Method for forming a conjugate between a sulfonamide and a polypeptide.

Abstract

A method for forming a conjugate of a sulfonamide and a polypeptide, comprising: a) providing an activated sulfonamide, the activated sulfonamide corresponding to Formula (I); b) providing an aqueous solution of a polypeptide having a free amino group, the aqueous solution optionally containing an alcohol; c) contacting the aqueous solution of b) with the activated sulfonamide of a); and d) reacting the activated sulfonamide with the polypeptide having a free amino group to obtain a solution containing a conjugate of the sulfonamide and the polypeptide, wherein the sulfonamide is covalently bonded to the polypeptide. Also, related conjugates, processes, procedures, proinsulin, etc.

Description

Technical Field

[0001] Polypeptides are used in various application fields. Polypeptides have several functional groups, such as the NH2 group at the N-terminus, the COOH group at the C-terminus, and usually one or more additional functional groups directly attached to the polypeptide chain or side chains, so coupling with other compounds is complex. Usually, by-products are formed that reduce the yield of the desired product and are difficult to separate from the desired product.

[0002] Human insulin is a polypeptide of 51 amino acid residues and is divided into two amino acid chains: an A chain having 21 amino acid residues and a B chain having 30 amino acid residues. These chains are connected to each other by two disulfide bridges. There is a third disulfide bridge between the cysteines at positions 6 and 11 of the A chain. Some of the products currently used for the treatment of type 1 diabetes are insulin analogs, i.e., insulin variants with a sequence different from that of human insulin due to one or more amino acid substitutions in the A chain and / or B chain.

[0003] Like many other peptide hormones, human insulin has a short in vivo half-life. Therefore, it is administered frequently, which is associated with discomfort for the patient. Thus, insulin analogs having an increased in vivo half-life and, consequently, an extended duration of action are desired.

[0004] Patent Document 1 discloses protease-stabilized insulin analogs.

[0005] In another approach, a long-chain fatty acid group is conjugated to the epsilon amino group of LysB29 of insulin. The presence of this group allows insulin to bind to serum albumin by a non-covalent, reversible bond. As a result, this insulin analog has a significantly extended time-action profile compared to human insulin (see, e.g., Non-Patent Document 1; or Patent Document 2). Patent Document 3 discloses another conjugate comprising an insulin analog and a covalent functional group that allows insulin to bind to serum albumin by a non-covalent, reversible bond: the document describes an acylated analog of human insulin, which is derivatized by acylation of the epsilon amino group of the lysine residue at position A22 with an [acyl]-[linker]-group, where the linker group is an amino acid chain consisting of 1 to 10 amino acid residues selected from gGlu (gamma glutamic acid residue) and / or OEG (8-amino-3,6-dioxaoctanoic acid residue).

[0006] One of the obstacles in the synthesis of such conjugates (i.e., in which an insulin analog is coupled with another molecule, resulting in the non-covalent, reversible binding of insulin (analog) to serum albumin) is that the coupling step often results in insufficient yield, and the yield is further reduced due to the need for numerous workup and purification steps. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] WO2008 / 034881A1 (Novo Nordisk, Nielsen) [Patent Document 2] WO2009 / 115469A1 (Novo Nordisk, Madsen) [Patent Document 3] WO2017 / 032798A1 (Novo Nordisk, Madsen) [Non-patent literature]

[0008] [Non-Patent Document 1] Mayer et al., Inc. Biopolymers (Pept Sci) 88:687~713, 2007 [Overview of the Initiative] [Means for solving the problem]

[0009] This specification provides a method for forming conjugates of sulfonamides and polypeptides that enables relatively high overall yields in conjugate synthesis, i.e., yields of more than 20%, 30%, 40%, or 50%, depending on the polypeptide used. This method is described in Section A below.

[0010] Furthermore, a process for producing a conjugate of an albumin binder and an insulin polypeptide is provided, comprising: a) preparing proinsulin comprising an insulin B chain, a linker peptide, and an insulin A chain from the N-terminus to the C-terminus; b) cleaving the proinsulin prepared in step a) between the last amino acid of the insulin B chain and the first amino acid of the linker peptide with a first protease, thereby producing an insulin precursor, wherein the insulin precursor comprises an N-terminally extended A chain including an insulin B chain, a linker peptide, and an A chain; c) contacting the insulin precursor with an albumin binder, thereby producing a conjugate of the albumin binder and the insulin precursor; and d) cleaving the N-terminally extended A chain of the insulin precursor contained in the conjugate between the last amino acid of the linker peptide and the first amino acid of the A chain with a second protease, thereby producing a conjugate of the albumin binder and mature insulin. This process is described in Section B below. [Brief explanation of the drawing]

[0011] [Figure 1] This figure shows the blood glucose level (y axis) [percentage of placebo] as time (x axis) progresses over time intervals after subcutaneous administration of insulin conjugates 1-5. [Figure 2] This figure shows the curves for insulin conjugates 1-5, with the y-axis representing normalized plasma concentration [ng / ml] and the x-axis representing time [hours]. [Figure 3] This figure shows insulin conjugate 1 (see Example 4 for further details). The sequences of the A chain (SEQ ID NO: 47) and B chain (SEQ ID NO: 48) are shown by three-letter codes, except for the last amino acid of the B chain (lysine at position B29). The structure of the lysine residue is shown. The lysine residue is covalently bonded to the binder (via the epsilon amino acid of the lysine residue). [Figure 4]This figure shows insulin conjugate 2. Except for the last amino acid of chain B (lysine at position B29), the sequences of chain A (SEQ ID NO: 47) and chain B (SEQ ID NO: 48) are shown with three-letter codes. The structure of the lysine residue is shown. The lysine residue is covalently bonded to the binder (via the epsilon amino acid of the lysine residue). [Figure 5] This figure shows insulin conjugate number 3. Except for the last amino acid of the B chain (lysine at position B29), the sequences of the A chain (sequence number: 77) and the B chain (sequence number: 78) are shown with three-letter codes. The structure of the lysine residue is shown. The lysine residue is covalently bonded to the binder (via the epsilon amino acid of the lysine residue). [Figure 6] This figure shows insulin conjugate number 4. Except for the last amino acid of the B chain (lysine at position B29), the sequences of the A chain (sequence number: 43) and B chain (sequence number: 44) are shown with three-letter codes. The structure of the lysine residue is shown. The lysine residue is covalently bonded to the binder (via the epsilon amino acid of the lysine residue). [Figure 7] This figure shows the sequences of insulin precursors (B chain: SEQ ID NO: 48, N-terminally elongated A chain: SEQ ID NO: 107). [Figure 8] This figure shows a conjugate containing an insulin precursor and a sulfonamide (B chain: SEQ ID NO: 48, N-terminally extended A chain: SEQ ID NO: 107). [Modes for carrying out the invention]

[0012] Section A: Methods for forming conjugates of sulfonamides and polypeptides To increase the yield of conjugate synthesis, the number of required steps and the number and ratio of by-products play a significant role. Therefore, a more preferable synthetic route is needed that reduces the number of steps and at least increases the ratio of desired products to unwanted by-products.

[0013] Surprisingly, it has been found that using a specifically activated sulfonamide in coupling reactions with polypeptides helps improve the ratio of the desired product to unwanted by-products. Furthermore, the use of polypeptide precursors in combination with the use of specific enzymes enables a so-called "one-pot" reaction, meaning that the coupling of the activated sulfonamide and the cleavage of the polypeptide precursor to obtain the final polypeptide can be performed in a single reaction vessel without the need for separation or intermediate purification steps. This avoids the use of additional protecting groups for the sulfonamide, which also reduces the need for intermediate separation or purification steps. The desired product, namely the conjugate of sulfonamide and polypeptide, can be obtained in a yield of 50% or more.

[0014] Therefore, in a first embodiment, this specification provides a method for forming a conjugate of a sulfonamide and a polypeptide: a) A step of preparing an activated sulfonamide, wherein the activated sulfonamide is of formula (I): [ka] [In the formula, A is selected from the group consisting of an oxygen atom, a -CH2CH2- group, an -OCH2- group, and a -CH2O- group; E represents a -C6H3R- group, where R is a hydrogen atom or a halogen atom, where the halogen atom is selected from the group consisting of fluorine, chlorine, bromine, and iodine atoms; X represents a nitrogen atom or a -CH- group; m is an integer between 5 and 17; n is an integer between 0 and 3; p is either 0 or 1; q is either 0 or 1; r is an integer in the range of 1 to 6; s is either 0 or 1; t is either 0 or 1; R 1 These include hydrogen atoms, halogen atoms, C1-C3 alkyl groups, and halogenated C1-C3 alkyl groups. Represents at least one residue selected from the group of 3 alkyl groups; R 2 This represents at least one residue selected from the group consisting of a hydrogen atom, a halogen atom, a C1-C3 alkyl group, and a halogenated C1-C3 alkyl group; R x This represents an activating group; Here, the combination where s is 1, p is 0, n is 0, A is an oxygen atom, and t is 1 corresponds to the process that is excluded in formula (I); b) A step of preparing an aqueous solution of polypeptide, wherein the aqueous solution may contain alcohol; c) The step of contacting the aqueous solution of b) with the activated sulfonamide of a); and d) A step of reacting an activated sulfonamide with a polypeptide to obtain a solution containing a conjugate of the sulfonamide and a pharmaceutical active ingredient or diagnostic compound, wherein the sulfonamide is covalently bonded to the polypeptide. A method including this is provided.

[0015] In at least one embodiment, the activated sulfonamide of formula (I) is R in the non-coupling state of the activated sulfonamide of formula (I). x The terminal carboxyl group supporting the group is covalently bonded to the polypeptide by covalently bonding to an appropriate functional group of the polypeptide, such as an amino group or a hydroxyl group of the polypeptide.

[0016] A "polypeptide" is a peptide containing at least two amino acid residues. In some embodiments, the peptide contains at least 10 amino acid residues, or at least 20 amino acid residues. In some embodiments, the peptide contains 1000 or fewer amino acid residues, such as 500 or fewer amino acid residues, for example, 100 or fewer amino acid residues.

[0017] As used herein, the term “polypeptide” includes any diagnostic chemical or biological polypeptide, a pharmaceutically active chemical or biological polypeptide, and any pharmaceutically acceptable salt of a diagnostic polypeptide or a pharmaceutically active polypeptide, as well as any mixture thereof, which produce any diagnostic or pharmacological effect and are used for the diagnosis, treatment, or prevention of a disease.

[0018] A "polypeptide" is a mature polypeptide or its precursor.

[0019] In at least one embodiment, the polypeptide is selected from the group consisting of antidiabetic polypeptides, anti-obesity polypeptides, appetite-regulating polypeptides, antihypertensive polypeptides, polypeptides for the treatment and / or prevention of complications caused by or associated with diabetes, polypeptides for the treatment and / or prevention of complications and disorders caused by or associated with obesity, and precursors of any one of these polypeptides.

[0020] In at least one embodiment of this method, the polypeptide is an antidiabetic polypeptide or its precursor. In some embodiments, the polypeptide is GLP-1, a GLP-1 analog, a GLP-1 receptor agonist; a dual GLP-1 receptor / glucagon receptor agonist; human FGF21, an FGF21 analog, an FGF21 derivative; insulin (e.g., human insulin), an insulin analog, an insulin derivative, or a precursor of any one of these polypeptides.

[0021] According to at least one embodiment of the present method, the polypeptide is selected from the group consisting of insulin, insulin analogs, GLP-1, and GLP-1 analogs (e.g., GLP(-1) agonists), as well as a precursor of any one of these polypeptides.

[0022] As used herein, the term "GLP-1 analog" refers to a polypeptide having a molecular structure that can be formally derived from the structure of a naturally occurring glucagon-like peptide-1 (GLP-1), for example, the structure of human GLP-1, by the deletion and / or replacement of at least one amino acid residue that occurs in naturally occurring GLP-1, and / or the addition of at least one amino acid residue. The amino acid residue to be added and / or replaced may be an encoding amino acid residue, another naturally occurring residue, or a purely synthetic amino acid residue.

[0023] As used herein, the term “GLP(-1) receptor agonist” refers to an analog of GLP(-1) that activates the glucagon-like peptide-1 receptor (GLP-1 receptor). Examples of GLP(-1) agonists include, but are not limited to, lixisenatide, exenatide / exendin-4, semaglutide, taspoglutide, albiglutide, and dulaglutide.

[0024] Lixisenatide has the following amino acid sequence (SEQ ID NO: 98): His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-Lys-Lys-Lys-Lys-Lys-NH2

[0025] Exenatide has the following amino acid sequence (SEQ ID NO: 99): His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0026] The albumin-binding agent coupled to semaglutide-Lys(20) has the following amino acid sequence (SEQ ID NO: 100): His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEAc-AEEAc-γ-Glu-17-carboxyheptadecanoyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly

[0027] Dulaglutide (GLP1(7-37) coupled to the fc fragment via a peptide linker) has the following amino acid sequence (SEQ ID NO: 101): His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly

[0028] As used herein, the term "FGF-21" means "fibroblast growth factor 21." FGF-21 compounds may be human FGF-21, an analog of FGF-21 (referred to as "FGF-21 analog"), or a derivative of FGF-21 (referred to as "FGF-21 derivative").

[0029] According to at least one embodiment of this method, the polypeptide is insulin, an insulin analog, or a precursor of insulin or an insulin analog. As used herein, the term "insulin analog" is derived from the structure of naturally occurring insulin (also referred herein as "parent insulin," e.g., human insulin). This refers to peptides having a molecular structure that can be formally derived by the deletion and / or substitution of at least one amino acid residue, and / or the addition of at least one amino acid residue, in existing insulin. The amino acid residue added and / or replaced may be an encoding amino acid residue, another naturally occurring residue, or a purely synthetic amino acid residue. The analogs referred to herein are capable of lowering blood glucose levels in vivo, for example, in human subjects.

[0030] In at least one embodiment, “insulin analog” refers to an analog of human insulin (human insulin analog) whose sequence differs from that of human insulin by one or more amino acid substitutions in the A chain and / or B chain.

[0031] In some embodiments, insulin, insulin analogs, or precursors of insulin or insulin analogs have an epsilon amino group of lysine present in insulin or insulin analogs or precursors of insulin or insulin analogs, or the N-terminal amino group of the B chain of insulin, insulin analogs, insulin precursors, or precursors of insulin analogs. For example, insulin or insulin analogs or their precursors have one lysine in the A chain and / or B chain. In some embodiments, insulin, insulin analogs, insulin precursors, or precursors of insulin analogs have one lysine in the A and B chains. In some embodiments, the activated sulfonamide of formula (I) is R in the pre-coupling state of the activated sulfonamide of formula (I). x The terminal carboxyl group supporting the group forms an amide bond with the amino group, thereby covalently bonding to the lysine of the polypeptide, for example, to the epsilon-amino group of lysine.

[0032] In some embodiments, the insulin analogs provided herein include two peptide chains, namely chain A and chain B. Typically, the two chains are connected by disulfide crosslinks between cysteine ​​residues. For example, in some embodiments, the insulin analogs provided herein include three disulfide crosslinks: one between cysteine ​​at positions A6 and A11, one between cysteine ​​at position A7 of chain A and cysteine ​​at position B7 of chain B, and one between cysteine ​​at position A20 of chain A and cysteine ​​at position B19 of chain B. Thus, the insulin analogs provided herein may include cysteine ​​residues at positions A6, A7, A11, A20, B7, and B19.

[0033] Insulin mutations, i.e., mutations in parent insulin, are indicated herein by reference to the chain of the analog, i.e., either the A chain or the B chain, the position of the mutated amino acid residue in the A chain or B chain (e.g., A14, B16, and B25), and the three-letter code of the amino acid that substitutes the native amino acid in parent insulin. The term "desB30" refers to an analog in which the B30 amino acid is missing from the parent insulin (i.e., the amino acid at position B30 is absent). For example, Glu(A14)Ile(B16)desB30 human insulin is an analog of human insulin in which the amino acid residue at position 14 (A14) of the A chain of human insulin is substituted with glutamic acid, the amino acid residue at position 16 (B16) of the B chain is substituted with isoleucine, and the amino acid at position 30 of the B chain is deleted (i.e., absent).

[0034] The insulin analogs that can be used in the methods described herein include at least one mutation (amino acid substitution, deletion, or addition) to the parent insulin. The term “at least one” as used herein means one or more than one, for example, “at least two,” “at least three,” “at least four,” “at least five,” etc. In some embodiments, the insulin analogs provided herein include at least one The insulin analog contains at least one mutation in the B chain and at least one mutation in the A chain. In a further embodiment, the insulin analog provided herein contains at least two mutations in the B chain and at least one mutation in the A chain.

[0035] In some embodiments, the insulin analog comprises an A chain and a B chain, wherein the A chain contains at least one mutation from the A chain of parent insulin (e.g., human insulin), and / or the B chain contains at least one mutation from the parent insulin (e.g., human insulin). For example, the at least one mutation from the A chain of human insulin is a substitution at position A14, such as a substitution with an amino acid selected from the group consisting of glutamic acid (Glu), aspartic acid (Asp), and histidine (His), and / or a substitution at position A21, such as a substitution with glycine (Gly). For example, mutations in the B chain of human insulin can include substitutions at position B16, such as substitutions with amino acids selected from the group consisting of valine (Val), isoleucine (Ile), leucine (Leu), alanine (Ala), or histidine (His); substitutions at position B25, such as substitutions with valine (Val), isoleucine (Ile), leucine (Leu), alanine (Ala), or histidine (His); and / or deletions at position B30.

[0036] In some embodiments, the insulin analog includes a deletion at position B30. In some embodiments, the insulin analog may include a substitution at position B16, a deletion at position B30, and a substitution at position A14. In some embodiments, the insulin analog may include a substitution at position B25, a deletion at position B30, and a substitution at position A14. In some embodiments, the insulin analog may include a substitution at position B16, a substitution at position B25, a deletion at position B30, and a substitution at position A14.

