[go: up one dir, main page]

WO2022097216A1 - Method for producing recombinant insulin-like growth factor i - Google Patents

Method for producing recombinant insulin-like growth factor i Download PDF

Info

Publication number
WO2022097216A1
WO2022097216A1 PCT/JP2020/041283 JP2020041283W WO2022097216A1 WO 2022097216 A1 WO2022097216 A1 WO 2022097216A1 JP 2020041283 W JP2020041283 W JP 2020041283W WO 2022097216 A1 WO2022097216 A1 WO 2022097216A1
Authority
WO
WIPO (PCT)
Prior art keywords
sumo
rigf
elution
eluate
igf
Prior art date
Application number
PCT/JP2020/041283
Other languages
French (fr)
Japanese (ja)
Inventor
裕幸 川嶋
政博 高栖
光▲隆▼ 薛
Original Assignee
シミックホールディングス株式会社
株式会社オーファンパシフィック
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by シミックホールディングス株式会社, 株式会社オーファンパシフィック filed Critical シミックホールディングス株式会社
Priority to JP2022560551A priority Critical patent/JP7570428B2/en
Priority to CN202080106934.7A priority patent/CN116368149A/en
Priority to KR1020237018335A priority patent/KR20230098304A/en
Priority to PCT/JP2020/041283 priority patent/WO2022097216A1/en
Publication of WO2022097216A1 publication Critical patent/WO2022097216A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2

