [1] A manufacturing method, which is a method for manufacturing recombinant insulin-like growth factor (rIGF-1), comprising: (1) obtaining rIGF-1 as a fusion protein (SUMO-IGF-1) with ubiquitin-like protein SUMO (small ubiquitin-related modifier); (2) treating SUMO-IGF-1 with SUMO protease; and (3) applying the treatment solution of step (2) to cation exchange chromatography, and obtaining a precipitate containing rIGF-1 by gradient elution. [2] The manufacturing method of [1], wherein the gradient elution comprises: (i) a elution step in a elution solution with a salt concentration of 200 mM or more but less than 300 mM, comprising a column volume or more; (ii) a elution step in a elution solution with a salt concentration of 300 mM or more but less than 500 mM, comprising a column volume or more; and (iii) a elution step in a elution solution with a salt concentration of 500 mM or more, wherein rIGF-1 is recovered from the elution solution of the above-mentioned elution step (ii). [3] The manufacturing method of [2], wherein the elution conditions of the elution step (1) are as follows: elution in a 1.5-6 column volume (CV) at a salt concentration of 200 mM, followed by elution in a 3-12 CV at a salt concentration of 250 mM, and further elution in a 1.5-3 CV of a linear concentration gradient from 250 mM to 300 mM. [4] Dissolution step (ii) involves dissolution at a salt concentration of 300 mM at 1.5-6 CV, followed by dissolution at a linear concentration gradient of 1.5-3 CV from 300 mM to 400 mM. [5] The manufacturing method of any of [2]-[4], wherein the dissolution conditions of dissolution step (iii) are as follows: dissolution at a salt concentration of 1 M at 1.5-6 CV.[6] The manufacturing method of any one of [1]-[5], wherein step (1) includes: dissolving host cells expressing SUMO-IGF-1 to obtain a first sample solution (1-1); and applying the first sample solution to Ni-NTA (nickel-nitrilotriacetic acid) affinity chromatography to obtain a first precipitate containing SUMO-IGF-1 (1-2); step (2) is to treat the first precipitate with SUMO protease to obtain a second sample solution containing SUMO-IGF-1, SUMO protease, SUMO and rIGF-1; and step (3) is to apply the second sample solution to cation exchange chromatography and separate rIGF-1 from SUMO-IGF-1, SUMO protease and SUMO by gradient chromatography to obtain a third sample solution containing rIGF-1. [7] A manufacturing method of any one of [1]-[6], wherein, after step (2), a separation step by Ni-NTA affinity chromatography is not included. [8] A manufacturing method of any one of [1]-[7], wherein a separation step of misfolded proteins by reversed-phase high-performance liquid chromatography is not included. [9] A manufacturing method of any one of [1]-[8], wherein, in the third sample solution, the ratio of correctly folded rIGF-1 to the total amount of protein is 60% or more, preferably 70% or more, more preferably 80% or more, further preferably 90% or more, and particularly preferably 95% or more.

[10] A manufacturing method of any one of [1]-[9], wherein the host cell is Escherichia coli.

[0009]

[11] A purification method for recombinant insulin-like growth factor 1 (rIGF-1) includes: (1) obtaining rIGF-1 as a fusion protein (SUMO-IGF-1) with a ubiquitin-like protein SUMO (small ubiquitin-related modifier); (2) treating SUMO-IGF-1 with SUMO protease; and (3) applying the treatment solution of step (2) to cation exchange chromatography and obtaining a precipitate containing rIGF-1 by gradient elution.

[0010] In this invention, "gradient dissolution" refers to salt concentration gradient dissolution. Gradient dissolution only needs to include a portion of the steps in which the salt concentration changes linearly or stepwise to perform dissolution; it is not necessary to change the salt concentration in all steps. That is, gradient dissolution can also include a portion of the steps in which the salt concentration is fixed to perform dissolution. [Effects of the Invention]

[0011] According to the present invention, an improved method is provided for obtaining rIGF-1 that corrects folding errors in rIGF-1 with high purity.