[0037] The insulin analogs provided herein may include mutations in addition to those described above. In some embodiments, the number of mutations does not exceed a certain number. In some embodiments, the insulin analog contains fewer than 12 mutations (i.e., deletions, substitutions, additions) relative to parent insulin. In another embodiment, the analog contains fewer than 10 mutations relative to parent insulin. In another embodiment, the analog contains fewer than 8 mutations relative to parent insulin. In another embodiment, the analog contains fewer than 7 mutations relative to parent insulin. In another embodiment, the analog contains fewer than 6 mutations relative to parent insulin. In another embodiment, the analog contains fewer than 5 mutations relative to parent insulin. In another embodiment, the analog contains fewer than 4 mutations relative to parent insulin. In another embodiment, the analog contains fewer than 3 mutations relative to parent insulin.

[0038] As used herein, the term “parent insulin” refers to naturally occurring insulin, i.e., unmutated wild-type insulin. In some embodiments, parent insulin is animal insulin, such as mammalian insulin. For example, parent insulin may be human insulin, porcine insulin, or bovine insulin.

[0039] In some embodiments, the parent insulin is human insulin. The sequence of human insulin is well known in the art and is shown in Table 1 of the Examples section. Human insulin comprises an A chain having the amino acid sequence shown in SEQ ID NO: 1 (GIVEQCCTSICSLYQLENYCN) and a B chain having the amino acid sequence shown in SEQ ID NO: 2 (FVNQHLCGSHLVEALYLVCGERGFFYTPKT).

[0040] In another embodiment, the parent insulin is bovine insulin. The sequence of bovine insulin is well known in the art. Bovine insulin has an A chain having the amino acid sequence shown in SEQ ID NO: 81 (GIVEQCCASVCSLYQLENYCN) and the amino acid sequence shown in SEQ ID NO: 82 (FVNQHLCGSHLVEALYLVCGERGFFYTPKA) It includes a B chain having a sequence.

[0041] In another embodiment, the parent insulin is porcine insulin. The sequence of porcine insulin is well known in the art. Porcine insulin comprises an A chain having the amino acid sequence shown in SEQ ID NO: 83 (GIVEQCCTSICSLYQLENYCN) and a B chain having the amino acid sequence shown in SEQ ID NO: 84 (FVNQHLCGSHLVEALYLVCGERGFFYTPKA).

[0042] Human, bovine, and porcine insulin contains three disulfide crosslinks: one between cysteine ​​at positions A6 and A11, one between cysteine ​​at position A7 of chain A and cysteine ​​at position B7 of chain B, and one between cysteine ​​at position A20 of chain A and cysteine ​​at position B19 of chain B.

[0043] As used herein, the term “mature insulin” includes parent insulin, such as human insulin, and insulin analogs. In some embodiments, mature insulin is an insulin analog, such as those listed in Table 1 of the Examples section. For example, the insulin analog may be insulin analog 24 in the table.

[0044] The insulin analogs provided herein typically have an insulin receptor binding affinity that is reduced compared to that of the corresponding parent insulin, such as human insulin.

[0045] The insulin receptor can be any mammalian insulin receptor, such as the insulin receptor of a cow, pig, or human. In some embodiments, the insulin receptor is a human insulin receptor, for example, human insulin receptor isoform A or human insulin receptor isoform B (as used in the Examples section).

[0046] Conveniently, the human insulin analogs provided herein exhibit significantly reduced binding affinity to the human insulin receptor compared to the binding affinity of human insulin to the human insulin receptor (see Examples). Therefore, the insulin analogs have extremely low clearance rates, i.e., extremely low insulin receptor-mediated clearance rates.

[0047] In some embodiments, the insulin analog used in the method of the present invention has, i.e., exhibits, a binding affinity to the corresponding insulin receptor of less than 20% compared to its parent insulin. In another embodiment, the insulin analog provided herein has a binding affinity to the corresponding insulin receptor of less than 10% compared to its parent insulin. In yet another embodiment, the insulin analog provided herein has a binding affinity to the corresponding insulin receptor of less than 5% compared to its parent insulin, for example, less than 3% compared to its parent insulin. For example, the insulin analog provided herein may have a binding affinity to the corresponding insulin receptor of 0.1% to 10%, for example 0.3% to 5%, compared to its parent insulin. Furthermore, the insulin analog provided herein may have a binding affinity to the corresponding insulin receptor of 0.5% to 3%, for example 0.5% to 2%, compared to its parent insulin.

[0048] Methods for determining the binding affinity of insulin analogs to insulin receptors are well known in the art. For example, insulin receptor binding affinity is determined by the binding affinity of [125I]-labeled parent insulin to the insulin receptor, such as [125I]-labeled human insulin. This can be determined by a scintillation proximity assay based on the evaluation of competitive binding between thrin and an (unlabeled) insulin analog. The insulin receptor may be present on the membrane of cells overexpressing recombinant insulin receptors, such as CHO (Chinese hamster ovary) cells. In one embodiment, the insulin receptor binding affinity is determined as described in the Examples section.

[0049] When naturally occurring insulin or insulin analogs bind to the insulin receptor, the insulin signaling pathway is activated. The insulin receptor possesses tyrosine kinase activity. When insulin binds to its receptor, a conformational change is induced that stimulates autophosphorylation of the receptor at tyrosine residues. Autophosphorylation of the insulin receptor stimulates the receptor's tyrosine kinase activity toward intracellular substrates involved in signal transduction. Therefore, autophosphorylation of the insulin receptor by an insulin analog is considered a measure of the signal transduction induced by that analog.

[0050] Therefore, insulin analogs that can be used in the methods of the present invention may have low binding activity and consequently low receptor-mediated clearance rates, but may still be able to induce relatively high signal transduction. Accordingly, the insulin analogs provided herein can be used as long-acting insulin. In some embodiments, the insulin analogs provided herein can induce 1-10%, for example 2-8%, of insulin receptor autophosphorylation compared to parent insulin (e.g., human insulin). Furthermore, in some embodiments, the insulin analogs provided herein can induce 3-7%, for example 5-7%, of insulin receptor autophosphorylation compared to parent insulin (e.g., human insulin). The insulin receptor autophosphorylation compared to parent insulin can be determined as described in the Examples section.

[0051] As disclosed above, the activated sulfonamide of formula (I) has an R in the uncoupled state of the activated sulfonamide of formula (I) x The terminal carboxy group bearing the group is covalently attached to the polypeptide by covalently attaching to a suitable functional group of the polypeptide, such as an amino group or a hydroxyl group of the polypeptide. According to at least one embodiment of the method, the amino group of the polypeptide to which the activated sulfonamide of formula (I) is covalently attached is that of human insulin, a human insulin analog, a precursor of human insulin, or a precursor of a human insulin analog, such as that of a human insulin analog or a precursor of a human insulin analog, at positions B26 to B29 of the B chain, for example, the epsilon amino group of lysine present at position B29. In some embodiments, the polypeptide and the activated sulfonamide of formula (I) have an R in the pre-coupling state of the activated sulfonamide of formula (I) x The terminal carboxy group bearing the group and an amino group of the polypeptide, such as that of human insulin, a human insulin analog, a precursor of human insulin, or a precursor of a human insulin analog, such as that of a human insulin analog or a precursor of a human insulin analog, at positions B26 to B29 of the B chain, for example, the epsilon amino group of lysine present at position B29, are connected by an amide bond formed therebetween. Of course, in the case of an amide bond, the carboxy group bearing the R x group is present as a carbonyl group -C(=O)- in the conjugate formed, i.e., the amide bond is -C(=O)-NH- as shown below, wherein all residues E, A, R 1 、R 2 、X, and the indices m, s, p, n, t, r and q have the meanings shown above for formula (I), and the NH---- group is already the remaining part from the amino group of the peptide:

Chemical formula

[0052] An exemplary embodiment of the first aspect is a method for forming a conjugate of a sulfonamide and a polypeptide: a) A step of preparing an activated sulfonamide, wherein the activated sulfonamide is of formula (I): [ka] [In the formula, A is selected from the group consisting of an oxygen atom, a -CH2CH2- group, an -OCH2- group, and a -CH2O- group; E represents a -C6H3R- group, where R is a hydrogen atom or a halogen atom, where the halogen atom is selected from the group consisting of fluorine, chlorine, bromine, and iodine atoms; X represents a nitrogen atom or a -CH- group; m is an integer between 5 and 17; n is an integer between 0 and 3; p is either 0 or 1; q is either 0 or 1; r is an integer in the range of 1 to 6; s is either 0 or 1; t is either 0 or 1; R 1 This represents at least one residue selected from the group consisting of a hydrogen atom, a halogen atom, a C1-C3 alkyl group, and a halogenated C1-C3 alkyl group; R 2 This represents at least one residue selected from the group consisting of a hydrogen atom, a halogen atom, a C1-C3 alkyl group, and a halogenated C1-C3 alkyl group; R x This represents an activating group; Here, the combination where s is 1, p is 0, n is 0, A is an oxygen atom, and t is 1 corresponds to the process that is excluded in formula (I); b) A step of preparing an aqueous solution of a polypeptide having free amino groups, wherein the aqueous solution may contain alcohol; c) The step of contacting the aqueous solution of b) with the activated sulfonamide of a); and d) A step of reacting an activated sulfonamide with a polypeptide having a free amino group to obtain a solution containing a conjugate of sulfonamide and polypeptide, wherein the sulfonamide is covalently bonded to the polypeptide, A method is provided in which the polypeptide is an insulin polypeptide.

[0053] In some embodiments, for example, the method involves a sulfonamide and an insulin polypeptide In the case of a method for forming a conjugate, the activated sulfonamide is an activated albumin binder.

[0054] With respect to the activated sulfonamide of formula (I): In some embodiments, s is 0, where the remaining residues and indices have the meanings set forth above for formula (I).

[0055] According to at least one embodiment of the present method, the polypeptide having free amino groups is a mature polypeptide or its precursor, each having a free amino group, and the precursor of the mature polypeptide includes an additional sequence of one or more further amino acid residues compared to the mature polypeptide. In some embodiments, the “insulin polypeptide” is mature insulin or a precursor of mature insulin, and the precursor of mature insulin includes an additional sequence of one or more further amino acid residues compared to mature insulin. The term “mature insulin” as used herein includes parent insulin, such as human insulin, and insulin analogs. In some embodiments, the mature insulin is an insulin analog, such as the insulin analogs listed in Table 1 of the Examples section. For example, the insulin analog may be insulin analog 24 in Table 1.

[0056] In at least one embodiment of this method, the aqueous solution prepared in a) has a pH value in the range of 9 to 12, or in the range of 9.5 to 11.5, or in the range of 10 to 11, the pH value being determined using a pH-sensitive glass electrode in accordance with ASTM E 70:2007. In some embodiments, the pH value is adjusted within each range by adding a base such as a base selected from the group consisting of alkali hydroxides (lithium hydroxide, sodium hydroxide, potassium hydroxide), alkylamines, and mixtures of two or more thereof. In some embodiments, the base is selected from the group consisting of tertiary alkylamines N(C1-C5 alkyl)3, primary alkylamines H2N-C(C1-C5 alkyl)3, and mixtures of two or more thereof (where the C1-C5 alkyl groups of the tertiary and primary amines are each independently selected from branched or linear C1-C5 alkyl groups, and where each C1-C5 alkyl group has at least one substituent selected from the group consisting of a hydrogen atom, a hydroxyl group, and a carboxyl group). In some embodiments, the base is selected from the group of tertiary alkylamines N(C1-C3 alkyl)3, primary alkylamines H2N-C(C1-C3 alkyl)3, and two or more mixtures thereof (where the C1-C3 alkyl groups of the tertiary and primary amines are each independently selected from branched or linear C1-C3 alkyl groups, where each C1-C3 alkyl group has at least one substituent selected from the group of hydrogen atoms, hydroxyl groups, and carboxyl groups). In some embodiments, the base is selected from the group of bicine, trimethylamine, triethylamine, tris(hydroxymethyl)aminomethane, and two or more mixtures thereof. In some embodiments, the base includes at least triethylamine.

[0057] In at least one embodiment of this method, contacting the aqueous solution of b) with the activated sulfonamide of a) according to step c) is performed by adding the activated sulfonamide of a) to the aqueous solution of b) as a solution of the activated sulfonamide. In some embodiments, the solution of the activated sulfonamide is an organic solution such as a solution containing the activated sulfonamide and a polar aprotic organic solvent. In some embodiments, the polar aprotic organic solvent has an octanol / water partition coefficient (K) in the range of 1 to 5 under standard conditions (T: 20 to 25°C, p: 10¹³ mbar). OW ) has. In some embodiments, the polar aprotic organic solvent has an octanol / water partition coefficient (K) in the range of 2 to 4 under standard conditions (T: 20 to 25°C, p: 10¹³ mbar). OW ) has. In some embodiments, the polar aprotic organic solvent is selected from the group consisting of tetrahydrofuran, acetonitrile, dimethylformamide, and mixtures of two or more of these. Therefore, the polar aprotic organic solvent is selected from the group consisting of tetrahydrofuran, acetonitrile, and mixtures of tetrahydrofuran and acetonitrile.

[0058] In at least one embodiment of this method, contacting the aqueous solution of b) with the activated sulfonamide of a) according to step c) is performed by adding the activated sulfonamide of a) to the aqueous solution of b) in solid form. In some embodiments, the activated sulfonamide of a) is added in at least partially crystalline form. In some embodiments, the activated sulfonamide of a) is added such that at least 90% by weight is in crystalline form.

[0059] In at least one embodiment of this method, step d) is: d.1) A step of reacting an activated sulfonamide with a mature polypeptide precursor having a free amino group at a pH in the range of 9 to 12 to obtain a preconjugate containing the sulfonamide and the mature polypeptide precursor, wherein the sulfonamide is covalently bonded to the mature polypeptide precursor by an amide bond C(=O)-NH- formed between the -C(=O)-O(R) of the (activated) sulfonamide of formula (I) and the amino group of the mature polypeptide precursor; d.2) The process includes the step of obtaining a solution containing a conjugate of sulfonamide and a mature polypeptide by enzymatic digestion of the preconjugate mature insulin precursor obtained according to d.1).

[0060] In some embodiments, the reaction of the activated sulfonamide with the precursor of a mature polypeptide having a free amino group according to d.1) is carried out at a pH in the range of 9.5 to 11.5. In some embodiments, the reaction of the activated sulfonamide with the precursor of a mature polypeptide having a free amino group according to d.1) is carried out at a pH in the range of 10 to 11. In some embodiments, the enzymatic digestion according to d.2) is carried out at a pH in the range of less than 9. In some embodiments, the enzymatic digestion according to d.2) is carried out at a pH in the range of 7 to 9.

[0061] In at least one embodiment, the method is: e) The step of isolating the conjugate of sulfonamide and mature polypeptide from the solution obtained in d) or d.2). It also includes.

[0062] According to at least one embodiment, the activated sulfonamide has formula (I-1), [ka] During the ceremony: E represents a -C6H3R- group, where R is a hydrogen atom or a halogen atom, where the halogen atom is selected from the group consisting of fluorine, chlorine, bromine, and iodine atoms, for example, a fluorine atom; X represents a nitrogen atom or a -CH- group; p is either 0 or 1; q is either 0 or 1; r is an integer in the range of 1 to 6; R 1 represents at least one residue selected from the group of hydrogen atoms and halogen atoms, where the halogen atom is, for example, a fluorine or chlorine atom; R 2 represents at least one residue selected from the group consisting of a hydrogen atom, a C1-C3 alkyl group, and a halogenated C1-C3 alkyl group, where the C1-C3 alkyl group is, for example, a methyl group, and the halogenated C1-C3 alkyl group is perhaled, such as a trifluoromethyl group; R x This represents an activating group; If p is 0, m is an integer between 5 and 15, or if p is 1, m is an integer between 7 and 15.

[0063] In some embodiments, the activated sulfonamide residue R 1 and R 2 is a hydrogen atom. In some embodiments, residue X of the activated sulfonamide represents a nitrogen atom. In some embodiments, HOOC-(CH2) of formula (I) of the activated sulfonamide m -(O) s -(E) p -(CH2) n -(A) t -Base or formula (I-1) HOOC-(CH2) m -(E) p The -O- group is located at the meta or para position of the phenyl ring Ph relative to the -S(O)2- group. In some embodiments, when p is 1, HOOC-(CH2) m -(O) s - group and - (CH2) n -(A) t - The group is (E) of formula (I) of the activated sulfonamide. pIt is located at the meta or para position of HOOC-(CH2) m -Base and -O- are (E) of formula (I-1) p It is located at the meta or para position. In some embodiments, the index q of the activated sulfonamide is 0.

[0064] In some embodiments, the activated sulfonamide has formula (I-1-2), [ka] In the formula, X is a nitrogen atom or a -CH- group, for example a nitrogen atom; m is an integer in the range of 5 to 15; r is an integer in the range of 1 to 6; q is 0 or 1, for example 0; R x is an activating group; HOOC-(CH2) m The -O- group is located at the meta or para position of the phenyl ring Ph relative to the -S(O)2- group.

[0065] According to some embodiments, the activated sulfonamide has formula (I-1-2a), [ka] In the formula, R x This is an activating group.

[0066] In at least one embodiment of this method, the activating group R of the activated sulfonamide of formula (I) x These are 7-azabenzotriazole, 4-nitrobenzene, and N-succinate. Selected from the group consisting of imidyl groups. 7-Azabenzotriazole may be derived from HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) or HBTU (3-[bis(dimethylamino)methyliumyl]-3H-benzotriazole-1-oxide hexafluorophosphate). In some embodiments, R x This is an N-succinimidyl group.

