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WO2013015697A1 - Protéine recombinante, polynucléotide codant pour celle-ci ainsi que procédé d'obtention d'insuline ou de son analogue - Google Patents

Protéine recombinante, polynucléotide codant pour celle-ci ainsi que procédé d'obtention d'insuline ou de son analogue Download PDF

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Publication number
WO2013015697A1
WO2013015697A1 PCT/PL2011/050030 PL2011050030W WO2013015697A1 WO 2013015697 A1 WO2013015697 A1 WO 2013015697A1 PL 2011050030 W PL2011050030 W PL 2011050030W WO 2013015697 A1 WO2013015697 A1 WO 2013015697A1
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WIPO (PCT)
Prior art keywords
amino
insulin
acid sequence
asp
analogue
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PCT/PL2011/050030
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English (en)
Inventor
Sławomir JAROS
Tadeusz PIETRUCHA
Maciej Wieczorek
Original Assignee
Mabion S.A.
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.)
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Publication date
Application filed by Mabion S.A. filed Critical Mabion S.A.
Priority to PCT/PL2011/050030 priority Critical patent/WO2013015697A1/fr
Publication of WO2013015697A1 publication Critical patent/WO2013015697A1/fr

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Classifications

    • 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
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • a recombinant protein a polynucleotide encoding it as well as a method of obtaining insulin or its an analogue
  • the present invention relates to a method of producing insulin or its an analogue as well as a recombinant protein and a polynucleotide encoding it which may be used in this method.
  • the disclosed present invention is useful in biotechnology as well as the pharmaceutical industry.
  • diabetes a disease caused by disruptions in the functioning of the pancreas evidenced by the insufficient production of the hormone that regulates blood sugar levels, insulin (type I diabetes, called insulin-dependent), or the inability of an organism to utilize the produced insulin (type II diabetes).
  • Type I diabetes accounts for about 10% of all diabetes cases, which means that in 2009 22 million people suffered from it (WHO, Diabetes Fact sheet no 312. November 2009). In the case of type II diabetes, though it is not directly connected with the absence of the hormone, insulin is also commonly used in therapy.
  • WHO Diabetes Fact sheet no 312. November 2009.
  • insulin is also commonly used in therapy.
  • the above epidemiological and technical data unequivocally show how significant the development of recombinant therapeutic proteins is, without which the therapy of diseases such as diabetes would be less effective.
  • the technology of human insulin production via conversion on a commercial scale was developed by Novo Nordisk and consisted of substituting alanine at position B-30 of porcine insulin for threonine in five steps (Ladish M.R., Kohlmann K.L., Recombinant Human Insulin, Biotechnol.Prog., 1992. 8 (6), 469478).
  • the insulin is extracted from frozen porcine pancreases, and then the purified porcine insulin is converted into human in a medium containing a small amount of water and trypsin as well as large quantities of organic solvents and threonine esters.
  • the trypsin hydrolyses insulin between positions Lys29-AlaB30 and simultaneously catalyses a reaction, in which a threonine ester replaces alanine at position B30.
  • Chromatographic purification is performed following the enzymatic reaction to remove proinsulin as well as other reagents, and then product formulation and portioning under sterile conditions are performed.
  • the most common method of obtaining insulin is to use recombinant DNA technology. The pioneers in this respect were Eli Lilly and Co. (Indianapolis) and Genentech (San Francisco) which jointly developed the first recombinant insulin. In 1978 the hormone was first expressed in Escherichia coli K12. using the expression vector pBR322.
  • the fusion protein molecules were digested with CNBr which occurred at the methionine that separated the ⁇ -galactosidase from the insulin chain, and then purified.
  • the next step was to mix chains A and B at a 2:1 ratio (S-sulphonated forms) in the presence of a mercaptan (Chance R.E., Kroeff E.P., Hoffman J.A., W: Insulins, Growth Hormone and Recombinant DNA Technology, Raven Press, New York, 1981 , pp. 71 -85).
  • the newest technological approach is the production of recombinant proinsulin, composed of peptides A, B and C from one cell clone, and then enzymatic treatment to alter the proinsulin into insulin.
  • This has many benefits.
  • a single fermentation step suffices, and then a single protein purification process following fermentation instead of two separate ones for the A and B chains.