Implementation Method

[0013] Hereinafter, suitable embodiments for carrying out the present invention will be described. Furthermore, the embodiments described below are merely examples illustrating representative embodiments of the present invention and are not intended to narrowly interpret the scope of the present invention.

[0014] The present invention relates to a method for manufacturing and purifying recombinant insulin-like growth factor 1 (rIGF-1), comprising the following steps (1)-(3). Step (1): Obtaining rIGF-1 as a fusion protein (SUMO-IGF-1) with the ubiquitin-like protein SUMO (small ubiguitin-related modifier). Step (2): Treating SUMO-IGF-1 with SUMO protease. Step (3): Applying the treatment solution from step (2) to cation exchange chromatography and obtaining a precipitate containing rIGF-1 by gradient chromatography.

[0015] [Step (1)] This step is to obtain rIGF-1 as a fusion protein (SUMO-IGFE-1) with the ubiquitin-like protein SUMO (small ubiquitin-related modifier). This step includes: step (1-1) of dissolving host cells expressing SUMO-IGF-1 to obtain a first sample solution; and step (1-2) of applying the first sample solution to Ni-NTA (nickel-nitrilotriacetic acid) affinity chromatography to obtain a first precipitate containing SUMO-IGF-1.

[0016] [Step (1-1)] This step involves dissolving the host cells expressing SUMO-IGF-1 to obtain the first sample solution. The gene construct used to express SUMO-IGF-1 can be prepared using conventional methods (e.g., the method described in Non-Patent Document 1). The SUMO-IGF-1 gene and expression plasmid used to prepare the gene construct can be made using commercially available DNA synthesis genes and common vectors. There are no particular limitations on the host cells, but Escherichia coli is suitable, and commercially available E. coli cell lines can be used. The transformation of the host cells constructed using the gene and the culture of the transformant can also be performed using conventional methods. After culture, the cells are collected by centrifugation or the like, the cells are broken up using a high-pressure homogenizer or the like, and insoluble matter is removed by centrifugation or the like to obtain the first sample solution.

[0017] [Steps (1-2)] This step involves adapting the first sample solution to Ni-NTA (nickel-nitrilotriacetic acid) affinity chromatography to obtain a first precipitate containing SUMO-IGF-1. The first sample solution is loaded into an equilibrated Ni-NTA affinity chromatography column. SUMO-IGF-1 is maintained on the column carrier via a histidine tag attached to SUMO. After washing the column with an appropriate buffer, precipitate is performed using an imidazole concentration gradient to dissolve the SUMO-IGF-1 maintained on the column carrier, obtaining the first precipitate. Imidazole concentration gradient dissolution, for example, can be carried out by using a linear concentration gradient dissolution with an initial buffer (50 mM Tris, 300 mM NaCl, 8 M Urea, 20 mM Imidazole, pH 7.8) and a dissolution buffer (50 mM Tris, 300 mM NaCl, 600 mM Imidazole, pH 7.8).

[0018] [Step (2)] This step involves treating SUMO-IGF-1 with SUMO protease. By treating the first elution solution with SUMO protease, a second sample solution containing SUMO-IGF-1, SUMO protease, SUMO, and rIGF-1 is obtained. SUMO protease treatment can be performed using conventional methods (e.g., the method described in Non-Patent Document 1). After SUMO protease treatment, the treatment solution containing SUMO-IGF-1, SUMO protease, SUMO, and rIGF-1 can also be appropriately concentrated and the buffer solution replaced to obtain the second sample solution.

