COMPOSITION FOR CONVERTING INSULIN PRECURSOR INTO INSULIN ENZYME AND METHOD FOR CONVERTING INSULIN PRECURSOR INTO INSULIN BY USING SAME

Abstract

The present invention relates to: a composition for an enzymatic conversion reaction for converting proinsulin or a proinsulin analogue into insulin or an insulin analogue, the composition comprises one or more selected from the group consisting of calcium chloride, glycine, and proline, and trypsin and/or carboxypeptidase B; and a method for converting proinsulin or a proinsulin analogue into insulin or an insulin analogue by using the composition. The present invention increases the structural stability of an insulin protein and the stability of the enzyme at the same time so as to significantly improve the enzymatic conversion yield and the purity of insulin, thereby being useful in insulin production processes.

Claims

1. A composition for enzymatically converting an insulin precursor into insulin, comprising: one or more selected from the group consisting of calcium chloride, glycine, and proline; and trypsin and/or carboxypeptidase B.

2. The composition according to claim 1, further comprising a buffer solution.

3. The composition according to claim 2, wherein the buffer solution is 1-100 mM Tris-HCl or 1-100 mM borate.

4. The composition according to claim 1, wherein the trypsin is comprised at a weight ratio of 1/1,000 to 1/40,000 relative to the insulin precursor, and/or the carboxypeptidase B is comprised at a weight ratio of 1/600 to 1/20,000 relative to the insulin precursor.

5. The composition according to claim 1, wherein the insulin is insulin glargine.

6. The composition according to claim 1, wherein the composition comprises calcium chloride, glycine, and proline.

7. The composition according to claim 6, wherein calcium chloride is comprised at 5-30 mM, glycine is comprised at 3-60 mM, and proline is comprised at 20-80 mM.

8. A method for converting an insulin precursor into insulin, comprising adding an insulin precursor to the composition according to claim 1 and carrying out a reaction.

9. The method according to claim 8, wherein calcium chloride is comprised at 5-30 mM, glycine is comprised at 3-60 mM, and proline is comprised at 20-80 mM.

Description

4. MODE FOR INVENTION

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those typically understood by those skilled in the art to which the present invention belongs. Generally, the nomenclature used herein and the test method described below are well known in the art and are typical.

[0016] In the present invention, when calcium chloride and an amino acid (proline or glycine) are added alone or in combination to a composition for an enzymatic conversion reaction converting an insulin glargine precursor into insulin glargine, it is confirmed that the yield of enzymatic conversion is increased and the purity of insulin is also increased.

[0017] Accordingly, an aspect of the present invention pertains to a composition for enzymatic converting an insulin precursor into insulin, comprising one or more selected from the group consisting of calcium chloride, glycine, and proline; and trypsin and/or carboxypeptidase B.

[0018] As used herein, the term “insulin precursor” refers to a single-stranded peptide including an insulin A-chain and an insulin B-chain, with a C-peptide therebetween, and may be used interchangeably with “proinsulin”. In the present invention, the insulin precursor conceptually comprises all precursor forms such as natural insulin precursors, insulin analogue precursors, and derivatives thereof. The insulin precursor may be prepared by those of ordinary skill in the art with reference to methods disclosed in documents such as EP 0,211,299, EP 0,227,938, EP 0,229,998, EP 0,286,956, or KR 10-0158197.

[0019] As used herein, the term “insulin” refers to a protein that controls blood sugar in the body. Natural insulin is a hormone secreted by the pancreas, and typically promotes intracellular glucose uptake and inhibits the breakdown of fat, and thus plays a role in controlling blood sugar in the body. In the present invention, insulin conceptually comprises all forms such as natural insulin, insulin analogues, and derivatives thereof.

[0020] For insulin, an insulin precursor (proinsulin) having no blood sugar control function is processed into insulin having a blood sugar control function. Insulin is composed of two polypeptide chains, particularly an A-chain and a B-chain, each comprising 21 and 30 amino acid residues, which are linked by two disulfide bridges. The A-chain and B-chain of natural insulin may comprise the following amino acid sequences, respectively.

TABLE-US-00001 A-chain: (SEQ ID NO: 1) Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser- Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn B-chain: (SEQ ID NO: 2) Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Pro-Lys-Thr

[0021] The insulin precursor and insulin used in the present invention may be derived from human, but the present invention is not limited thereto.