Definitions

  • the present invention relates to a method for producing recombinant insulin-like growth factor I (rIGF-1).
  • rIGF-1 insulin-like growth factor I
  • SUMO-rIGF-1 a fusion protein
  • HPLC reverse phase high performance liquid chromatography
  • rIGF-1 is purified by a nickel column (Ni-NTA resin column). Specifically, a solution obtained by treating the expressed SUMO-rIGF-1 with SUMO protease (including SUMO-rIGF-1, SUMO protease, SUMO and rIGF-1) is subjected to Ni-NTA (nickel-nitrilotriacetic acid) affinity chromatography.
  • Ni-NTA nickel-nitrilotriacetic acid
  • the main object of the present invention is to provide an improved method for obtaining a correctly folded rIGF-1 with high purity with respect to a misfolded rIGF-1.
  • [1] A method for producing recombinant insulin-like growth factor I (rIGF-1).
  • rIGF-1 as a fusion protein (SUMO-IGF-1) with a ubiquitin-like protein SUMO (small ubiquitin-related modifier), and
  • the step of treating SUMO-IGF-1 with SUMO protease and (3) A step of applying the treatment solution of step (2) to cation exchange chromatography to obtain an eluate containing rIGF-1 by gradient elution, and a step of obtaining an eluate.
  • Manufacturing method including.
  • Step (1) is a step (1-1) of lysing a host cell expressing SUMO-IGF-1 to obtain a first sample solution, and Ni-NTA (nickel-) for the first sample solution. nitrilotriacetic acid) Including the step (1-2) of obtaining a first eluate containing SUMO-IGF-1 by applying to affinity chromatography.
  • Step (2) is a step of treating the first eluate with SUMO protease to obtain a second sample solution containing SUMO-IGF-1, SUMO protease, SUMO and rIGF-1.
  • step (3) the second sample solution is applied to cation exchange chromatography to separate rIGF-1 from SUMO-IGF-1, SUMO protease and SUMO by gradient elution, and the third step contains rIGF-1.
  • the process of obtaining a sample solution [1] The production method according to any one of [5]. [7] The production method according to any one of [1] to [6], which does not include a separation step by Ni-NTA affinity chromatography after the step (2). [8] The production method according to any one of [1] to [7], which does not include a step of separating misfolding proteins by reverse phase high performance liquid chromatography.
  • the ratio of the correctly folded rIGF-1 amount to the total amount of protein is 60% or more, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more.
  • a method for purifying recombinant insulin-like growth factor I (rIGF-1). (1) A step of obtaining rIGF-1 as a fusion protein (SUMO-IGF-1) with a ubiquitin-like protein SUMO (small ubiquitin-related modifier), and (2) The step of treating SUMO-IGF-1 with SUMO protease and (3) A step of applying the treatment solution of step (2) to cation exchange chromatography to obtain an eluate containing rIGF-1 by gradient elution, and a step of obtaining an eluate. Purification method including.
  • gradient elution means salt concentration gradient elution.
  • the gradient elution may include a step of elution in which the salt concentration is changed linearly (liner) or stepwise (stepwise), and the salt concentration is changed in all the steps. Does not need to be done. That is, the gradient elution may include a step of elution with a constant salt concentration.
  • the present invention provides an improved method for obtaining high-purity rIGF-1 correctly folded with respect to misfolded rIGF-1.
  • the chromatogram of the third sample solution obtained in Example 1 is shown.
  • the horizontal axis shows the elution time and the vertical axis shows the absorbance (OD 220 nm).
  • the black arrow indicates the peak of rIGF-1 (correctly folded rIGF-1), and the white arrow indicates the misfolded rIGF-1 (mis-folded isomer).
  • the chromatogram of the eluate obtained in the elution step (i) of Example 2 is shown.
  • the horizontal axis shows the elution time and the vertical axis shows the absorbance (OD 220 nm).
  • the black arrow indicates the peak of rIGF-1, and the white arrow indicates mis-folded isomer.
  • the chromatogram of the eluate (third sample solution) obtained in the elution step (ii) of Example 2 is shown.
  • the horizontal axis shows the elution time and the vertical axis shows the absorbance (OD 220 nm).
  • the black arrow indicates the peak of rIGF-1.
  • the chromatogram of the sample solution obtained in the elution / washing step of Comparative Example 1 is shown.
  • the horizontal axis shows the elution time and the vertical axis shows the absorbance (OD 220 nm).
  • the black arrow indicates the peak of rIGF-1
  • the white arrow indicates mis-folded isomer
  • the diagonal arrow indicates SUMO.
  • the method for producing and purifying recombinant insulin-like growth factor I (rIGF-1) according to the present invention includes the following steps (1)-(3).
  • Step (1) A step of obtaining rIGF-1 as a fusion protein (SUMO-IGF-1) with a ubiquitin-like protein SUMO (small ubiquitin-related modifier).
  • Step (2) A step of treating SUMO-IGF-1 with SUMO protease.
  • rIGF-1 is obtained as a fusion protein (SUMO-IGF-1) with a ubiquitin-like protein SUMO (small ubiquitin-related modifier).
  • This step is a step of lysing a host cell expressing SUMO-IGF-1 to obtain a first sample solution (1-1), and a Ni-NTA (nickel-nitrilotriacetic acid) affinity chromatograph of the first sample solution. It comprises a step (1-2) of obtaining a first eluate containing SUMO-IGF-1 by applying it to a graph.
  • Step (1-1) In this step, host cells expressing SUMO-IGF-1 are lysed to obtain a first sample solution.
  • the gene construct for SUMO-IGF-1 expression may be prepared by a conventionally known method (for example, the method described in Non-Patent Document 1). Commercially available DNA synthesis genes and general-purpose vectors can be used for the SUMO-IGF-1 gene and the expression plasmid for producing the gene construct.
  • the host cell is not particularly limited, but Escherichia coli is preferably used, and a commercially available Escherichia coli cell line can be used. Transformation of host cells with a gene construct and culturing of transformants can also be performed by conventionally known methods. After culturing, the cells are collected from the culture solution by centrifugation or the like, the cells are crushed using a high-pressure crusher or the like, and the insoluble matter is removed by centrifugation or the like to obtain a first sample solution.
  • Step (1-2) the first sample solution is applied to Ni-NTA (nickel-nitrilotriacetic acid) affinity chromatography to obtain a first eluate containing SUMO-IGF-1.
  • Ni-NTA nickel-nitrilotriacetic acid
  • SUMO-IGF-1 is retained on the column carrier via a histidine tag attached to SUMO.
  • elution with a concentration gradient of imidazole is performed to elute SUMO-IGF-1 retained on the column carrier to obtain a first eluate.
  • imidazole concentration gradient elution for example, linear concentration gradient elution using an initiating buffer (50mM Tris, 300mM NaCl, 8M Urea, 20mM Imidazole, pH 7.8) and an elution buffer (50mM Tris, 300mM NaCl, 600mM Imidazole, pH 7.8). Can be done by.
  • an initiating buffer 50mM Tris, 300mM NaCl, 8M Urea, 20mM Imidazole, pH 7.8
  • elution buffer 50mM Tris, 300mM NaCl, 600mM Imidazole, pH 7.8.
  • Step (2) This step is a step of treating SUMO-IGF-1 with SUMO protease.
  • Treatment of the first eluate with SUMO protease gives a second sample solution containing SUMO-IGF-1, SUMO protease, SUMO and rIGF-1.
  • the SUMO protease treatment may be carried out by a conventionally known method (for example, the method described in Non-Patent Document 1). After the SUMO protease treatment, the treatment solution containing SUMO-IGF-1, SUMO protease, SUMO and rIGF-1 may be appropriately concentrated or replaced with a buffer solution to obtain a second sample solution.
  • Step (3) is a step of applying the second sample solution to cation exchange chromatography to obtain an eluate containing rIGF-1 by gradient elution.
  • a SUMO protease-treated solution containing SUMO-IGF-1, SUMO protease, SUMO and rIGF-1 is applied again to Ni-NTA affinity chromatography, and His-tag is applied.
  • SUMO-rIGF-1, SUMO protease and SUMO are adsorbed on the column and only rIGF-1 is eluted to purify rIGF-1 (see Comparative Example 1).
  • the method according to the present invention purifies rIGF-1 by using cation exchange chromatography instead of Ni-NTA affinity chromatography.
  • the second sample solution is cation exchange chromatography.
  • To obtain a third sample solution containing rIGF-1 separate rIGF-1 from SUMO-IGF-1, SUMO protease and SUMO by gradient elution.
  • the method according to the present invention does not include a separation step by Ni-NTA affinity chromatography after the step (2).
  • a third sample solution is obtained by eluting rIGF-1 retained on the column carrier by performing salt concentration gradient elution.
  • the salt concentration gradient elution can be performed, for example, by linear concentration gradient elution using the above buffer as a starting buffer and also using an elution buffer (20 mM Ammonium acetate, 200 mM Imidazole, 1M NaCl, pH 4.0).
  • the elution volume and elution rate can be adjusted as appropriate.
  • the eluate is, for example, a 4-column volume (CV), 1-16 CV, preferably 2-8 CV, more preferably 3-6 CV.
  • CV 4-column volume
  • rIGF-1 without SUMO contamination can be obtained with high purity and high yield as compared with the conventional method (see Comparative Example 1) (Example). 1).
  • the salt concentration gradient elution preferably comprises the following elution steps (i)-(iii).
  • misfolding rIGF-1 is mainly eluted.
  • the eluate for example, an eluate containing ammonium acetate as a buffer and a salt (NaCl or the like) at the above concentration is used.
  • the salt concentration in the eluate may be constant in the above concentration range, or may change linearly (liner) or stepwise (stepwise).
  • the eluate may contain imidazole at an appropriate concentration (eg, 200 mM).
  • the elution volume and elution rate can be adjusted as appropriate.
  • the eluate is, for example, 12 CV, in the range of 3-48 CV, preferably 6-24 CV, more preferably 9-15 CV.
  • elution step (i) specifically, elution of 1.5-6 column volume (CV) at a salt concentration of 200 mM, then elution of 3-12 CV at a salt concentration of 250 mM, and a linear concentration gradient from a salt concentration of 250 mM to 300 mM. It is said that 1.5-3 CV is eluted.
  • rIGF-1 is mainly eluted.
  • highly purified rIGF-1 can be obtained.
  • the eluate for example, an eluate containing ammonium acetate as a buffer and a salt (NaCl or the like) at the above concentration is used.
  • the salt concentration in the eluate may be constant in the above concentration range, or may change linearly (liner) or stepwise (stepwise).
  • the eluate may contain imidazole at an appropriate concentration (eg, 200 mM). The elution volume and elution rate can be adjusted as appropriate.
  • the elution solution is, for example, 6 CV, preferably in the range of 1.5-24 CV, preferably 3-12 CV, more preferably 4.5-8 CV.
  • the elution step (ii) is specifically defined as elution of 1.5-6 CV at a salt concentration of 300 mM and then elution of 1.5-3 CV with a linear concentration gradient from a salt concentration of 300 mM to 400 mM.
  • SUMO-IGF1, SUMO protease and SUMO are mainly eluted.
  • the eluate for example, an eluate containing ammonium acetate as a buffer and a salt (NaCl or the like) at the above concentration is used.
  • the salt concentration in the eluate may be constant in the above concentration range, or may change linearly (liner) or stepwise (stepwise).
  • the eluate may contain imidazole at an appropriate concentration (eg, 200 mM). The elution volume and elution rate can be adjusted as appropriate.
  • the elution solution is, for example, 3 CV, in the range of 0.75-12 V, preferably 1.5-6 CV, more preferably 2-4 CV.
  • the elution step (iii) is specifically elution of 1.5-6 CV at a salt concentration of 1 M.
  • rIGF-1 reverse phase high performance liquid chromatography
  • a misfolding rIGF-1 is used by using an inexpensive cation exchange chromatography column (step (3)) without using an expensive HPLC column.
  • RIGF-1 which is correctly folded, can be obtained with high purity and high recovery rate. It should be noted that it is not excluded that further HPLC separation is performed following the process of the present invention for the purpose of removing MetO 59 -IGF-1 among the correctly folded rIGF-1.
  • the ratio of the correctly folded rIGF-1 amount to the total amount of protein in the eluate (third sample solution) of step (3) is 60%.
  • it can be preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, and particularly preferably 95% or more.
  • Example 1 (1) Material (1-1) SUMO-IGF-1 DNA synthesis gene A commercially available DNA synthesis gene (MISSION BIOTECH, GS3075) was used for the SUMO-IGF-1 gene.
  • pET22b Expression regulatory plasmid
  • pET22b EMD Millipore, 69744-3CN
  • pET22b EMD Millipore, 69744-3CN
  • pET22b EMD Millipore, 69744-3CN
  • 1-3 Gene expression construct (pET22b SUMO-IGF-1) The SUMO-IGF-1 gene expression construct (pET22b SUMO-IGF-1) was constructed by integrating the SUMO-IGF-1 DNA synthesis gene into pET22b.
  • (1-4) Host cell As the host cell, a cell line BL21 (DE3) derived from Escherichia coli B strain was used. (1-5) Preparation of seed cell line for preparation of master working cell bank A mecasermin (genetical recombination) gene expression construct was introduced into a host cell BL21 (BD3) strain by an Escherichia coli transformation method. Eight single colonies were selected by culturing at 37 ° C. on LB plate medium containing ampicillin. These single colonies were cultured in LB medium containing ampicillin at 37 ° C. to a culture turbidity (OD 600 nm) of 0.6-0.8, and 1 mM isopropyl- ⁇ -thiogalactopyranoside for 2.5 hours.
  • a culture turbidity OD 600 nm
  • Highly expressed cell lines were selected by inducing at 28 ° C. (1-6) Preparation of Master Working Cell Bank One selected colony was inoculated into LB medium containing ampicillin and cultured at 37 ° C. with a turbidity (OD 600 nm) of 0.6 to 0.8. After culturing, centrifuge, discard the medium, add the LB medium containing ampicillin so that the turbidity (OD 600 nm) is 2, add sterile glycerol to a final concentration of 20%, and suspend. , The final turbidity (OD 600 nm) was adjusted to 1.2. The cell suspension was dispensed into cryotubes in 1 mL increments.
  • Ni-NTA affinity chromatography column (height 25 cm) equilibrated with a cell lysis buffer. After washing the column with cell lysis buffer, use nickel buffer B (50 mM Tris, 300 mM NaCl, 8M Urea, 20 mM Imidazole, pH 7.8) as the starting buffer and nickel buffer C (50 mM Tris, 300 mM NaCl, 600 mM).
  • a first eluate containing SUMO-IGF-1 was obtained by linear concentration gradient elution (linear velocity 60 cm / h) using Imidazole, pH 7.8) as the elution buffer.
  • SUMO Protease Treatment A 1/2000 times volume of SUMO protease solution was added to the first eluate, and the mixture was reacted at 15 ° C. or higher for 1 hour.
  • the reaction solution was concentrated with an ultrafiltration membrane (molecular weight cut off of 2 kDa).
  • the buffer was replaced with TFF buffer B (20 mM Ammonium acetate, 200 mM Imidazole, pH 4.0) in an amount of 7 times or more that of the concentrate. Then, it was concentrated again.
  • the concentrate was recovered with TFF buffer B to give a second sample solution containing SUMO-IGF-1, SUMO protease, SUMO and rIGF-1.
  • the chromatogram of the obtained third sample solution is shown in FIG.
  • the horizontal axis shows the elution time and the vertical axis shows the absorbance (OD 220 nm).
  • the black arrow is the peak of rIGF-1 (correctly folded rIGF-1), and the white arrow is the misfolded rIGF-1 (mis-folded isomer).
  • the ratios of rIGF-1 and mis-folded isomers to the total amount of protein calculated from the peak intensity were 64.2% and 28.9%, respectively (see Table 1).
  • the amount of rIGF-1 (recovery rate) of the third sample solution was 80% with respect to the amount of rIGF-1 in the second sample solution. In the third sample solution, rIGF-1 without SUMO contamination could be obtained with high purity (64.2%) and high recovery rate (80%).
  • Example 2 Purification of cation exchange chromatography The entire amount of the second sample solution obtained in the same manner as in Example 1 was placed on a cation exchange chromatography column (SP cellulose, height 20 cm) equilibrated with SP buffer A. Loaded. Then, gradient elution consisting of the following elution steps (i)-(iii) was performed.
  • Dissolution step (i) SP buffer A 33% / SP buffer B1 (20 mM ammonium acetate, 200 mM imidazole, 300 mM NaCl, pH 4.0) 67% eluate (salt concentration 200 mM) was eluted with 3 CV (linear velocity 60 cm / h). Then, elution was performed with 6 CV of eluate (salt concentration 250 mM) of SP buffer A 17% / SP buffer B1 83% (linear velocity 60 cm / h).
  • the horizontal axis shows the elution time and the vertical axis shows the absorbance (OD 220 nm).
  • the black arrow is rIGF-1.
  • the ratios of rIGF-1 and mis-folded isomers to the total amount of protein calculated from the peak intensities were 95.7% and 0.4%, respectively (see Table 1).
  • the recovery rate of rIGF-1 was 70%.
  • rIGF-1 In the eluate (third sample solution) of the elution step (ii), rIGF-1 can be obtained with high purity (95.7%) and high recovery rate (70%) with respect to mis-folded isomer (0.4%). rice field.
  • Example 1 (1) Ni-NTA affinity chromatography purification 2 The second sample solution obtained in the same manner as in Example 1 was fully loaded onto a Ni-NTA affinity chromatography column (linear velocity 60 cm / h). The eluate eluted during full loading and the eluate by subsequent washing (20 mM Tris buffer, 300 mM NaCl, pH 7.8) were obtained as a sample solution containing IGF-1.
  • the chromatogram of the obtained sample solution is shown in FIG.
  • the horizontal axis shows the elution time and the vertical axis shows the absorbance (OD 220 nm).
  • the black arrow is the peak of rIGF-1 (correctly folded rIGF-1), the white arrow is the misfolded rIGF-1 (mis-folded isomer), and the diagonal arrow is SUMO.
  • the ratios of rIGF-1 and mis-folded isomers to the total amount of protein calculated from the peak intensity were 49.6% and 21.5%, respectively, and SUMO was included in a high ratio of 21.7% (see Table 1).
  • the rIGF-1 recovery rate was also low at 31%.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Endocrinology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Diabetes (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Provided is a method for producing recombinant insulin-like growth factor I (rIGF-1) as an improved method for obtaining correctly folded rIGF-1 with high purity relative to misfolded rIGF-1, the method comprising: (1) a step for obtaining rIGF-1 as a fusion protein (SUMO-IGF-1) of rIGF-1 and a ubiquitin-like protein, small ubiquitin-related modifier (SUMO); (2) a step for treating SUMO-IGF-1 with SUMO protease; and (3) a step for applying the treatment solution in step (2) to cation exchange chromatography to obtain an eluate containing rIGF-1 by means of gradient elution.