[0019] [Step (3)] This step involves applying the second sample solution to cation exchange chromatography and obtaining a precipitate containing rIGF-1 by gradient elution. Conventional methods (e.g., the method described in Non-Patent Document 1) involve applying a SUMO protease treatment solution containing SUMO-IGF-1, SUMO protease, SUMO, and rIGF-1 to Ni-NTA affinity chromatography again, so that the His-tag SUMO-rIGF-1, SUMO protease, and SUMO are adsorbed onto the column, and only rIGF-1 is eluted, thus purifying IGF-1 (see Comparative Example 1). The method related to this invention uses cation exchange chromatography instead of Ni-NTA affinity chromatography for the purification of rIGF-1. This step involves applying the second sample solution to cation exchange chromatography and separating rIGF-1 from SUMO-IGF-1, SUMO protease, and SUMO via gradient dissolution, obtaining a third sample solution containing rIGF-1. The method of this invention does not include a separation step using Ni-NTA affinity chromatography after step (2).

[0020] The second sample solution was loaded into a cation exchange chromatography column (SP cellulose) equilibrated with an appropriate buffer (e.g., 20 mM Ammonium acetate, 200 mM Imidazole, pH 4.0). A salt gradient was performed to dissolve the rIGF-1 maintained on the column support, yielding the third sample solution.

[0021] Salt concentration gradient dissolution can be performed, for example, by using the above-mentioned buffer as a starting buffer and combining a linear concentration gradient of dissolution buffers (20 mM Ammonium acetate, 200 mM Imidazole, 1 M NaCl, pH 4.0). The dissolution volume-dissolution rate can be appropriately adjusted. The dissolution solution is, for example, 4 column volumes (CV), 1-16 CV, preferably 2-8 CV, more preferably 3-6 CV. By using a salt concentration gradient dissolution with a cation exchange chromatography column, compared with conventional methods (see Comparative Example 1), SUMO-free rIGF-1 can be obtained in high purity and high yield (see Example 1).

[0022] Salt concentration gradient dissolution is preferably composed of the following dissolution steps (i)-(iii): (i) a dissolution step with a salt concentration of 200 mM to 300 mM (or 200 mM to 300 mM) or more, using a column volume or more. (ii) a dissolution step with a salt concentration of 300 mM to 500 mM or more, using a column volume or more. (iii) a dissolution step with a salt concentration of 500 mM or more.

[0023] The dissolution step (i) primarily involves dissolving the misfolded rIGF-1. For example, ammonium acetate can be used as a buffer in the dissolution solution, containing salts (such as NaCl) at the concentrations described above. The salt concentration in the dissolution solution can be fixed within the aforementioned concentration range, or it can vary linearly or stepwise. The dissolution solution may also contain an appropriate concentration of imidazole (e.g., 200 mM). The dissolution volume-dissolution rate can be appropriately adjusted. The dissolution solution is, for example, in the range of 12 CV, 3-48 CV, preferably 6-24 CV, and more preferably 9-15 CV. Specifically, the dissolution step (i) is dissolution at a salt concentration of 200 mM in 1.5–6 column volumes (CV), followed by dissolution at a salt concentration of 250 mM in 3–12 CV, and further by dissolution at a linear concentration gradient of 1.5–3 CV from 250 mM to 300 mM.

[0024] The dissolution step (ii) primarily involves dissolving the correctly folded rIGF-1. High-purity refined rIGF-1 can be obtained by recovering this dissolution fraction. The dissolution solution can use, for example, ammonium acetate as a buffer, containing salts (such as NaCl) at the concentrations described above. The salt concentration in the dissolution solution can be fixed, linear, or stepwise within the aforementioned concentration range. The dissolution solution may also contain an appropriate concentration of imidazole (e.g., 200 mM). The dissolution volume-dissolution rate can be appropriately adjusted. The dissolution solution is, for example, in the range of 6 CV, 1.5-24 CV, preferably 3-12 CV, and more preferably 4.5-8 CV. Specifically, step (ii) is a dissolution at a salt concentration of 300 mM with a concentration of 1.5-6 CV, followed by a dissolution at a linear concentration gradient of 1.5-3 CV from 300 mM to 400 mM.