[0022] In the present invention, the insulin analogue comprises one in which the amino acid of the B-chain or the A-chain is mutated compared to the native type. The in-vivo blood sugar control function of the insulin analogue may be the same as or may correspond to that of natural insulin. Specifically, the insulin analogue precursor or insulin analogue may be configured such that at least one amino acid of natural insulin is subjected to any variation selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof, but the present invention is not limited thereto.

[0023] The insulin analogue that may be used in the present invention comprises an insulin analogue made by genetic recombination technology, and the insulin analogue conceptually comprises inverted insulin, insulin variants, insulin fragments, and the like.

[0024] The derivative has a blood sugar control function in the body, exhibits homology to each of the amino acid sequences of the A-chain and B-chain of the natural insulin or insulin analogue, and comprises a peptide in a form in which some groups of one amino acid residue are chemically substituted (e.g. alpha-methylation, alpha-hydroxylation), removed (e.g. deamination), or modified (e.g. N-methylation). The insulin fragment is a form in which at least one amino acid is added to or deleted from insulin, and the added amino acid may be an amino acid that does not exist in nature (e.g. a D-type amino acid), and such an insulin fragment plays a blood sugar control function in the body.

[0025] The insulin variant is a peptide having a sequence in which at least one amino acid is different from that of insulin, and plays a blood sugar control function in the body.

[0026] The insulin analogue, derivative, fragment and variant of the present invention may be used independently or in combination. For example, a peptide, which has a sequence in which at least one amino acid is different, in which the amino-terminal amino acid residue is subjected to deamination, and which plays a blood sugar control function in the body, is also comprised in the scope of the present invention.

[0027] Specifically, in the present invention, the insulin analogue may be insulin glargine. Insulin glargine is stabilized by substituting asparagine, which is the 21″ amino acid of the A-chain of insulin, with glycine, and is also made soluble at a weakly acidic pH by adding two arginines to the carboxy terminus of the B-chain. Here, insulin glargine is an insulin analogue developed such that it forms a microprecipitate in subcutaneous tissue when administered with an acidic solution (pH 4.0) and is slowly dissolved and released from the microprecipitate, which is an insulin glargine hexamer, whereby the action time is prolonged up to 24 hours. The A-chain and B-chain of insulin glargine may comprise the following amino acid sequences (U.S. Pat. No. 5,656,722).

TABLE-US-00002 A-chain: (SEQ ID NO: 3) Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser- Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Gly B-chain: (SEQ ID NO: 4) Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Pro-Lys-Thr-Arg-Arg

[0028] In the present invention, the term “enzymatic conversion” means converting an insulin precursor into insulin by digesting the insulin precursor with an enzyme. Specifically, enzymatic conversion serves to convert an insulin precursor (proinsulin) comprising a C-peptide between the A-chain and the B-chain into insulin using an enzyme, and the enzyme used for the enzymatic conversion may be selected from among trypsin, carboxypeptidase B, and combinations thereof. The term “enzymatic conversion” may be used interchangeably with terms such as “enzyme cleavage” or “enzyme digestion”.

[0029] In the present invention, the composition may further comprise a buffer solution. The buffer solution for the enzymatic reaction may be 1 mM to 100 mM Tris-HCl or 1 mM to 100 mM borate, and preferably about 50 mM Tris-HCl or borate, but is not limited thereto.

[0030] The enzyme that is used in the present invention is preferably selected from among trypsin, carboxypeptidase B, and combinations thereof, but is not limited thereto.

[0031] In the present invention, the concentration of the insulin precursor that is allowed to react with the composition is 1 to 15 mg/mL, but is not limited thereto.

[0032] In the present invention, trypsin may be used at a weight ratio (w/w) of 1/1,000 to 1/40,000, preferably 1/1000 to 1/30,000 (w/w), more preferably 1/1,000 to 1/20,000 (w/w), and still more preferably 1/5,000 to 1/10,000 (w/w) relative to the insulin precursor, but the present invention is not limited thereto.

[0033] In the present invention, carboxypeptidase B may be used at a ratio of 1/600 to 1/20,000 (w/w), and preferably 1/600 to 1/15,000 (w/w) relative to the insulin precursor, but the present invention is not limited thereto.

[0034] Calcium chloride, glycine, and proline may be added each alone or in combination to the buffer solution of the enzymatic reaction of the present invention.

[0035] In the present invention, calcium chloride may be added at a concentration of 1 to 50 mM, preferably 5 to 40 mM, more preferably 10 to 40 mM, still more preferably 15 to 40 mM, even more preferably 20 to 35 mM, and most preferably 25 to 35 mM, but the present invention is not limited thereto.

