阅读说明：本技术 使用梭菌蛋白酶制备长效胰岛素类似物缀合物的活性形式的方法 (Method for preparing active forms of long-acting insulin analogue conjugates using clostripain ) 是由 安炅勋 郑先敬 尹彩夏 于 2019-07-19 设计创作，主要内容包括：本发明涉及一种制备长效胰岛素类似物缀合物的活性形式的方法,其中胰岛素B链上位置22处的氨基酸从精氨酸(Arg)变为赖氨酸(Lys),由此即使与梭菌蛋白酶反应,所述胰岛素也可以在所述胰岛素B链不被切割的情况下转化为活性形式。根据本发明的制备方法克服了在将胰岛素原转化为活性形式中使用胰蛋白酶的常规方法的问题,即由于白蛋白结合结构域的切割,难以将长效胰岛素类似物缀合物转化为活性形式。因此,本发明的方法可以有利地用于产生糖尿病长效治疗剂。(The present invention relates to a method for preparing an active form of a long acting insulin analogue conjugate wherein the amino acid at position 22 on the insulin B-chain is changed from arginine (Arg) to lysine (Lys) whereby the insulin can be converted to the active form without the insulin B-chain being cleaved even if reacted with clostripain. The preparation method according to the present invention overcomes the problem of the conventional method using trypsin in converting proinsulin into active form, i.e. the difficulty of converting long-acting insulin analogue conjugates into active form due to cleavage of the albumin binding domain. Thus, the methods of the invention can be advantageously used to produce long-acting therapeutics for diabetes.)

1. A method for producing an active form of a long acting insulin analogue derivative in which an insulin analogue comprising an insulin B-chain variant represented by the amino acid sequence of SEQ ID NO:2 with an arginine (Arg) at amino acid position 22 of the insulin B-chain substituted with a lysine (Lys) in native insulin is fused to an albumin binding domain, comprising the step of reacting the insulin analogue derivative with clostripain.

2. The method of claim 1, wherein the insulin analogue further comprises one or more amino acid substitutions selected from the group consisting of:

a valine (Val) to leucine (Leu) substitution at amino acid position 3 of the insulin A chain represented by the amino acid sequence of SEQ ID NO. 4;

a threonine (Thr) to aspartic acid (Asp) substitution at amino acid position 8 of the insulin A chain represented by the amino acid sequence of SEQ ID NO: 4;

an isoleucine (Ile) to lysine (Lys) substitution at amino acid position 10 of the insulin A chain represented by the amino acid sequence of SEQ ID NO. 4;

a tyrosine (Tyr) to glutamic acid (Glu) substitution at amino acid position 14 of the insulin A chain represented by the amino acid sequence of SEQ ID NO: 4;

a tyrosine (Tyr) to phenylalanine (Phe) substitution at amino acid position 19 of the insulin A chain represented by the amino acid sequence of SEQ ID NO. 4;

a histidine (His) to threonine (Thr) substitution at amino acid position 5 of the insulin B chain variant represented by the amino acid sequence of SEQ ID NO: 2;

a serine (Ser) to aspartic acid (Asp) substitution at amino acid position 9 of an insulin B chain variant represented by the amino acid sequence of SEQ ID NO: 2;

a glutamic acid (Glu) to alanine (Ala) substitution at amino acid position 13 of an insulin B chain variant represented by the amino acid sequence of SEQ ID NO: 2;

a leucine (Leu) to glutamine (Gln) substitution at amino acid position 17 of an insulin B chain variant represented by the amino acid sequence of SEQ ID NO: 2; and

the phenylalanine (Phe) to serine (Ser) substitution at amino acid position 24 of the insulin B chain variant represented by the amino acid sequence of SEQ ID NO: 2.

3. The method of claim 1, wherein the albumin binding domain comprises an albumin binding motif represented by the amino acid sequence:

GVSDFYKKLIX_aKAKTVEGVEALKX_bX_cI

wherein

X_aIndependently selected from the group consisting of D and E,

X_bis independently selected from D and E, and

X_cindependently selected from A and E.

4. The method of claim 3, wherein the albumin binding domain comprises the amino acid sequence:

LAX₃AKX₆X₇ANX₁₀ELDX₁₄Y-[BM]-LX₄₃X₄₄LP

wherein

[ BM ] is an albumin binding motif as defined in claim 3,

X₃independently selected from C, E, Q and S;

X₆independently selected from C, E and S;

X₇independently selected from A and S;

X₁₀independently selected from A, R and S;

X₁₄independently selected from A, C, K and S;

X₄₃independently selected from A and K; and is

X₄₄Independently selected from A, E and S.

5. The method of claim 4, wherein the albumin binding domain is represented by an amino acid sequence selected from the group consisting of SEQ ID NOs 6 to 13.

6. The method of claim 5, wherein the albumin binding domain is represented by the amino acid sequence of SEQ ID NO 6.

7. The method of claim 1, wherein the insulin analogue and the albumin binding domain are fused to each other by: a peptide bond; a polypeptide linker; or a non-peptidyl linker selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, biodegradable polymer, fatty acid, nucleotide, lipopolymer, chitin, hyaluronic acid, and a combination thereof.

8. The method of claim 7, wherein the polypeptide linker comprises (GGGGS)_n(where n is an integer ranging from 1 to 6).

9. The method of claim 8, wherein the peptide linker is represented by the amino acid sequence of SEQ ID NO 5.

10. The method according to claim 1, wherein a reducing agent is added during the reaction with the clostripain.

11. The method of claim 10, wherein the reducing agent is selected from cysteine, beta mercaptoethanol, TCEP (tris (2-carboxyethyl) phosphine hydrochloride), GSH (glutathione), and DTT (dithiothreitol).

12. The method according to claim 10, wherein 0.1 to 0.5mM DTT is added as the reducing agent.

13. The method of claim 12, wherein the DTT is added at an initial stage of the reaction of the insulin analogue derivative with clostripain and is further added 3 to 6 hours after the reaction.

14. The method according to claim 1, wherein the insulin analogue derivative is also reacted with carboxypeptidase B (CpB), which is added during or after the reaction with clostripain.

15. The method according to claim 14, wherein the clostripain and/or carboxypeptidase b (cpb) are reacted at a pH of 6.0-9.0.

16. The method according to claim 15, wherein the clostripain and/or carboxypeptidase b (cpb) are reacted at a pH of 6.5-7.5.

17. The method of claim 14, wherein the clostripain and/or carboxypeptidase b (cpb) are reacted at a temperature of 4 ℃ -40 ℃.

18. An active form of a long acting insulin analogue derivative produced by the method according to any one of claims 1 to 17.