[0025] The dissolution step (iii) primarily involves the dissolution of SUMO-IGF1, SUMO protease, and SUMO. The dissolution buffer can be, for example, ammonium acetate as a buffer, containing salts (such as NaCl) at the concentrations described above. The salt concentration in the dissolution buffer can be fixed, linear, or stepwise within the aforementioned concentration range. The dissolution buffer may also contain an appropriate concentration of imidazole (e.g., 200 mM). The dissolution volume-dissolution rate can be appropriately adjusted. The dissolution solution is, for example, in the range of 3 CV, 0.75-12 V, preferably 1.5-6 CV, and more preferably 2-4 CV. Particularly specifically, dissolution step (iii) is a dissolution at a salt concentration of 1 M at 1.5-6 CV.

[0026] In the past, reverse-phase high-performance liquid chromatography (HPLC) was used to separate correctly folded rIGF-1 from incorrectly folded rIGF-1. According to the rIGF-1 manufacturing and purification methods of the present invention, instead of using expensive HPLC columns, inexpensive cation exchange chromatography columns are used (step (3)), and correctly folded rIGF-1 can be obtained with high purity and high recovery relative to incorrectly folded rIGF-1. Furthermore, it is not excluded that, for the purpose of removing MetO59-IGF-1, in particular, from correctly folded rIGF-1, further HPLC separation is performed following the steps of the present invention. According to the rIGF-1 manufacturing and purification methods of the present invention, in the elution solution (third sample solution) of step (3), the ratio of correctly folded rIGF-1 to the total protein content is 60% or more, preferably 70% or more, more preferably 80% or more, further preferably 90% or more, and particularly preferably 95% or more. [Example]

[0027] [Example 1] (1) Materials (1-1) SUMO-IGF-1 DNA Synthesis Gene The SUMO-IGF-1 gene used was a commercially available DNA synthesis gene (MISSION BIOTECH, GS3075). (1-2) Expression Regulation Plastid (pET22b) The SUMO-IGF-1 expression plastid was pET22b (EMD Millipore, 69744-3CN). (1-3) Gene Expression Component (pET22b SUMO-IGF-1) The SUMO-IGF-1 gene expression component (pET22b SUMO-IGF-1) was constructed by inserting the SUMO-IGF-1 DNA synthesis gene into pET22b. (1-4) Host Cell The host cell used was the BL21(DE3) cell line from Escherichia coli strain B. (1-5) Preparation of seed cell lines for the master-working cell bank: Mecasermin (recombinant gene) expression constructs were introduced into host cell BL21(BD3) via E. coli transfection. The cells were cultured on LB agar containing ampicillin at 37°C, and eight single colonies were selected. These single colonies were then cultured on LB agar containing ampicillin at 37°C until the turbidity (OD 600nm) of the culture medium reached 0.6–0.8. Induction was then performed with 1 mM isopropyl-β-thiogalactoside at 28°C for 2.5 hours, and cell lines exhibiting high expression were selected. (1-6) Preparation of the master-working cell bank: One selected colony was inoculated onto LB medium containing ampicillin and incubated at 37°C until the turbidity (OD 600nm) reached 0.6–0.8. After incubation, centrifugation was performed, the medium was discarded, and sterilized glycerol was added to the LB medium containing ampicillin at a final concentration of 20% to achieve an OD 600nm turbidity of 1.2. This cell suspension was then aliquoted into cryotubes at 1 mL each.

[0028] (2) Expression of SUMO-IGF-1 (2-1) Pre-culture 1 Add 0.5 mL of thawed working-cell bank to 5 mL of pre-culture medium and incubate at 37°C and 120 rpm for 2-8 hours. Then, transfer to 195 mL of pre-culture medium and incubate at 37°C and 120 rpm until the turbidity is above 5. (2-2) Pre-culture 2 Add the culture obtained from pre-culture 1 to 3.1 L of pre-culture medium, 400 mL of glucose solution, 1025 μL of trace metal solution and 3.5 mL of ampicillin solution, and incubate at 37°C until the turbidity is above 10. (2-3) Main culture Add the culture obtained from pre-culture 2 to the main culture medium, glucose solution, trace metal solution and ampicillin solution, and incubate at 37°C until the turbidity is 50. Add IPTG solution and continue culturing to express SUMO-IGF-1 in the cells.