  • This approach was first used for industrial scale production in 1986 (Chance R., Glazer N., wishner K., Insulin Lispro (Humalog). W: Walsh G., Murphy B. (red) Biopharmaceuticals, an industrial perspective, Kluwer, Dordrecht 1999, pp 149-172).
  • Enzymatic processing usually consists of the use of two enzymes, trypsin and carboxypeptidase B.
  • Trypsin is a serine protease that cleaves peptide bonds on the C- side of positively charged amino-acids (arginine and lysine) if the next amino-acid is not proline.
  • trypsin cleaves the peptide bond between lysine and arginine on the C-end of recombinant proinsulin; carboxypeptidase B cleaves basic aminoacids at the C-end which arises follwing trypsin activity.
  • the drawback of this approach is that the cleavage results in various insulin derivatives that constitute contaminants, A21 -desamide insulin, desthreonine-(B30) insulin, arginyl-(AO) insulin and diarginyl-(B31 ,B32) insulin (Son YJ, Kim CK, Kim YB, Kweon DH, Park YC, Seo JH, Effects of Citraconylation on Enzymatic Modification of Human Proinsulin Using Trypsin and Carb 1070).
  • proinsulin Structure of proinsulin, according to Son VJ, Kim CK, Kim VB, Kweon DH, Park VC, Seo JH, Effects of Citraconylation on Enzymatic Modification of Human Proinsulin Using Trypsin and Carboxypeptidase B, 2009, Biotechnol. Prog., 25(4), 1064-1070.
  • Lispro (Eli Lilly) which was first marketed in 1996, Aspart (NovoNordisk, 1999) as well as Insulinum Glargine (Sanofi Aventis, 2000).
  • Lispro is an analogue with accelerated activity, characterized by an inverted amino-acid order at positions B28 (proline) and B29 (lysine).
  • Aspart is characterized by the replacement of proline B28 with aspartic acid, and is also an accelerated activity an analogue.
  • Insulinum Glargine possesses a substitution of asparagine A21 for glycine, as well as having its chain B extended by two arginines. Its main advantage is extended activity, which results in increased patient comfort by reducing the injection number to once daily.
  • Insulin an analogues are made using recombinant DNA technology. Amino-acid order changes or substitutions pose no major problem. Simple sequence engineering suffices, and the rest of the process can remain unchanged. It is definitely more difficult to obtain an analogues with amino-acids added on the ends, particularly basic amino-acids, i.e. insulinum Glargine. In this case it is necessary to use trypsin and then subsequent chemical modifications and further arginine synthesis (Habermann P., Zocher F., Method for producing insulin an analogue s having a dibascic chain B terminus, US Patent Application Publication no US 2009/0192073 A 1 ).
  • the subject of the present invention is a recombinant protein with the general formula:
  • X3 denotes any given amino-acid sequence
  • - E1 denotes an amino-acid sequence recognized by the first protease
  • - A denotes the amino-acid sequence of chain A of insulin or its an analogue
  • - B denotes the amino-acid sequence of chain B of insulin or its an analogue
  • X1 , X2 and X3 are selected from a group encompassing known sequences used in purification using affinity chromatography, particularly His6 or they are omitted.
  • X2 denotes the amino-acid sequence of chain C of proinsulin or its an analogue.
  • a protein according to the present invention contains an amino- acid sequence selected from among the sequences with identification numbers from 1 to 6.
  • the next subject of the present invention is a polynucleotide encoding the above described proteins according to the present invention. It preferably contains a nucleotide sequence selected from among sequences with the identification numbers from 7 to 12.
  • the next subject of the present invention is a method of producing insulin or its an analogue, characterized in that:
  • a recombinant protein is produced with the general formula X1 -E1 -B-E2-X2-E1 -A-X3, where:
  • X3 denotes any given amino-acid sequence
  • - E1 denotes an amino-acid sequence recognized by the first protease
  • - A denotes the amino-acid sequence of chain A of insulin or its an analogue
  • - B denotes the amino-acid sequence of chain B of insulin or its an analogue
  • first protease does not exhibit trypsin or carboxypeptidase C activity, preferably constitutes enterokinase
  • second protease does not exhibit trypsin or carboxypeptidase C activity, preferably constitutes endopeptidase Asp-N
  • the protein produced is subjected to the activity of the first protease, preferably an enterokinase,
  • the protein produced is subjected to the activity of the second protease, preferably endopeptidase Asp-N,
  • a protein according to the present invention defined above is produced, wherein preferably an expression host is cultured that has been transformed with a polynucleotide according to the present invention defined above.