Technical Field

The present invention relates to a method of producing an active form of a long acting insulin analogue derivative using clostripain, and more particularly to a method of producing an active form of a long acting insulin analogue derivative in which the amino acid at position 22 of the insulin B-chain is substituted from arginine (Arg) to lysine (Lys) so that the insulin analogue can be converted to the active form without the B-chain being cleaved even when reacted with clostripain.

Background

Diabetes mellitus is a metabolic disease characterized by high blood glucose levels and develops from the combined action of genetic and environmental factors. Diabetes includes type 1 diabetes, type 2 diabetes, gestational diabetes, and other conditions that cause hyperglycemia. Diabetes mellitus means a metabolic disorder in which the pancreas produces insufficient amounts of insulin, or in which the human cells fail to respond appropriately to insulin, and thus their ability to absorb glucose is impaired. As a result, glucose accumulates in the blood.

Type 1 diabetes, also known as Insulin Dependent Diabetes Mellitus (IDDM) and juvenile onset diabetes, is caused by β cell destruction, resulting in absolute insulin deficiency. On the other hand, type 2 diabetes, known as non-insulin dependent diabetes mellitus (NIDDM) and adult-onset diabetes, is associated with major insulin resistance, and thus with relative insulin deficiency and/or major insulin secretion deficiency with insulin resistance.

In particular, diabetes is associated with various complications such as cardiovascular diseases and retinopathy, and is therefore a disease that becomes very troublesome if not managed properly (e.g., blood sugar control). The world market for diabetes drugs is expected to expand from $ 417 billion in 2015 to $ 661 billion in 2022 and is expected to be the second largest market next to the anticancer drug market. Worldwide, 4.22 billion adults in 2014 have diabetes, accounting for 8.5% of the overall adult population, which has nearly doubled compared to 4.5% in 1980 (WHO, 2016). Further, the global health expenditure for diabetes is estimated to amount to $ 6730 billion, and it is expected that the number of diabetic patients of 20-79 years will increase to about 6.2 billion by 2040 years.

The most representative treatment methods for treating diabetes include a method of administering insulin to control the blood glucose level of a patient to a normal level. Insulin is a hormone secreted by the human pancreas that regulates blood glucose. Its function is to transfer excess glucose in the blood to the cells, to provide energy to the cells, and to maintain blood glucose levels at normal levels.

Insulin undergoes various post-translational modifications along the production pathway. Production and secretion are largely independent; the prepared insulin is stored to be secreted. Both C-peptide and mature insulin are biologically active.

In mammals, insulin is synthesized in pancreatic beta cells. Insulin is composed of two polypeptide chains (a and B chains) linked to each other by disulfide bonds. However, insulin is first synthesized in pancreatic beta cells as a single polypeptide called preproinsulin. The preproinsulin contains a 24 amino acid signal peptide that directs nascent polypeptide chains to the rough endoplasmic reticulum. The signal peptide translocates into the lumen of the rough endoplasmic reticulum and is then cleaved, thereby forming proinsulin. In the rough endoplasmic reticulum, the proinsulin folds into the correct conformation and forms three disulfide bonds. After 5 to 10 minutes of assembly in the endoplasmic reticulum, the proinsulin is transported to the reverse golgi apparatus where immature granules are formed.

Proinsulin is matured to active insulin by the action of intracellular peptidases called prohormone convertases (PC1 and PC2) and carboxypeptidase E, which is an exoprotease. The endopeptidase cleaves at 2 positions, releasing a fragment called the C peptide and leaving 2 peptide chains linked by 2 disulfide bonds: b chain and A chain. The cleavage sites are located behind a pair of basic residues, lysine (Lys) -64 and arginine (Arg) -65, and arginine (Arg) -31 and arginine (Arg) -32, respectively. After cleavage of the C peptide, these 2 pairs of basic residues are removed by carboxypeptidase. The C peptide is located in the central portion of proinsulin, and the order of the primary sequence of proinsulin is "B-C-A" (B and A chains are identified on a mass basis, and C peptide is later discovered).

The resulting mature insulin (active insulin) is packaged within the mature particle, awaiting metabolic signals (e.g., leucine (Leu), arginine (Arg), glucose, and mannose) and vagus nerve stimulation for extracellular secretion from the cell into the circulation.

For the treatment of diabetes, active insulin is administered. Techniques for producing active insulin using gene recombination techniques are as follows. First, the method adopted by Eli Lilly corp comprises the following steps: the a chain and the B chain are separately expressed using e.coli, and mixed in vitro to form a disulfide bridge, thereby connecting the a chain and the B chain to each other via a disulfide bond. However, this method has a problem in that the production efficiency is low. Then, Eli Lilly corp. a method has been developed comprising the following steps: expressing proinsulin; disulfide bond formation in vitro; and then cleaving peptide C from the product with trypsin and carboxypeptidase B, thereby producing insulin.

A method has been developed by Novo Nordisk corp, comprising the steps of: expressing in yeast a mini-proinsulin comprising a B chain and an A chain linked via two basic amino acids; and then treating the mini-proinsulin with trypsin to produce insulin. This method has advantages in that disulfide bonds are formed during the expression and secretion of miniproinsulin, and since miniproinsulin is secreted into the medium, it is easy to isolate and purify. However, this method is difficult to apply to large-scale production comparable to production using E.coli.

Since then, a novel method for producing insulin by gene recombination technology has been actively developed. Hoechst AG developed a method comprising the steps of: expressing a novel insulin derivative or preproinsulin in E.coli; and disulfide bonds are formed in vitro, followed by treatment with lysyl endopeptidase or clostripain and carboxypeptidase B, thereby producing insulin. Bio-Technology General Corp. A method has been developed in which a fusion protein comprising superoxide dismutase (SOD) linked to proinsulin is expressed in E.coli to improve in vitro expression efficiency and disulfide bond formation efficiency. Proinsulin is converted to insulin using trypsin and carboxypeptidase B. As described above, many methods for producing insulin by gene recombination techniques have been tried and improved in terms of expression efficiency, disulfide bond formation efficiency and conversion process of proinsulin to insulin (KR 10-2001) -7013921).

The inventors have made extensive efforts to develop a production method suitable for a novel long-acting insulin analog derivative developed by the inventors, which has an increased half-life in vivo, resulting in an improvement in long-acting effect as compared with natural insulin, and as a result, found that the use of clostripain enables efficient production of the novel long-acting insulin analog derivative developed by the inventors, thereby completing the present invention.

Disclosure of Invention

Technical problem

It is an object of the present invention to provide a method for producing active forms of novel long acting insulin analogue derivatives.