Description

組み換えインスリン様成長因子Ｉの製造方法Method for Producing Recombinant Insulin-like Growth Factor I
　本発明は、組み換えインスリン様成長因子Ｉ（rIGF-1）の製造方法に関する。 The present invention relates to a method for producing recombinant insulin-like growth factor I (rIGF-1).
　大腸菌を用いた組み換えタンパク質の生産には、発現させたタンパク質の不溶化とミスフォールディングの問題がある。組み換えインスリン様成長因子Ｉ（rIGF-1）に関しては、不溶化の問題に対処するため、rIGF-1をユビキチン様タンパク質SUMO（small ubiquitin-related modifier）との融合タンパク質（SUMO-rIGF-1）として発現させる技術が報告されている（非特許文献１参照）。また、正しくフォールディングされたrIGF-1をミスフォールディングのrIGF-1と分離するために、逆相高速液体クロマトグラフィー（HPLC）が用いられている（特許文献１参照）。 Production of recombinant proteins using E. coli has problems of insolubilization and misfolding of expressed proteins. Regarding recombinant insulin-like growth factor I (rIGF-1), rIGF-1 is expressed as a fusion protein (SUMO-rIGF-1) with the ubiquitin-like protein SUMO (small ubiquitin-related modifier) in order to address the problem of insolubilization. (See Non-Patent Document 1). In addition, reverse phase high performance liquid chromatography (HPLC) is used to separate correctly folded rIGF-1 from misfolded rIGF-1 (see Patent Document 1).
　非特許文献１に記載されるSUMO-rIGF-1の発現によるrIGF-1の生産では、ニッケルカラム（Ni-NTA樹脂カラム）によるrIGF-1の精製が行われている。具体的には、発現させたSUMO-rIGF-1をSUMOプロテアーゼにより処理した溶液（SUMO-rIGF-1、SUMOプロテアーゼ、SUMO及びrIGF-1を含む）をNi-NTA（nickel-nitrilotriacetic acid）アフィニティークロマトグラフィーに適用し、ヒスチジンタグを有するSUMO-rIGF-1、SUMOプロテアーゼ及びSUMOをカラムに吸着させrIGF-1のみを溶出させることによりrIGF-1を精製している。 In the production of rIGF-1 by expressing SUMO-rIGF-1 described in Non-Patent Document 1, rIGF-1 is purified by a nickel column (Ni-NTA resin column). Specifically, a solution obtained by treating the expressed SUMO-rIGF-1 with SUMO protease (including SUMO-rIGF-1, SUMO protease, SUMO and rIGF-1) is subjected to Ni-NTA (nickel-nitrilotriacetic acid) affinity chromatography. It is applied to chromatography to purify rIGF-1 by adsorbing SUMO-rIGF-1, SUMO protease and SUMO having a histidine tag on a column and eluting only rIGF-1.
特表平０７－５０１７０４号公報Special Table No. 07-501704
　本発明は、ミスフォールディングのrIGF-1に対して正しくフォールディングされたrIGF-1を高純度に得るための改善された手法を提供することを主な目的とする。 The main object of the present invention is to provide an improved method for obtaining a correctly folded rIGF-1 with high purity with respect to a misfolded rIGF-1.
　上記課題解決のため、本発明は、以下の［１］－［１１］を提供する。
［１］　組み換えインスリン様成長因子Ｉ（rIGF-1）の製造方法であって、
（１）rIGF-1をユビキチン様タンパク質SUMO（small ubiquitin-related modifier）との融合タンパク質（SUMO-IGF-1）として得る工程と、
（２）SUMO-IGF-1をSUMOプロテアーゼにより処理する工程と、
（３）工程（２）の処理液を陽イオン交換クロマトグラフィーに適用し、グラジエント溶出によりrIGF-1を含む溶出液を得る工程と、
を含む製造方法。
［２］　前記グラジエント溶出が、
（ｉ）塩濃度200 mM以上300 mM未満の溶出液での１カラム容積以上の溶出工程、
（ｉｉ）塩濃度300 mM以上500 mM未満の溶出液での１カラム容積以上の溶出工程、
（ｉｉｉ）塩濃度500 mM以上の溶出液での溶出工程、
を含み、
rIGF-1を前記溶出工程（ｉｉ）の溶出液中に回収する、［１］の製造方法。
［３］　溶出工程（ｉ）の溶出条件が次のとおりである、［２］の製造方法。
　塩濃度200mMで1.5-6カラム容積（CV）の溶出、次いで塩濃度250mMで3-12CVの溶出、さらに塩濃度250mMから300mMまでの直線濃度勾配による1.5-3CVの溶出。
［４］　溶出工程（ｉｉ）
　塩濃度300mMで1.5-6CVの溶出、次いで塩濃度300mMから400mMまでの直線濃度勾配による1.5-3CVの溶出。
［５］　溶出工程（ｉｉｉ）の溶出条件が次のとおりである、［２］－［４］のいずれかの製造方法。
　塩濃度1Mでの1.5-6CVの溶出。
［６］　工程（１）は、SUMO-IGF-1を発現する宿主細胞を溶解して第１の試料溶液を得る工程（１－１）と、第１の試料溶液をNi-NTA（nickel-nitrilotriacetic acid）アフィニティークロマトグラフィーに適用して、SUMO-IGF-1を含む第１の溶出液を得る工程（１－２）を含み、
工程（２）は、前記第１の溶出液をSUMOプロテアーゼにより処理して、SUMO-IGF-1、SUMOプロテアーゼ、SUMO及びrIGF-1を含む第２の試料溶液を得る工程であり、
工程（３）は、第２の試料溶液を陽イオン交換クロマトグラフィーに適用してグラジエント溶出によりSUMO-IGF-1、SUMOプロテアーゼ及びSUMOからrIGF-1を分離してrIGF-1を含む第３の試料溶液を得る工程である、
［１］［５］のいずれかの製造方法。
［７］　工程（２）よりも後段に、Ni-NTAアフィニティークロマトグラフィーによる分離工程を含まない、［１］－［６］のいずれかの製造方法。
［８］　逆相高速液体クロマトグラフィーによるミスフォールディングタンパクの分離工程を含まない、［１］－［７］のいずれかの製造方法。
［９］　前記第３の試料溶液中において、タンパク質全量に占める正しくフォールディングされたrIGF-1量の比率が６０％以上、好ましくは７０％以上、より好ましくは８０％以上、さらに好ましくは９０％以上、特に好ましくは９５％以上である、［１］－［８］のいずれかの製造方法。
［１０］　前記宿主細胞が、大腸菌である、［１］－［９］のいずれかの製造方法。 In order to solve the above problems, the present invention provides the following [1]-[11].
[1] A method for producing recombinant insulin-like growth factor I (rIGF-1).
(1) A step of obtaining rIGF-1 as a fusion protein (SUMO-IGF-1) with a ubiquitin-like protein SUMO (small ubiquitin-related modifier), and
(2) The step of treating SUMO-IGF-1 with SUMO protease and
(3) A step of applying the treatment solution of step (2) to cation exchange chromatography to obtain an eluate containing rIGF-1 by gradient elution, and a step of obtaining an eluate.
Manufacturing method including.
[2] The gradient elution
(I) Elution step of 1 column volume or more with an eluate having a salt concentration of 200 mM or more and less than 300 mM.
(Ii) Elution step of 1 column volume or more with an eluate having a salt concentration of 300 mM or more and less than 500 mM.
(Iii) Elution step with an eluate having a salt concentration of 500 mM or more,
Including
The production method of [1], wherein rIGF-1 is recovered in the eluate of the elution step (ii).
[3] The production method of [2], wherein the elution conditions of the elution step (i) are as follows.
Elution of 1.5-6 column volume (CV) at salinity 200 mM, then elution of 3-12 CV at salinity 250 mM, and elution of 1.5-3 CV with a linear concentration gradient from salinity 250 mM to 300 mM.
[4] Dissolution step (ii)
Elution of 1.5-6 CV at a salinity of 300 mM, followed by elution of 1.5-3 CV with a linear concentration gradient from salinity 300 mM to 400 mM.
[5] The production method according to any one of [2] to [4], wherein the elution conditions in the elution step (iii) are as follows.
Elution of 1.5-6 CV at a salt concentration of 1 M.
[6] Step (1) is a step (1-1) of lysing a host cell expressing SUMO-IGF-1 to obtain a first sample solution, and Ni-NTA (nickel-) for the first sample solution. nitrilotriacetic acid) Including the step (1-2) of obtaining a first eluate containing SUMO-IGF-1 by applying to affinity chromatography.
Step (2) is a step of treating the first eluate with SUMO protease to obtain a second sample solution containing SUMO-IGF-1, SUMO protease, SUMO and rIGF-1.
In step (3), the second sample solution is applied to cation exchange chromatography to separate rIGF-1 from SUMO-IGF-1, SUMO protease and SUMO by gradient elution, and the third step contains rIGF-1. The process of obtaining a sample solution,
[1] The production method according to any one of [5].
[7] The production method according to any one of [1] to [6], which does not include a separation step by Ni-NTA affinity chromatography after the step (2).
[8] The production method according to any one of [1] to [7], which does not include a step of separating misfolding proteins by reverse phase high performance liquid chromatography.
[9] In the third sample solution, the ratio of the correctly folded rIGF-1 amount to the total amount of protein is 60% or more, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more. The production method according to any one of [1] to [8], which is particularly preferably 95% or more.
[10] The production method according to any one of [1] to [9], wherein the host cell is Escherichia coli.
［１１］　組み換えインスリン様成長因子Ｉ（rIGF-1）の精製方法であって、
（１）rIGF-1をユビキチン様タンパク質SUMO（small ubiquitin-related modifier）との融合タンパク質（SUMO-IGF-1）として得る工程と、
（２）SUMO-IGF-1をSUMOプロテアーゼにより処理する工程と、
（３）工程（２）の処理液を陽イオン交換クロマトグラフィーに適用し、グラジエント溶出によりrIGF-1を含む溶出液を得る工程と、
を含む精製方法。 [11] A method for purifying recombinant insulin-like growth factor I (rIGF-1).
(1) A step of obtaining rIGF-1 as a fusion protein (SUMO-IGF-1) with a ubiquitin-like protein SUMO (small ubiquitin-related modifier), and
(2) The step of treating SUMO-IGF-1 with SUMO protease and