[0036] In the present invention, glycine may be added at a concentration of 3 to 60 mM, preferably 10 to 60 mM, more preferably 20 to 60 mM, still more preferably 30 to 60 mM, and most preferably 40 to 60 mM, but the present invention is not limited thereto.

[0037] In the present invention, proline may be added at a concentration of 20 to 80 mM, preferably 20 to 70 mM, more preferably 20 to 60 mM, still more preferably 20 to 50 mM, and most preferably 20 to 40 mM, but the present invention is not limited thereto.

[0038] In another embodiment of the present invention, the composition may comprise 5-30 mM calcium chloride, 3-60 mM glycine, and 20-80 mM proline, each alone or in combination, but is not limited thereto.

[0039] Another aspect of the present invention pertains to a method for converting an insulin precursor into insulin comprising adding an insulin precursor to the composition and carrying out a reaction.

[0040] When the enzymatic conversion of the present invention, the pH of the reaction solution is not particularly limited, so long as effective conversion of the insulin precursor into insulin is possible, and the pH may be set to 7.5 to 9.5, and preferably 8.5 to 9.0, but is not limited thereto.

[0041] The reaction temperature in the enzymatic conversion of the present invention may be 4.0 to 25.0° C., but is not limited thereto.

[0042] The reaction time in the enzymatic conversion of the present invention may be 0.5 to 24 hours, but is not limited thereto.

[0043] A better understanding of the present invention may be obtained through the following examples. These examples are merely set forth to illustrate the present invention, and are not to be construed as limiting the scope of the present invention.

5. EXAMPLES

Example 1: Preparation of Insulin Precursor

[0044] 1-1. Refolding of Insulin Glargine Precursor

[0045] In order to solubilize the insulin glargine precursor manufactured by the present applicant, 3.75 g of the insulin glargine precursor was added with a 0.2 M Tris-HCl buffer solution so that the volume thereof was adjusted to 250 mL, followed by stirring at room temperature for 1 hour. The solubilized solution was diluted 10-fold with a 0.2 M Tris-HCl buffer solution containing 0.4 mM cysteine as an oxidizing agent for refolding, and was then stirred at a low temperature for 10 hours. After completion of stirring, the pH of the resulting solution was lowered to 9.0 using hydrochloric acid.

[0046] 1-2. Anion Exchange Chromatography Purification

[0047] The solution containing the insulin glargine precursor prepared in Example 1-2 was bound to a POROS 50D (Thermo Scientific) ion resin equilibrated with a 50 mM Tris-HCl (pH 9.0) buffer solution, after which the insulin glargine precursor was eluted with a linear concentration gradient such that the concentration thereof was 10-14% using a 50 mM Tris-HCl (pH 9.0) buffer solution containing 0.5 M sodium chloride.

[0048] 1-3. Citraconylation

[0049] This process is an optional process for reducing the amount of impurities that may occur during enzymatic conversion of the insulin glargine precursor, and was performed before enzyme treatment using the insulin glargine precursor purified in Example 1-2. The purified glargine precursor was diluted to a concentration of 1-15 mg/mL using a 50 mM Tris-HCl solution. Thereafter, citraconic anhydride was added to the diluted insulin glargine precursor solution such that the molar concentration ratio of citraconic anhydride to insulin glargine precursor was 1:9.9-266.5, and the pH was titrated to 8.5-9.0 using an aqueous sodium hydroxide solution, followed by stirring for 2 hours.

Comparative Example 1. Enzymatic Conversion without Addition of Salt or Amino Acid

[0050] The solution obtained in Example 1-3 was subjected to enzyme treatment to conduct an experiment for converting the insulin glargine precursor into insulin glargine. The insulin glargine precursor in the concentration range of 1 to 15 mg/mL was added with trypsin corresponding to 1/100,000-1/1,000 of the weight ratio of the protein, followed by stirring for 2 hours. After completion of stirring, the pH was titrated to 2.5 using acetic acid to complete the reaction. After completion of the reaction, the purity and yield of insulin glargine were analyzed using RP-HPLC.

Example 2: Example 2. Improvement in Yield of Enzymatic Conversion Reaction by Addition of Calcium Chloride

[0051] In order to confirm the effect of the addition of calcium chloride on the yield of the enzymatic conversion reaction, the insulin glargine precursor obtained in Example 1-3 in the concentration range of 1 to 15 mg/mL was added with trypsin corresponding to 1/40,000-1/5,000 of the weight ratio of the protein, followed by stirring for 2 hours. Here, the reaction solution was 50 mM Tris-HCl, calcium chloride was added thereto in the concentration range of 1 to 20 mM, and the pH was titrated to 8.5-9.0. After completion of stirring, the pH was titrated to 2.5 using acetic acid to complete the reaction.