Technical scheme

To achieve the above object, the present invention provides a method for producing an active form of a long acting insulin analogue derivative, the method comprising the step of reacting an insulin analogue derivative comprising an insulin analogue comprising an insulin B-chain variant represented by the amino acid sequence of SEQ ID NO:2 with an albumin binding domain fused thereto, the arginine (Arg) at amino acid position 22 of the insulin B-chain being substituted by lysine (Lys) in native insulin, with clostripain.

The invention also provides a long acting insulin analogue derivative produced by the method.

Drawings

Figure 1 shows the results of analysis of the stability of long-acting insulin analogue derivatives constructed by substitution of arginine (Arg) at position 22 of the insulin B-chain with lysine (Lys) by treatment with clostripain. Mass spectrometry analysis showed that cleavage of the insulin B chain occurred when it contained arginine (Arg) at position 22, but did not occur when arginine (Arg) was replaced by lysine (Lys).

Figure 2 shows the results of analysis of the expression of long-acting insulin analogue derivatives 1 to 10 in recombinant e.coli by SDS-PAGE. Even if one or more mutations are introduced in the insulin sequence, protein expression in E.coli is retained.

Figure 3 shows the results of monitoring solubilization and refolding of insulin analogue 4 fused to ABD by RP-HPLC. When the protein structure is unfolded by solubilization and refolding is subsequently induced, a shift in retention time is observed in RP-HPLC due to the formation of three-dimensional structures.

Figure 4 shows the results of SDS-PAGE performed to analyze the efficiency of production of the active form of insulin analogue 4 fused to ABD by clostripain treatment according to the addition of DTT.

Figure 5 shows the results of SDS-PAGE performed to analyze the efficiency of the generation of the active form of insulin analogue 4 fused to ABD, depending on the time of treatment with clostripain and CpB.

Figure 6 shows the results of SDS-PAGE performed to determine the pH range over which the active form of ABD fused insulin analogue 4 was produced with high efficiency when treated simultaneously with clostripain and CpB.

Figure 7 shows the results of SDS-PAGE performed to analyze the efficiency of production of the active form of insulin analogue 4 fused to ABD according to further addition of clostripain and DTT.

Figure 8 shows the results confirming the effect of increasing the efficiency of conversion to the active form of insulin analogue 4 fused to ABD and reducing impurities (molecular weight of the main impurities: 10296 Da; molecular weight of the active form of insulin: 11238Da) under optimal enzymatic reaction conditions according to an embodiment of the present invention.

Figure 9 shows the results of SDS-PAGE performed to determine the temperature range over which the active form of ABD fused insulin analogue 4 was produced with high efficiency when treated simultaneously with clostripain and CpB.

Figure 10 shows the results of analysis of insulin analogue 4 fused to ABD by SDS-PAGE after digestion and purification with clostripain and CpB.

Figure 11 shows the results of a size exclusion chromatography analysis performed to examine whether hexamers will be formed when zinc and phenol are added to insulin analogue 4 of the fused ABD according to an embodiment of the present invention.

Fig. 12 shows the results of evaluating the hypoglycemic ability of an insulin analogue fused to ABD according to an embodiment of the present invention.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein is well known and commonly employed in the art.

Insulin analogs obtained by substituting one or more amino acids of natural insulin have increased half-lives in vivo and, therefore, can be used as basic insulin therapeutics. When this insulin analogue is combined with albumin in humans, its in vivo half-life can be further increased so that the insulin analogue can be used as a weekly formulation.

The inventors can find that the in vivo half-life of insulin can be increased when one or more amino acids of natural insulin are substituted, and that when the albumin binding domain is additionally fused thereto, the insulin can bind to albumin in vivo, and thus its half-life can be further increased. However, when the albumin binding domain is fused to an insulin analogue comprising one or more amino acid substitutions, and when trypsin as used in conventional techniques is used in the process of converting the insulin analogue to an active form, the problem does arise that the albumin binding domain itself is cleaved, making it difficult to produce an active form that lasts for a long period of time.

Thus, in the present invention, it is attempted to use clostripain as an enzyme that enables the conversion of insulin to the active form without inducing cleavage of the albumin binding domain. However, clostripain induces cleavage of insulin itself, and for this reason, amino acids of insulin are substituted, thereby preventing cleavage of insulin itself.

Thus, in one aspect, the invention relates to a method for producing an active form of a long acting insulin analogue derivative in which an insulin analogue comprising an insulin B-chain variant represented by the amino acid sequence of SEQ ID NO:2 and having an arginine (Arg) at amino acid position 22 of the insulin B-chain substituted with a lysine (Lys) in native insulin is fused to an albumin binding domain, comprising the step of reacting the insulin analogue derivative with clostripain.

In the present invention, "an insulin analog comprising an insulin B-chain variant represented by the amino acid sequence of SEQ ID NO:2 and having arginine (Arg) at amino acid position 22 of the insulin B-chain replaced with lysine (Lys) in natural insulin" means that the insulin analog may further comprise other amino acid variants in the insulin a-chain or insulin B-chain in addition to the substitution of arginine (Arg) to lysine (Lys) at amino acid position 22 of the natural insulin B-chain.

In the present invention, the insulin analogue may further comprise one or more amino acid substitutions selected from the group consisting of: a valine (Val) to leucine (Leu) substitution at amino acid position 3 of the insulin A chain represented by the amino acid sequence of SEQ ID NO. 4; a threonine (Thr) to aspartic acid (Asp) substitution at amino acid position 8 of the insulin A chain represented by the amino acid sequence of SEQ ID NO: 4; an isoleucine (Ile) to lysine (Lys) substitution at amino acid position 10 of the insulin A chain represented by the amino acid sequence of SEQ ID NO. 4; a tyrosine (Tyr) to glutamic acid (Glu) substitution at amino acid position 14 of the insulin A chain represented by the amino acid sequence of SEQ ID NO: 4; a tyrosine (Tyr) to phenylalanine (Phe) substitution at amino acid position 19 of the insulin A chain represented by the amino acid sequence of SEQ ID NO. 4; a histidine (His) to threonine (Thr) substitution at amino acid position 5 of the insulin B chain variant represented by the amino acid sequence of SEQ ID NO: 2; a serine (Ser) to aspartic acid (Asp) substitution at amino acid position 9 of an insulin B chain variant represented by the amino acid sequence of SEQ ID NO: 2; a glutamic acid (Glu) to alanine (Ala) substitution at amino acid position 13 of an insulin B chain variant represented by the amino acid sequence of SEQ ID NO: 2; a leucine (Leu) to glutamine (Gln) substitution at amino acid position 17 of an insulin B chain variant represented by the amino acid sequence of SEQ ID NO: 2; and a phenylalanine (Phe) to serine (Ser) substitution at amino acid position 24 of the insulin B chain variant represented by the amino acid sequence of SEQ ID NO:2, but is not limited thereto.