  • the disclosed process for obtaining proteins from their precursors, including insulins and their an analogues using one or two enzymes other than trypsin and carboxypeptidase is based on introducing individual or a greater number of amino- acids recognized by proteases/peptidases into the precursor, and then using these enzymes to remove superfluous fragments and then forming the correct target protein.
  • the benefits stemming from the present invention are as follows:
  • Example 1 Production of human insulin from modified human proinsulin.
  • nucleotides encoding amino-acids recognized by Enterokinase cleavage: - Asp - Asp - Asp - Asp - Lys -1 1 - X; cleavage at the C-end of the recognized sequences
  • Endopeptidase Asp-N cleavage of Asp from the N-end
  • the amino-acid sequence of the recombinant precursor is shown as the sequence with ID No. 1 as well as in Fig. 1 .
  • the example nucleotide sequence encoding the precursor MabionHM is shown as sequence No. 7.
  • a comparison of the sequences of human proinsulin and the precursor Mabion HI1 is also shown in Fig. 1 .
  • the precursor MabionHM was produced in E. coli cells and purified chromatographically. In this case, affinity chromatography was used with a His Trap gel (GE Healthcare) gel, which interacts with a poly-His fusion peptide (6xHis) added in front of the actual precursor molecule.
  • His Trap gel GE Healthcare
  • 6xHis poly-His fusion peptide
  • the subject technology of the patent makes it possible to remove this peptide, or any other fusion peptide or amino-acid, during the enzymatic processing of proinsulin to insulin, without any other additional steps.
  • 10 g of the precursor MabionHM was subjected to digestion using for this purpose 0.0012 units of an Enterokinase solution (Sigma) over 12 hours, the digestion temperature was 25°C.
  • Example 2 Production of human insulin from a precursor not being proinsulin
  • nucleotides encoding amino-acids recognized by Enterokinase cleavage: - Asp - Asp - Asp - Asp - Lys -1 1 - X; cleavage at the C-end of the recognized sequences
  • Endopeptidase Asp-N cleavage of Asp at the N-end
  • the amino-acid sequence of the recombinant precursor is shown as the sequence with ID No. 2 as well as in Fig. 2.
  • the example nucleotide sequence encoding precursor MabionHI 2 is shown as sequence No. 8.
  • the precursor MabionHI2 was produced in E. coli cells and purified chromatographically. In this case, affinity chromatography was used with a His Trap gel (GE Healthcare) which interacts with the fusion peptide poly-His (6xHis) added in front of the actual precursor molecule. 10 g of the precursor MabionHI2 were subjected to digestion using a total of 0.002 units of Enterokinase solution (Sigma) over 12 hours, the digestion temperature was 25°C. In this way, amino-acids preceding the the actual precursor molecule were cleaved off as well as separating chains B and A of the precursor.
  • the precursor was subjected to a further digestion, this time using endopeptidase Asp-N (New England Biolabs) at a rate of 8 g, the digestion time was was 6 h, the digestion temperature was 37°C.
  • the resulting recombinant human insulin molecule was purified chromatographically.
  • Example 3 Production of an analogue of human insulin with prolonged activity, corresponding to Insulinum glargine, from a precursor based on human proinsulin.
  • nucleotides encoding amino-acids recognized by Enterokinase cleavage: - Asp - Asp - Asp - Asp - Lys -1 1 - X; cleavage at the C-end of the recognized sequences
  • Endopeptidase Asp-N cleavage of Asp at the N-end
  • the amino-acid sequence of the recombinant precursor is shown as the sequence with ID No. 3 as well as in Fig. 3.
  • the example nucleotide sequence encoding precursor MabionHI2 is shown as sequence No. 9.
  • a comparison of the sequences of human proinsulin and the precursor MabionHI2 is also shown in Fig. 3.
  • the precursor MabionlGI was produced in E. coli cells and purified chromatographically. In this case, affinity chromatography was used with a His Trap gel (GE Healthcare) which interacts with the fusion peptide poly-His (6xHis) added in front of the actual precursor molecule.
  • Precursor MabionlGI at a rate of 40 g was subjected to digestion using a total of 0.00475 units of Enterokinase solution (Sigma) over 24 hours, the digestion temperature was 25°C. In this way, amino-acids preceding the the actual precursor molecule were cleaved off as well as separating chains C and A of the modified proinsulin. The results are shown in Fig. 4.
  • the precursor was subjected to a further digestion, this time using endopeptidase Asp-N (New England Biolabs).
  • the sample was digested using the enzyme at a ratio of 1/50 by mass to the substrate, the digestion time was 22 h, the digestion temperature was 25°C.