(3) A step of applying the treatment solution of step (2) to cation exchange chromatography to obtain an eluate containing rIGF-1 by gradient elution, and a step of obtaining an eluate.
Purification method including.
　本発明において「グラジエント溶出」とは、塩濃度勾配溶出のことを意味する。グラジエント溶出は、塩濃度が直線的（ライナー）にあるいは段階的（ステップワイズ）に変化させて溶出を行う工程が一部に含まれていればよく、その全工程で塩濃度を変化させた溶出が行われることを必要としない。すなわち、グラジエント溶出は、塩濃度を一定として溶出を行う工程を一部に含んでいてもよい。 In the present invention, "gradient elution" means salt concentration gradient elution. The gradient elution may include a step of elution in which the salt concentration is changed linearly (liner) or stepwise (stepwise), and the salt concentration is changed in all the steps. Does not need to be done. That is, the gradient elution may include a step of elution with a constant salt concentration.
　本発明により、ミスフォールディングのrIGF-1に対して正しくフォールディングされたrIGF-1を高純度に得るための改善された手法が提供される。 The present invention provides an improved method for obtaining high-purity rIGF-1 correctly folded with respect to misfolded rIGF-1.
実施例１で得られた第３の試料溶液のクロマトグラムを示す。図中、横軸は溶出時間、縦軸は吸光度（OD 220nm）を示す。黒塗りの矢印がrIGF-1（正しくフォールディングされたrIGF-1）のピークを、白抜きの矢印がミスフォールディングのrIGF-1（mis-folded isomer）を示す。The chromatogram of the third sample solution obtained in Example 1 is shown. In the figure, the horizontal axis shows the elution time and the vertical axis shows the absorbance (OD 220 nm). The black arrow indicates the peak of rIGF-1 (correctly folded rIGF-1), and the white arrow indicates the misfolded rIGF-1 (mis-folded isomer). 実施例２の溶出工程（ｉ）で得られた溶出液のクロマトグラムを示す。図中、横軸は溶出時間、縦軸は吸光度（OD 220nm）を示す。黒塗りの矢印がrIGF-1のピークを、白抜きの矢印がmis-folded isomerを示す。The chromatogram of the eluate obtained in the elution step (i) of Example 2 is shown. In the figure, the horizontal axis shows the elution time and the vertical axis shows the absorbance (OD 220 nm). The black arrow indicates the peak of rIGF-1, and the white arrow indicates mis-folded isomer. 実施例２の溶出工程（ｉｉ）で得られた溶出液（第３の試料溶液）のクロマトグラムを示す。図中、横軸は溶出時間、縦軸は吸光度（OD 220nm）を示す。黒塗りの矢印がrIGF-1のピークを示す。The chromatogram of the eluate (third sample solution) obtained in the elution step (ii) of Example 2 is shown. In the figure, the horizontal axis shows the elution time and the vertical axis shows the absorbance (OD 220 nm). The black arrow indicates the peak of rIGF-1. 比較例１の溶出・洗浄工程で得られた試料溶液のクロマトグラムを示す。図中、横軸は溶出時間、縦軸は吸光度（OD 220nm）を示す。黒塗りの矢印がrIGF-1のピークを、白抜きの矢印がmis-folded isomerを、斜線の矢印がSUMOを示す。The chromatogram of the sample solution obtained in the elution / washing step of Comparative Example 1 is shown. In the figure, the horizontal axis shows the elution time and the vertical axis shows the absorbance (OD 220 nm). The black arrow indicates the peak of rIGF-1, the white arrow indicates mis-folded isomer, and the diagonal arrow indicates SUMO.
　以下、本発明を実施するための好適な形態について説明する。なお、以下に説明する実施形態は、本発明の代表的な実施形態の一例を示したものであり、これにより本発明の範囲が狭く解釈されることはない。 Hereinafter, a suitable mode for carrying out the present invention will be described. It should be noted that the embodiments described below show an example of a typical embodiment of the present invention, and the scope of the present invention is not narrowly interpreted by this.
　本発明に係る組み換えインスリン様成長因子Ｉ（rIGF-1）の製造方法及び精製方法は、以下の工程（１）－（３）を含む。
　工程（１）：rIGF-1をユビキチン様タンパク質SUMO（small ubiquitin-related modifier）との融合タンパク質（SUMO-IGF-1）として得る工程。
　工程（２）：SUMO-IGF-1をSUMOプロテアーゼにより処理する工程。
　工程（３）：工程（２）の処理液を陽イオン交換クロマトグラフィーに適用し、グラジエント溶出によりrIGF-1を含む溶出液を得る工程。 The method for producing and purifying recombinant insulin-like growth factor I (rIGF-1) according to the present invention includes the following steps (1)-(3).
Step (1): A step of obtaining rIGF-1 as a fusion protein (SUMO-IGF-1) with a ubiquitin-like protein SUMO (small ubiquitin-related modifier).
Step (2): A step of treating SUMO-IGF-1 with SUMO protease.
Step (3): A step of applying the treatment solution of the step (2) to cation exchange chromatography to obtain an eluate containing rIGF-1 by gradient elution.
［工程（１）］
　本工程では、rIGF-1をユビキチン様タンパク質SUMO（small ubiquitin-related modifier）との融合タンパク質（SUMO-IGF-1）として得る。本工程は、SUMO-IGF-1を発現する宿主細胞を溶解して第１の試料溶液を得る工程（１－１）と、第１の試料溶液をNi-NTA（nickel-nitrilotriacetic acid）アフィニティークロマトグラフィーに適用して、SUMO-IGF-1を含む第１の溶出液を得る工程（１－２）とを含む。 [Step (1)]
In this step, rIGF-1 is obtained as a fusion protein (SUMO-IGF-1) with a ubiquitin-like protein SUMO (small ubiquitin-related modifier). This step is a step of lysing a host cell expressing SUMO-IGF-1 to obtain a first sample solution (1-1), and a Ni-NTA (nickel-nitrilotriacetic acid) affinity chromatograph of the first sample solution. It comprises a step (1-2) of obtaining a first eluate containing SUMO-IGF-1 by applying it to a graph.
［工程（１－１）］
　本工程では、SUMO-IGF-1を発現する宿主細胞を溶解して第１の試料溶液を得る。
　SUMO-IGF-1発現のための遺伝子コンストラクトは従来公知の手法（例えば非特許文献１に記載の方法）により作製すればよい。遺伝子コンストラクト作製のためのSUMO-IGF-1遺伝子及び発現プラスミドには市販のDNA合成遺伝子及び汎用ベクターを利用できる。
　宿主細胞は、特に限定されないが、大腸菌が好適に用いられ、市販の大腸菌細胞株を利用できる。遺伝子コンストラクトによる宿主細胞の形質転換及び形質転換体の培養も従来公知の手法により行うことができる。
　培養後、培養液から遠心分離等によって細胞を回収し、高圧破砕機等を用いて細胞を破砕し、遠心分離等により不溶物を取り除くことで、第１の試料溶液を得る。 [Step (1-1)]
In this step, host cells expressing SUMO-IGF-1 are lysed to obtain a first sample solution.
The gene construct for SUMO-IGF-1 expression may be prepared by a conventionally known method (for example, the method described in Non-Patent Document 1). Commercially available DNA synthesis genes and general-purpose vectors can be used for the SUMO-IGF-1 gene and the expression plasmid for producing the gene construct.
The host cell is not particularly limited, but Escherichia coli is preferably used, and a commercially available Escherichia coli cell line can be used. Transformation of host cells with a gene construct and culturing of transformants can also be performed by conventionally known methods.
After culturing, the cells are collected from the culture solution by centrifugation or the like, the cells are crushed using a high-pressure crusher or the like, and the insoluble matter is removed by centrifugation or the like to obtain a first sample solution.
［工程（１－２）］
　本工程では、第１の試料溶液をNi-NTA（nickel-nitrilotriacetic acid）アフィニティークロマトグラフィーに適用して、SUMO-IGF-1を含む第１の溶出液を得る。
　平衡化したNi-NTAアフィニティクロマトグラフィーカラムに第１の試料溶液をロードする。SUMO-IGF-1は、SUMOに付加されたヒスチジンタグ（His-tag）を介してカラム担体に保持される。適当な緩衝液でカラムを洗浄した後、イミダゾールの濃度勾配による溶出を行ってカラム担体に保持されたSUMO-IGF-1を溶出させて第１の溶出液を得る。イミダゾール濃度勾配溶出は、例えば、開始バッファー（50mM Tris, 300mM NaCl, 8M Urea, 20mM Imidazole, pH 7.8）と、溶出バッファー（50mM Tris, 300mM NaCl, 600mM Imidazole, pH 7.8）を用いた直線濃度勾配溶出により行うことができる。 [Step (1-2)]
In this step, the first sample solution is applied to Ni-NTA (nickel-nitrilotriacetic acid) affinity chromatography to obtain a first eluate containing SUMO-IGF-1.
Load the first sample solution onto an equilibrated Ni-NTA affinity chromatography column. SUMO-IGF-1 is retained on the column carrier via a histidine tag attached to SUMO. After washing the column with an appropriate buffer solution, elution with a concentration gradient of imidazole is performed to elute SUMO-IGF-1 retained on the column carrier to obtain a first eluate. For imidazole concentration gradient elution, for example, linear concentration gradient elution using an initiating buffer (50mM Tris, 300mM NaCl, 8M Urea, 20mM Imidazole, pH 7.8) and an elution buffer (50mM Tris, 300mM NaCl, 600mM Imidazole, pH 7.8). Can be done by.
［工程（２）］
　本工程は、SUMO-IGF-1をSUMOプロテアーゼにより処理する工程である。第１の溶出液をSUMOプロテアーゼにより処理することにより、SUMO-IGF-1、SUMOプロテアーゼ、SUMO及びrIGF-1を含む第２の試料溶液を得る。
　SUMOプロテアーゼ処理は、従来公知の手法（例えば非特許文献１に記載の方法）により行えばよい。SUMOプロテアーゼ処理後に、SUMO-IGF-1、SUMOプロテアーゼ、SUMO及びrIGF-1を含む処理液を適宜濃縮や緩衝液置換して第２の試料溶液としてもよい。 [Step (2)]