[0029] (3) Cell Recovery - After the main culture is completed, the culture medium is centrifuged to recover the cells. 10 mL of cell lysis buffer (50 mM Tris, 300 mM NaCl, 8 M Urea, pH 7.8) is added relative to 1 g of cells to suspend the cells. The cells are then broken up using a high-pressure grinder (850 bar), centrifuged to remove insoluble matter, and the first sample solution is obtained.

[0030] (4) Purification by Ni-NTA affinity chromatography: The entire volume of the first sample solution was loaded into a Ni-NTA affinity chromatography column (25 cm high) equilibrated with cell lysis buffer. After washing the column with cell lysis buffer, a linear concentration gradient elution (60 cm / h) was performed using nickel buffer B (50 mM Tris, 300 mM NaCl, 8 M Urea, 20 mM Imidazole, pH 7.8) as the starting buffer and nickel buffer C (50 mM Tris, 300 mM NaCl, 600 mM Imidazole, pH 7.8) as the elution buffer to obtain the first elution solution containing SUMO-IGF-1.

[0031] (5) SUMO protease treatment: Add 1 / 2000 volume of SUMO protease solution to the first precipitate and react at 15°C or higher for 1 hour. Concentrate the reaction solution using an ultrafiltration membrane (molecular weight of fraction 2 kDa). Replace the buffer solution with more than 7 times the volume of TFF buffer B (20 mM Ammonium acetate, 200 mM Imidazole, pH 4.0). Then, concentrate again. Recover the concentrate using TFF buffer B to obtain the second sample solution containing SUMO-IGF-1, SUMO protease, SUMO, and rIGF-1.

[0032] (6) Purification by cation exchange chromatography: The entire volume of the second sample solution was loaded into a cation exchange chromatography column (SP cellulose, height 20 cm) equilibrated with SP buffer A (20 mM Ammonium acetate, 200 mM Imidazole, pH 4.0). A third sample solution containing IGF-1 was obtained by linear concentration gradient dissolution (4 column volumes (CV), linear velocity 60 cm / h) using SP buffer A as the starting buffer and SP buffer B (20 mM Ammonium acetate, 200 mM Imidazole, 1 M NaCl, pH 4.0) as the dissolution buffer.

[0033] The chromatogram of the obtained third sample solution is shown in Figure 1. In the figure, the horizontal axis represents the dissolution time, and the vertical axis represents the absorbance (OD 220nm). The black arrows represent the peaks of rIGF-1 (correctly folded rIGF-1), and the white arrows represent misfolded rIGF-1 (misfolded isomer). The ratios of rIGF-1 and misfolded isomers to the total protein content, calculated from the peak intensities, are 64.2% and 28.9%, respectively (refer to Table 1). Furthermore, the amount of rIGF-1 in the third sample solution relative to the amount of rIGF-1 in the second sample solution (recovery rate) is 80%. High purity (64.2%) and high recovery rate (80%) of SUMO-free rIGF-1 in the third sample solution can be obtained.

[0034] [Table 1] IGF-1 misfolded isomer SUMO Other peaks Recovery rate Example 1 64.2% 28.9% 0% 6.9% 80% Example 2 95.7% 0.4% 0% 3.9% 70% Comparative Example 1 49.6% 21.5% 21.7% 7.2% 31%

[0035] [Example 2] (1) Purification by cation exchange chromatography The second sample solution obtained in the same manner as in Example 1 was loaded in its entirety into a cation exchange chromatography column (SP cellulose, height 20 cm) equilibrated with SP buffer A. Then, a gradient elution consisting of the following elution steps (i)-(iii) was performed.