In an example of the invention, the albumin binding domain may comprise an albumin binding motif represented by the amino acid sequence:

GVSDFYKKLIX_aKAKTVEGVEALKX_bX_cI

wherein

X_aIndependently selected from the group consisting of D and E,

X_bis independently selected from D and E, and

X_cindependently selected from A and E.

In the examples of the present invention, X_aIs D, X_bIs D, and X_cIs A.

In an embodiment of the invention, the albumin binding domain may comprise the amino acid sequence:

LAX₃AKX₆X₇ANX₁₀ELDX₁₄Y-[BM]-LX₄₃X₄₄LP

wherein

[ BM ] is an albumin binding motif as defined in the preceding paragraph,

X₃independently selected from C, E, Q and S;

X₆independently selected from C, E and S;

X₇independently selected from A and S;

X₁₀independently selected from A, R and S;

X₁₄independently selected from A, C, K and S;

X₄₃independently selected from A and K; and is

X₄₄Independently selected from A, E and S.

The albumin binding domain may be represented by an amino acid sequence selected from the group consisting of SEQ ID NOS 6 to 13, but is not limited thereto. The albumin binding domain may preferably be represented by the amino acid sequence of SEQ ID NO 6.

In the present invention, it is preferred that the albumin binding domain is fused to the C-terminus of the a-chain in insulin or an insulin analogue having a B-chain-C-chain in terms of protein refolding efficiency, enzyme reaction efficiency and activity of the resulting insulin derivative or insulin analogue derivative. In this case, a linker may be introduced between the insulin (or insulin analogue) and the albumin binding domain.

In the present invention, a polynucleotide encoding an insulin analog and a nucleotide encoding an albumin binding domain can be introduced into a recombinant vector so that the insulin analog and the albumin binding domain can be expressed as a fusion protein.

The function of the insulin analogue derivative according to the invention varies according to the three-dimensional structure of the derivative. Thus, small changes in the amino acid sequence of the long acting insulin analogue derivatives according to the invention can be made without affecting the function of the derivatives. Thus, the present invention includes variants of albumin binding domains or long acting insulin analogue derivatives which retain albumin binding properties or high resistance to enzymatic cleavage. For example, an amino acid residue belonging to a particular functional group (e.g., hydrophobic, hydrophilic, polar, etc.) of an amino acid residue can be replaced with another amino acid residue belonging to the same functional group.

As used herein, the terms "albumin binding" and "binding affinity for albumin" refer to the properties of a polypeptide or protein that can be tested using surface plasmon resonance techniques (e.g., Biacore instruments). For example, albumin binding affinity can be tested in an experiment in which albumin or a fragment thereof is immobilized on a sensor chip of the instrument and a sample containing the polypeptide or protein to be tested is passed through the chip.

Alternatively, the polypeptide or protein to be tested is immobilized on a sensor chip of the instrument and a sample containing albumin or a fragment thereof is passed through the chip. In this regard, the albumin may be a serum albumin of mammalian origin, such as human serum albumin. The results obtained by such experiments can be interpreted by the skilled person to establish a quantitative measure of the binding affinity of a polypeptide or protein to albumin. If quantitative measurements are required, e.g. to determine the K of the interaction_DAlso, a surface plasmon resonance method may be used. Binding values can be defined, for example, in a Biacore 2000 instrument (Biacore AB). Albumin is suitably immobilized on a measurement sensor chip and a polypeptide or protein sample to be affinity determined is prepared by serial dilution and injected in random order. K can then be calculated from the results using a 1:1 Langmuir (Langmuir) binding model, such as the BIAevaluation 4.1 software provided by the instrument manufacturer_DThe value is obtained.

In one embodiment of the invention, the albumin to which the insulin analogue derivative binds is selected from the group consisting of human serum albumin, rat serum albumin, cynomolgus serum albumin and mouse serum albumin, but is not limited thereto.

In a particular embodiment, the albumin to which the insulin analogue derivative binds is human serum albumin.

Meanwhile, the description of the albumin binding motif and albumin binding domain (or albumin binding polypeptide) is explained to include the contents disclosed in Korean patent laid-open No. 10-2015-0058454 corresponding to WO 2014048977.

The insulin analogue and albumin binding domain are linked to each other by: a peptide bond; a polypeptide linker; or a non-peptidyl linker selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran (dextran), polyvinyl ethyl ether, biodegradable polymer, fatty acid, nucleotide, lipid polymer, chitin, hyaluronic acid, and a combination thereof, but not limited thereto.

In the present invention, a peptide (GGGGS) may be inserted between the insulin analog and the albumin binding domain_n(wherein n is an integer ranging from 1 to 6) sequence. This is based on experimental results showing that the refolding rate is lower when no linker is introduced, but there is no significant difference in refolding rate when two or more repeats of the (GGGGS) sequence are introduced. At the same time, the rate of conversion to the active form of insulin by clostripain increases with increasing number of repeats (n) of the sequence, but the in vivo pharmacokinetics of the insulin analogue is not significantly affected. Taken together, the linker preferably consists of 2 to 4 repeats of the GGGGS sequence.

Thus, in the present invention, the insulin analogue and the albumin binding domain may be linked to each other by a polypeptide linker, and the polypeptide linker may comprise (GGGGS)_n(where n is an integer ranging from 1 to 6, but is not limited thereto). Preferably, the peptide linker may be represented by the amino acid sequence of SEQ ID NO 5.

In the present invention, the reducing agent may be added during the reaction with the clostripain. The reducing agent may be selected from cysteine, beta mercaptoethanol, TCEP, GSH, and DTT, but is not limited thereto. As reducing agent, preferably 0.1 to 0.5mM DTT is added. More preferably, 0.1 to 0.4mM DTT is added.

DTT may be added only once at the initial stage of the reaction of the insulin analogue derivative with clostripain, i.e. at the point in time when the reaction starts, and DTT may be added 2 to 5 more times after the point in time when the reaction starts. However, in view of the convenience and efficiency of the production process, it is most preferable to add DTT at the initial stage of the reaction of the insulin analog derivative with clostripain and further add it 3 to 6 hours after the reaction. Clostripain can be added at a concentration of 0.1 to 5 units, preferably 0.5 to 2 units per mg protein, but is not limited thereto.

In the present invention, the insulin analogue derivative may also be reacted with carboxypeptidase B (cpb), which is added during or after the reaction with clostripain. In this case, CpB may be added with the clostripain when the reaction is started.