[0067] In at least one embodiment of this method, the aqueous solution of the polypeptide having a free amino group according to b) comprises an alcohol selected from the group consisting of C1-C4 monoalcohols and mixtures of two or more thereof. In some embodiments, the aqueous solution of the polypeptide having a free amino group according to b) comprises an alcohol selected from the group consisting of methanol, ethanol, propan-2-ol, propan-1-ol, butan-1-ol, and mixtures of two or more thereof. In some embodiments, the aqueous solution of the polypeptide having a free amino group according to b) comprises an alcohol selected from the group consisting of ethanol, propan-2-ol, propan-1-ol, and mixtures of two or more thereof.

[0068] In at least one embodiment of this method, the aqueous solution according to b) contains alcohol, and the amount of alcohol in the aqueous solution is in the range of 0.0001 to 35 vol% based on the total volume of water and alcohol. In some embodiments, the aqueous solution according to b) contains alcohol, and the amount of alcohol in the aqueous solution is in the range of 0.001 to 30 vol% based on the total volume of water and alcohol. In some embodiments, the aqueous solution according to b) contains alcohol, and the amount of alcohol in the aqueous solution is in the range of 0.01 to 25 vol% based on the total volume of water and alcohol. In some embodiments, the aqueous solution according to b) contains alcohol, and the amount of alcohol in the aqueous solution is in the range of 0.1 to 20 vol% based on the total volume of water and alcohol.

[0069] In at least one embodiment of this method, the enzymatic digestion according to d.2) includes the use of at least one enzyme selected from the group consisting of trypsin, TEV protease (tobacco etch virus protease), and a mixture of two or more of these.

[0070] In at least one embodiment of the present method, the mature polypeptide is mature insulin comprising an A chain and a B chain, wherein the A chain contains at least one mutation of the A chain of human insulin, and / or the B chain contains at least one mutation of the A chain of human insulin. In some embodiments, the at least one mutation of the A chain of human insulin is a substitution at the A14 position, such as a substitution with an amino acid selected from the group consisting of glutamic acid (Glu), aspartic acid (Asp), and histidine (His), and / or a substitution at the A21 position, such as a substitution with glycine (Gly). In some embodiments, mutations in the B chain of human insulin include substitutions at position B16, such as substitutions with amino acids selected from the group consisting of valine (Val), isoleucine (Ile), leucine (Leu), alanine (Ala), or histidine (His); substitutions at position B25, such as substitutions with valine (Val), isoleucine (Ile), leucine (Leu), alanine (Ala), or histidine (His); and / or deletions at position B30.

[0071] In some embodiments, insulin analogs contain a mutation at the B16 position, which is substituted with a hydrophobic amino acid. Thus, the amino acid at the B16 position (tyrosine in human, bovine, and porcine insulin) is replaced with a hydrophobic amino acid.

[0072] In another embodiment, the insulin analog contains a mutation at position B25, which is substituted with a hydrophobic amino acid. Thus, the amino acid at position B25 (phenylalanine in human, bovine, and porcine insulin) is replaced with a hydrophobic amino acid.

[0073] In another embodiment, the insulin analog includes a mutation at position B16 substituted with a hydrophobic amino acid, and a mutation at position B25 substituted with a hydrophobic amino acid. The hydrophobic amino acid can be any hydrophobic amino acid. For example, the hydrophobic amino acid can be an aliphatic amino acid such as a branched-chain amino acid.

[0074] In some embodiments, the hydrophobic amino acids used for substitution at the B16 and / or B25 positions are isoleucine, valine, leucine, alanine, tryptophan, methionine, proline, glycine, phenylalanine, or tyrosine. In some embodiments, the hydrophobic amino acids used for substitution at the B16 and / or B25 positions are isoleucine, valine, leucine, for example, valine. Furthermore, it is assumed that the amino acids at the B16 and / or B25 positions are substituted with histidine.

[0075] The insulin analog may contain further mutations. For example, the insulin analog may contain further mutations at position A14. Such mutations are known to increase protease stability (see, for example, WO2008 / 034881A1 [Novo Nordisk, Nielsen]). In some embodiments, the amino acid at position A14 is substituted with glutamic acid (Glu). In some embodiments, the amino acid at position A14 is substituted with aspartic acid (Asp). In some embodiments, the amino acid at position A14 is substituted with histidine (His).

[0076] Furthermore, the insulin analog may contain a mutation at position B30. In one embodiment, the B30 mutation is a deletion of threonine at position B30 of the parent insulin (also called a Des(B30) mutation).

[0077] Furthermore, the insulin analog of the present invention may further include a mutation at the B3 position substituted with glutamic acid (Glu) and / or a mutation at the A21 position substituted with glycine (Gly).

[0078] In some embodiments, the insulin analog includes substitutions at position A14, substitutions at position B25, and deletions at position B30 (i.e., the amino acid at position B30 is absent).

[0079] In some embodiments, the A chain of the insulin analog has the following sequence: GIVEQCCTSICSL Xaa9 QLENYCN (Sequence ID: 109) The B chain of an insulin analog contains or consists of the following sequence: FVNQHLCGSHLVEAL Xaa10 LVCGERGF Xaa11 YTPK (Sequence ID: 110) including or consisting of In the formula, Xaa9 is glutamic acid (Glu), aspartic acid (Asp), or histidine (His). In the formula, Xaa10 is tyrosine (Tyr), valine (Val), isoleucine (Ile), leucine (Leu), alanine (Ala), or histidine (His), and / or In the formula, Xaa11 is phenylalanine (Phe), valine (Val), isoleucine (Ile), leucine (Leu), alanine (Ala), or histidine (His).

[0080] In some embodiments, Xaa9 is glutamic acid (Glu), Xaa10 is tyrosine (Tyr), and Xaa11 is valine (Val), isoleucine (Ile), or leucine (Leu). In some embodiments, Xaa9 is glutamic acid Xaa10 is tyrone (Glu), Xaa10 is tyrosine (Tyr), and Xaa11 is valine (Val).

[0081] In some embodiments, mature insulin is Leu(B16)-human insulin, Val(B16) - Human insulin, Ile(B16) - Human insulin, Leu(B16)Des(B30)-Human insulin, Val(B16)Des(B30)-Human Insulin Ile(B16)Des(B30)-Human insulin, Leu(B25) - Human insulin, Val(B25) - Human insulin, Ile(B25) - Human insulin, Leu(B25)Des(B30)-Human Insulin Val(B25)Des(B30)-Human Insulin Ile(B25)Des(B30)-Human insulin, Glu(A14)Leu(B16)Des(B30)-Human insulin, Glu(A14)Ile(B16)Des(B30)-Human insulin, Glu(A14)Val(B16)Des(B30)-Human insulin, Glu(A14)Leu(B16)-Human insulin, Glu(A14)Ile(B16)-Human insulin, Glu(A14)Val(B16)-Human Insulin, Glu(A14)Leu(B25)Des(B30)-Human insulin, Glu(A14)Ile(B25)Des(B30)-Human insulin, Glu(A14)Val(B25)Des(B30)-Human insulin, Glu(A14)Leu(B25)-Human insulin, Glu(A14)Ile(B25)-Human Insulin, Glu(A14)Val(B25)-Human insulin, Glu(A14)Gly(A21)Glu(B3)Val(B25)Des(B30)-Human insulin, Glu(A14)Ile(B16)Ile(B25)Des(B30)-Human Insulin Glu(A14)Glu(B3)Ile(B16)Ile(B25)Des(B30)-Human insulin, Glu(A14)Ile(B16)Val(B25)Des(B30)-Human Insulin Glu(A14)Gly(A21)Glu(B3)Ile(B16)Val(B25)Des(B30)-Human insulin, Glu(A14)Val(B16)Ile(B25)Des(B30)-Human insulin, Glu(A14)Val(B16)Val(B25)Des(B30)-Human insulin, Glu(A14)Glu(B3)Val(B16)Val(B25)Des(B30)-Human insulin, Glu(A14)Gly(A21)Glu(B3)Val(B16)Val(B25)Des(B30)-Human insulin, Glu(A14)Gly(A21)Glu(B3)Val(B25)-Human Insulin Glu(A14)Ile(B16)Ile(B25)-Human Insulin Glu(A14)Glu(B3)Ile(B16)Ile(B25)-Human Insulin , Glu(A14)Ile(B16)Val(B25)-Human Insulin Glu(A14)Gly(A21)Glu(B3)Ile(B16)Val(B25)-Human Insulin Glu(A14)Val(B16)Ile(B25)-Human Insulin Glu(A14)Val(B16)Val(B25)-Human Insulin Glu(A14)Glu(B3)Val(B16)Val(B25)-Human insulin, and Glu(A14)Gly(A21)Glu(B3)Val(B16)Val(B25)-Human Insulin 、 Glu(A14)His(B25)Des(B30) human insulin, and The group is selected from Glu(A14), His(B16), His(B25), and Des(B30) human insulins.

[0082] In some embodiments, the amino acid residues referred to herein are L-amino acid residues (such as L-isoleucine, L-valine, or L-leucine). Thus, the amino acid residues (or derivatives thereof) used for substitutions at positions B16, B25, A14, and / or A21, for example, are typically L-amino acid residues.

[0083] In another embodiment, the insulin analog is Leu(B25)Des(B30)-insulin (e.g., Leu(B25)Des(B30)-human insulin). The sequence of this analog is shown, for example, in Table 1 in the Examples section (see Analogue 11).

[0084] In another embodiment, the insulin analog is Val(B25)Des(B30)-insulin (e.g., Val(B25)Des(B30)-human insulin). The sequence of this analog is shown, for example, in Table 1 in the Examples section (see Analogue 12).

[0085] In another embodiment, the insulin analog is Glu(A14)Ile(B25)Des(B30)-insulin (e.g., Glu(A14)Ile(B25)Des(B30)-human insulin). The sequence of this analog is shown, for example, in Table 1 in the Examples section (see Analogue 22).

[0086] In another embodiment, the insulin analog is Glu(A14)Val(B25)Des(B30)-insulin (e.g., Glu(A14)Val(B25)Des(B30)-human insulin). The sequence of this analog is shown, for example, in Table 1 in the Examples section (see Analogue 24).

[0087] In another embodiment, the insulin analog is Glu(A14)Gly(A21)Glu(B3)Val(B25)Des(B30)-insulin (e.g., Glu(A14)Gly(A21)Glu(B3)Val(B25)Des(B30)-human insulin). The sequence of this analog is shown, for example, in Table 1 in the Examples section (see Analogue 25).

[0088] In another embodiment, the insulin analog is Glu(A14)Ile(B16)Ile(B25)Des(B30)-insulin (e.g., Glu(A14)Ile(B16)Ile(B25)Des(B30)-human insulin). The sequence of this analog is shown, for example, in Table 1 in the Examples section (see Analogue 29).

[0089] In another embodiment, the insulin analog is Glu(A14)Glu(B3)Ile(B16)Ile(B25)Des(B30)-insulin (e.g., Glu(A14)Glu(B3)Ile(B16)Ile(B25)Des(B30)-human insulin). The sequence of this analog is shown, for example, in Table 1 in the Examples section (see Analogue 30). (FVEQHLCGSHLVEALILVCGERGFIYTPK).

[0090] In another embodiment, the insulin analog is Glu(A14)Ile(B16)Val(B25)Des(B30)-insulin (e.g., Glu(A14)Ile(B16)Val(B25)Des(B30)-human insulin). The sequence of this analog is shown, for example, in Table 1 in the Examples section (see Analogue 32).

[0091] In another embodiment, the insulin analog is Glu(A14)Gly(A21)Glu(B3)Ile(B16)Val(B25)Des(B30)-insulin (e.g., Glu(A14)Gly(A21)Glu(B3)Ile(B16)Val(B25)Des(B30)-human insulin). The sequence of this analog is shown, for example, in Table 1 in the Examples section (see Analogue 33).

[0092] In another embodiment, the insulin analog is Glu(A14)Val(B16)Ile(B25)Des(B30)-insulin (e.g., Glu(A14)Val(B16)Ile(B25)Des(B30)-human insulin). The sequence of this analog is shown, for example, in Table 1 in the Examples section (see Analogue 35).

[0093] In another embodiment, the insulin analog is Glu(A14)Val(B16)Val(B25)Des(B30)-insulin (e.g., Glu(A14)Val(B16)Val(B25)Des(B30)-human insulin). The sequence of this analog is shown, for example, in Table 1 in the Examples section (see Analogue 38).

[0094] In another embodiment, the insulin analog is Glu(A14)Glu(B3)Val(B16)Val(B25)Des(B30)-insulin (e.g., Glu(A14)Glu(B3)Val(B16)Val(B25)Des(B30)-human insulin). The sequence of this analog is shown, for example, in Table 1 in the Examples section (see Analogue 39).

[0095] In another embodiment, the insulin analog is This is Glu(A14)Gly(A21)Glu(B3)Val(B16)Val(B25)Des(B30)-insulin (e.g., Glu(A14)Gly(A21)Glu(B3)Val(B16)Val(B25)Des(B30)-human insulin). The sequence of this analog is shown, for example, in Table 1 in the Examples section (see Analogue 40).

[0096] In another embodiment, the insulin analog is Glu(A14)His(B25)Des(B30) human insulin.

[0097] In another embodiment, the insulin analog is Glu(A14)His(B16)His(B25)Des(B30) human insulin.

[0098] In at least one embodiment of the method, the precursor of the mature polypeptide comprises the sequence described in detail above herein for mature insulin, and includes the A chain and the B chain, as well as an additional linker peptide having a length of at least two amino acid residues, wherein the precursor is mature insulin. It is a precursor of [the linker peptide]. In some cases, the linker peptide has a length in the range of 2 to 30 amino acid residues, for example, in the range of 4 to 9 amino acid residues. In some embodiments, the precursor is the precursor as defined in Section B of this application.

[0099] In at least one embodiment of the present method, the first amino acid of the linker peptide is selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine residues. For example, the first amino acid of the linker peptide is selected from alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. In some embodiments, the first amino acid of the linker peptide is a threonine residue, a phenylalanine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue. Regarding the first amino acids of the linker peptide, at least these amino acid residues are amino acid residues that have an amino acid with low nucleophilicity at the N-terminus. This results in the activated carboxyl group of the sulfonamide -COOR x The reactivity related to the reaction with is reduced.

[0100] In at least one embodiment of this method, the last amino acid of the linker peptide is an arginine residue.

[0101] In at least one embodiment of this method, the linker peptide is the following sequence Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Arg (Sequence number: 106) The formula includes or consists of, where Xaa1 to Xaa8 can be: Xaa1 can be selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, Xaa1 is threonine, phenylalanine, glutamine, glutamic acid, asparagine, or aspartic acid. Xaa2 can be selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. In some embodiments, Xaa2 is glutamic acid. Alternatively, Xaa2 may not exist. Xaa3 can be selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, Xaa3 is glycine. Alternatively, Xaa3 may not exist. Xaa4 can be selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Alternatively, Xaa4 may not exist. Xaa5 can be selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Alternatively, Xaa5 may not exist. Xaa6 can be selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Alternatively, Xaa6 may not exist. Xaa7 can be selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Alternatively, Xaa7 may not exist. Xaa8 can be selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Alternatively, Xaa8 may not exist.

[0102] In at least one embodiment of this method, the linker peptide has the sequence TEGR (SEQ ID NO: 112). A preconjugate comprising an exemplary sulfonamide, a precursor of a mature polypeptide [where the linker peptide has the sequence TEGR (SEQ ID NO: 112)], and an exemplary sulfonamide is shown in Figure 8.

[0103] In at least one embodiment of the present method, the linker peptide is a linker peptide as defined in Section B of this application.

[0104] In at least one embodiment of this method, the sulfonamide is the -C(=O)-O(R) of the (activated) sulfonamide of formula (I). X The free amino group of the mature polypeptide and its precursor is covalently bonded to the mature polypeptide and its precursor by an amide bond C(=O)-NH- formed between the free amino group of the mature polypeptide and its precursor. In some embodiments, the free amino group of the polypeptide is the amino group of lysine contained in the mature polypeptide and its precursor. In some embodiments, the free amino group of the polypeptide is the amino group of terminal lysine. In some embodiments, the free amino group of the polypeptide is the amino group of lysine located at the C-terminus of the mature polypeptide and its precursor. In some embodiments, the free amino group of the polypeptide is the amino group of lysine located at the C-terminus of the B chain.

[0105] (Cleavable) linker peptides, particularly the linker peptide TEGR (SEQ ID NO: 112), protect the N-terminus of the A chain from coupling with the activated sulfonamide of formula (I). Peptide TEGR (SEQ ID NO: 112) is the activated carboxyl group of the sulfonamide - COOR x No reaction, or a small reaction of less than 1% degree Since it only reacts with sulfonamide, the required excess sulfonamide is reduced. The cleavage of TEGR (SEQ ID NO: 112) after coupling with sulfonamide can be achieved in one pot by adjusting the pH to a value in the range of less than 9, and then adding trypsin, TEV protease (tobacco etch virus protease), or a mixture of these two enzymes. In some embodiments, the pH is adjusted to a value in the range of 7 to 9. In some embodiments, the pH is adjusted to a value of approximately 8. Since the linker peptide, typically the TEGR (SEQ ID NO: 112) peptide, protects the A1 amino acid, i.e., its free NH2- group, no A1-acylation byproduct is formed. Thus, the separation of the desired compound is simplified, because the A1-acylation byproduct exhibits a retention time similar to the most desired product, in which only lysine B29 is coupled to the sulfonamide conjugate.

[0106] In Section B of this specification, exemplary conjugates of a sulfonamide of formula (I) and a polypeptide, such as an insulin analog, which can be obtained or obtained by the methods described above, are disclosed; exemplary conjugates are shown in Figures 3–6.