[0036] Dissolution Step (i) Dissolution was performed at 3 CV (linear velocity 60 cm / h) with a 67% (salt concentration 200 mM) precipitate of SP buffer A 33% / SP buffer B1 (20 mM ammonium acetate, 200 mM imidazole, 300 mM NaCl, pH 4.0). Next, dissolution was performed at 6 CV (linear velocity 60 cm / h) with a 17% (salt concentration 250 mM) precipitate of SP buffer A 17% / SP buffer B1 83% precipitate. Furthermore, linear concentration gradient dissolution was performed from the SP buffer A 17% / SP buffer A 83% condition (salt concentration 250 mM) to the SP buffer A 0% / SP buffer B1 100% condition (salt concentration 300 mM). The chromatogram of the resulting precipitate is shown in Figure 2. In the figure, the horizontal axis represents dissolution time, and the vertical axis represents absorbance (OD 220nm). The black arrows represent the rIGF-1 peak, and the white arrows represent misfolded isomers. The percentage of rIGF-1 in the total protein mass, calculated from the peak intensity, is 27.6%.

[0037] Dissolution Step (ii) Next, dissolution was performed at 3CV (linear velocity 60 cm / h) with 100% SP buffer B1 (salt concentration 300 mM) to obtain the third sample solution containing IGF-1. Furthermore, linear concentration gradient dissolution was performed from 70% SP buffer A / 30% SP buffer B2 (20 mM ammonium acetate, 200 mM imidazole, 1 M NaCl, pH 4.0) (salt concentration 300 mM) to 60% SP buffer / 40% SP buffer B2 (salt concentration 400 mM) (salt concentration 400 mM) (3CV, linear velocity 60 cm / h). The chromatogram of the obtained third sample solution is shown in Figure 3. In the figure, the horizontal axis represents dissolution time, and the vertical axis represents absorbance (OD 220 nm). The black arrow represents rIGF-1. The percentages of rIGF-1 and misfolded isomers, calculated from peak intensity, were 95.7% and 0.4% of the total protein content, respectively (see Table 1). Furthermore, the rIGF-1 recovery rate was 70%.

[0038] Dissolution step (iii) Finally, dissolve the sample in SP buffer B2 100% (salt concentration 1M) at 3CV (linear velocity 60cm / h).

[0039] In the precipitate (sample solution 3) of the precipitate step (ii), rIGF-1 can be obtained with high purity (95.7%) and high recovery (70%) relative to mis-folded isomer (0.4%).

[0040] [Comparative Example 1] (1) Purification by Ni-NTA Affinity Chromatography 2 The second sample solution obtained in the same manner as in Example 1 was loaded entirely into a Ni-NTA affinity chromatography column (linear velocity 60 cm / h). The precipitate containing the entire amount of the precipitate and the precipitate obtained by subsequent washing (20 mM Tris buffer, 300 mM NaCl, pH 7.8) was used as the sample solution containing IGF-1.

[0041] The chromatogram of the obtained sample solution is shown in Figure 4. In the figure, the horizontal axis represents the dissolution time, and the vertical axis represents the absorbance (OD 220nm). The black arrows represent the peaks of rIGF-1 (correctly folded rIGF-1), the white arrows represent misfolded rIGF-1 (mis-folded isomer), and the diagonal arrows represent SUMO. The ratios of rIGF-1 and mis-folded isomers, calculated from the peak intensities, were 49.6% and 21.5% of the total protein, respectively, with a high SUMO content of 21.7% (refer to Table 1). Furthermore, the rIGF-1 recovery rate was as low as 31%. [Simplified Explanation of the Diagram]