  • Graph A shows a HPLC separation of a pharmaceutical separation containing insulinum glargine (under these conditions, the identified signal was about 22.446 min)
  • graph B a separation of a mixture following the digestion of the precursor MabionlG2 with Enterokinase and peptidase Asp-N, mixed with insulinum glargine (under these conditions, the identified signals were about 22.245 and 22.501 min). It is easily observable that the digestion product as well as the reference molecule migrate through the column together.
  • nucleotides encoding amino-acids recognized by Enterokinase cleavage: - Asp - Asp - Asp - Asp - Lys -1 1 - X; cleavage at the C-end of the recognized sequences
  • Endopeptidase Asp-N cleavage of Asp at the N-end
  • amino-acid sequence of the recombinant precursor is shown as the sequence with ID No. 4 as well as in Fig. 6.
  • the example nucleotide sequence encoding the precursor MabionlG2 is shown as sequence No. 10.
  • Precursor MabionlG2 was produced in E. coli cells and purified chromatographically. In this case, affinity chromatography was used with a His Trap gel (GE Healthcare) which interacts with the fusion peptide poly-His (6xHis) added in front of the actual precursor molecule.
  • Precursor MabionlG2 at a rate of 40 g was subjected to digestion using a total of 0.00475 units of Enterokinase solution (Sigma) over 24 hours, the digestion temperature was 25°C. In this way, amino-acids preceding the the actual precursor molecule were cleaved off as well as separating chains B and A of the modified proinsulin.
  • the precursor was subjected to a further digestion, this time using endopeptidase Asp-N (New England Biolabs).
  • the sample divided into five equal portions, was digested using 10 g of the enzyme, the digestion time was 3 h, the digestion temperature was 37°C.
  • Example 5 Production of an analogue of human insulin with accelerated activity, corresponding to the analogue Lispro, from a precursor based on human proinsulin. Additional nucleotides encoding amino-acids recognized by Enterokinase (cleavage: - Asp - Asp - Asp - Asp - Lys -1 1 - X; cleavage at the C-end of the recognized sequences) as well as Endopeptidase Asp-N (cleavage of Asp at the N-end) were inserted into sequences encoding human proinsulin. In the sequence of chain A, the order was changed (in comparison with human insulin) of amino-acids at positions B28 (proline) and B29 (lysine).
  • amino-acid sequence of the recombinant precursor is shown as the sequence with ID No. 5 as well as in Fig. 7.
  • the example nucleotide sequence encoding precursor MabionLPI is shown as sequence No. 1 1 .
  • the precursor MabionLPI was produced in E. coli cells and purified chromatographically.
  • the precursor MabionLPI at a rate of 1 pg, was subjected to digestion using a total of 0.02 units of Enterokinase solution (Genscript) over 22 hours, the digestion temperature was 22°C.
  • the digestion temperature was 22°C.
  • amino-acids preceding the the actual precursor molecule were cleaved off as well as separating chains C and A of the modified proinsulin.
  • the precursor was subjected to a further digestion, this time using endopeptidase Asp-N (New England Biolabs) at a rate of 0.6 g, the digestion time was 2 h, the digestion temperature was 37°C.
  • the resulting molecule of a recombinant analogue of human insulin was purified chromatographically.
  • Example 6 Production of an analogue of human insulin with accelerated activity, corresponding to the analogue Lispro, from a precursor not being a derivative of human proinsulin.
  • nucleotides encoding amino-acids recognized by Enterokinase cleavage: - Asp - Asp - Asp - Asp - Lys -1 1 - X; cleavage at the C-end of the recognized sequences
  • Endopeptidase Asp-N cleavage of Asp at the N-end
  • the amino-acid sequence of the recombinant precursor (precursor MabionLP2) is shown as the sequence with ID No. 6 as well as in Fig. 8.
  • the example nucleotide sequence encoding the precursor Mabionl_P2 is shown as sequence No. 12.
  • the precursor Mabionl_P2 was produced in E. coli cells and purified chromatographically.
  • the precursor Mabionl_P2 at a rate of 1 pg, was subjected to digestion using a total of 0.03 units of Enterokinase solution (Genscript) over 23 hours, the digestion temperature was 22°C. In this way, amino-acids preceding the the actual precursor molecule were cleaved off as well as separating chains B and A of the modified proinsulin.
  • the precursor was subjected to a further digestion, this time using endopeptidase Asp-N (New England Biolabs) at a rate of 0.95 g, the digestion time was 2 h, the digestion temperature was 37°C.
  • the resulting molecule of the recombinant human insulin analogue was purified chromatographically.
  • Cys Thr Ser lie Cys Ser Leu Tyr Gin Leu Glu Asn Tyr Cys Gly