[0107] In at least one embodiment of this method, the activated sulfonamide of formula (I) is the protected activated sulfonamide of formula (0). [ka] (In the formula, A, E, X, m, n, p, q, r, s, t, R 1 , R 2 , and R x (The terms have the meanings defined herein above) and the protected activated sulfonamide of formula (0) may be deprotected by the addition of one or more acids, for example, by the addition of at least trifluoroacetic acid.

[0108] In a second aspect, a conjugate is provided which can be obtained or obtained from any one of the embodiments described herein.

[0109] In a third aspect, a precursor of a mature polypeptide is provided, comprising the sequence of a mature polypeptide according to the embodiments described herein and an additional linker peptide, as defined in any one of the embodiments described herein, covalently bonded to the N-terminus of the A chain of the mature polypeptide.

[0110] In the fourth aspect, the activated sulfonamide corresponding to formula (I) [ka] (In the formula, A, E, X, m, n, p, q, r, s, t, R 1 , R 2 , and R xA procedure for crystallizing (which has the meaning defined above with respect to a method for manufacturing a conjugate), A) A step of preparing a solution containing an activated sulfonamide and an organic solvent; B) A step of removing the organic solvent, for example by distillation, to obtain an activated sulfonamide phase having a reduced amount of organic solvent compared to the solution prepared in A); The step of adding an organic solvent to the phase obtained in C)B) to obtain a solution of activated sulfonamide; and A step in which step B) is repeated with the solution obtained in D)C) to obtain an activated sulfonamide phase having a reduced amount of organic solvent compared to the solution obtained in C); E) A process in which steps C) and D) are repeated at least one more time if applicable. The procedure including the following is provided.

[0111] The "phase of activated sulfonamide having a reduced amount of organic solvent compared to the solution prepared in A)" includes a solution of activated sulfonamide (i.e., liquid phase) having a reduced amount of organic solvent compared to the solution prepared in A), an oily phase of activated sulfonamide, and a solid phase of activated sulfonamide. In at least one embodiment, the procedure for crystallizing the activated sulfonamide corresponding to formula (I) is an additional step: F) Adding an organic solvent to the activated sulfonamide phase obtained in D) and / or the activated sulfonamide phase obtained in E), and maintaining the resulting solution at a temperature in the range of 5 to 40°C for at least 1 hour to obtain a precipitate containing activated sulfonamide in solid form. The precipitate obtained according to (F) can be separated from the solution by means known in the art, for example, by filtration.

[0112] In some embodiments, the solution obtained in F) is kept at a temperature in the range of 10 to 35°C. In some embodiments, the solution obtained in F) is kept at a temperature in the range of 20 to 30°C. In some embodiments, the solution obtained in F) is kept at each temperature for 1 to 72 hours. In some embodiments, the solution obtained in F) is kept at each temperature for 10 to 48 hours. In some embodiments, the solution obtained in F) is kept at each temperature for 15 to 30 hours. In some embodiments, the precipitate obtained in F) contains activated sulfonamide in at least partially crystalline form. In some embodiments, the precipitate obtained in F) contains activated sulfonamide in at least 90% by weight in crystalline form.

[0113] In at least one embodiment of the procedure for crystallizing the activated sulfonamide corresponding to formula (I), the solution comprising the activated sulfonamide and the organic solvent prepared in A) further comprises trifluoroacetic acid. In at least one embodiment, the organic solvent is selected from the group of organic solvents capable of forming an azeotropic mixture with trifluoroacetic acid. In at least one embodiment of the procedure for crystallizing the activated sulfonamide corresponding to formula (I), the organic solvent is a polar aprotic organic solvent. In some embodiments, the polar aprotic organic solvent has an octanol / water partition coefficient (K) in the range of 1 to 5 under standard conditions (temperature: 20 to 25°C, pressure: 10¹³ mbar). OW ) has. In some embodiments, the polar aprotic organic solvent has an octanol / water partition coefficient (K) in the range of 2 to 4 under standard conditions (temperature: 20 to 25°C, pressure: 10¹³ mbar). OW ) has. In some embodiments, the organic solvent is selected from the group consisting of acetonitrile, tetrahydrofuran, and mixtures of acetonitrile and tetrahydrofuran. In some embodiments, the organic solvent contains at least acetonitrile.

[0114] For example, for synthetic reasons, the activated sulfonamide corresponding to formula (I) contains trace amounts of trifluoroacetic acid (less than 5% by weight based on the weight of the activated sulfonamide). However, these trace amounts prevent the solidification and crystallization of the activated sulfonamide, respectively, resulting in the activated sulfonamide existing in an oily form. Repeated addition of organic solvents and their subsequent distillation removal allows for azeotropic removal of trifluoroacetic acid, resulting in solidification / crystallization of the activated sulfonamide following reduction and removal of the amount of trifluoroacetic acid, respectively. According to Dortmunder Databank, the octanol / water partition coefficient of acetonitrile (K OW ) is 2, and the K of tetrahydrofuran (THF) OW It is 4, and the K of dimethylformamide OW It is 4.

[0115] The organic solvent used to prepare the solution in A), the organic solvent added in C), the optional organic solvent used in E), and the organic solvent used in F) are either the same or different, and are independently selected from the group of organic solvents capable of forming an azeotropic mixture with trifluoroacetic acid, and / or from the group of polar aprotic organic solvents, and these are within the range of 1 to 5 K OW It may have a polar aprotic organic solvent in the range of 2 to 4 K. OW It has the following characteristics. In some embodiments, the same organic solvent is used in preparing the solution in A), in C), and optionally in E), and F).

[0116] In the fifth aspect, the activated sulfonamide corresponding to formula (I) is in solid form. [ka] (In the formula, A, E, X, m, n, p, q, r, s, t, R 1 , R 2 , and R x A (meaning as defined above in this specification) is provided. In some embodiments, the activating binder is crystalline.

[0117] In one embodiment, an exemplary sulfonamide of formula (I) is produced by coupling two constituent units A and B as shown in Scheme 1 below, where the coupling of constituent units A and B yields an exemplary sulfonamide called pyrimidine-bis-OEG acid:

[0118] [ka]

[0119] Scheme 2 shows the synthesis of constituent unit A, and scheme 3 shows the synthesis of constituent unit B:

[0120] [ka]

[0121] [ka]

[0122] The definitions and descriptions provided in Section A above in this Specification are in accordance with the following sections of this Specification. The embodiments described in B shall apply mutatis mutandis.

[0123] Section B As described in Section A, the presence of one or more additional amino acid residues at the N-terminus of the A chain, such as the TEGR (SEQ ID NO: 112) peptide, protects the A1 amino acid of the A chain, i.e., the free NH2- group of the A chain. Therefore, no A1-acylation byproduct is formed when preparing a conjugate of the albumin-binding agent with an insulin precursor containing the N-terminally extended A and B chains. After conjugation, the one or more additional peptides at the N-terminus of the A chain can be conveniently removed, for example, by proteolytic cleavage with trypsin, thereby producing a conjugate of mature insulin (e.g., an insulin analog) with the albumin-binding agent. The A1-acylation byproduct exhibits a similar retention time to the desired product, where only the lysine at B29 is coupled to the albumin-binding agent conjugate, thus simplifying the separation of the desired conjugate.

[0124] Furthermore, if the first amino acid of the N-terminal extended A chain has lower nucleophilicity than the A1 amino acid, the amount of binder used for conjugation may be reduced. This allows for the activation of the albumin binder's carboxyl group -COOR x The reactivity with respect to the reaction is reduced. For example, threonine has lower nucleophilicity than glycine, which is frequently found at the A1 position of insulin analogs.

[0125] An insulin precursor containing N-terminally elongated A and B chains can be produced by cleaving proinsulin, which includes an insulin B chain, a linker peptide, and an insulin A chain from the N-terminus to the C-terminus, between the last amino acid of the insulin B chain and the first amino acid of the linker peptide using a protease. The resulting N-terminally elongated A chain contains a linker peptide and an A chain from the N-terminus to the C-terminus. The linker peptide then protects the A1 amino acid of the A chain in a subsequent conjugation step.

[0126] Therefore, the present invention relates to a process for producing a conjugate of an albumin-binding agent and mature insulin, wherein the process is a) A step of preparing proinsulin containing an insulin B chain, a linker peptide, and an insulin A chain from the N-terminus to the C-terminus, b) A step of cleaving the proinsulin prepared in step a) between the last amino acid of the insulin B chain and the first amino acid of the linker peptide using a first protease, thereby generating an insulin precursor, wherein the insulin precursor includes an insulin B chain, a linker peptide and an N-terminally elongated A chain including the A chain, c) A step of contacting the insulin precursor with an albumin binder, wherein the albumin binder comprises a functional group capable of binding to albumin; This process generates a conjugate of an albumin binder and an insulin precursor. d) The N-terminal extended A chain of the insulin precursor contained in the conjugate is cleaved between the last amino acid of the linker peptide and the first amino acid of the A chain by a second protease, thereby generating a conjugate of albumin-binding agent and mature insulin. Includes.

[0127] An "albumin binder" is a compound that can non-covalently bind to albumin, such as human albumin, in a blood sample, for example.

[0128] In some cases, the albumin binder is an activated albumin binder. The mine binder is an activated carboxyl group -COOR x (In the formula, R x It may contain an activating group R. x In at least one embodiment, is selected from the group consisting of, for example, 7-azabenzotriazole derived from HATU or HBTU, 4-nitrobenzene, and an N-succinimidyl group. Optionally, R x This is an N-succinimidyl group.

[0129] In at least one embodiment, the albumin binder comprises a functional group capable of binding to albumin, such as human serum albumin. The functional group capable of binding to albumin may be a carboxyl group or a bioequivalent of a carboxyl group. Depending on the circumstances, functional groups that can bind to albumin are selected from the group consisting of carboxyl groups, hydroxamic acid groups, hydroxamic acid ester groups, phosphonic acid groups, phosphinic acid groups, sulfonic acid groups, sulfinic acid groups, sulfonamide groups, acylsulfonamide groups, sulfonylurea groups, acyllurea groups, tetrazole groups, thiazolininidine groups, oxazolinidine groups, oxadiazole-5(4H)-one groups, thiadiazole-5(4H)-one groups, oxathiadiazole-2-oxide groups, oxadiazole-5(4H)-thione groups, isoxazole groups, tetramic acid groups, cyclopentane 1,3-dione, cyclopentane 1,2-dione, squamic acid derivatives, substituted phenols, -CO-Asp, -CO-Glu, -CO-Gly, -CO-Sar(-CO-sarcosine), -CH(COOH)2, and -N(CH2COOH)2. Depending on the circumstances, functional groups that can bind to albumin may be selected from the group consisting of carboxyl groups, -CO-Asp, -CO-Glu, -CO-Gly, -CO-Sar, -CH(COOH)2, -N(CH2COOH)2, sulfonic acid groups (-SO3H), and phosphonic acid groups (-PO3H).

[0130] In some cases, the albumin binder includes an acyl moiety. For example, the acyl moiety has the general formula Acy-AA1n-AA2m-AA3p-(formula N), where: n is an integer between 0 and 3; m is an integer between 0 and 10; p is an integer between 0 and 10; Acy is a fatty acid or fatty diacid containing approximately 8 to 24 carbon atoms; AA1 is a neutral linear or cyclic amino acid residue; AA2 is an acidic amino acid residue; AA3 is a neutral alkylene glycol-containing amino acid residue.

[0131] In formula N, the order in which AA1, AA2, and AA3 appear in the formula can be independently interchanged; AA2 may occur multiple times along the formula (e.g., Acy-AA2-AA3rAA2-); AA2 may occur multiple times along the formula independently (and as different species) (e.g., Acy-AA2-AA3-AA2-). In formula N, the bonds between Acy, AA1, AA2, and / or AA3 are amide (peptide) bonds. Depending on the circumstances, AA1 may be selected from the group consisting of:Gly, D-Ala or L-Ala, β-Ala, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, D-Glu-α-amide or L-Glu-α-amide, D-Glu-γ-amide or L-Glu-γ-amide, D-Asp-α-amide or L-Asp-α-amide, D-Asp-β-amide or L-Asp-β-amide, 7-aminoheptanoic acid, and 8-aminooctanoic acid. Depending on the circumstances, AA2 may be selected from the group consisting of L-Glu or D-Glu, L-Asp or D-Asp, or L-homoGlu or D-homoGlu. In some cases, the neutral cyclic amino acid residue denoted as AA1 is an amino acid containing a saturated six-membered carbocyclic ring, which may contain a nitrogen heteroatom, and this ring may be a cyclohexane ring or a piperidine ring. In some cases, the molecular weight of this neutral cyclic amino acid is in the range of approximately 100 to approximately 200 Da.

[0132] An acidic amino acid residue denoted as AA2 may be an amino acid with a maximum molecular weight of approximately 200 Da, containing two carboxylic acid groups and one primary or secondary amino group. Alternatively, an acidic amino acid residue denoted as AA2 may be an amino acid with a maximum molecular weight of approximately 250 Da, containing one carboxylic acid group and one primary or secondary sulfonamide group. The neutral alkylene glycol-containing amino acid residue denoted as AA3 is an alkylene glycol portion, which may contain a carboxylic acid functional group at one end and an amino group functional group at the other end, and is either an oligoalkylene glycol portion or a polyalkylene glycol portion. In this specification, the term alkylene glycol moiety includes monoalkylene glycol moieties and oligoalkylene glycol moieties. Monoalkylene glycols and oligoalkylene glycols include monoethylene glycol and oligoethylene glycol bases, monopropylene glycol and oligopropylene glycol bases, and monobutylene glycol and oligobutylene glycol base chains, i.e., chains based on repeating units -CH2CH2O-, -CH2CH2CH2O-, or -CH2CH2CH2CH2O-. Alkylene glycol moieties may be monodispersible (having a distinct length / molecular weight). Monoalkylene glycol moieties include -OCH2CH2O-, -OCH2CH2CH2O-, or -OCH2CH2CH2CH2O-, each containing a different group at its end.

[0133] The connections between the Acy, AA1, AA2, and / or AA3 segments are formally obtained by the formation of amide bonds (peptide bonds) (-CONH-) by the removal of water from the parent compounds from which they are formally constructed.

[0134] For example, a suitable albumin binder containing an acyl moiety is eicosanediol-gGlu-(OEG)2. In eicosanediol-gGlu-(OEG)2, the functional group capable of binding to albumin is the terminal COOH group of the eicosanediol group, and the albumin binder can be coupled to the insulin polypeptide via the terminal OEG group. HOOC-(CH2) 18 -C(=O)-NH-CH(COOH)-(CH2)2-C(=O)-NH-(CH2)2-O-(CH2)2-O-CH2-C(=O)-NH-(CH2)2-O-(CH2)2-OC(=O)-NH-insulin polypeptide.

[0135] The appropriate acile section is described on pages 27, line 13 to 43 of WO2009 / 115469A1 (Novo Nordisk, published September 24, 2009).

[0136] In some cases, the albumin binder is a sulfonamide of formula (I), as detailed in Section A above.

[0137] According to step a) of the process of the present invention described in Section B, proinsulin is prepared. In some embodiments, the prepared proinsulin is produced by expressing a polynucleotide encoding the proinsulin in host cells. The proinsulin thus produced may be subsequently purified from the culture medium in which the host cells were cultured.

[0138] The proinsulin according to the present invention comprises an insulin B chain, a linker peptide, and an insulin A chain from the N-terminus to the C-terminus. Thus, the proinsulin includes the B chain fused to the linker peptide followed by the A chain at the C-terminus. The insulin B chain, linker peptide, and insulin A chain are linked via peptide bonds, typically without intervening amino acid residues. Suitable proinsulins are described below in this specification.

[0139] According to the present invention, the linker peptide will have a length of at least one amino acid residue, such as at least two amino acid residues. For example, the linker peptide may have a length in the range of 1 to 30 amino acid residues, particularly in the range of 2 to 30 amino acid residues. In some embodiments, the linker will have a length in the range of 4 to 9 amino acid residues. In some embodiments, the linker peptide will have a length of 4 amino acid residues.

[0140] In at least one embodiment, the first amino acid of the linker peptide is selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine residues.

[0141] In some embodiments, the first amino acid of the linker peptide is a threonine, phenylalanine, glutamine, glutamic acid, asparagine, or aspartic acid residue. The aforementioned amino acid residues have low nucleophilicity. For example, the nucleophilicity of the aforementioned amino acid residues is lower than that of glycine, which is found at position A1 in many insulin analogs. Therefore, the first amino acid of the insulin A chain is a glycine residue.

[0142] In some embodiments, the first amino acid of the linker peptide is threonine. Furthermore, it is assumed that the last amino acid of the linker peptide is arginine. The presence of an arginine residue at this position makes it possible to remove the linker peptide with trypsin in step d) of the above method.

[0143] Therefore, the linker peptide, i.e., the linker peptide between the B chain and the A chain, may contain the following sequence, or in particular, consist of the following sequence: Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Arg (Sequence number: 106) During the ceremony, Xaa1 is any naturally occurring amino acid residue, for example, here Xaa1 is threonine, phenylalanine, glutamine, glutamic acid, asparagine, or aspartic acid. Xaa2 is either any naturally occurring amino acid residue, or Xaa2 is absent. In some embodiments, Xaa2 is glutamic acid. Xaa3 is any naturally occurring amino acid residue, in particular, where Xaa3 is glycine, or where Xaa3 is absent. Xaa4 is either any naturally occurring amino acid residue, or Xaa4 does not exist. Xaa5 is either any naturally occurring amino acid residue, or Xaa5 does not exist. Xaa6 is either any naturally occurring amino acid residue, or Xaa6 does not exist. Xaa7 is either any naturally occurring amino acid residue, or Xaa7 does not exist. Xaa8 is either any naturally occurring amino acid residue or Xaa8 does not exist. do not have.