In the present invention, the clostripain and/or carboxypeptidase B (CpB) are reacted at a pH of 6.0 to 9.0, preferably 6.5 to 7.5, and at a temperature of 4 ℃ to 40 ℃, preferably 30 ℃ to 40 ℃, but is not limited thereto.

CpB may be added at a concentration of 0.001 to 1 unit, preferably 0.001 to 0.1 unit per mg of protein, but is not limited thereto.

In another aspect, the invention relates to an active form of an insulin analogue derivative produced by the above method.

In the present invention, the insulin analogue derivative may be produced in a recombinant microorganism by lysis and purification of a cultured recombinant microorganism, wherein the recombinant vector comprises a polynucleotide encoding the analogue derivative.

The recombinant vector according to the present invention may be constructed as a vector for conventional cloning or expression, and may be constructed as a vector using prokaryotic or eukaryotic cells as host cells.

As used herein, the term "vector" refers to a recombinant vector capable of expressing a target protein in an appropriate host cell, which is a genetic construct comprising the necessary regulatory factors operably linked to enable expression of the nucleic acid insert. The present invention makes it possible to prepare recombinant vectors comprising nucleic acids encoding insulin analogue derivatives, and the insulin analogue derivatives of the present invention can be obtained by transforming or transfecting the recombinant vectors into host cells.

In the present invention, nucleic acids encoding insulin analogues and analogue derivatives thereof are operably linked to nucleic acid expression control sequences. As used herein, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, a ribosome binding site, a transcription termination sequence, etc.) and another nucleotide sequence, and thus the control sequence can control the transcription and/or translation of the other nucleotide sequence.

As used herein, the term "promoter" refers to an untranslated nucleic acid sequence located upstream of a coding region that includes a polymerase binding site and has the activity to initiate transcription of a gene located downstream of the promoter into mRNA, i.e., a DNA site where the polymerase binds to and initiates transcription of the gene, and which is located 5' to the mRNA transcription initiation region.

For example, when the vector of the present invention is a recombinant vector and a prokaryotic cell is used as a host cell, a strong promoter capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL lambda promoter, pR lambda promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, trc promoter, phoA promoter, araBAD promoter, T5 promoter and T7 promoter), a ribosome binding site for initiating translation and a transcription/translation termination sequence are usually included.

In addition, a vector that can be used in the present invention can be prepared by manipulating a plasmid (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pPICZ α series, pUC19, etc.), a phage (e.g., λ gt4 λ B, λ -Charon, λ Δ z1, M13, etc.), or a virus (e.g., SV40, etc.) that is commonly used in the art, but is not limited thereto.

Meanwhile, when the vector of the present invention is a recombinant vector and a eukaryotic cell is used as a host cell, a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter), a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and HSV tk promoter) may be used, and generally, the vector includes a polyadenylation sequence (e.g., bovine growth hormone terminator and polyadenylation sequence derived from SV 40) as a transcription termination sequence.

In addition, the recombinant vector of the present invention includes antibiotic resistance genes commonly used in the art as a selection marker, and may include genes having resistance to, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

The recombinant vector of the present invention may additionally include a different sequence to make it easy to purify the collected target protein, i.e., insulin and/or its analogs. The additionally included sequence may be a tag sequence for protein purification, such as glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), 6-histidine, etc., but the kind of sequence necessary for purification of the target protein is not limited thereto. The fusion protein expressed from the recombinant vector comprising the above tag sequence can be purified by affinity chromatography. For example, when glutathione S-transferase is fused, glutathione (which is a substrate of the enzyme) may be used, and when a 6-histidine tag is used, a desired target protein may be easily collected through a Ni-NTA column. Recombinant microorganisms transformed with the vectors can be constructed using recombinant vectors comprising polynucleotides encoding insulin analogs and/or analog derivatives.

The term "transformation" as used herein refers to the process of introducing DNA into a host cell and making the DNA replicable therein as a chromosomal factor or by completing chromosomal integration and making the DNA replicable therein, which is a phenomenon of artificially causing genetic changes by introducing foreign DNA into the cell.

The transformation method used in the present invention may be any transformation method, and may be easily performed according to a conventional method used in the art. Examples of commonly used transformation methods may include CaCl₂Precipitation method using Dimethylsulfoxide (DMSO) as CaCl₂Hanahan method for improving efficiency by precipitation of reducing agent in process, electroporation, CaPO₄Precipitation, protoplast fusion, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, transformation using PEG, dextran sulfate, lipofectamine anddry/inhibition mediated transformation, and the like.

The method for transforming a recombinant vector comprising a nucleic acid encoding an insulin analogue and/or analogue derivative according to the invention may not be limited to these methods, but any transformation or transfection method commonly used in the art may be used without limitation.

The recombinant transformant of the present invention can be obtained by introducing a recombinant vector comprising a nucleic acid encoding an insulin analog derivative into a host cell. An appropriate host used in the present invention may not be particularly limited as long as it can express the nucleic acid of the present invention. Examples of suitable hosts may include bacteria belonging to the genus Escherichia (e.g., Escherichia coli), bacteria belonging to the genus Bacillus (e.g., Bacillus subtilis), bacteria belonging to the genus Pseudomonas (e.g., Pseudomonas putida), yeast (e.g., Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe), insect cells (e.g., Spodoptera frugiperda (SF9)), and animal cells (e.g., CHO, COS, and BSC), but are not limited thereto.

The term "active form" as used herein means the mature form of insulin, insulin analogue or insulin analogue derivative capable of regulating blood glucose levels in vivo and refers to insulin, insulin analogue or insulin analogue derivative obtained by removing the C-peptide from the proinsulin form and comprising the insulin a-chain and B-chain.

Examples

Hereinafter, the present invention will be described in further detail with reference to examples. It will be apparent to those of ordinary skill in the art that these examples are for illustrative purposes only and should not be construed to limit the scope of the present invention.

In the following examples, an insulin analogue fused to ABD is used as having the same meaning as the long-acting insulin analogue derivative in the present invention.

Example 1: construction of ABD-fused insulin expression vector and strain

Human insulin is synthesized as preproinsulin, the pro sequence is cleaved in the endoplasmic reticulum, and proinsulin is processed in the golgi and endoplasmic reticulum to form mature insulin. Based on this fact, proinsulin was designed for the production of recombinant insulin by a method of expressing proinsulin protein in E.coli and then removing the C chain by trypsin treatment. To improve the expression efficiency and purification efficiency of proinsulin in E.coli, a fusion tag was inserted into the N-terminus and codon-optimized.