This step is a step of treating SUMO-IGF-1 with SUMO protease. Treatment of the first eluate with SUMO protease gives a second sample solution containing SUMO-IGF-1, SUMO protease, SUMO and rIGF-1.
The SUMO protease treatment may be carried out by a conventionally known method (for example, the method described in Non-Patent Document 1). After the SUMO protease treatment, the treatment solution containing SUMO-IGF-1, SUMO protease, SUMO and rIGF-1 may be appropriately concentrated or replaced with a buffer solution to obtain a second sample solution.
［工程（３）］
　本工程は、第２の試料溶液を陽イオン交換クロマトグラフィーに適用し、グラジエント溶出によりrIGF-1を含む溶出液を得る工程である。
　従来手法（例えば非特許文献１に記載の方法）では、SUMO-IGF-1、SUMOプロテアーゼ、SUMO及びrIGF-1を含むSUMOプロテアーゼ処理液を再度Ni-NTAアフィニティークロマトグラフィーに適用し、His-tagを有するSUMO-rIGF-1、SUMOプロテアーゼ及びSUMOをカラムに吸着させrIGF-1のみを溶出させることによりrIGF-1を精製している（比較例１参照）。
　本発明に係る方法は、Ni-NTAアフィニティークロマトグラフィーに替えて陽イオン交換クロマトグラフィーを用いてrIGF-1の精製を行うものであり、本工程では、第２の試料溶液を陽イオン交換クロマトグラフィーに適用し、グラジエント溶出によりSUMO-IGF-1、SUMOプロテアーゼ及びSUMOからrIGF-1を分離してrIGF-1を含む第３の試料溶液を得る。本発明に係る方法では、工程（２）よりも後段にNi-NTAアフィニティークロマトグラフィーによる分離工程が含まれない。 [Step (3)]
This step is a step of applying the second sample solution to cation exchange chromatography to obtain an eluate containing rIGF-1 by gradient elution.
In the conventional method (for example, the method described in Non-Patent Document 1), a SUMO protease-treated solution containing SUMO-IGF-1, SUMO protease, SUMO and rIGF-1 is applied again to Ni-NTA affinity chromatography, and His-tag is applied. SUMO-rIGF-1, SUMO protease and SUMO are adsorbed on the column and only rIGF-1 is eluted to purify rIGF-1 (see Comparative Example 1).
The method according to the present invention purifies rIGF-1 by using cation exchange chromatography instead of Ni-NTA affinity chromatography. In this step, the second sample solution is cation exchange chromatography. To obtain a third sample solution containing rIGF-1, separate rIGF-1 from SUMO-IGF-1, SUMO protease and SUMO by gradient elution. The method according to the present invention does not include a separation step by Ni-NTA affinity chromatography after the step (2).
　第２の試料溶液を、適当な緩衝液（例えば、20mM Ammonium acetate, 200mM Imidazole, pH 4.0）で平衡化した陽イオン交換クロマトグラフィーカラム（SPセルロース）にロードする。塩濃度勾配溶出を行ってカラム担体に保持されたrIGF-1を溶出させて第３の試料溶液を得る。 Load the second sample solution onto a cation exchange chromatography column (SP cellulose) equilibrated with a suitable buffer (eg, 20 mM Ammonium acetate, 200 mM Imidazole, pH 4.0). A third sample solution is obtained by eluting rIGF-1 retained on the column carrier by performing salt concentration gradient elution.
　塩濃度勾配溶出は、例えば、上記緩衝液を開始バッファーとし、溶出バッファー（20mM Ammonium acetate, 200mM Imidazole, 1M NaCl, pH 4.0）を併せて用いた直線濃度勾配溶出により行うことができる。溶出容積・溶出速度は、適宜調整され得る。溶出溶液は、例えば4カラム容積（CV）、1-16CV、好ましくは2-8CV、より好ましくは3-6CVである。
　陽イオン交換クロマトグラフィーカラムを用いた塩濃度勾配溶出により、従来方法（比較例１参照）に比して、SUMOの混入のないrIGF-1を高純度かつ高収量で得ることができる（実施例１参照）。 The salt concentration gradient elution can be performed, for example, by linear concentration gradient elution using the above buffer as a starting buffer and also using an elution buffer (20 mM Ammonium acetate, 200 mM Imidazole, 1M NaCl, pH 4.0). The elution volume and elution rate can be adjusted as appropriate. The eluate is, for example, a 4-column volume (CV), 1-16 CV, preferably 2-8 CV, more preferably 3-6 CV.
By salt concentration gradient elution using a cation exchange chromatography column, rIGF-1 without SUMO contamination can be obtained with high purity and high yield as compared with the conventional method (see Comparative Example 1) (Example). 1).
　塩濃度勾配溶出は、好ましくは以下の溶出工程（ｉ）－（ｉｉｉ）からなるものとされる。
（ｉ）塩濃度200 mM以上300 mM以下（あるいは200 mM以上300 mM未満）の溶出液での１カラム容積以上の溶出工程。
（ｉｉ）塩濃度300 mM以上500 mM未満の溶出液での１カラム容積以上の溶出工程。
（ｉｉｉ）塩濃度500 mM以上の溶出液での溶出工程。 The salt concentration gradient elution preferably comprises the following elution steps (i)-(iii).
(I) Elution step of 1 column volume or more with an eluate having a salt concentration of 200 mM or more and 300 mM or less (or 200 mM or more and less than 300 mM).
(Ii) Elution step of 1 column volume or more with an eluate having a salt concentration of 300 mM or more and less than 500 mM.
(Iii) Elution step with an eluate having a salt concentration of 500 mM or more.
　溶出工程（ｉ）では、ミスフォールディングのrIGF-1が主として溶出される。
　溶出液には、例えば、酢酸アンモニウムをバッファーとし、上記濃度で塩（NaClなど）を含む溶出液が用いられる。溶出液中の塩濃度は、上記濃度範囲で一定であっても、直線的（ライナー）に又は段階的（ステップワイズ）に変化してもよい。溶出液はイミダゾールを適当な濃度（例えば200mM）で含んでいてもよい。溶出容積・溶出速度は、適宜調整され得る。溶出溶液は、例えば12CVであり、3-48CV、好ましくは6-24CV、より好ましくは9-15CVの範囲とされる。
　溶出工程（ｉ）は、特に具体的には、塩濃度200mMで1.5-6カラム容積（CV）の溶出、次いで塩濃度250mMで3-12CVの溶出、さらに塩濃度250mMから300mMまでの直線濃度勾配による1.5-3CVの溶出とされる。 In the elution step (i), misfolding rIGF-1 is mainly eluted.
As the eluate, for example, an eluate containing ammonium acetate as a buffer and a salt (NaCl or the like) at the above concentration is used. The salt concentration in the eluate may be constant in the above concentration range, or may change linearly (liner) or stepwise (stepwise). The eluate may contain imidazole at an appropriate concentration (eg, 200 mM). The elution volume and elution rate can be adjusted as appropriate. The eluate is, for example, 12 CV, in the range of 3-48 CV, preferably 6-24 CV, more preferably 9-15 CV.
In the elution step (i), specifically, elution of 1.5-6 column volume (CV) at a salt concentration of 200 mM, then elution of 3-12 CV at a salt concentration of 250 mM, and a linear concentration gradient from a salt concentration of 250 mM to 300 mM. It is said that 1.5-3 CV is eluted.
　溶出工程（ｉｉ）では、正しくフォールディングされたrIGF-1が主として溶出される。この溶出分画を回収することで高純度に精製されたrIGF-1が得られる。
　溶出液には、例えば、酢酸アンモニウムをバッファーとし、上記濃度で塩（NaClなど）を含む溶出液が用いられる。溶出液中の塩濃度は、上記濃度範囲で一定であっても、直線的（ライナー）に又は段階的（ステップワイズ）に変化してもよい。溶出液はイミダゾールを適当な濃度（例えば200mM）で含んでいてもよい。溶出容積・溶出速度は、適宜調整され得る。溶出溶液は、例えば6CVであり、1.5-24CV、好ましくは3-12CV、より好ましくは4.5-8CVの範囲とされる。
　溶出工程（ｉｉ）は、特に具体的は、塩濃度300mMで1.5-6CVの溶出、次いで塩濃度300mMから400mMまでの直線濃度勾配による1.5-3CVの溶出とされる。 In the elution step (ii), correctly folded rIGF-1 is mainly eluted. By recovering this elution fraction, highly purified rIGF-1 can be obtained.
As the eluate, for example, an eluate containing ammonium acetate as a buffer and a salt (NaCl or the like) at the above concentration is used. The salt concentration in the eluate may be constant in the above concentration range, or may change linearly (liner) or stepwise (stepwise). The eluate may contain imidazole at an appropriate concentration (eg, 200 mM). The elution volume and elution rate can be adjusted as appropriate. The elution solution is, for example, 6 CV, preferably in the range of 1.5-24 CV, preferably 3-12 CV, more preferably 4.5-8 CV.
The elution step (ii) is specifically defined as elution of 1.5-6 CV at a salt concentration of 300 mM and then elution of 1.5-3 CV with a linear concentration gradient from a salt concentration of 300 mM to 400 mM.
　溶出工程（ｉｉｉ）では、SUMO-IGF1、SUMOプロテアーゼ及びSUMOが主として溶出される。
　溶出液には、例えば、酢酸アンモニウムをバッファーとし、上記濃度で塩（NaClなど）を含む溶出液が用いられる。溶出液中の塩濃度は、上記濃度範囲で一定であっても、直線的（ライナー）に又は段階的（ステップワイズ）に変化してもよい。溶出液はイミダゾールを適当な濃度（例えば200mM）で含んでいてもよい。溶出容積・溶出速度は、適宜調整され得る。溶出溶液は、例えば3CVであり、0.75-12V、好ましくは1.5-6CV、より好ましくは2-4CVの範囲とされる。
　溶出工程（ｉｉｉ）は、特に具体的には、塩濃度1Mでの1.5-6CVの溶出とされる。 In the elution step (iii), SUMO-IGF1, SUMO protease and SUMO are mainly eluted.