[0144] Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, and Xaa8 may be any naturally occurring amino acid residue, in particular alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. In some embodiments, Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, and Xaa8 are not lysine and cysteine. In this case, Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, and Xaa8 are (independently) selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0145] Therefore, Xaa1 to Xaa8 can be as follows: Xaa1 can be selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, Xaa1 is threonine, phenylalanine, glutamine, glutamic acid, asparagine, or aspartic acid. Xaa2 can be selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. In some embodiments, Xaa2 is glutamic acid. Alternatively, Xaa2 may not exist. Xaa3 can be selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, Xaa3 is glycine. Alternatively, Xaa3 may not exist. Xaa4 can be selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Alternatively, Xaa4 may not exist. Xaa5 can be selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Alternatively, Xaa5 may not exist. Xaa6 can be selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Alternatively, Xaa6 may not exist. Xaa7 can be selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Alternatively, Xaa7 may not exist. Xaa8 contains alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, and phenylalanine. You can choose from the group consisting of lanin, proline, serine, threonine, tryptophan, tyrosine, and valine. Alternatively, Xaa8 does not exist.

[0146] In some embodiments, the linker peptide consists of the sequence Xaa1-Arg, where Xaa1 has the meaning described above, for example, Xaa1 is threonine, phenylalanine, glutamine, glutamic acid, asparagine, or aspartic acid. In some embodiments, the linker peptide consists of the sequence Thr-Arg.

[0147] In an alternative embodiment, the linker peptide consists of the sequence Xaa1-Xaa2-Arg, for example Thr-Xaa2-Arg, for example Thr-Glu-Arg.

[0148] In an alternative embodiment of the present invention, the linker peptide consists of the sequence Xaa1-Xaa2-Xaa-3-Arg (SEQ ID NO: 114), for example Thr-Xaa2-Xaa3-Arg (SEQ ID NO: 115), for example Thr-Glu-Gly-Arg (SEQ ID NO: 112). Thus, the linker peptide may be TEGR (SEQ ID NO: 112).

[0149] As described above, the proinsulin prepared in step a) of the process described in this section will also include an insulin A chain and an insulin B chain. For example, the insulin A chain and insulin B chain may be the insulin A chain and insulin B chain of the insulin analog described in Section A of this application. In some embodiments, the insulin A chain is the A chain of any one of the insulin analogs listed in Table 1, and the insulin B chain is the corresponding insulin B chain. For example, the proinsulin may include the A chain and B chain of the insulin analog 24 shown in Table 1.

[0150] In some embodiments, proinsulin comprises the following sequence, or more specifically, the A chain consisting of the following sequence: GIVEQCCTSICSL Xaa9 QLENYCN (Sequence ID: 109) In the formula, Xaa9 is glutamic acid (Glu), aspartic acid (Asp), or histidine (His).

[0151] Furthermore, proinsulin may contain, or in particular, a B chain consisting of the following sequence: FVNQHLCGSHLVEAL Xaa10 LVCGERGF Xaa11 YTPK (Sequence ID: 110) In the formula, Xaa10 is tyrosine (Tyr), valine (Val), isoleucine (Ile), leucine (Leu), alanine (Ala), or histidine (His), and / or In the formula, Xaa11 is phenylalanine (Phe), valine (Val), isoleucine (Ile), leucine (Leu), alanine (Ala), or histidine (His).

[0152] In some embodiments, Xaa9 is glutamic acid (Glu), Xaa10 is tyrosine (Tyr), and Xaa11 is valine (Val), isoleucine (Ile), or leucine (Leu).

[0153] In some embodiments, the proinsulin prepared in step a) of the above process has the following sequence: [ka] In the formula, Xaa1 to Xaa11 have the meanings described above.

[0154] In the sequence shown above, the B chain is shown in bold, the linker peptide is shown in italics, and the A chain is underlined.

[0155] In some embodiments, proinsulin comprises an A chain consisting of the sequence GIVEQCCTSICSLEQLENYCN (SEQ ID NO: 47) and a B chain consisting of the sequence shown in FVNQHLCGSHLVEALYLVCGERGFVYTPK (SEQ ID NO: 48). Furthermore, the linker peptide between the B chain and the A chain may have the above sequence, for example, as shown in SEQ ID NO: 106. In some embodiments, the linker peptide has the sequence TEGR (SEQ ID NO: 112). For example, the proinsulin prepared in step a) of the above process may have the following sequence: [ka]

[0156] Here again, the B chain is shown in bold, the linker peptide is shown in italics, and the A chain is underlined.

[0157] According to step b) of the process described in Section B, the proinsulin prepared in step a) is cleaved by a first protease between the last amino acid of the insulin B chain and the first amino acid of the linker peptide. Cleavage of proinsulin with a protease produces an insulin precursor comprising an insulin B chain and an N-terminally elongated A chain. The N-terminally elongated A chain contains, from the N-terminus to the C-terminus, a linker peptide and an A chain (fused via peptide bonds). Thus, the N-terminally elongated A chain contains, at its N-terminus, 2 to 30 additional amino acid residues, such as 4 to 9 additional amino acid residues, depending on the length of the linker peptide, compared to the A chain of mature insulin.

[0158] In some embodiments, the N-terminally extended A chain contains the entire linker peptide at its N-terminus. Therefore, the first amino acid of the linker peptide is the first amino acid of the N-terminally extended A chain.

[0159] As described above, the insulin precursor produced by cleavage by the first protease will contain i) the insulin B chain, and ii) the N-terminally elongated A chain, which includes the linker peptide and the A chain. In the N-terminally elongated A chain, the linker peptide is still fused to the A chain via peptide bonds, but the B chain is no longer bound to the linker peptide via peptide bonds. However, the B chain and the N-terminally elongated A chain are linked by disulfide crosslinks between cysteine ​​residues, for example, between the cysteine ​​at position A7 of the A chain and position B7 of the B chain. They may be connected by one disulfide bridge between cysteine ​​and another disulfide bridge between the cysteine ​​at position A20 of chain A and the cysteine ​​at position B19 of chain B.

[0160] According to the process described in this section, the first protease will be able to cleave the peptide bond between the B chain and the linker peptide in the prepared proinsulin, that is, the peptide bond between the last amino acid residue of the B chain and the first amino acid residue of the linker peptide of the A chain. Therefore, the first protease should be selected to enable the cleavage of said peptide bond.

[0161] As used herein, the term “protease” is synonymous with peptidase or proteinase. This term refers to a protein that catalyzes the cleavage of peptide bonds in peptides / polypeptides. Examples of proteases include trypsin, TEV protease (tobacco etch virus protease), and endoproteinase Lys-C.

[0162] In one embodiment of the process of the present invention, the first protease is endoproteinase Lys-C. Endoproteinase Lys-C is a serine endoproteinase that cleaves the peptide bond on the carboxyl side of lysine. Therefore, in order to enable cleavage of proinsulin by endoproteinase Lys-C in step b) of the process described in Section B, the last amino acid of the B chain, i.e., the C-terminal amino acid, is assumed to be lysine. For example, the B chain is assumed to contain lysine at position B29 but lack the amino acid at position B30. Therefore, the B chain contained in the proinsulin referred to in step a) of the proinsulin described above may be a des(B30)B chain.

[0163] It should be understood that the cleavage by the first protease and the cleavage by the second protease in steps b) and d) of the process described in Section B are carried out under conditions that enable cleavage. Such conditions are well known in the art and can be easily selected by those skilled in the art.

[0164] The insulin precursors referred to in Section B of this specification will contain a free amino group. In some embodiments, the free amino group is an amino group of lysine contained in the precursor, such as terminal lysine, for example, a lysine located at the C-terminus of the B chain, such as at position B29 of the B chain.

[0165] In some embodiments, terminal lysine is the only lysine residue present in the insulin precursor produced by the cleavage of proinsulin by the first protease.

[0166] Following cleavage by the first protease, the resulting insulin precursor comes into contact with an albumin binder in step c) of the process described in Section B). The albumin binder is as defined above in relation to the process of the present invention.

[0167] In step c) of the process of the present invention as described in Section B, a conjugate of an albumin binder and an insulin precursor is produced. In some embodiments, a conjugate like that described in the method of the present invention in Section A is produced. Thus, the optionally activated albumin binder used in step c) of the process of the present invention as described in Section B may be an activated albumin binder, as described above in Section A.

[0168] According to step c), a conjugate of albumin binder and insulin precursor is produced. In at least one embodiment, the insulin precursor is covalently bound to an albumin binder. For example, the albumin binder is an activated carboxyl group in the pre-coupling state -COOR x If it is an activated albumin binder containing R in the non-conjugated state x The terminal carboxyl group supporting the group covalently bonds to an appropriate functional group of the insulin precursor, for example, to an amino group or hydroxyl group of the insulin precursor. Naturally, in the case of an amide bond formed with an amino group of the insulin precursor, the R in the pre-coupling state x The carboxyl group supporting the group exists as a carbonyl group -C(=O)- in the formed conjugate, that is, an amide bond -C(=O)-NH- is formed, where -C(=O) is COOR xThe remaining part of the group, -NH-, is the remaining part of the amino group of the insulin precursor. For example, the amino group is the lysine located at the C-terminus of the B chain, such as at position B29 of the B chain. After producing a conjugate of the albumin binder and the insulin precursor by step c) of the process of the present invention as described in Section B), the produced conjugate is contacted with a second protease in step d). Step d) enables proteolytic cleavage of the N-terminally extended A chain of the insulin precursor contained in the conjugate produced in step c) between the last amino acid of the linker peptide and the first amino acid of the A chain, thereby producing a conjugate of the albumin binder and mature insulin, in particular a conjugate of the albumin binder and an insulin analog as described herein, for example, an insulin analog disclosed in Section A.

[0169] Therefore, the N-terminally extended A chain becomes cleavable between the linker peptide and the A chain by the second protease, thus containing a cleavage site for the second protease between the last amino acid of the linker peptide and the first amino acid of the A chain. When the N-terminally extended A chain is cleaved by the second protease, the A chain and the linker peptide are obtained, where the A chain and the linker peptide are no longer covalently bonded via peptide bonds.

[0170] In some embodiments, the second protease used in step b) of the process described in Section B of this specification is trypsin or TEV protease (tobacco etch virus protease). Thus, the first protease may be endoproteinase Lys-C, and the second protease may be trypsin or TEV protease (tobacco etch virus protease).

[0171] In some embodiments, the second protease is trypsin. Trypsin (EC 3.4.21.4) Trypsin is a serine protease of the PA crane superfamily, found in the digestive systems of many vertebrates, and hydrolyzes proteins in the digestive system. In some embodiments, trypsin is a vertebrate trypsin, such as mammalian trypsin, e.g., porcine trypsin. Other names for trypsin include α-trypsin; β-trypsin, pseudotrypsin, tryptase, tripcellim, and sperm receptor hydrolase. Trypsin cleaves peptide bonds after arginine residues or lysine residues (Arg-|-Xaa, Lys-|-Xaa). The trypsins used herein may be of any source, such as bovine pancreas trypsin, human pancreas trypsin, porcine pancreas trypsin, recombinant trypsin, or mutant trypsin (e.g., trypsins described in WO2006015879A1 [Roche, Hoess] or WO2007031187A1 [Sanofi-Aventis, Geipel]), as long as they are capable of cleaving peptides or polypeptides, such as the N-terminally elongated A chain described herein, after an arginine residue and / or after a lysine residue. Thus, the last amino acid of the linker peptide is typically an arginine residue or a lysine residue. In some embodiments, the last amino acid of the linker peptide is an arginine residue (e.g., (If protease 1 is LysC).

[0172] According to the present invention, it is contemplated that the proinsulin prepared in step a) of the process described in section B may further include a signal peptide, for example, a signal peptide that enables the secretion of the proinsulin produced by the host cell into the culture medium. Suitable signal peptides are known in the art and are selected according to the selected expression host. In particular, the proinsulin may include a signal peptide at the N-terminus of the proinsulin, i.e., on the N-terminal side of the insulin B chain. Thus, the order is as follows (from the N-terminus to the C-terminus): signal peptide, B chain, linker peptide, A chain (all linked via peptide bonds). The signal peptide contained in the proinsulin is removed by cleavage with a first protease in step b). Thus, the first protease further cleaves the proinsulin between the last amino acid of the signal peptide and the first amino acid of the B chain. Thus, the proinsulin contains two cleavage sites by the first protease, i.e., one cleavage site between the signal peptide and the B chain, and one cleavage site between the B chain and the linker peptide. By cleavage with the first protease in step b) of the above-described process of the present invention, a signal peptide, and an insulin precursor containing the B chain and the N-terminally extended A chain (as described elsewhere in this specification) are generated. Thus, the signal peptide and the B chain are no longer covalently linked by a peptide bond. The same applies to the B chain and the N-terminally extended A chain.

[0173] As described above, the first protease can be endoproteinase Lys-C. Thus, the last amino acid of the signal peptide can be a lysine residue. In some embodiments, the last amino acid of the signal peptide and the last amino acid of the B chain are lysine residues. The presence of lysine residues at these positions enables cleavage by endoproteinase Lys-C.

[0174] The generated signal peptide is no longer needed and may be removed. The generated insulin precursor may be further processed later in step c). Thus, the precursor is contacted with an activated albumin binder as described elsewhere in this specification.

[0175] In step d) of the process of the invention, a conjugate of an albumin binder and mature insulin is provided. Thus, a conjugate of an albumin binder and an insulin analog (e.g., an insulin analog as disclosed in Section A or Table 4) is provided. In some embodiments, the conjugate is the conjugate shown in Figure 3. In some embodiments, the conjugate is the conjugate shown in Figure 4. In some embodiments, the conjugate is the conjugate shown in Figure 5. In some embodiments, the conjugate is the conjugate shown in Figure 6.

[0176] The definitions set forth above in this specification in connection with the processes described in this section are applicable mutatis mutandis to the following.

[0177] The present invention also relates to proinsulin comprising, from the N-terminus to the C-terminus: (a) an insulin B chain, (b) a linker peptide, and (c) an insulin A chain wherein the proinsulin comprises a cleavage site for endoprotease Lys-C between the last amino acid of the insulin B chain and the first amino acid of the linker peptide, and a cleavage site for trypsin between the last amino acid of the linker peptide and the first amino acid of the insulin A chain. Thus, the last amino acid residue of the insulin B chain is a lysine residue. Further, the last amino acid of the linker peptide is an arginine residue. In some embodiments, the first amino acid of the A chain is a glycine residue.

[0178]

[0179] ​ In some embodiments, proinsulin further includes a signal peptide at the N-terminal end of the insulin B chain. Thus, proinsulin includes a further cleavage site of endoproteinase Lys-C located between the last amino acid of the signal peptide and the first amino acid of the B chain. Therefore, the last amino acid residue of the signal peptide is a lysine residue.

[0180] The present invention also relates to a polynucleotide encoding proinsulin according to the present invention. In some embodiments, the polynucleotide is operably ligated to a promoter, which enables the expression of the polynucleotide in a host cell. In some embodiments, the promoter is heterogeneous to the polynucleotide.

[0181] The present invention further relates to a vector comprising the polynucleotide of the present invention. In some embodiments, the vector is an expression vector.

[0182] The present invention also relates to a host cell comprising the proinsulin of the present invention, the polynucleotide of the present invention, and / or the vector of the present invention. In some embodiments, the host cell is a bacterial cell, such as a cell belonging to the genus Escherichia, e.g., an E. coli cell. In other embodiments, the host cell is a yeast cell, such as a Pichia pastoris cell or a Kluyveromyces lactis cell.

[0183] The present invention also relates to the N-terminally elongated insulin A chain described above in step a) of the process of the present invention as described in Section B of this specification. Specifically, the N-terminally elongated insulin A chain is formed from the N-terminus to the C-terminus: (a) linker peptide, and (b) Containing insulin A chain, The proinsulin contains a trypsin cleavage site located between the last amino acid of the linker peptide and the first amino acid of the A chain.

[0184] Typically, the last amino acid of a linker peptide is an arginine residue. Furthermore, the first amino acid of the insulin A chain is a glycine residue.

[0185] In some embodiments, the insulin A chain comprises or consists of the sequence GIVEQCCTSICSL Xaa9 QLENYCN (SEQ ID NO: 109) [wherein Xaa9 is glutamic acid (Glu), aspartic acid (Asp), or histidine (His), for example, in the formula Xaa9 is glutamic acid (Glu)], and the linker peptide comprises or consists of the sequence Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-arginine (SEQ ID NO: 106) [wherein Xaa1 to Xaa8 have the meanings described above in this section herein].

[0186] In some embodiments, the N-terminally elongated A chain is sequence Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 R GIVEQCCTSICSL Xaa9 QLENYCN (SEQ ID NO: 113) (wherein Xaa1 to Xaa9 have the meanings described above) includes, or in particular consists of. In some embodiments, the N-terminally extended A chain is sequence Contains or consists of TEGRGIVEQCCTSICSLEQLENYCN (Sequence ID: 107).

[0187] The present invention further relates to insulin precursors comprising the N-terminally elongated insulin A chain and insulin B chain of the present invention.

[0188] The insulin B chain contained in the precursor is assumed not to be bound to the N-terminal extended insulin A chain via a peptide bond. Further, it can be any B chain described herein. In some embodiments, the insulin B chain is a des(B30)B chain. Thus, there is no amino acid at position 30. In some embodiments, the N-terminal amino acid is lysine at position B29.

[0189] In some embodiments, the B chain comprises or consists of the following sequence: FVNQHLCGSHLVEAL Xaa10 LVCGERGF Xaa11 YTPK (SEQ ID NO: 110), wherein Xaa10 is tyrosine (Tyr), valine (Val), isoleucine (Ile), leucine (Leu), alanine (Ala), or histidine (His), and / or wherein Xaa11 is phenylalanine (Phe), valine (Val), isoleucine (Ile), leucine (Leu), alanine (Ala), or histidine (His).