Theoretically, the number of Albumin Binding Domains (ABDs) that can be fused to an insulin site is four. However, the N-terminus of the a chain is a position important for insulin activity and is therefore excluded from the fusion site. Although the N-terminus of the B chain is important for the formation of the insulin hexamer, it is contained at the fusion site because the activity of insulin can be maintained. If ABD is fused between the B and C chains, it may have an effect on protein folding. Thus, insulin constructs were designed to have either the B-C-A sequence or the A-C-B sequence as candidate structures. Finally, the genetic structure of insulin was designed for expression of the following three forms of fused ABD:

NdeI-fusion label-B chain-C chain-A chain-connector-ABD-EcoRI,

NdeI-fusion tag-ABD-linker-B chain-C chain-A chain-EcoRI, and

NdeI-fusion label-A chain-C chain-B chain-connector-ABD-EcoRI.

The pJ401(DNA2.0) vector was used as an expression vector. The vector was digested with restriction enzymes NdeI and EcoRI, and then the DNA fragments were separated by electrophoresis on a 1% agarose gel. The gene construct for expressing the ABD fusion protein and the DNA fragment obtained from the expression vector as described above were ligated to each other using T4DNA ligase, thereby constructing a plasmid. Then, each plasmid was transformed into E.coli BL21(DE3) by the calcium chloride method. A transformant strain resistant to kanamycin was selected, and DNA was isolated therefrom. Whether the DNA was correctly inserted was determined by an analytical method based on restriction enzyme digestion.

When the gene was designed to have a structure of NdeI-fusion tag-ABD-linker-B chain-C chain-A chain-EcoRI, it was considered to be inappropriate because the protein refolding rate was low. In addition, when the gene is designed to have an NdeI-fusion tag-a chain-C chain-B chain-linker-ABD-EcoRI structure, insulin fused to ABD has insulin activity, but it shows low refolding rate and enzyme treatment rate, and thus is considered to be unsuitable as a method for preparing an active form of insulin. Thus, among the three forms of ABD fused insulin, the NdeI-fusion tag-B chain-C chain-a chain-linker-ABD-EcoRI structure was chosen and used in subsequent experiments.

Between the a chain and the Albumin Binding Domain (ABD), a linker consisting of 1 to 6 repeats of the (GGGGS) sequence is inserted. In this case, when no linker is introduced, the refolding rate is low, but when a linker consisting of two or more repeats of the (GGGGS) sequence is introduced, there is no significant difference in the refolding rate. At the same time, with (GGGGS)_nThe increase in n in the sequence, through clostripain into insulin active form rate increase, but insulin pharmacokinetics in vivo is not significantly affected. Taken together, the linker was designed to consist of 2 to 4 repeats of the GGGGS sequence.

The amino acid sequence of each part of the insulin fused to ABD used in the present invention is shown in table 1 below.

TABLE 1

Example 2: construction of fused ABD insulin analogs with modified insulin amino acid sequences

In order to produce insulin using recombinant E.coli, a method of converting proinsulin into an active form by using trypsin is necessary. However, trypsin cleaves dibasic amino acids with high efficiency and also cleaves single amino acids such as lysine (Lys) or arginine (Arg), which makes it difficult to produce the desired active form of insulin. In addition, the ABD sequence also includes many lysine (Lys) and arginine (Arg) residues, which makes it difficult to further generate insulin with the desired fusion ABD active by using trypsin.

For this reason, clostripain is used as an enzyme capable of replacing trypsin in order to induce conversion into an active form. In this case, when clostripain reacts with insulin fused to ABD, cleavage of arginine (Arg) at position 22 of the insulin B chain occurs. To solve this problem, lysine (Lys) was substituted for position 22 of the B chain. In this case, the cleavage of the insulin B chain at position 22 by clostripain is significantly reduced and therefore the active form of insulin fused to ABD can be efficiently produced (figure 1).

In addition to the mutations described above, additional mutations are introduced into the insulin fused to the ABD to further improve the stability and in vivo half-life of the insulin fused to the ABD. Five amino acids of each of the a chain and the B chain were substituted, and the substituted positions are shown in table 2 below.

TABLE 2

Analogues	Modified sequences
		Analog 1	V → L at position 3 of A chain
Analog 2	T → D at position 8 of A chain
		Analogue 3	I → K at position 10 of chain A
Analog 4	Y → E at position 14 of chain A
		Analog 5	Y → F at position 19 of chain A
Analog 6	H → T at position 5 of B chain
		Analog 7	S → D at position 9 of B chain
Analog 8	E → A at position 13 of chain B
		Analog 9	L → Q at position 17 of the B chain
Analog 10	F → S at position 24 of chain B
		Analog 11	No additional mutations in the A or B chain

The GeneArt algorithm was used to codon optimize gene expression in e.coli and synthesize the gene to have substituted amino acids.

The plasmid constructed as described above was introduced into E.coli BL21(DE3) in the same manner as described in example 1, thereby constructing an E.coli strain.

Example 3: expression of insulin analogs fused to ABD

To express the insulin analog of the fused ABD, each recombinant e.coli strain was inoculated in 100mL of LB medium and cultured with shaking at 37 ℃ for 16 hours, and the culture was used as a seed culture. 2L of LB medium was added to a 7L fermentor (New Brunswick BioFlo), sterilized, and then the seed culture was inoculated. The culture was carried out at a temperature of 35 ℃ and an air flow rate of 3vvm with a stirring speed of 1,000rpm, and the pH during the culture was maintained at 6.8 with ammonia and phosphoric acid. At the point in the medium where the carbon source was depleted, the feed was started and protein expression was induced simultaneously with IPTG. After induction of expression, cultivation was further carried out for 10 hours, and the recombinant strain was recovered using a centrifuge.

The insulin analogs fused to ABD were expressed as inclusion bodies in e.coli strains and even when amino acid mutations were introduced into the insulin domain, the expression levels appeared in the vector system used in the present invention (figure 2).

Example 4: induction of cell lysis and solubilization/refolding

Each strain expressing an ABD-fused insulin or ABD-fused insulin analog was suspended in lysis buffer (20mM Tris, 10mM EDTA, 10% sucrose, 0.2M NaCl, pH 8.0) and the cells were lysed using a high pressure homogenizer. The lysed cells were centrifuged in a high speed centrifuge at 7,000rpm and the soluble proteins and some cell debris were removed, thereby separating the pellet including the inclusion bodies. The isolated inclusion bodies were washed with a buffer (containing 1% Triton X-100, 0.2M NaCl and 1M urea) and then centrifuged at 7,000 rpm. The precipitated inclusion bodies were washed two additional times with distilled water and then stored at-80 ℃ until use.