As the eluate, for example, an eluate containing ammonium acetate as a buffer and a salt (NaCl or the like) at the above concentration is used. The salt concentration in the eluate may be constant in the above concentration range, or may change linearly (liner) or stepwise (stepwise). The eluate may contain imidazole at an appropriate concentration (eg, 200 mM). The elution volume and elution rate can be adjusted as appropriate. The elution solution is, for example, 3 CV, in the range of 0.75-12 V, preferably 1.5-6 CV, more preferably 2-4 CV.
The elution step (iii) is specifically elution of 1.5-6 CV at a salt concentration of 1 M.
　従来、正しくフォールディングされたrIGF-1をミスフォールディングのrIGF-1と分離するために、逆相高速液体クロマトグラフィー（HPLC）が用いられている。
　本発明に係るrIGF-1の製造方法及び精製方法によれば、高価なHPLCカラムを用いることなく、安価な陽イオン交換クロマトグラフィーカラム（工程（３））を用いて、ミスフォールディングのrIGF-1に対して正しくフォールディングされたrIGF-1を高純度でかつ高い回収率で取得することができる。なお、正しくフォールディングされたrIGF-1のうち特にMetO59-IGF-1を除去することを目的として本発明の工程に続けてさらにHPLC分離を行うことは排除されない。
　本発明に係るrIGF-1の製造方法及び精製方法によれば、工程（３）の溶出液（第３の試料溶液）において、タンパク質全量に占める正しくフォールディングされたrIGF-1量の比率を６０％以上、好ましくは７０％以上、より好ましくは８０％以上、さらに好ましくは９０％以上、特に好ましくは９５％以上とできる。 Conventionally, reverse phase high performance liquid chromatography (HPLC) has been used to separate correctly folded rIGF-1 from misfolded rIGF-1.
According to the method for producing and purifying rIGF-1 according to the present invention, a misfolding rIGF-1 is used by using an inexpensive cation exchange chromatography column (step (3)) without using an expensive HPLC column. RIGF-1, which is correctly folded, can be obtained with high purity and high recovery rate. It should be noted that it is not excluded that further HPLC separation is performed following the process of the present invention for the purpose of removing MetO 59 -IGF-1 among the correctly folded rIGF-1.
According to the method for producing and purifying rIGF-1 according to the present invention, the ratio of the correctly folded rIGF-1 amount to the total amount of protein in the eluate (third sample solution) of step (3) is 60%. As mentioned above, it can be preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, and particularly preferably 95% or more.
［実施例１］
（１）材料
（１－１）SUMO-IGF-1 DNA合成遺伝子
　SUMO-IGF-1遺伝子には、市販のDNA合成遺伝子（MISSION BIOTECH、GS3075）を用いた。
（１－２）発現調節用プラスミド（pET22b）
　SUMO-IGF-1の発現プラスミドには、pET22b（EMD Millipore、69744-3CN）を用いた。
（１－３）遺伝子発現構成体（pET22b SUMO-IGF-1）
　SUMO-IGF-1遺伝子発現構成体（pET22b SUMO-IGF-1）は、SUMO-IGF-1 DNA合成遺伝子をpET22bに組み込むことにより構築した。
（１－４）宿主細胞
　宿主細胞には、大腸菌（Escherichia coli）B株由来の細胞株BL21(DE3)を用いた。
（１－５）マスター・ワーキング・セル・バンク作製のための種細胞株の調製
　宿主細胞BL21（BD3）株に大腸菌形質転換法でメカセルミン（遺伝子組換え）遺伝子発現構成体を導入した。アンピシリンを含むLB平板培地で、37℃で培養し、8つのシングルコロニーを選択した。これらのシングルコロニーを、アンピシリンを含むLB培地で、37℃で培養液の濁度（OD 600 nm）が0.6～0.8まで培養し、1 mMのイソプロピル-β-チオガラクトピラノシドで2.5時間、28℃で誘導させ、高発現の細胞株を選択した。
（１－６）マスター・ワーキング・セル・バンクの調製
　選択したひとつのコロニーを、アンピシリンを含むLB培地に接種し37℃で、濁度（OD 600nm）が0.6～0.8まで培養した。培養後、遠心分離し、培地を廃棄し、アンピシリンを含むLB培地の濁度（OD 600nm）が2になるように添加し、最終濃度が20%となるよう滅菌済みグリセロールを添加し、懸濁し、最終濁度（OD 600 nm）が1.2になるように調整した。この細胞懸濁液をクライオチューブに1 mLずつ分注した。 [Example 1]
(1) Material (1-1) SUMO-IGF-1 DNA synthesis gene A commercially available DNA synthesis gene (MISSION BIOTECH, GS3075) was used for the SUMO-IGF-1 gene.
(1-2) Expression regulatory plasmid (pET22b)
As the expression plasmid for SUMO-IGF-1, pET22b (EMD Millipore, 69744-3CN) was used.
(1-3) Gene expression construct (pET22b SUMO-IGF-1)
The SUMO-IGF-1 gene expression construct (pET22b SUMO-IGF-1) was constructed by integrating the SUMO-IGF-1 DNA synthesis gene into pET22b.
(1-4) Host cell As the host cell, a cell line BL21 (DE3) derived from Escherichia coli B strain was used.
(1-5) Preparation of seed cell line for preparation of master working cell bank A mecasermin (genetical recombination) gene expression construct was introduced into a host cell BL21 (BD3) strain by an Escherichia coli transformation method. Eight single colonies were selected by culturing at 37 ° C. on LB plate medium containing ampicillin. These single colonies were cultured in LB medium containing ampicillin at 37 ° C. to a culture turbidity (OD 600 nm) of 0.6-0.8, and 1 mM isopropyl-β-thiogalactopyranoside for 2.5 hours. Highly expressed cell lines were selected by inducing at 28 ° C.
(1-6) Preparation of Master Working Cell Bank One selected colony was inoculated into LB medium containing ampicillin and cultured at 37 ° C. with a turbidity (OD 600 nm) of 0.6 to 0.8. After culturing, centrifuge, discard the medium, add the LB medium containing ampicillin so that the turbidity (OD 600 nm) is 2, add sterile glycerol to a final concentration of 20%, and suspend. , The final turbidity (OD 600 nm) was adjusted to 1.2. The cell suspension was dispensed into cryotubes in 1 mL increments.
（２）SUMO-IGF-1の発現
（２－１）前培養１
　融解したワーキング・セル・バンク0.5mLを前培養培地5mLに加え、37℃、2～8時間、120rpmで培養した。その後、195mLの前培養培地に移し、濁度が5以上になるまで37℃、120rpmで培養した。
（２－２）前培養２
　前培養１で得られた培養液を、前培養培地3.1L、グルコース溶液400 mL、微量金属溶液1025μL及びアンピシリン溶液3.5mLに加え、濁度が10以上となるまで37°Cで培養した。
（２－３）本培養
　前培養２で得られた培養液を、本培養培地、グルコース溶液、微量金属溶液及びアンピリシン溶液に加え、濁度が50になるまで37℃で培養した。IPTG溶液を添加して培養を継続し、菌体内にSUMO-IGF-1を発現させた。 (2) Expression of SUMO-IGF-1 (2-1) Preculture 1
0.5 mL of thawed working cell bank was added to 5 mL of preculture medium and cultured at 37 ° C. for 2-8 hours at 120 rpm. Then, the cells were transferred to 195 mL of preculture medium and cultured at 37 ° C. and 120 rpm until the turbidity became 5 or more.
(2-2) Preculture 2
The culture solution obtained in preculture 1 was added to 3.1 L of preculture medium, 400 mL of glucose solution, 1025 μL of trace metal solution and 3.5 mL of ampicillin solution, and cultured at 37 ° C until the turbidity became 10 or more.
(2-3) Main culture The culture solution obtained in preculture 2 was added to the main culture medium, glucose solution, trace metal solution and ampyricin solution, and cultured at 37 ° C. until the turbidity reached 50. The IPTG solution was added and the culture was continued to express SUMO-IGF-1 in the cells.
（３）菌体回収・溶解
　本培養終了後、培養液を遠心分離し、菌体を回収した。菌体1gに対して菌体溶解緩衝液（50mM Tris, 300mM NaCl, 8M Urea, pH 7.8）10 mLを加えて、菌体を懸濁した。高圧破砕機（850 bar）により菌体を破砕した後、遠心分離を行って不溶物を取り除き、第１の試料溶液を得た。 (3) Recovery and lysis of bacterial cells After the completion of the main culture, the culture solution was centrifuged to recover the bacterial cells. 10 mL of cell lysis buffer (50 mM Tris, 300 mM NaCl, 8M Urea, pH 7.8) was added to 1 g of the cell to suspend the cell. After crushing the cells with a high-pressure crusher (850 bar), centrifugation was performed to remove insoluble matter, and a first sample solution was obtained.
（４）Ni-NTAアフィニティークロマトグラフィー精製
　菌体溶解緩衝液で平衡化したNi-NTAアフィニティクロマトグラフィーカラム（高さ25cm）に第１の試料溶液を全量負荷した。菌体溶解緩衝液でカラムを洗浄した後、ニッケル緩衝液B（50mM Tris, 300mM NaCl, 8M Urea, 20mM Imidazole, pH 7.8）を開始バッファーとし、ニッケル緩衝液C（50mM Tris, 300mM NaCl, 600 mM Imidazole, pH 7.8）を溶出バッファーとする直線濃度勾配溶出（線速度60cm/h）により、SUMO-IGF-1を含む第１の溶出液を得た。 (4) Purification of Ni-NTA Affinity Chromatography The entire amount of the first sample solution was loaded on a Ni-NTA affinity chromatography column (height 25 cm) equilibrated with a cell lysis buffer. After washing the column with cell lysis buffer, use nickel buffer B (50 mM Tris, 300 mM NaCl, 8M Urea, 20 mM Imidazole, pH 7.8) as the starting buffer and nickel buffer C (50 mM Tris, 300 mM NaCl, 600 mM). A first eluate containing SUMO-IGF-1 was obtained by linear concentration gradient elution (linear velocity 60 cm / h) using Imidazole, pH 7.8) as the elution buffer.
（５）SUMOプロテアーゼ処理