[0190] In some embodiments, the insulin precursor comprises a B chain that comprises or consists of the sequence FVNQHLCGSHLVEALYLVCGERGFVYTP (SEQ ID NO: 48).

[0191] Thus, the insulin precursor can have the sequence shown in FIG. 7 (B chain: SEQ ID NO: 48, N-terminal extended A chain: SEQ ID NO: 107):

[0192] The present invention further relates to a conjugate comprising an insulin precursor and a sulfonamide as described in section B herein. In some embodiments, the conjugate is as shown in FIG. 8.

[0193] Finally, the present invention relates to a process for generating an insulin precursor, the process comprising a) A step of preparing proinsulin containing an insulin B chain, a linker peptide, and an insulin A chain from the N-terminus to the C-terminus, b) A step of cleaving the proinsulin prepared in step a) between the last amino acid of the insulin B chain and the first amino acid of the linker peptide with a first protease, thereby producing an insulin precursor, wherein the insulin precursor includes an insulin B chain, a linker peptide and an N-terminally elongated A chain, and Includes.

[0194] The present invention is further described by the following embodiments and combinations of embodiments indicated by their respective dependencies and backreferences. In particular, note that in each instance in which the scope of an embodiment is described, all embodiments within this scope are intended to be clearly disclosed to those skilled in the art, for example, in the context of terms such as "the process described in any one of Embodiments 1 to 4." That is, the expression of this term should be understood to those skilled in the art as synonymous with "the process described in any one of Embodiments 1, 2, 3, and 4." Furthermore, the following set of embodiments is not a set of claims that determines the scope of protection, but rather a well-structured description of the schematic and exemplary aspects of the present invention. Clearly note that this represents a simplified portion.

[0195] 1. A method for forming a conjugate between a sulfonamide and a polypeptide: a) Step of preparing the activated sulfonamide of formula (I): [ka] [In the formula, A is selected from the group consisting of an oxygen atom, a -CH2CH2- group, an -OCH2- group, and a -CH2O- group; E represents a -C6H3R- group, where R is a hydrogen atom or a halogen atom, where the halogen atom is selected from the group consisting of fluorine, chlorine, bromine, and iodine atoms; X represents a nitrogen atom or a -CH- group; m is an integer between 5 and 17; n is an integer between 0 and 3; p is either 0 or 1; q is either 0 or 1; r is an integer in the range of 1 to 6; s is either 0 or 1; t is either 0 or 1; R 1 This represents at least one residue selected from the group consisting of a hydrogen atom, a halogen atom, a C1-C3 alkyl group, and a halogenated C1-C3 alkyl group; R 2 This represents at least one residue selected from the group consisting of a hydrogen atom, a halogen atom, a C1-C3 alkyl group, and a halogenated C1-C3 alkyl group; R x This represents an activating group; Here, the combination where s is 1, p is 0, n is 0, A is an oxygen atom, and t is 1 is excluded from equation (I). b) A step of preparing an aqueous solution of a polypeptide having free amino groups, wherein the aqueous solution may contain alcohol; c) The step of contacting the aqueous solution of b) with the activated sulfonamide of a); and d) A step of reacting an activated sulfonamide with a polypeptide having a free amino group to obtain a solution containing a conjugate of sulfonamide and polypeptide, wherein the sulfonamide is covalently bonded to the polypeptide. A method that includes this.

[0196] 2. The polypeptide having free amino groups is a polypeptide or its precursor having free amino groups, wherein the polypeptide precursor comprises an additional sequence of one or more further amino acid residues compared to the polypeptide, according to Embodiment 1.

[0197] 3. The method according to Embodiment 1 or 2, wherein the aqueous solution prepared in a) has a pH value in the range of 9 to 12, or in the range of 9.5 to 11.5, or in the range of 10 to 11, and the pH value is determined using a pH-sensitive glass electrode in accordance with ASTM E 70:2007.

[0198] 4. The pH value is selected within each range from the group consisting of bases, or alkali hydroxides (lithium hydroxide, sodium hydroxide, potassium hydroxide), alkylamines, and mixtures of two or more of these; or from the group consisting of tertiary alkylamine N(C1-C5 alkyl)3, primary alkylamine H2N-C(C1-C5 alkyl)3, and mixtures of two or more of these (where the C1-C5 alkyl groups of the tertiary and primary amines are each independently selected from branched or linear C1-C5 alkyl groups, where each C1-C5 alkyl group has at least one substituent selected from the group consisting of a hydrogen atom, a hydroxyl group, and a carboxyl group); or from the group consisting of tertiary alkylamine N(C1 A C1-C3 alkyl group is selected from the group consisting of (C1-C3 alkyl)3, primary alkylamine H2N-C(C1-C3 alkyl)3, and two or more mixtures thereof (wherein the C1-C3 alkyl groups of the tertiary and primary amines are each independently selected from branched or linear C1-C3 alkyl groups, where each C1-C3 alkyl group has at least one substituent selected from the group consisting of a hydrogen atom, a hydroxyl group, and a carboxyl group); or is prepared by the addition of a base selected from the group consisting of bicine, trimethylamine, triethylamine, tris(hydroxymethyl)aminomethane, and two or more mixtures thereof; the method according to any one of Embodiments 1-3, wherein the base particularly contains at least triethylamine.

[0199] 5. The method according to any one of Embodiments 1 to 4, wherein contacting the aqueous solution of b) with the activated sulfonamide of a) according to step c) is performed by adding the activated sulfonamide of a) to the aqueous solution of b) as a solution of the activated sulfonamide.

[0200] 6. The method according to Embodiment 5, wherein the solution of the activated sulfonamide is an organic solution.

[0201] 7. The method according to Embodiment 6, wherein the organic solution comprises an activated sulfonamide and a polar aprotic organic solvent.

[0202] 8. Polar aprotic organic solvents have an octanol / water partition coefficient (K) in the range of 1 to 5 under standard conditions (T: 20-25°C, p: 10¹³ mbar). OW ) having; or the polar aprotic organic solvent is selected from the group consisting of tetrahydrofuran, acetonitrile, dimethylformamide, and mixtures of two or more thereof; in particular, the method according to Embodiment 7, selected from the group consisting of tetrahydrofuran, acetonitrile, and mixtures of tetrahydrofuran and acetonitrile.

[0203] 9. The method according to any one of Embodiments 1 to 8, wherein contacting the aqueous solution of b) with the activated sulfonamide of a) according to step c) is carried out by adding the activated sulfonamide of a) to the aqueous solution of b) in solid form, or at least partially in crystalline form, or at least 90% by weight in crystalline form.

[0204] 10. Step d) is: d.1) Reacting an activated sulfonamide with a polypeptide precursor having a free amino group at a pH in the range of 9 to 12 to produce a product containing a sulfonamide and a polypeptide precursor. A step to obtain a preconjugate, wherein the sulfonamide is covalently bonded to the polypeptide precursor by an amide bond C(=O)-NH- formed between the -C(=O)-O(R) of the (activated) sulfonamide of formula (I) and the amino group of the polypeptide precursor; d.2) Enzymatic digestion of the preconjugated polypeptide precursor obtained according to d.1) at a pH in the range of less than 9 to obtain a solution containing a conjugate of sulfonamide and polypeptide. The method according to any one of embodiments 2 to 9, including the method described above.

[0205] 11. A method according to any one of embodiments 2 to 10: e) The step of isolating the sulfonamide and polypeptide conjugate from the solution obtained in d) or d.2). A method that further includes this.

[0206] 12. Activating group R of the activated sulfonamide of formula (I) x is selected from the group consisting of 7-azabenzotriazole (e.g., derived from HATU or HBTU), 4-nitrobenzene, and an N-succinimidyl group, or R x The method according to any one of Embodiments 1 to 11, wherein is an N-succinimidyl group.

[0207] 13. The method according to any one of Embodiments 1 to 12, wherein the aqueous solution of the polypeptide having a free amino group according to b) comprises an alcohol selected from the group consisting of C1-C4 monoalcohols and mixtures of two or more thereof, or methanol, ethanol, propan-2-ol, propan-1-ol, butan-1-ol, and mixtures of two or more thereof, or ethanol, propan-2-ol, propan-1-ol, and mixtures of two or more thereof.

[0208] 14. The method according to any one of Embodiments 1 to 13, wherein the aqueous solution according to (b) contains alcohol, and the amount of alcohol present in the aqueous solution is within the range of 0.0001 to 35 vol% or 0.001 to 30 vol% or 0.01 to 25 vol% or 0.1 to 20 vol% based on the total volume of water and alcohol, respectively.

[0209] 15. The method according to any one of Embodiments 6 to 14, wherein the enzymatic digestion according to d.2) comprises the use of at least one enzyme selected from the group consisting of trypsin, TEV protease (tobacco etch virus protease), and a mixture of trypsin and TEV protease.

[0210] 16. The polypeptide is mature insulin comprising an A chain and a B chain, wherein the A chain contains at least one mutation relative to the A chain of human insulin, and / or the B chain contains at least one mutation relative to human insulin. For example, at least one mutation in the A chain of human insulin is a substitution at position A14, such as a substitution with an amino acid selected from the group consisting of glutamic acid (Glu), aspartic acid (Asp), and histidine (His), and / or a substitution at position A21, such as a substitution with glycine (Gly). For example, mutations in the B chain of human insulin include valine (Val) and isoleucine (I Substitutions include substitutions at position B16, such as substitutions with amino acids selected from the group consisting of le), leucine (Leu), alanine (Ala), or histidine (His); substitutions at position B25, such as substitutions with valine (Val), isoleucine (Ile), leucine (Leu), alanine (Ala), or histidine (His); and / or deletions at position B30. In particular, mature insulin, Leu(B16)-human insulin, Val(B16) - Human insulin, Ile(B16) - Human insulin, Leu(B16)Des(B30)-Human insulin, Val(B16)Des(B30)-Human Insulin Ile(B16)Des(B30)-Human insulin, Leu(B25) - Human insulin, Val(B25) - Human insulin, Ile(B25) - Human insulin, Leu(B25)Des(B30)-Human Insulin Val(B25)Des(B30)-Human Insulin Ile(B25)Des(B30)-Human insulin, Glu(A14)Leu(B16)Des(B30)-Human insulin, Glu(A14)Ile(B16)Des(B30)-Human insulin, Glu(A14)Val(B16)Des(B30)-Human insulin, Glu(A14)Leu(B16)-Human insulin, Glu(A14)Ile(B16)-Human insulin, Glu(A14)Val(B16)-Human Insulin, Glu(A14)Leu(B25)Des(B30)-Human insulin, Glu(A14)Ile(B25)Des(B30)-Human insulin, Glu(A14)Val(B25)Des(B30)-Human insulin, Glu(A14)Leu(B25)-Human insulin, Glu(A14)Ile(B25)-Human Insulin, Glu(A14)Val(B25)-Human insulin, Glu(A14)Gly(A21)Glu(B3)Val(B25)Des(B30)-Human insulin, Glu(A14)Ile(B16)Ile(B25)Des(B30)-Human Insulin Glu(A14)Glu(B3)Ile(B16)Ile(B25)Des(B30)-Human insulin, Glu(A14)Ile(B16)Val(B25)Des(B30)-Human Insulin Glu(A14)Gly(A21)Glu(B3)Ile(B16)Val(B25)Des(B30)-Human insulin, Glu(A14)Val(B16)Ile(B25)Des(B30)-Human insulin, Glu(A14)Val(B16)Val(B25)Des(B30)-Human insulin, Glu(A14)Glu(B3)Val(B16)Val(B25)Des(B30)-Human insulin, Glu(A14)Gly(A21)Glu(B3)Val(B16)Val(B25)Des(B30)-Human insulin, Glu(A14)Gly(A21)Glu(B3)Val(B25)-Human Insulin Glu(A14)Ile(B16)Ile(B25)-Human Insulin Glu(A14)Glu(B3)Ile(B16)Ile(B25)-Human insulin, Glu(A14)Ile(B16)Val(B25)-Human Insulin Glu(A14)Gly(A21)Glu(B3)Ile(B16)Val(B25)-Human Insulin Glu(A14)Val(B16)Ile(B25)-Human Insulin Glu(A14)Val(B16)Val(B25)-Human Insulin Glu(A14)Glu(B3)Val(B16)Val(B25)-Human insulin, and Glu(A14)Gly(A21)Glu(B3)Val(B16)Val(B25)-Human Insulin 、 Glu(A14)His(B25)Des(B30) human insulin, and The method according to any one of Embodiments 1 to 15, selected from the group consisting of Glu(A14)His(B16)His(B25)Des(B30) human insulin.

[0211] 17. Sulfonamides are the (activated) sulfonamides of formula (I) -C(=O)-O(R xThe method according to any one of Embodiments 1 to 16, wherein an amide bond C(=O)-NH- is formed between the free amino group of the polypeptide and its precursor, and the free amino group of the polypeptide is, in some cases, an amino group of lysine contained in mature insulin and its precursor, such as terminal lysine, in particular lysine present at the C-terminus of the B chain, or lysine present at the C-terminus of mature insulin and its precursor.

[0212] 18. The activated sulfonamide of formula (I) is the protected activated sulfonamide of formula (0). [ka] (In the formula, A, E, X, m, n, p, q, r, s, t, R 1 , R 2 , and R x (The meaning of is defined in Embodiment 1) is obtained from or can be obtained from, and the protected activated sulfonamide of formula (0) is deprotected by the addition of one or more acids or at least trifluoroacetic acid. The method according to any one of Embodiments 1 to 17.

[0213] 19. The method according to any one of Embodiments 1 to 18, wherein the polypeptide precursor comprises the sequence according to Embodiment 12 and an additional linker peptide having a length of at least 2 amino acid residues, or a length in the range of 2 to 30 amino acid residues, or a length in the range of 4 to 9 amino acid residues.

[0214] 20. The first amino acids of a linker peptide are alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, and threonine. The method according to Embodiment 19, wherein the first amino acid of the linker peptide is selected from tryptophan, tyrosine, or valine residues, and is, for example, a threonine, phenylalanine, glutamine, glutamic acid, asparagine, or aspartic acid residue.

[0215] twenty one. The method according to Embodiment 19 or 20, wherein the last amino acid of the linker peptide is an arginine residue.

[0216] twenty two. The linker peptide has the following sequence Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Arg (Sequence number: 106) (In the formula, Xaa1 is any naturally occurring amino acid residue, and in some cases, here Xaa1 is threonine, phenylalanine, glutamine, glutamic acid, asparagine, or aspartic acid. Xaa2 is any naturally occurring amino acid residue, where Xaa2 is glutamic acid, or where Xaa2 is absent. Xaa3 is any naturally occurring amino acid residue, in particular, where Xaa3 is glycine, or where Xaa3 is absent. Xaa4 is either any naturally occurring amino acid residue, or, in this case, Xaa4 is not present. Xaa5 is either any naturally occurring amino acid residue, or, in this case, Xaa5 is not present. Xaa6 is either any naturally occurring amino acid residue, or, in this case, Xaa6 is not present. Xaa7 is either any naturally occurring amino acid residue, or, in this case, Xaa7 is not present. Xaa8 is either any naturally occurring amino acid residue or, in this case, Xaa8 does not exist. The method according to any one of embodiments 19 to 21, including the method described above.

[0217] twenty three. The linker peptide has the sequence TEGR (SEQ ID NO: 112), according to any one of embodiments 19 to 22.

[0218] twenty four. A conjugate obtained or obtainable from the method described in any one of Embodiments 1 to 23.

[0219] twenty five. A polypeptide precursor comprising the sequence of mature insulin according to Embodiment 16 and an additional linker peptide, as defined in any one of Embodiments 19-23, covalently bonded to the N-terminus of the mature insulin A chain.

[0220] 26. Activated sulfonamide corresponding to formula (I)

[0221] [ka] (In the formula, A, E, X, m, n, p, q, r, s, t, R 1 , R 2 , and R x A procedure for crystallizing (which has the meaning defined in Embodiment 1), A) A step of preparing a solution containing an activated sulfonamide and an organic solvent; B) A step of removing the organic solvent, for example by distillation, to obtain an activated sulfonamide phase having a reduced amount of organic solvent compared to the solution prepared in A); The step of adding an organic solvent to the phase obtained in C)B) to obtain a solution of activated sulfonamide; and A step in which step B) is repeated with the solution obtained in D)C) to obtain an activated sulfonamide phase having a reduced amount of organic solvent compared to the solution obtained in C); E) A process in which steps C) and D) are repeated at least one more time if applicable. Procedures including the above.

[0222] 27. The procedure according to Embodiment 26, wherein the solution prepared in A) further comprises trifluoroacetic acid.

[0223] 28. The procedure according to Embodiment 26 or 27, wherein the organic solvent is selected from the group of organic solvents capable of forming an azeotropic mixture with trifluoroacetic acid.

[0224] 29. For example, under standard conditions (temperature: 20-25°C, pressure: 10¹³ mbar), the octanol / water partition coefficient (K) is in the range of 1-5. OW The procedure according to any one of Embodiments 26 to 28, wherein the organic solvent is a polar aprotic organic solvent having ), and the organic solvent is selected from the group of, for example, acetonitrile, tetrahydrofuran, and a mixture of acetonitrile and tetrahydrofuran, and the organic solvent particularly contains at least acetonitrile.

[0225] 30. The activated sulfonamide in solid form, corresponding to formula (I).

[0226] [ka] (In the formula, A, E, X, m, n, p, q, r, s, t, R 1 , R 2 , and R x (This has the meaning defined in Embodiment 1).