[0012] [Figure 1] shows the chromatography diagram of the third sample solution obtained in Example 1. In the figure, the horizontal axis represents the dissolution time, and the vertical axis represents the absorbance (OD 220nm). The black arrows represent the peaks of rIGF-1 (correctly folded rIGF-1), and the white arrows represent misfolded rIGF-1 (mis-folded isomer). [Figure 2] shows the chromatography diagram of the precipitate obtained in dissolution step (i) of Example 2. In the figure, the horizontal axis represents the dissolution time, and the vertical axis represents the absorbance (OD 220nm). The black arrows represent the peaks of rIGF-1, and the white arrows represent mis-folded isomers. [Figure 3] shows the chromatography diagram of the precipitate (third sample solution) obtained in dissolution step (ii) of Example 2. In the figure, the horizontal axis represents the dissolution time, and the vertical axis represents the absorbance (OD 220nm). The black arrows represent the peaks of rIGF-1. [Figure 4] shows the chromatography of the sample solution obtained in the dissolution-washing step of Comparative Example 1. In the figure, the horizontal axis represents the dissolution time, and the vertical axis represents the absorbance (OD 220nm). The black arrows represent the peak of rIGF-1, the white arrows represent the misfolded isomer, and the diagonal arrows represent SUMO.

Claims

1. A method for manufacturing recombinant insulin-like growth factor (rIGF-1), comprising: (1) obtaining rIGF-1 in the form of a fusion protein (SUMO-IGF-1) formed by fusing rIGF-1 with a ubiquitin-like protein SUMO (small ubiquitin-related modifier); (2) treating SUMO-IGF-1 with SUMO protease; and (3) applying the treatment solution from step (2) to cation exchange chromatography by gradient elution to obtain a elution solution containing rIGF-1, wherein the gradient elution is performed using a buffer containing salt, and the concentration gradient of the salt in the buffer is used to separate misfolded rIGF-1 from correctly folded rIGF-1, wherein... The gradient elution described above includes: (i) a elution step with a salt concentration of 200 mM to 300 mM or more in a column volume or more; (ii) a elution step with a salt concentration of 300 mM to 500 mM or more in a column volume or more; and optionally (iii) a elution step with a salt concentration of 500 mM or more in a elution solution, wherein rIGF-1 is recovered from the elution solution of the above-mentioned elution step (ii).

2. The manufacturing method as described in claim 1, wherein, Step (1) includes: dissolving host cells expressing SUMO-IGF-1 to obtain a first sample solution (1-1); applying the first sample solution to Ni-NTA (nickel-nitrilotriacetic acid) affinity chromatography to obtain a first precipitate containing SUMO-IGF-1 (1-2); step (2) treating the first precipitate with SUMO protease to obtain a second sample solution containing SUMO-IGF-1, SUMO protease, SUMO, and rIGF-1; and step (3) applying the second sample solution to cation exchange chromatography to separate correctly folded rIGF-1 from SUMO-IGF-1, SUMO protease, SUMO, and misfolded rIGF-1 by gradient chromatography to obtain a third sample solution containing rIGF-1.

3. The manufacturing method as described in claim 1, wherein, After step (2), a separation step by Ni-NTA affinity chromatography is not included.

4. The manufacturing method as described in claim 1, wherein, The separation steps for misfolded proteins by reverse-phase high-performance liquid chromatography are not included.

5. The manufacturing method as described in claim 1, wherein, In the above-mentioned third sample solution, the proportion of correctly folded rIGF-1, which accounts for more than 60% of the total protein content, is above 60%.

6. The manufacturing method as described in claim 1, wherein, In the above-mentioned third sample solution, the proportion of correctly folded rIGF-1, which accounts for more than 70% of the total protein content, is above 70%.

7. The manufacturing method as described in claim 1, wherein, In the above-mentioned third sample solution, the proportion of correctly folded rIGF-1, which accounts for more than 80% of the total protein content, is above 80%.

8. The manufacturing method as described in claim 1, wherein, In the above-mentioned third sample solution, the proportion of correctly folded rIGF-1, which accounts for more than 90% of the total protein content, is above 90%.

9. The manufacturing method as described in claim 1, wherein, In the above-mentioned third sample solution, the proportion of correctly folded rIGF-1, which accounts for more than 25% of the total protein content, is above 25%.

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