　第１の溶出液に1/2000倍容量のSUMOプロテアーゼ溶液を添加し、15℃以上で1時間反応させた。反応液を限外ろ過膜（分画分子量2kDa）で濃縮した。濃縮液の7倍量以上のTFF緩衝液B（20mM Ammonium acetate, 200mM Imidazole, pH 4.0）で緩衝液置換した。その後、再度濃縮した。濃縮物をTFF緩衝液Bにより回収し、SUMO-IGF-1、SUMOプロテアーゼ、SUMO及びrIGF-1を含む第２の試料溶液を得た。 (5) SUMO Protease Treatment A 1/2000 times volume of SUMO protease solution was added to the first eluate, and the mixture was reacted at 15 ° C. or higher for 1 hour. The reaction solution was concentrated with an ultrafiltration membrane (molecular weight cut off of 2 kDa). The buffer was replaced with TFF buffer B (20 mM Ammonium acetate, 200 mM Imidazole, pH 4.0) in an amount of 7 times or more that of the concentrate. Then, it was concentrated again. The concentrate was recovered with TFF buffer B to give a second sample solution containing SUMO-IGF-1, SUMO protease, SUMO and rIGF-1.
（６）陽イオン交換クロマトグラフィー精製
　第２の試料溶液を、SP緩衝液A（20mM Ammonium acetate, 200mM Imidazole, pH 4.0）で平衡化した陽イオン交換クロマトグラフィーカラム（SPセルロース、高さ20 cm）に全量負荷した。
　SP緩衝液Aを開始バッファーとし、SP緩衝液B（20mM Ammonium acetate, 200mM Imidazole, 1M NaCl, pH 4.0）を溶出バッファーとする直線濃度勾配溶出（4カラム容積（CV）、線速度60 cm/h）により、IGF-1を含む第３の試料溶液を得た。 (6) Cation Exchange Chromatography Purification A cation exchange chromatography column (SP cellulose, height 20 cm) in which the second sample solution was equilibrated with SP buffer A (20 mM Ammonium acetate, 200 mM Imidazole, pH 4.0). Was fully loaded.
Linear concentration gradient elution (4 column volume (CV), linear velocity 60 cm / h) with SP buffer A as the starting buffer and SP buffer B (20 mM Ammonium acetate, 200 mM Imidazole, 1M NaCl, pH 4.0) as the elution buffer. ) To obtain a third sample solution containing IGF-1.
　得られた第３の試料溶液のクロマトグラムを図１に示す。図中、横軸は溶出時間、縦軸は吸光度（OD 220nm）を示す。黒塗りの矢印がrIGF-1（正しくフォールディングされたrIGF-1）のピークであり、白抜きの矢印がミスフォールディングのrIGF-1（mis-folded isomer）である。ピーク強度から算出したタンパク質全量に占めるrIGF-1及びmis-folded isomerの比率は、それぞれ64.2％及び28.9％であった（表１参照）。また、第２の試料溶液中のrIGF-1量に対する第３の試料溶液のrIGF-1量（回収率）は80％であった。第３の試料溶液においてSUMOの混入のないrIGF-1を高純度（64.2％）でかつ高い回収率（80％）で取得できた。 The chromatogram of the obtained third sample solution is shown in FIG. In the figure, the horizontal axis shows the elution time and the vertical axis shows the absorbance (OD 220 nm). The black arrow is the peak of rIGF-1 (correctly folded rIGF-1), and the white arrow is the misfolded rIGF-1 (mis-folded isomer). The ratios of rIGF-1 and mis-folded isomers to the total amount of protein calculated from the peak intensity were 64.2% and 28.9%, respectively (see Table 1). The amount of rIGF-1 (recovery rate) of the third sample solution was 80% with respect to the amount of rIGF-1 in the second sample solution. In the third sample solution, rIGF-1 without SUMO contamination could be obtained with high purity (64.2%) and high recovery rate (80%).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
［実施例２］
（１）陽イオン交換クロマトグラフィー精製
　実施例１と同様にして得た第２の試料溶液を、SP緩衝液Aで平衡化した陽イオン交換クロマトグラフィーカラム（SPセルロース、高さ20 cm）に全量負荷した。その後、以下の溶出工程（ｉ）－（ｉｉｉ）からなるグラジエント溶出を行った。 [Example 2]
(1) Purification of cation exchange chromatography The entire amount of the second sample solution obtained in the same manner as in Example 1 was placed on a cation exchange chromatography column (SP cellulose, height 20 cm) equilibrated with SP buffer A. Loaded. Then, gradient elution consisting of the following elution steps (i)-(iii) was performed.
溶出工程（ｉ）
　SP緩衝液A 33％/SP緩衝液B1（20mM ammonium acetate, 200mM imidazole, 300mM NaCl, pH 4.0）67％の溶出液（塩濃度200mM）3CVで溶出を行った（線速度60 cm/h）。
　次いで、SP緩衝液A 17％/SP緩衝液B1 83％の溶出液（塩濃度250mM）6CVで溶出を行った（線速度60 cm/h）。
　さらに、SP緩衝液A 17％/SP緩衝液A 83％の条件（塩濃度250mM）から、SP緩衝液A 0％/SP緩衝液B1 100％の条件（塩濃度300mM）まで、直線濃度勾配溶出（3CV、線速度60 cm/h）を行った。
　得られた溶出液のクロマトグラムを図２に示す。図中、横軸は溶出時間、縦軸は吸光度（OD 220nm）を示す。黒塗りの矢印がrIGF-1のピークであり、白抜きの矢印がmis-folded isomerである。ピーク強度から算出したタンパク質全量に占めるrIGF-1の比率は、27.6％であった。 Dissolution step (i)
SP buffer A 33% / SP buffer B1 (20 mM ammonium acetate, 200 mM imidazole, 300 mM NaCl, pH 4.0) 67% eluate (salt concentration 200 mM) was eluted with 3 CV (linear velocity 60 cm / h).
Then, elution was performed with 6 CV of eluate (salt concentration 250 mM) of SP buffer A 17% / SP buffer B1 83% (linear velocity 60 cm / h).
Furthermore, a linear concentration gradient elution is performed from the condition of SP buffer A 17% / SP buffer A 83% (salt concentration 250 mM) to the condition of SP buffer A 0% / SP buffer B1 100% (salt concentration 300 mM). (3 CV, linear velocity 60 cm / h) was performed.
The chromatogram of the obtained eluate is shown in FIG. In the figure, the horizontal axis shows the elution time and the vertical axis shows the absorbance (OD 220 nm). The black arrow is the peak of rIGF-1, and the white arrow is the mis-folded isomer. The ratio of rIGF-1 to the total amount of protein calculated from the peak intensity was 27.6%.
溶出工程（ｉｉ）
　続いて、SP緩衝液B1 100％の溶出液（塩濃度300mM）3CVで溶出を行い（線速度60 cm/h）、IGF-1を含む第３の試料溶液を得た。
　さらに、SP緩衝液A 70%/SP緩衝液B2（20mM ammonium acetate, 200mM imidazole, 1M NaCl, pH 4.0）30％の条件（塩濃度300mM）から、SP緩衝液60%/SP緩衝液B2 40％の条件（塩濃度400mM）まで、直線濃度勾配溶出（3CV、線速度60 cm/h）を行った。
　得られた第３の試料溶液のクロマトグラムを図３に示す。図中、横軸は溶出時間、縦軸は吸光度（OD 220nm）を示す。黒塗りの矢印がrIGF-1である。ピーク強度から算出したタンパク質全量に占めるrIGF-1及びmis-folded isomerの比率は、それぞれ95.7％及び0.4％であった（表１参照）。また、rIGF-1回収率は70％であった。 Dissolution step (ii)
Subsequently, elution was performed with 3 CV of SP buffer solution B1 100% eluate (salt concentration 300 mM) (linear velocity 60 cm / h) to obtain a third sample solution containing IGF-1.
Furthermore, from the condition of SP buffer A 70% / SP buffer B2 (20 mM ammonium acetate, 200 mM imidazole, 1M NaCl, pH 4.0) 30% (salt concentration 300 mM), SP buffer 60% / SP buffer B2 40% Linear concentration gradient elution (3 CV, linear velocity 60 cm / h) was performed up to the condition (salt concentration 400 mM).
The chromatogram of the obtained third sample solution is shown in FIG. In the figure, the horizontal axis shows the elution time and the vertical axis shows the absorbance (OD 220 nm). The black arrow is rIGF-1. The ratios of rIGF-1 and mis-folded isomers to the total amount of protein calculated from the peak intensities were 95.7% and 0.4%, respectively (see Table 1). The recovery rate of rIGF-1 was 70%.
溶出工程（ｉｉｉ）
　最後に、SP緩衝液B2 100％の溶出液（塩濃度1M）3CVで溶出を行った（線速度60 cm/h）。 Dissolution step (iii)
Finally, elution was performed with 3 CV of SP buffer solution B2 100% eluate (salt concentration 1 M) (linear velocity 60 cm / h).
　溶出工程（ｉｉ）の溶出液（第３の試料溶液）において、mis-folded isomer（0.4％）に対してrIGF-1を高純度（95.7％）でかつ高い回収率（70％）で取得できた。 In the eluate (third sample solution) of the elution step (ii), rIGF-1 can be obtained with high purity (95.7%) and high recovery rate (70%) with respect to mis-folded isomer (0.4%). rice field.
［比較例１］
（１）Ni-NTAアフィニティークロマトグラフィー精製２
　実施例１と同様にして得た第２の試料溶液を、Ni-NTAアフィニティクロマトグラフィーカラムに全量負荷した（線速度60cm/h）。全量負荷中に溶出する溶出液及びその後の洗浄（20mM Tris buffer, 300mM NaCl, pH 7.8）による溶出液を、IGF-1を含む試料溶液として取得した。 [Comparative Example 1]
(1) Ni-NTA affinity chromatography purification 2
The second sample solution obtained in the same manner as in Example 1 was fully loaded onto a Ni-NTA affinity chromatography column (linear velocity 60 cm / h). The eluate eluted during full loading and the eluate by subsequent washing (20 mM Tris buffer, 300 mM NaCl, pH 7.8) were obtained as a sample solution containing IGF-1.
　得られた試料溶液のクロマトグラムを図４に示す。図中、横軸は溶出時間、縦軸は吸光度（OD 220nm）を示す。黒塗りの矢印がrIGF-1（正しくフォールディングされたrIGF-1）のピークであり、白抜きの矢印がミスフォールディングのrIGF-1（mis-folded isomer）であり、斜線の矢印がSUMOである。ピーク強度から算出したタンパク質全量に占めるrIGF-1及びmis-folded isomerの比率はそれぞれ49.6％及び21.5％であり、SUMOが21.7％と高い比率で含まれた（表１参照）。また、rIGF-1回収率も31％と低かった。
 