[0052] The purity and yield of the completed insulin glargine were analyzed using RP-HPLC as in Comparative Example 1. When calcium chloride was added during the enzymatic conversion reaction, the yield of the enzymatic conversion reaction was increased than when not added, and this effect was observed to be the highest when calcium chloride was added at a concentration of 20 mM (Table 1).

[0053] Table 1 below shows the yield of the enzymatic conversion reaction depending on the concentration of calcium chloride during the enzymatic conversion reaction of the insulin glargine precursor.

TABLE-US-00003 TABLE 1 Concentration (mM) Purity (%) Yield (%) Comparative — 33.4 39.5 Example 1 Calcium 1 35.5 41.6 chloride 5 36.4 43.0 10 37.0 43.3 15 37.2 43.7 20 37.5 44.1

[0054] As shown in Table 1, both the yield and the purity were increased depending on the concentration of calcium chloride. The purity was increased from 33.4% when calcium chloride was not added to 35.5-37.5% depending on the concentration thereof, and the yield was increased from 39.5% when calcium chloride was not added to 41.6-44.1% depending on the concentration thereof. From these results, it was found that the yield and purity were effectively increased when calcium chloride was added during the enzymatic conversion reaction.

Example 3: Improvement in Yield of Enzymatic Conversion Reaction by Addition of Glycine

[0055] In order to confirm the effect of the addition of glycine on the yield of the enzymatic conversion reaction, the insulin glargine precursor obtained in Example 1-3 in the concentration range of 1 to 15 mg/mL was added with trypsin corresponding to 1/100,000-1/1,000 of the weight ratio of the protein, followed by stirring for 2 hours. Here, the reaction solution was 50 mM Tris-HCl, glycine was added thereto in the concentration range of 20 to 140 mM, and the pH was titrated to 8.5-9.0. After completion of stirring, the pH was titrated to 2.5 using acetic acid to complete the reaction.

[0056] The purity and yield of the completed insulin glargine were analyzed using RP-HPLC as in Comparative Example 1. When glycine was added to the enzymatic conversion reaction, the yield of the enzymatic conversion reaction was generally increased than when not added, and this effect was observed to be the highest when glycine was added at a concentration of 60 mM (Table 2).

[0057] Table 2 below shows the yield of the enzymatic conversion reaction depending on the concentration of glycine during the enzymatic conversion reaction of the insulin glargine precursor.

TABLE-US-00004 TABLE 2 Concentration (mM) Purity (%) Yield (%) Comparative — 30.3 42.4 Example 1 Glycine 20 31.6 43.1 60 31.4 43.3 100 30.8 42.8 140 30.8 42.2

[0058] As shown in Table 2, the yield and purity were varied depending on the concentration of glycine. Specifically, the purity was increased from 30.3% before the addition of glycine to 31.6% when added at a concentration of 20 mM and to 31.4% when added at a concentration of 60 mM, but the extent of increase in purity was slightly decreased when added at concentrations of 100 mM and 140 mM. In addition, the yield was also increased from 42.4% before the addition of glycine to 43.1% when added at a concentration of 20 mM and to 43.3% when added at a concentration of 60 mM, but the extent of increase in yield was slightly decreased when added at concentrations of 100 mM and 140 mM. From these results, it was found that the yield and purity were effectively increased when glycine was added at an appropriate concentration during the enzymatic conversion reaction.

Example 4: Improvement in Yield of Enzymatic Conversion Reaction by Addition of Proline

[0059] In order to confirm the effect of the addition of proline on the yield of the enzymatic conversion reaction, the insulin glargine precursor obtained in Example 1-3 in the concentration range of 1 to 15 mg/mL was added with trypsin corresponding to 1/100,000-1/1,000 of the weight ratio of the protein, followed by stirring for 2 hours. Here, the reaction solution was 50 mM Tris-HCl, proline was added thereto in the concentration range of 20 to 80 mM, and the pH was titrated to 8.5-9.0. After completion of stirring, the pH was titrated to 2.5 using acetic acid to complete the reaction.

[0060] The purity and yield of the completed insulin glargine were analyzed using RP-HPLC as in Comparative Example 1. When proline was added during the enzymatic conversion reaction, the yield of the enzymatic conversion reaction was generally increased than when not added, and this effect was observed to be the highest when proline was added at a concentration of 60 mM (Table 3).