The cryopreserved inclusion bodies were dissolved in solubilization buffer (25mM Tris, 8M Urea, 30mM cysteine-HCl, pH 10.5) and then diluted in refolding buffer (25mM Tris-HCl, pH 10.5) and refolded at 4 ℃ for 16 hours. Whether refolding occurred was determined by RP-HPLC analysis. When the solubilized solution was analyzed by RP-HPLC, a protein peak was observed at about 20 minutes due to high hydrophobicity, but when refolding was performed, the peak was observed to shift to 16 minutes (fig. 3). This is because the hydrophobicity decreases as refolding progresses compared to the hydrophobicity in the solubilizing solution. Refolding continued until no more protein peak shifts were observed during analysis by RP-HPLC.

Example 5: conversion to the active form by use of an enzyme

In order to produce insulin using recombinant E.coli strains, it is necessary to convert proinsulin into the active form by using trypsin. However, ABD-fused insulin or ABD-fused insulin analogues have multiple trypsin cleavage sites in their sequence and for this reason clostripain is used as an enzyme capable of replacing trypsin in order to induce conversion to the active form.

When the conversion to the active form is induced using clostripain, cleavage occurs at position 22 of the insulin B chain, making it difficult to produce the desired form of insulin. For this reason, insulin analogs fused to ABD obtained by substituting arginine (Arg) for lysine (Lys) at B-chain position 22 are used to produce the active form of insulin. Meanwhile, the ABD fused insulin analogue 4 was used in all experiments unless otherwise stated.

Clostripain contains a cysteine (Cys) in its active site and therefore requires reducing conditions to exhibit enzymatic activity. Furthermore, in the case of treatment of refolding solutions with reducing agents to maintain clostridial protease activity, the disulfide bonds in the insulin region are most likely to be disrupted. For this reason, it is required to deduce conditions for improving the yield of the enzymatic reaction without disrupting disulfide bonds. Therefore, experiments were conducted on the treatment concentration and other treatments using the reducing agent DTT contained in the enzymatic reaction solution.

In order to convert the insulin analogue of the fused ABD in its precursor form into the active form, carboxypeptidase b (cpb) is additionally required. CpB is an enzyme that cleaves basic amino acids at the carboxy terminus, and detailed conditions were also tested. Specifically, the time of treatment with clostripain and CpB, the possibility of simultaneous treatment, the amount of enzyme treatment, the temperature of enzyme treatment, and the appropriate pH level were evaluated.

Since clostripain exists as pro-form (pro-form), it is activated by inducing auto-cleavage. For this purpose, freeze-dried clostripain (Waxinton, USA) was dissolved in distilled water and then activated for 30 minutes at 4 ℃ after addition of activation buffer (500mM Tris, 50mM DTT, 25mM CaCl2 pH 7.8). Activated clostripain is added to the refolded protein at a concentration of 0.1 to 5 units per mg of protein and allowed to react for 2 to 8 hours at 25 ℃ to 40 ℃. After the clostripain reaction, CpB was added at a concentration of 0.001 to 1 unit per 1mg protein and allowed to react. The enzymatic reaction was stopped by lowering the pH to 3.5 or less using HCl.

5-1: evaluation of the efficiency of conversion to active form after addition of DTT

To examine the enzymatic reaction conditions of clostripain and CpB, DTT was added to the refolding solution to a concentration of 0 to 0.4mM, and then the active form of insulin produced by clostripain was observed by SDS-PAGE. It was shown that the disulfide bond between the insulin a and B chains was broken when more than 0.5mM DTT was added (data not shown).

Meanwhile, as can be seen in fig. 4, the rate of conversion of the insulin analog fused to ABD into the active form was low without addition of DTT in the enzymatic reaction solution, but the efficiency of conversion into the active form increased with increasing concentration of DTT.

5-2: evaluation of efficiency of conversion to insulin active form at different time points of CpB addition

To convert the proinsulin analog fused to ABD to the active form, the proinsulin analog should be treated with both clostripain and CpB. In this regard, the point in time at which CpB was added was determined. To cleave the insulin C-peptide and fusion tag, treatment with clostripain should be performed first, and then treatment with CpB should be performed to remove arginine (Arg) from the cleavage site. However, to shorten the production time, treatment with CpB 2 to 4 hours after treatment with clostripain was compared with simultaneous treatment with clostripain and CpB.

As a result, as can be seen in fig. 5, the enzymatic reaction pattern in the simultaneous treatment with clostripain and CpB was not different from that in the case where the treatment with clostripain was performed first. Therefore, to increase the efficiency of the process, simultaneous treatment with clostripain and CpB was chosen.

5-3: pH conditions in simultaneous treatment with clostripain and CpB

Clostripain is known to exhibit optimal activity at pH 7.4 to 7.8, and CpB exhibits optimal activity at pH 9.0. Since the optimal pH ranges of the two enzymes are different from each other, the pH range showing the highest efficiency of conversion into the active form of insulin was determined by simultaneous treatment with the two enzymes. The theoretical pI values of all the ABD fused insulin analogues shown in table 2 according to the present invention are close to 6 and thus involve precipitation. Thus, a pH of 6.0 or lower is excluded. Further, at a pH of 9.0 or higher, aggregates increase. For these reasons, the evaluation was carried out at a pH between 6.0 and 9.0.

Simultaneous treatment with both enzymes at a pH range of 6.5 to 8.5 showed high efficiency of conversion to the active form of insulin. Furthermore, as shown in fig. 6, the efficiency of conversion to the insulin active form is particularly high in the pH range of 6.5 to 7.5.

5-4: evaluation of the efficiency of conversion to the active form of insulin after additional addition of clostripain and DTT

As shown in example 5-1, the efficiency of conversion to the active form of insulin increases when DTT is added. However, the half-life of DTT is known to decrease rapidly at high pH and temperature, and thus it is considered difficult to maintain the activity of clostripain for a long time in the enzymatic reaction. Therefore, a method capable of maintaining clostripain activity for a long period of time was studied.

For this reason, the method in which 1U/mg clostripain was additionally added 4.5 hours after the start of the enzymatic reaction was evaluated, compared with the method in which 0.2mM DTT was additionally added. As a result, as shown in fig. 7, the efficiency of conversion into the active form was not significantly increased even when clostripain was additionally added, but the conversion into the active form was significantly increased even when DTT was additionally added, even when clostripain was not additionally added. Furthermore, when both clostripain and DTT were additionally added, a relatively small amount of unconverted form was observed, and the efficiency of conversion to the active form was increased.