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Abstract

La présente invention concerne un procédé d'obtention de protéines issues de précurseurs à l'aide d'un traitement enzymatique, la construction des précurseurs et des séquences d'ADN, codant pour de tels précurseurs, étant démontrée par l'exemple des insulines et de leurs analogues.
PCT/PL2011/050030 2011-07-28 2011-07-28 Protéine recombinante, polynucléotide codant pour celle-ci ainsi que procédé d'obtention d'insuline ou de son analogue WO2013015697A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113773395A (zh) * 2020-06-10 2021-12-10 宁波鲲鹏生物科技有限公司 一种地特胰岛素的制备方法
CN114380903A (zh) * 2021-12-28 2022-04-22 上海仁会生物制药股份有限公司 一种胰岛素或其类似物前体
CN115216463A (zh) * 2022-06-15 2022-10-21 武汉瀚海新酶生物科技有限公司 具有稳定性的重组胰蛋白酶及其制备方法和应用
WO2023225534A1 (fr) * 2022-05-18 2023-11-23 Protomer Technologies Inc. Composés contenant du bore aromatiques et analogues d'insuline associés

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WO2009054754A1 (fr) * 2007-10-22 2009-04-30 Obschestvo S Ogranichennoi Otvetstvennostyu 'gerofarm' Plasmide recombinant phins21 codant une protéine hybride avec la proinsuline humaine, souche de bactéries escherichia coli jm109/ phins21 productrice de la protéine hybride avec la proinsuline humaine et procédé de fabrication de proinsuline humaine
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WO2007031187A1 (fr) * 2005-09-14 2007-03-22 Sanofi-Aventis Deutschland Gmbh Clivage de precurseurs d'insulines par un variant de la trypsine
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WO2009054754A1 (fr) * 2007-10-22 2009-04-30 Obschestvo S Ogranichennoi Otvetstvennostyu 'gerofarm' Plasmide recombinant phins21 codant une protéine hybride avec la proinsuline humaine, souche de bactéries escherichia coli jm109/ phins21 productrice de la protéine hybride avec la proinsuline humaine et procédé de fabrication de proinsuline humaine

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113773395A (zh) * 2020-06-10 2021-12-10 宁波鲲鹏生物科技有限公司 一种地特胰岛素的制备方法
CN114380903A (zh) * 2021-12-28 2022-04-22 上海仁会生物制药股份有限公司 一种胰岛素或其类似物前体
CN114380903B (zh) * 2021-12-28 2023-07-25 上海仁会生物制药股份有限公司 一种胰岛素或其类似物前体
WO2023225534A1 (fr) * 2022-05-18 2023-11-23 Protomer Technologies Inc. Composés contenant du bore aromatiques et analogues d'insuline associés
CN115216463A (zh) * 2022-06-15 2022-10-21 武汉瀚海新酶生物科技有限公司 具有稳定性的重组胰蛋白酶及其制备方法和应用
CN115216463B (zh) * 2022-06-15 2023-08-15 武汉瀚海新酶生物科技有限公司 具有稳定性的重组胰蛋白酶及其制备方法和应用

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