[0227] 31. A process for producing a conjugate of albumin-binding agent and mature insulin, a) A step of preparing proinsulin containing an insulin B chain, a linker peptide, and an insulin A chain from the N-terminus to the C-terminus, b) A step of cleaving the proinsulin prepared in step a) between the last amino acid of the insulin B chain and the first amino acid of the linker peptide using a first protease, thereby generating an insulin precursor, wherein the insulin precursor includes an insulin B chain, a linker peptide and an N-terminally elongated A chain including the A chain, c) A step of contacting the insulin precursor with an albumin binder, wherein the albumin binder is It contains a functional group capable of binding to albumin; This process generates a conjugate of an albumin binder and an insulin precursor. d) The N-terminal extended A chain of the insulin precursor contained in the conjugate is cleaved between the last amino acid of the linker peptide and the first amino acid of the A chain by a second protease, thereby generating a conjugate of albumin-binding agent and mature insulin. The process including the process.

[0228] 32. The process according to Embodiment 31, wherein the last amino acid of the insulin B chain is a lysine residue.

[0229] 33. The process according to embodiment 31 or 32, wherein the first amino acid of chain A is a glycine residue.

[0230] 34. The process according to any one of embodiments 31 to 33, wherein the linker peptide has a length of at least two amino acid residues.

[0231] 35. The linker peptide has a length of 2 to 30 amino acid residues, such as a length of 4 to 9 amino acid residues, in the process according to Embodiment 34.

[0232] 36. The process according to any one of embodiments 31 to 35, wherein the first amino acid of the linker peptide is selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and tyrosine, for example, the process according to any one of embodiments 31 to 35, wherein the first amino acid of the linker peptide is selected from alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and tyrosine.

[0233] 37. The process according to Embodiment 36, in which the first amino acid of the linker peptide is a threonine residue, a phenylalanine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue, for example, the first amino acid of the linker peptide is a threonine residue.

[0234] 38. The process according to any one of embodiments 31 to 37, wherein the last amino acid of the linker peptide is an arginine residue.

[0235] 39. The process according to any one of embodiments 31 to 38, wherein the first protease is endoproteinase Lys-C and / or the second protease is trypsin or TEV protease (tobacco etch virus protease).

[0236] 40. The process according to any one of embodiments 31 to 39, wherein the proinsulin prepared in step a) further contains a signal peptide at the N-terminal end of the insulin B chain, and the first protease further cleaves the proinsulin between the last amino acid of the signal peptide and the first amino acid of the B chain.

[0237] 41. The process according to Embodiment 40, wherein the last amino acid of the signal peptide is a lysine residue.

[0238] 42. From the N-terminus to the C-terminus: (a) Insulin B chain, (b) Linker peptide, and (c) Insulin A chain Proinsulin containing, The proinsulin comprises an endoproteinase Lys-C cleavage site between the last amino acid of the insulin B chain and the first amino acid of the linker peptide, and a trypsin cleavage site between the last amino acid of the linker peptide and the first amino acid of the insulin A chain.

[0239] 43. The proinsulin according to Embodiment 42, further comprising a signal peptide at the N-terminal end of the insulin B chain, wherein the proinsulin includes a cleavage site of endoproteinase Lys-C located between the last amino acid of the signal peptide and the first amino acid of the B chain.

[0240] 44. The linker peptide has the following sequence Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Arg (Sequence number: 106) (In the formula, Xaa1 is any naturally occurring amino acid residue, and in some cases, here Xaa1 is threonine, phenylalanine, glutamine, glutamic acid, asparagine, or aspartic acid. Xaa2 is any naturally occurring amino acid residue, where Xaa2 is glutamic acid, or where Xaa2 is absent. Xaa3 is any naturally occurring amino acid residue, in particular, where Xaa3 is glycine, or where Xaa3 is absent. Xaa4 is either any naturally occurring amino acid residue, or, in this case, Xaa4 is not present. Xaa5 is either any naturally occurring amino acid residue, or, in this case, Xaa5 is not present. Xaa6 is either any naturally occurring amino acid residue, or, in this case, Xaa6 is not present. Xaa7 is either any naturally occurring amino acid residue, or, in this case, Xaa7 is not present. Xaa8 is either any naturally occurring amino acid residue or, in this case, Xaa8 does not exist. Proinsulin according to Embodiment 43, including the above.

[0241] 45. The A chain has the following sequence: GIVEQCCTSICSL Xaa9 QLENYCN (Sequence ID: 109) [wherein Xaa9 is glutamic acid (Glu), aspartic acid (Asp), or histidine (His)] and / or The B chain has the following sequence: FVNQHLCGSHLVEAL Xaa10 LVCGERGF Xaa11 YTPK (Sequence ID: 110) [In the formula, Xaa10 is tyrosine (Tyr), valine (Val), isoleucine (Ile), leucine (Leu), alanine (Ala), or histidine (His), and / or The proinsulin according to any one of embodiments 42 to 44, wherein Xaa11 is phenylalanine (Phe), valine (Val), isoleucine (Ile), leucine (Leu), alanine (Ala), or histidine (His) in the formula.

[0242] 46. The linker peptide is proinsulin according to any one of embodiments 42 to 45, having the sequence TEGR (Sequence ID: 112).

[0243] 47. Proinsulin is in the following sequence FVNQHLCGSHLVEAL Xaa10 LVCGERGF Xaa11 YTPK Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 R GIVEQCCTSICSL Xaa9 QLENYCN (SEQ ID NO: 111) [wherein Xaa1 to Xaa8 have the meanings described in Embodiment 40 or Section B) above, and Xaa9 to Xaa11 have the meanings described in Embodiment 41] For example, proinsulin has the following sequence FVNQHLCGSHLVEALYLVCGERGFVYTPKTEGRGIVEQCCTSICSLEQLENYCN(Sequence ID: 108) Proinsulin according to Embodiment 46, including the above.

[0244] 48. A polynucleotide encoding proinsulin according to any one of embodiments 42 to 47.

[0245] 49. A vector comprising polynucleotides as described in Embodiment 48.

[0246] 50. A host cell comprising proinsulin according to any one of embodiments 42 to 47, polynucleotide according to embodiment 44, and / or the vector according to embodiment 45.

[0247] 51. From the N-terminus to the C-terminus: (a) linker peptide, and (b) Insulin A chain An N-terminally elongated insulin A chain containing, The N-terminally elongated insulin A chain, comprising a trypsin cleavage site between the last amino acid of the linker peptide and the first amino acid of the A chain.

[0248] 52. An insulin precursor comprising an N-terminally elongated insulin A chain and an insulin B chain as described in Embodiment 51.

[0249] 53. A conjugate comprising an insulin precursor and a sulfonamide as described in Embodiment 52.

[0250] 54. The conjugate is as follows: (B chain: SEQ ID NO: 48, N-terminally extended A chain: SEQ ID NO: 107), and is the conjugate shown in Figure 8, as described in Embodiment 53.

[0251] 55. The activated sulfonamide is in crystalline form, as described in Embodiment 30.

[0252] 56. A method for forming a conjugate of a sulfonamide and an insulin polypeptide, wherein the activated sulfonamide is an activated albumin binder, according to any one of Embodiments 1 to 23.

[0253] The present invention will be further explained by the following examples. [Examples]

[0254] I. Compound Synthesis and Conjugate Preparation All pH measurements were performed using pH-sensitive glass electrodes in accordance with ASTM E 70:2007.

[0255] The yield from the HPLC data was calculated from the overall relationship between the extract and the product.

[0256] 1 16-[4-[[5-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylcarbamoyl]pyrimidine-2-yl]sulfamoyl]phenoxy]hexadecanoate

[0257] 1.1 Synthesis 2-[2-[2-[[2-[2-[[2-[[4-(16-tert-butoxy-16-oxo-hexadecoxy)phenyl]sulfonylamino]pyrimidine-5-cal 32.5 g of vonylaminoethoxyethoxyacetylaminoethoxyethoxyacetic acid (a viscous syrup) was dissolved in 305 ml of anhydrous acetonitrile. 305 ml of anhydrous ethyl acetate, 16.25 g (63.4 mmol) of N,N-disuccinimidyl carbonate, and 3.25 g (26.6 mmol) of dimethylaminopyridine were added under the argon layer with stirring at room temperature (25°C). After 90 minutes, the solvent was removed by distillation using a rotary evaporator, and the remaining oily product was dissolved in 1.5 liters of ethyl acetate. This ethyl acetate solution was extracted three times with 300 ml of 0.1 N HCl and 300 ml of saturated NaCl solution each time. After distillation of the solvent using a rotary evaporator, an oily product remained. The oily product was dissolved in 0.5 liters of ethyl acetate, the precipitated NaCl was filtered off, and the ethyl acetate was removed by distillation. Next, 0.5 liters of ethyl acetate were added, and the solvent was removed by distillation using a rotary evaporator. This procedure, i.e., the addition of 0.5 liters of ethyl acetate and the removal of the solvent by distillation, was repeated three times to obtain an oily product. The oily product was dissolved in 325 ml of methylene chloride. 163 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature (25°C) for 80 minutes. The solvent and trifluoroacetic acid were removed by distillation using a rotary evaporator. 100 ml of acetonitrile was added to the remaining oily product, and the solvent was removed by distillation using a rotary evaporator. This procedure, i.e., the addition of 100 ml of acetonitrile and the removal of the solvent by distillation, was repeated six times. Next, 1.5 liters of acetonitrile were added, the solution was covered with argon, and kept overnight at a temperature between 2°C and 8°C. The product formed in the acetonitrile solution under argon was stable for a period of less than 3 days. The yield in acetonitrile solution was calculated to be 78% from HPLC data, based on the amount of 2-[2-[[[2-[[2-[[2-[[2-[[4-(16-tert-butoxy-16-oxo-hexadecoxy)phenyl]sulfonylamino]pyrimidine-5-carbonyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid used.

[0258] 1.2 Crystallization 174.4 g (194.6 mmol) of 2-[2-[2-[[2-[2-[2-[[2-[[4-(16-tert-butoxy-16-oxo-hexadecoxy)phenyl]sulfonylamino]pyrimidine-5-carbonyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid (a viscous syrup) was dissolved in 2.3 liters of ethyl acetate at 45°C. 3.47 g (28.4 mmol) of dimethylaminopyridine was added to the reaction vessel, and then an ethyl acetate solution of 2-[2-[2-[[2-[2-[[2-[[4-(16-tert-butoxy-16-oxo-hexadecoxy)phenyl]sulfonylamino]pyrimidine-5-carbonyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid was added at room temperature (25°C). Next, 3.7 liters of acetonitrile solution of 98.7 g (385.3 mmol) of N,N-disuccinimidyl carbonate was added with stirring at room temperature (25°C). After 90 minutes, the solvent was removed by distillation using a rotary evaporator, and the remaining oily product was dissolved in 2.3 liters of ethyl acetate. This ethyl acetate solution was extracted three times with 470 ml of 0.1 N HCl and 470 ml of saturated NaCl solution each time. After removing the solvent by distillation using a rotary evaporator, an oily product remained. The oily product was dissolved in 1.4 liters of ethyl acetate. Next, the solvent was removed by distillation using a rotary evaporator, followed by the addition of 2.7 liters of ethyl acetate. After filtering off the precipitated NaCl and removing the ethyl acetate by distillation, an oily product remained. The oily product was dissolved in 2 liters of methylene chloride. 450 ml of trifluoroacetic acid was added, and the solution was stirred at room temperature (25°C) for 100 minutes. After removing the solvent and trifluoroacetic acid by distillation using a rotary evaporator, an oily product remained. 940 ml of acetonitrile was added to the oily product, followed by the removal of the solvent by distillation using a rotary evaporator. This procedure, i.e., the addition of 940 ml of acetonitrile and the removal of the solvent by distillation, was repeated four times. Next, 2.2 liters of acetonitrile were added. After adding the solution and stirring it overnight at room temperature (25°C), the product crystallized. The suspension was filtered, and the precipitate was vacuum-dried at room temperature for 3 days. The crystalline product, 16-[4-[[5-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylcarbamoyl]pyrimidine-2-yl]sulfamoyl]phenoxy]hexadecanoate, was stable at 2°C to 8°C for at least 6 months.

[0259] Yield 162g, based on the amount of 2-[2-[[[2-[2-[2-[[2-[[4-(16-tert-butoxy-16-oxo-hexadecoxy)phenyl]sulfonylamino]pyrimidine-5-carbonyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid used, which is 89%.

[0260] 2. Insulin analogs 2.1 Insulin Analog 1 Insulin analog 1 is based on human insulin with a mutation at position A14, a mutation at position B25, removal of an amino acid at position B30, and an additional amino acid sequence TEGR (SEQ ID NO: 112) at the start of the A chain: Glu(A14): In human insulin, the amino acid at position 14 of the A chain (Y, tyrosine, Tyr) is substituted with glutamic acid (E, Glu). Val(B25): In human insulin, the amino acid at position 25 of the B chain (F, phenylalanine, Phe) is substituted with valine (V, Val). Des(B30): Human insulin has a missing amino acid at position 30 of the B chain.

[0261] The complete amino acid sequences of insulin analog 1 for chains A and B are as follows: Chain A:TEGRGIVEQCCTSICSLEQLENYCN (Sequence ID: 107) Chain B:FVNQHLCGSHLVEALYLVCGERGFVYTPK (Sequence ID: 48)

[0262] The presence of one intrachain disulfide bridge and two interchain disulfide bridges is consistent with human insulin.

[0263] Using preproinsulin containing a signal peptide and the following proinsulin amino acid sequence: FVNQHLCGSHLVEALYLVCGERGFVYTPKTEGRGIVEQCCTSICSLEQLENYCN(Sequence ID: 108)

[0264] The preproinsulin solution obtained from the cation exchange chromatography capture step was adjusted to pH 8.5.

[0265] Endoproteinase Lys-C was added, and the solution was stirred for 2 hours. The mixture was then purified by cation exchange chromatography, followed by reverse-phase chromatography. The solution containing the product fraction was collected and lyophilized. The resulting white powder contained insulin analog 1.

[0266] 2.2 Insulin Analog 2 Insulin analog 2 is equivalent to insulin analog 1, except that it lacks the TEGR (sequence number: 112) group at the starting point of the A chain.

[0267] The complete amino acid sequences of insulin analog 2 for chains A and B are as follows: ru: Chain A: GIVEQCCTSICSLEQLENYCN (Sequence ID: 47) Chain B: FVNQHLCGSHLVEALHLVCGERGFHYTPK (Sequence ID: 48)

[0268] The presence of one intrachain disulfide bridge and two interchain disulfide bridges is consistent with human insulin.

[0269] 3. Conjugate Synthesis 3.2 Conjugate of insulin analog 2 with 16-[4-[[5-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylcarbamoyl]pyrimidine-2-yl]sulfamoyl]phenoxy]hexadecanoate in acetonitrile solution - Conjugation in water / acetonitrile 37.5 g (12.4 mmol) of insulin analog 1 according to Section 2.1 above was suspended in 938 ml of water, and the pH was adjusted to 11.1 with triethylamine; 1313 ml of acetonitrile was added. 600 ml (14.5 mmol) (20 g / liter) of acetonitrile solution of 16-[4-[[5-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylcarbamoyl]pyrimidine-2-yl]sulfamoyl]phenoxy]hexadecanoate according to Section 1.1 above was slowly added to the water / acetonitrile solution of insulin analog 1 over 90 minutes with stirring at room temperature (25°C). The pH was maintained within the range of 10.9 to 11 by the addition of triethylamine. When the in-process control by HPLC showed a complete reaction (54.4% purity), the pH was adjusted to 10.3 with 1N HCl, and acetonitrile was removed from the solution by distillation using a rotary evaporator. Next, 1 liter of water was added, the pH of the aqueous solution was adjusted to 8.3 with 0.1N HCl, and 3.75 ml of aqueous trypsin solution (11035 U / ml) was added, and the mixture was stirred overnight at room temperature (25°C). When the control by HPLC showed a complete reaction (50.7% purity), the solution was sent for chromatographic purification. The yield in aqueous solution was calculated to be 50% based on the amount of insulin analog 1 used, derived from HPLC data before purification.

[0270] HPLC analysis was performed using an Agilent 1100 HPLC at 210 nm during conjugation and after trypsin cleavage. - Column: Phenomenex Gemini C6-Phenyl; 3μm, 50×3mm - Mobile phase A: Water / trifluoroacetic acid 0.1% - Mobile phase B: Acetonitrile / trifluoroacetic acid 0.1% - Gradient: From 90% A / 10% B to 12% A / 82% B in 13 minutes

[0271] The retention time in the chromatogram was 7.34 minutes after conjugation and before trypsin cleavage, and 7.51 minutes after trypsin cleavage.

[0272] The conjugate of insulin analog 2 and 16-[4-[[5-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylcarbamoyl]pyrimidine-2-yl]sulfamoyl]phenoxy]hexadecanoate had the structure shown in Figure 3 (conjugate 1).

[0273] 3.3 Conjugate of insulin analog 2 with crystalline 16-[4-[[5-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylcarbamoyl]pyrimidine-2-yl]sulfamoyl]phenoxy]hexadecanoate - Conjugation in water / acetonitrile / tetrahydrofuran, reaction in solution 75 g (80 mmol) of crystalline 16-[4-[[5-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylcarbamoyl]pyrimidine-2-yl]sulfamoyl]phenoxy]hexadecanoate according to Section 1.2 above was dissolved in 1.8 liters of tetrahydrofuran and 3.8 liters of acetonitrile (binding agent solution). 250 g (41.2 mmol) of insulin analog 1 according to Section 2.1 above was suspended in 6.2 liters of water, and the pH was adjusted to 10.3 with triethylamine to dissolve insulin analog 1. The binding agent solution was added to the insulin analog 1 solution while stirring at room temperature (25°C). When the in-process control by HPLC showed a complete reaction (reaction turnover 99%; purity 80%), the organic solvent in the solution was removed by distillation using a rotary evaporator. Next, the pH of the aqueous solution was adjusted to a value in the range of 8.0 to 8.5 with 0.1N HCl. To remove the TEGR group from the A chain of insulin analog 1, 3 ml of aqueous trypsin solution (11035 U / ml) was added and stirred overnight at room temperature (25°C). When the in-process control by HPLC showed a complete reaction (reaction turnover 99.5%; purity 81%), the pH was adjusted to 6.5 with 1N HCl and the solution was sent for chromatographic purification. The yield in aqueous solution was calculated to be 80% based on the amount of insulin analog 1 used, derived from HPLC data before purification.