  The chromatogram of the obtained sample solution is shown in FIG. In the figure, the horizontal axis shows the elution time and the vertical axis shows the absorbance (OD 220 nm). The black arrow is the peak of rIGF-1 (correctly folded rIGF-1), the white arrow is the misfolded rIGF-1 (mis-folded isomer), and the diagonal arrow is SUMO. The ratios of rIGF-1 and mis-folded isomers to the total amount of protein calculated from the peak intensity were 49.6% and 21.5%, respectively, and SUMO was included in a high ratio of 21.7% (see Table 1). The rIGF-1 recovery rate was also low at 31%.

Claims (6)

  1. 　組み換えインスリン様成長因子Ｉ（rIGF-1）の製造方法であって、
    （１）rIGF-1をユビキチン様タンパク質SUMO（small ubiquitin-related modifier）との融合タンパク質（SUMO-IGF-1）として得る工程と、
    （２）SUMO-IGF-1をSUMOプロテアーゼにより処理する工程と、
    （３）工程（２）の処理液を陽イオン交換クロマトグラフィーに適用し、グラジエント溶出によりrIGF-1を含む溶出液を得る工程と、
    を含む製造方法。     A method for producing recombinant insulin-like growth factor I (rIGF-1).
    (1) A step of obtaining rIGF-1 as a fusion protein (SUMO-IGF-1) with a ubiquitin-like protein SUMO (small ubiquitin-related modifier), and
    (2) The step of treating SUMO-IGF-1 with SUMO protease and
    (3) A step of applying the treatment solution of step (2) to cation exchange chromatography to obtain an eluate containing rIGF-1 by gradient elution, and a step of obtaining an eluate.
    Manufacturing method including.
  2. 　前記グラジエント溶出が、
    （ｉ）塩濃度200 mM以上300 mM以下の溶出液での１カラム容積以上の溶出工程、
    （ｉｉ）塩濃度300 mM以上500 mM未満の溶出液での１カラム容積以上の溶出工程、
    （ｉｉｉ）塩濃度500 mM以上の溶出液での溶出工程、
    を含み、
    rIGF-1を前記溶出工程（ｉｉ）の溶出液中に回収する、請求項１に記載の製造方法。     The gradient elution
    (I) Elution step of 1 column volume or more with an eluate having a salt concentration of 200 mM or more and 300 mM or less.
    (Ii) Elution step of 1 column volume or more with an eluate having a salt concentration of 300 mM or more and less than 500 mM.
    (Iii) Elution step with an eluate having a salt concentration of 500 mM or more,
    Including
    The production method according to claim 1, wherein rIGF-1 is recovered in the eluate of the elution step (ii).
  3. 　工程（１）は、SUMO-IGF-1を発現する宿主細胞を溶解して第１の試料溶液を得る工程（１－１）と、第１の試料溶液をNi-NTA（nickel-nitrilotriacetic acid）アフィニティークロマトグラフィーに適用して、SUMO-IGF-1を含む第１の溶出液を得る工程（１－２）を含み、
    工程（２）は、前記第１の溶出液をSUMOプロテアーゼにより処理して、SUMO-IGF-1、SUMOプロテアーゼ、SUMO及びrIGF-1を含む第２の試料溶液を得る工程であり、
    工程（３）は、第２の試料溶液を陽イオン交換クロマトグラフィーに適用してグラジエント溶出によりSUMO-IGF-1、SUMOプロテアーゼ及びSUMOからrIGF-1を分離してrIGF-1を含む第３の試料溶液を得る工程である、
    請求項１又は２に記載の製造方法。     Step (1) is a step (1-1) of lysing a host cell expressing SUMO-IGF-1 to obtain a first sample solution, and a first sample solution of Ni-NTA (nickel-nitrilotriacetic acid). Including a step (1-2) of applying to affinity chromatography to obtain a first eluate containing SUMO-IGF-1.
    Step (2) is a step of treating the first eluate with SUMO protease to obtain a second sample solution containing SUMO-IGF-1, SUMO protease, SUMO and rIGF-1.
    In step (3), the second sample solution is applied to cation exchange chromatography to separate rIGF-1 from SUMO-IGF-1, SUMO protease and SUMO by gradient elution, and the third step contains rIGF-1. The process of obtaining a sample solution,
    The manufacturing method according to claim 1 or 2.
  4. 　工程（２）よりも後段に、Ni-NTAアフィニティークロマトグラフィーによる分離工程を含まない、請求項１－３のいずれか一項に記載の製造方法。 The production method according to any one of claims 1-3, which does not include a separation step by Ni-NTA affinity chromatography after the step (2).
  5. 　逆相高速液体クロマトグラフィーによるミスフォールディングタンパクの分離工程を含まない、請求項１－４のいずれか一項に記載の製造方法。 The production method according to any one of claims 1-4, which does not include a step of separating misfolding proteins by reverse phase high performance liquid chromatography.
  6. 　前記第３の試料溶液中において、タンパク質全量に占める正しくフォールディングされたrIGF-1量の比率が６０％以上、好ましくは７０％以上、より好ましくは８０％以上、さらに好ましくは９０％以上、特に好ましくは９５％以上である、請求項１－５のいずれか一項に記載の製造方法。
          In the third sample solution, the ratio of the correctly folded rIGF-1 amount to the total amount of protein is 60% or more, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, particularly preferably. Is 95% or more, according to any one of claims 1-5.
PCT/JP2020/041283 2020-11-05 2020-11-05 Method for producing recombinant insulin-like growth factor i WO2022097216A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2022560551A JP7570428B2 (en) 2020-11-05 2020-11-05 Method for producing recombinant insulin-like growth factor I
CN202080106934.7A CN116368149A (en) 2020-11-05 2020-11-05 Method for producing recombinant insulin-like growth factor I
KR1020237018335A KR20230098304A (en) 2020-11-05 2020-11-05 Method for producing recombinant insulin-like growth factor I
PCT/JP2020/041283 WO2022097216A1 (en) 2020-11-05 2020-11-05 Method for producing recombinant insulin-like growth factor i

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/JP2020/041283 WO2022097216A1 (en) 2020-11-05 2020-11-05 Method for producing recombinant insulin-like growth factor i

Publications (1)

Publication Number Publication Date
WO2022097216A1 true WO2022097216A1 (en) 2022-05-12

Family

ID=81457004

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2020/041283 WO2022097216A1 (en) 2020-11-05 2020-11-05 Method for producing recombinant insulin-like growth factor i

Country Status (4)

Country Link
JP (1) JP7570428B2 (en)
KR (1) KR20230098304A (en)
CN (1) CN116368149A (en)
WO (1) WO2022097216A1 (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63109794A (en) * 1986-08-07 1988-05-14 インタ−ナシヨナル・ミネラルズ・アンド・ケミカル・コ−ポレイシヨン Purification and isolation of somatomedin c composed of formation of two ions
JPH06505010A (en) * 1991-01-16 1994-06-09 シビア・ニユーロサイエンシズ・インコーポレイテツド Method for purifying correctly folded and complete insulin-like growth factor-1
JPH11503733A (en) * 1995-04-14 1999-03-30 セファロン，インコーポレイテッド Methods for purifying IGF-I
JP2000500023A (en) * 1995-11-13 2000-01-11 フアーマシア・アンド・アツプジヨン・アー・ベー Method for producing correctly folded biologically active recombinant proteins

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5288931A (en) 1991-12-06 1994-02-22 Genentech, Inc. Method for refolding insoluble, misfolded insulin-like growth factor-I into an active conformation

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63109794A (en) * 1986-08-07 1988-05-14 インタ−ナシヨナル・ミネラルズ・アンド・ケミカル・コ−ポレイシヨン Purification and isolation of somatomedin c composed of formation of two ions
JPH06505010A (en) * 1991-01-16 1994-06-09 シビア・ニユーロサイエンシズ・インコーポレイテツド Method for purifying correctly folded and complete insulin-like growth factor-1
JPH11503733A (en) * 1995-04-14 1999-03-30 セファロン，インコーポレイテッド Methods for purifying IGF-I
JP2000500023A (en) * 1995-11-13 2000-01-11 フアーマシア・アンド・アツプジヨン・アー・ベー Method for producing correctly folded biologically active recombinant proteins

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HUI MIN, FUYONG LI, LU TANGA, LONG ZHENG, XUECHAO JIA, PENG LIN, LINNA LI, XIAOKUN LI, XIAOJIE WANG: "Soluble expression of human insulin-like growth factor-1 with the assistance of SUMO fusion partne r", JOURNAL OF CHEMICAL AND PHARMACEUTICAL RESEARCH, vol. 6, no. 4, 1 January 2014 (2014-01-01), pages 1059 - 1066, XP055938557 *

Also Published As

Publication number Publication date
JPWO2022097216A1 (en) 2022-05-12
JP7570428B2 (en) 2024-10-21
KR20230098304A (en) 2023-07-03
CN116368149A (en) 2023-06-30

Similar Documents

Publication Publication Date Title
US20200199641A1 (en) Method for enzymatic preparation of glutathione
CN108690854B (en) Method for producing L-glufosinate-ammonium by using chemical-enzymatic method
WO2022097216A1 (en) Method for producing recombinant insulin-like growth factor i
CN110791488A (en) Lipase for resolving chiral compounds, preparation method and application thereof
CN118085023B (en) Preparation method of short peptide
JP2007502609A (en) Methods for preparing recombinant polypeptides
CN108774635A (en) Recombinate production method of the TEV protease in Escherichia coli
JP4388698B2 (en) Methods for recovering and purifying recombinant proteins from cells
TW202219055A (en) Production method of recombinant insulin-like growth factor I including a rIGF-1 obtaining step, a SUMO-IGF-1 treatment step, and an eluent obtaining step
US11639515B2 (en) Genetically engineered strain for producing porcine myoglobin and food-grade fermentation and purification thereof
CN108570016B (en) PF1022A separation and purification method
RU2447149C1 (en) RECOMBINANT PLASMID DNA pMSIN4, CODING HYBRIDE POLYPEPTIDE - HUMAN INSULIN PRECURSOR, BL21(DE3)VpMSIN4-PRODUCER STRAIN OF RECOMBINANT HUMAN INSULIN, METHOD FOR PRODUCING RECOMBINANT HUMAN INSULIN
CN116693659A (en) A two-step mixed-mode chromatography method for the separation of recombinant human serum albumin
US20060003421A1 (en) Method for purifying a fermentation-derived product
WO2014009515A1 (en) Method for producing a recombinant protein of interest
CN110373440B (en) Method for preparing DL-serine by one-pot enzyme method
CN112812969A (en) System purification method for recombinant expression polypeptide in genetic engineering
CN106883286B (en) Extraction and purification method of tyrosine derivative
CN116236819B (en) Method for purifying polypeptide toxins in batches and composite double-layer chromatographic column
CN111172048A (en) Coarse and pure process of CA16 virus-like particles expressed by recombinant hansenula polymorpha, CA16 virus vaccine and preparation method thereof
CN111004318B (en) Purification method of rhPTH (1-34) protein stock solution
CN216321130U (en) Extraction and concentration device for clavulanic acid fermentation liquor
CN107573403B (en) Method for purifying ningnanmycin
CN116284222A (en) Didemnin B separation and purification method
CN104928327A (en) Method for preparing L-tryptophan

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20960769

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2022560551

Country of ref document: JP

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20237018335

Country of ref document: KR

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20960769

Country of ref document: EP

Kind code of ref document: A1