[0061] Table 3 below shows the yield of the enzymatic conversion reaction depending on the concentration of proline during the enzymatic conversion reaction of the insulin glargine precursor.

TABLE-US-00005 TABLE 3 Concentration (mM) Purity (%) Yield (%) Comparative — 35.8 41.6 Example 1 Proline 20 37.8 43.1 40 38.0 43.0 60 37.6 43.2 80 36.8 42.8

[0062] As shown in Table 3, when proline was added, the purity and yield of insulin glargine were increased. Accordingly, it was confirmed that the addition of proline had an influence on increasing the purity and yield in the enzymatic conversion reaction of insulin glargine.

Example 5: Improvement in Yield of Enzymatic Conversion Reaction by Addition of Calcium Chloride/Glycine/Proline

[0063] In order to confirm the effect of the addition of calcium chloride, glycine, and proline on the yield of the enzymatic conversion reaction, the insulin glargine precursor obtained in Example 1-3 in the concentration range of 1 to 15 mg/mL was added with trypsin corresponding to 1/100,000-1/1,000 of the weight ratio of the protein, followed by stirring for 2 hours. Here, the reaction solution was 50 mM Tris-HCl, the concentration of calcium chloride/glycine/proline complex salt was set as shown in Table 4 below, and the pH was titrated to 8.5-9.0. After completion of stirring, the pH was titrated to 2.5 using acetic acid to complete the reaction.

TABLE-US-00006 TABLE 4 Calcium chloride Proline Glycine Concentration concentration concentration (mM) (mM) (mM) 30.0 10.0 50.0 20.0 20.0 35.0 20.0 20.0 50.0 20.0 20.0 50.0 20.0 20.0 50.0 10.0 10.0 50.0 20.0 3.2 35.0 20.0 36.8 35.0 20.0 20.0 60.2 10.0 30.0 50.0 30.0 30.0 50.0 0.0 0.0 0.0

[0064] The purity and yield of the completed insulin glargine were analyzed using RP-HPLC as in Comparative Example 1. When the calcium chloride/glycine/proline complex salt was added during the enzymatic conversion reaction, the yield of the enzymatic conversion reaction was generally increased than when not added, and this effect was observed to be the highest when 30 mM calcium chloride, 30 mM proline, and 50 mM glycine were added (Table 5).

[0065] Table 5 below shows the yield of the enzymatic conversion reaction depending on the concentration of calcium chloride/glycine/proline during the enzymatic conversion reaction of the insulin glargine precursor.

TABLE-US-00007 TABLE 5 Calcium chloride Proline Glycine Concentration concentration concentration Purity Yield (mM) (mM) (mM) (%) (%) 30.0 10.0 50.0 39.8 44.8 20.0 20.0 35.0 38.8 44.8 20.0 20.0 50.0 39.0 44.9 20.0 20.0 50.0 39.2 44.6 20.0 20.0 50.0 39.1 44.9 10.0 10.0 50.0 38.6 44.5 20.0 3.2 35.0 39.1 44.8 20.0 36.8 35.0 39.8 44.9 20.0 20.0 60.2 39.9 44.8 10.0 30.0 50.0 38.6 44.4 30.0 30.0 50.0 39.7 45.0 0.0 0.0 0.0 33.2 39.6

[0066] As shown in Table 5, the yield and purity were increased depending on the concentration of the calcium chloride/glycine/proline complex salt. It was confirmed that the purity was increased by 5.4-6.8% and the yield was increased by 4.8-5.4% when three salts were added together than when added separately. Therefore, it was found that the addition of calcium chloride/glycine/proline was very effective at increasing the purity and yield of the enzymatic conversion reaction of insulin glargine.

[0067] From the above description, those skilled in the art to which the present invention belongs will appreciate that the present invention may be embodied in other specific forms without changing the technical spirit or essential features thereof. In this regard, the embodiments described above should be understood to be non-limiting and illustrative in every way. The scope of the present invention is defined by the claims below rather than the aforementioned detailed description, and all changes or modified forms that are capable of being derived from the meaning, range, and equivalent concepts of the appended claims should be construed as being comprised in the scope of the present invention.

6. INDUSTRIAL APPLICABILITY

[0068] According to the present invention, a composition for enzymatic conversion of an insulin precursor into insulin and a method for converting an insulin precursor into insulin using the same make it possible to simultaneously improve both the structural stability of an insulin protein and the stability of an enzyme, thereby notably increasing the yield of enzymatic conversion and the purity of insulin, and thus can be usefully applied to the process of production of insulin.

7. SEQUENCE LIST FREE TEXT

[0069] An electronic file is attached.