However, analysis of the enzymatic reaction solution by mass spectrometry showed that conversion to the active form of insulin was significantly increased when DTT was added, but non-specific cleavage of insulin was induced when DTT and clostripain were simultaneously added, resulting in an increase in impurities. Thus, it can be seen that the addition of the reducing agent DTT alone will favor the conversion to the active form of insulin (fig. 8).

5-5: evaluation of efficiency of conversion to active form of insulin at various temperatures

An evaluation was performed to determine the temperature range in which clostripain and CpB would show optimal activity. At this time, in view of the process operation on the production scale, temperatures of 4 ℃ or less and 40 ℃ or more were excluded and evaluation was performed at a temperature of 10 ℃ to 30 ℃. As a result, as shown in fig. 9, it can be seen that when the temperature is increased from 10 ℃ to 20 ℃ and 30 ℃, the amount of the unconverted form is smaller and the conversion to the active form proceeds faster. Furthermore, it was shown that the conversion to the active form proceeded faster with increasing concentration of DTT.

Example 6: purification of ABD-fused analogues of insulin

According to the manufacturer's instructions, use firstEMD COO- (M) (Merck) samples enzymatically treated with clostripain and CpB were purified by ion exchange resin chromatography and then used according to the manufacturer's instructionsP100 RP-18e (Merck) was further purified by reverse phase chromatography.

As a result, as can be seen in fig. 10, the active form of the fused ABD insulin analogue can be purified without impurities by both purification methods.

Example 7: measurement of binding affinity of insulin analogs fused to ABD for albumin

To measure the binding affinity of the insulin analogue protein fused to ABD for albumin, a surface plasmon resonance (SPR, BIACORE 3000, GE healthcare) analysis method was used. Recombinant human serum albumin was immobilized on a CM5 chip by amine coupling and ABD or ABD-fused insulin analogues diluted to five or more concentrations were allowed to bind to the recombinant human serum albumin and the affinity of the ABD or ABD-fused insulin analogue for human serum albumin was determined.

As a result, as can be seen in table 3 below, the affinity of insulin analogues to human serum albumin is maintained at pM levels, although it is lower than that of ABD itself.

TABLE 3

Example 8: comparison of the affinity of native insulin and ABD fused insulin analogs for the insulin receptor

To measure the binding affinity of native insulin and insulin analogs fused to ABD for the insulin receptor, a surface plasmon resonance (SPR, BIACORE 3000, GE healthcare) analysis method was used. The insulin receptor was immobilized on a CM5 chip by amine coupling, and each of natural insulin and an insulin analog fused to ABD diluted to five or more concentrations was bound to the insulin receptor, and the affinity of each of the natural insulin and the insulin analog fused to ABD for the insulin receptor was determined.

As a result, as can be seen in table 4 below, the affinity of the insulin analogs fused to ABD was reduced compared to native insulin. In particular, the affinity of analogue 3 showed the greatest decrease and decreased to a level of about 19.4% relative to native insulin.

TABLE 4

Analogues	Ka(1/Ms)	Kd(1/s)	KD(M)
				Analog 1	8.54×104	-	-
Analog 2	4.38×104	3.15×10-3	7.2×10-8
				Analogue 3	2.52×104	2.18×10-3	8.65×10-8
Analog 4	2.82×104	1.31×10-3	4.65×10-8
				Analog 5	4.07×104	2.34×10-3	5.75×10-8
Analog 6	5.23×104	2.27×10-3	4.33×10-8
				Analog 7	5.05×104	-	-
Analog 8	4.86×104	1.47×10-3	3.04×10-8
				Analog 9	2.13×104	1.29×10-3	6.06×10-8
Analog 10	3.83×104	-	-
				Insulin	1.03×105	1.72×10-3	1.68×10-8

The efficacy of insulin analogues fused to ABD was evaluated in comparison to insulin glargine in a model of type 1 diabetes induced by streptozotocin. Analogs 1, 3 and 10 were excluded from the candidates because they showed 50% or less of hypoglycemic capacity compared to insulin glargine.

Example 9: hexamer synthesis

Insulin binds to zinc in vivo, forming a stable hexamer structure. The hexameric nature of insulin is also used in formulation development and may play an important role in increasing the half-life in insulin. Thus, it was analyzed by size exclusion chromatography whether insulin analogs fused to ABD retain the property of forming hexamers. The results show that by adding zinc and phenol, analog 4 retained the ability to form hexamers (fig. 11). Hexamer formation was also observed in all analogues, excluding analogues 7, 9 and 10. Thus, among the analogs having the sequences shown in table 2, analogs 7 and 9 were further excluded from the candidates.

Example 10: in vivo pharmacokinetic and hypoglycemic Capacity assessment of ABD fused insulin analogs

To evaluate the in vivo pharmacokinetics of the six ABD-fused insulin analogs, normal SD rats (6 weeks old) were administered each insulin analog subcutaneously and then bled at 0, 1, 4, 8, 24, 48, 72, and 96 hours. The concentration of each of the insulin analogs fused to ABD remaining in the blood at each time point was measured using ELISA. Further, using a portion of the collected blood, a time-dependent blood glucose level is measured with a blood glucose monitoring device.

As a result, as can be seen in table 5 below, the insulin analogs fused to ABD showed significantly increased half-lives compared to the native insulin known to have a half-life of 5 minutes. Analogue 11, obtained by substituting only position 22 of the insulin B chain, showed a half-life of 7.2 hours, and analogue 4, which showed the greatest increase in half-life, was evaluated to have a half-life of 9.9 hours.

The time during which the blood glucose level of normal animals remained at a reduced level was analyzed. The results show that analogs 5, 6 and 8 have increased half-lives, but that blood glucose levels are maintained for a shorter period of time. Compared to these analogues, analogue 4 had the best ability to lower blood glucose levels and maintained its efficacy for the longest time (figure 12).

TABLE 5

Parameter(s)

Analog 2

Analog 4

Analog 5

Analog 6

Analog 8

Analog 11

T 1/2(hours)

9.4±3.4

9.9±0.1

9.1±0.7

8.4±1.7

8.8±0.4

7.2±2.3

MRT (hours)

17.0±2.2

22.1±1.3

21.2±0.3

15.9±1.5

19.8±1.5

16.3±0.9

Although the present invention has been described in detail with reference to specific features, it should be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Practicality of use

In the conventional method of converting proinsulin to the active form by using trypsin, the albumin binding domain of the novel long-acting insulin analog derivative newly developed by the inventors is also cleaved, thus making it difficult to convert the novel long-acting insulin analog derivative to the active form. To overcome this difficulty, the insulin analogue derivatives according to the invention are converted into the active form using clostripain. Thus, the methods of the present invention can be effectively used to produce long-acting therapeutic agents for the treatment of diabetes.

Text file

See the attached sequence listing.

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