[0274] After chromatographic purification, liberation analysis was performed using an Agilent 1100 HPLC at 210 nm: - Column: Waters X-Select CSH C18; 2.5 μm, 150 × 4.6 mm - Mobile phase A: Water / acetonitrile / trifluoroacetic acid 90% / 10% / 0.05% - Mobile phase B: Water / acetonitrile / trifluoroacetic acid 10% / 90% / 0.05% - Gradient: From 65% A / 35% B to 10% A / 90% B in 15 minutes

[0275] The purity of the conjugate of insulin analog 2 with 16-[4-[[5-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylcarbamoyl]pyrimidine-2-yl]sulfamoyl]phenoxy]hexadecanoate was 98.0%, and the retention time in the chromatogram was 7.65 minutes.

[0276] The conjugate of insulin analog 2 obtained in 3.3 and 16-[4-[[5-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylcarbamoyl]pyrimidine-2-yl]sulfamoyl]phenoxy]hexadecanoate had the structure shown in Figure 3 (conjugate 1).

[0277] 3.4 Insulin analog 2 and the crystalline form of 16-[4-[[5-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylcarbamoyl]pyrimidine-2-yl]sulfamoyl]phenoxy]hexadecanoate Conjugate - Conjugation in water, solid-phase conjugation 250 g (41.2 mmol) of insulin analog 1 according to Section 2.1 above was suspended in 10 liters of water, and the pH was adjusted with triethylamine to a value within the range of 10.6 to 10.8 to dissolve insulin analog 1. 70 g (74.7 mmol) of crystalline 16-[4-[[5-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylcarbamoyl]pyrimidine-2-yl]sulfamoyl]phenoxy]hexadecanoate according to Section 1.2 above was divided into 5 × 10 g and 4 × 5 g portions and added to the aqueous solution of insulin analog 1, and the pH was maintained with triethylamine to a value within the range of 10.6 to 10.8. When the in-process control by HPLC showed a complete reaction (reaction turnover 94%; purity 70%), the pH of the aqueous solution was adjusted to a value within the range of 8.2–8.4 with 1N HCl. To remove the TEGR group from the A chain of insulin, 3.25 ml of aqueous trypsin solution (11035 u / ml) was added and stirred overnight at room temperature (25°C). When the in-process control by HPLC showed a complete reaction (reaction turnover 100%; purity 72%), the pH was adjusted to 6.5 with 1N HCl and the solution was sent for chromatographic purification. The yield in the aqueous solution was calculated to be 72% based on the amount of insulin analog 1 used, from the HPLC data before purification.

[0278] After chromatographic purification, liberation analysis was performed using an Agilent 1100 HPLC at 210 nm: - Column: Waters X-Select CSH C18; 2.5 μm, 150 × 4.6 mm - Mobile phase A: Water / acetonitrile / trifluoroacetic acid 90% / 10% / 0.05% - Mobile phase B: Water / acetonitrile / trifluoroacetic acid 10% / 90% / 0.05% - Gradient: From 65% A / 35% B to 10% A / 90% B in 15 minutes

[0279] The purity of the conjugate of insulin analog 2 with 16-[4-[[5-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylcarbamoyl]pyrimidine-2-yl]sulfamoyl]phenoxy]hexadecanoate was 98.4%, and the retention time in the chromatogram was 7.89 minutes.

[0280] The conjugate of insulin analog 2 obtained in 3.4 and 16-[4-[[5-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylcarbamoyl]pyrimidine-2-yl]sulfamoyl]phenoxy]hexadecanoate had the structure shown in Figure 3 (Conjugate 1).

[0281] 3.5 Conjugation of insulin analog 2 with crystalline form of 16-[4-[[5-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylcarbamoyl]pyrimidine-2-yl]sulfamoyl]phenoxy]hexadecanoate - Conjugation in water / n-propanol, solid-phase conjugation 99.49 g of wet insulin analog 1, containing 23 g (3.8 mmol) of insulin analog 1 according to Section 2.1 above, was suspended in 688 ml of water, and the pH was adjusted to 10.9 with triethylamine to dissolve insulin analog 1. 231 ml of n-propanol was added, and the pH was adjusted to 10.6. Crystalline 6-[4-[[5-[2-[2-[2-[2-[2-[2-(2,5-dioxy) 6.25 g (6.68 mmol) of sopyrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylcarbamoyl]pyrimidine-2-yl]sulfamoyl]phenoxy]hexadecanoate was divided into 2 × 2 g, 2 × 1 g, and 1 × 250 mg portions and added to a water / n-propanol solution, maintaining the pH within the range of 10.6 to 10.8 with triethylamine. When the in-process control (reaction turnover 93%; purity 80%) showed a complete reaction by HPLC, the pH of the water / n-propanol solution was adjusted to 8.2 with 1N HCl, and 137.5 μl (18562 U / ml) of aqueous trypsin solution was added to remove the TEGR group from the A chain of insulin analog 1, and the mixture was stirred overnight at room temperature. When the in-process control by HPLC (reaction turnover 94%; purity 89%) showed a complete reaction, the pH was adjusted to 6.8 with 1N HCl, and the solution was sent for chromatographic purification.

[0282] The yield in aqueous solution was calculated to be 84% based on the amount of insulin analog 1 used, derived from HPLC data before purification.

[0283] After chromatographic purification, liberation analysis was performed using an Agilent 1100 HPLC at 210 nm: - Column: Waters X-Select CSH C18; 2.5 μm, 150 × 4.6 mm - Mobile phase A: Water / acetonitrile / trifluoroacetic acid 90% / 10% / 0.05% - Mobile phase B: Water / acetonitrile / trifluoroacetic acid 10% / 90% / 0.05% - Gradient: From 65% A / 35% B to 10% A / 90% B in 15 minutes

[0284] The purity of the conjugate of insulin analog 2 with 16-[4-[[5-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylcarbamoyl]pyrimidine-2-yl]sulfamoyl]phenoxy]hexadecanoate was 96.0%, and the retention time in the chromatogram was 8.12 minutes.

[0285] The conjugate of insulin analog 2 obtained in 3.5 and 16-[4-[[5-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylcarbamoyl]pyrimidine-2-yl]sulfamoyl]phenoxy]hexadecanoate had the structure shown in Figure 3 (conjugate 1).

[0286] 3.6 Conjugate of insulin analog 2 with 16-[4-[[5-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethyl - Comparative Examples The tert-butyl ester of 16-[4-[[5-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylcarbamoyl]pyrimidine-2-yl]sulfamoyl]phenoxy]hexadecanoate was reacted with insulin analog 2 to form an amide bond, and then the tert-butyl protecting group was removed as follows: A 400 mg solution of the insulin analog according to Section 2.2 above was suspended in 20 ml of water, and then 0.4 ml of triethylamine was added. To this clear solution, 20 ml of DMF was added, followed by tert-butyl16-[4-[[5-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylcarbamoyl]pyrimidine 5 ml of [-2-yl]sulfamoyl]phenoxy]hexadecanoate (17.04 mM in DMF) was added. The solution was stirred at room temperature for 2 hours. The reactants were analyzed using a Waters UPLC H-class at 214 nm in sodium chloride phosphate buffer. Waters BEH300 10cm. Insulin retention time: 2.643 minutes. The retention time of the insulin conjugate was 6.224 minutes. The product was purified by HPLC using an AKTA avant 25. Kinetex 5μm C18 100 A 250×21.2mm. Column volume (CV) 88ml. Solvent A: 0.5% aqueous acetic acid solution Solvent B: 0.5% aqueous acetic acid solution / acetonitrile 4:6 Gradient: 10CV from 80%A to 20%B to 20%A to 80%B

[0287] After freeze-drying the product, the powder was dissolved in 2 ml of trifluoroacetic acid. After 1 hour, the solution was neutralized with diluted sodium bicarbonate. A Purified by HPLC using a KTA avant 25. Kinetex 5μm C18 100 A 250×21.2mm column. Column volume (CV) 88 ml. Solvent A: 0.5% aqueous acetic acid solution Solvent B: 0.5% aqueous acetic acid solution / acetonitrile 4:6 Gradient: 8CV from 70%A 30%B to 30%A 70%B The reactants were analyzed in sodium chloride phosphate buffer using a Waters UPLC H-class at 214 nm. Waters BEH300 10cm. Insulin conjugate retention time: 5.121 minutes. The solution was freeze-dried to obtain the desired product. Based on 63 mg of insulin analog 2 used, the yield was 14%. Mass spectrum: 6453.9 g / mol.

[0288] The conjugate of insulin analog 2 obtained in 3.6 and 16-[4-[[5-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylcarbamoyl]pyrimidine-2-yl]sulfamoyl]phenoxy]hexadecanoate had the structure shown in Figure 3 (Conjugate 1).

[0289] 3.7 Conclusion The yields of the synthetic pathways in sections 3.2-3.6, in relation to the respective amounts of insulin analogs 1 and 2 used, can be summarized as follows:

[0290] [Table 1]

[0291] As can be seen from the yields summarized above, the method of the present invention for forming conjugates of sulfonamides and polypeptides (here, conjugates of a specific sulfonamide and a specific insulin analog) enables relatively high overall yields in conjugate synthesis, i.e., yields of over 20%, over 30%, over 40%, or over 50%, depending on the polypeptide / insulin analog used. When a specifically activated sulfonamide is used in combination with a TEGR-protected insulin analog, comparatively high yields are achieved. The yield is already increased by at least three times compared to the previous method; that is, the yield is at least 50%. The use of activated sulfonamide in solid form [in solid form (see 3.4 or 3.5) or dissolved in a solvent (see 3.3)] further increases the yield to over 70%. Optimized combinations of solid-phase reactions and suitable solvents, as described in 3.5, enable yields exceeding 80%.

[0292] II. In vivo and in vitro studies 1. Production of human insulin and insulin analogs For example, various insulin analogs with mutations at positions B16, B25, and / or A14 were generated. Table 1 provides an overview of the generated insulins.

[0293] [Table 2] [Table 3] [Table 4] [Table 5]

[0294] 2. Insulin receptor binding affinity assay / Insulin receptor autophosphorylation assay The insulin binding and signaling of various insulin analogs generated were determined by binding assays and receptor autophosphorylation assays.

[0295] A) Insulin receptor binding affinity assay The insulin receptor binding affinity of the analogs listed in Table 1 was determined as described by Hartmann et al. (Effect of the long-acting insulin analogs glargine and degludec on cardiomyocyte cell signaling and function. Cardiovasc Diabetol. 2016;15:96). Isolation and competitive binding experiments of insulin receptor embedded plasma membrane (M-IR) were performed as previously reported (Sommerfeld et al., PLoS One. 2010; 5(3): e9540). Briefly, CHO cells overexpressing IR were collected, resuspended in ice-cold 2.25 STM buffer (2.25 M sucrose, 5 mM Tris-HCl pH 7.4, 5 mM MgCl2, a complete protease inhibitor), and destroyed by Dounce homogenizer followed by sonication. The homogenate was covered with 0.8 STM buffer (0.8 M sucrose, 5 mM Tris-HCl pH 7.4, 5 mM MgCl2, complete protease inhibitor) and ultracentrifuged at 100,000 g for 90 minutes. The plasma membrane at the interface was collected and washed twice with phosphate-buffered saline (PBS). The final pellet was resuspended in dilution buffer (50 mM Tris-HCl pH 7.4, 5 mM MgCl2, complete protease inhibitor) and rehomogenized using a Dounce homogenizer. Competitive binding experiments were performed in a 96-well microplate in binding buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% BSA, complete protease inhibitor, adjusted to pH 7.8). In each well, 2 μg of isolated membrane was incubated with 0.25 mg of wheat germ agglutinin polyvinyltoluene polyethyleneimine scintillation proximity assay (SPA) beads. A constant concentration of [125I]-labeled human insulin (100 pM) and various concentrations of unlabeled insulin (0.001 to 1000 nM) were added at room temperature (23°C) for 12 hours. Radioactivity was measured using a microplate scintillation counter (Wallac Microbeta, Freiburg, Germany).) The measurements were taken under equilibrium conditions.

[0296] Table 2 shows the results of insulin receptor binding affinity assays for analogs compared to human insulin.

[0297] B) Insulin receptor autophosphorylation assay (as a measure of signal transduction) To determine the signaling pathway of insulin analogs that bind to insulin receptor B, autophosphorylation was measured in vitro. CHO cells expressing human insulin receptor isoform B (IR-B) were used for an IR autophosphorylation assay using in-cell Western technology, as previously reported (Sommerfeld et al., PLoS One. 2010; 5(3): e9540). For the analysis of IGF1R autophosphorylation, the receptor was overexpressed in mouse embryonic fibroblast 3T3 Tet-off cell line (BD Bioscience, Heidelberg, Germany) stably transfected with an IGF1R tetracycline-modulated expression plasmid. To determine the receptor tyrosine phosphorylation level, cells were seeded in 96-well plates and grown for 44 hours. Cells were serum-deficiented for 2 hours in serum-free Ham's F12 medium (Life Technologies, Darmstadt, Germany). Subsequently, cells were treated with either human insulin or an insulin analog at 37°C for 20 minutes at increasing concentrations. After incubation, discard the culture medium and immerse in 3.75% paraformaldehyde prepared immediately before use. Cells were fixed for 20 minutes. Cells were permeabilized with 0.1% Triton X-100 in PBS for 20 minutes. Blocking was performed at room temperature for 1 hour using Odyssey blocking buffer (LICOR, Bad Homburg, Germany). Anti-pTyr 4G10 (Millipore, Schwalbach, Germany) was incubated at room temperature for 2 hours. After incubation with the primary antibody, cells were washed with PBS + 0.1% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA). Secondary anti-mouse IgG-800-CW antibody (LICOR, Bad Homburg, Germany) was incubated for 1 hour. Results were normalized by DNA quantification with TO-PRO3 dye (Invitrogen, Karlsruhe, Germany). Data were obtained in relative units (RU).

[0298] Table 2 shows the results of insulin receptor autophosphorylation assays of analogs compared to human insulin.

[0299] [Table 6] [Table 7] [Table 8]

[0300] C) Conclusion As can be seen from Table 2, various hydrophobic substitutions at positions B16 and / or B25 were tested (tryptophan, alanine, valine, leucine, and isoleucine). To varying degrees, all tested insulin analogs with hydrophobic substitutions at these positions showed a decrease in insulin receptor binding activity. Compared to tryptophan substitutions (see, for example, analogs 4, 15, and 23), substitutions at aliphatic amino acid residues such as alanine, valine, leucine, and isoleucine showed a decrease in insulin receptor binding activity. The effects were strong. The strongest effects were observed for valine, leucine, and isoleucine, all of which are branched-chain amino acid residues. Substitutions at isoleucine, valine, and leucine resulted in a significant decrease in insulin receptor binding activity. Interestingly, insulin analogs with such substitutions at the B25 position (e.g., valine, leucine, or isoleucine substitutions at the B25 position, analogs 11, 12, 22, 24, 25, 29, 30, 32, 33, 35, 38, 39, 40) showed up to 6-fold enhancement of signal transduction than expected based on their IR-B binding affinity. Specifically, Leu(B25)Des(B30)-insulin and Val(B25)Des(B30)-insulin (analogs 11 and 12, respectively) showed only 1% insulin receptor B binding and only 6% autophosphorylation compared to human insulin. Similarly, a single leucine substitution at the B16 position (analog 3) also showed a similar, albeit slightly, enhancement of signal transduction. In comparison, with the exception of analog 26, analogs with histidine B25 substitutions (analogs 10, 13, 14, 21, and 28) also showed reduced receptor binding, but simultaneously reduced autophosphorylation.

[0301] In some cases (analogs 30, 32, 35, 38, 39), insulin receptor binding was 0%, although activity in autophosphorylation assays was still observed. All of these analogs shared a common combination of valine and / or isoleucine substitutions at positions B16 and B25, suggesting that this combination contributes to the further reduction in insulin receptor binding. Insulins with a B16 substitution but no B25 substitution exhibited slightly higher binding affinity compared to their autophosphorylation values ​​(analogs 3, 4, 16, 17, 18, 19, 20).

[0302] Alanine at position B25 exhibits similar effects to valine, leucine, or isoleucine substitutions (analogs 11, 12, and 22), albeit to a relatively low degree. The receptor binding affinity and autophosphorylation activity of analogs with valine, leucine, or isoleucine substitutions are lower than those of analogs with alanine substitutions.

[0303] 3. Generation of further conjugates - in vivo testing - evaluation of pharmacokinetic effects Insulin conjugates 1-4 were manufactured and tested. As a control, insulin conjugate 5, as described in WO2018109162A1 [Norrman, Novo Nordisk], was manufactured.

[0304] The manufactured insulin conjugates are summarized in the following table (Table 3). Furthermore, insulin conjugates 1-4 are shown in Figures 3-6.

[0305] [Table 9]

[0306] The in vivo pharmacodynamic and pharmacokinetic effects of ultra-long-acting insulin conjugates were evaluated in healthy, blood glucose-normal Göttingen miniature pigs (pigs aged 0.5–6 years, with body weights ranging from approximately 12–40 kg depending on age). Pigs were housed under standard laboratory animal housing conditions, fed once daily, and given ad libitum access to tap water. After overnight fasting, pigs were treated with a single subcutaneous injection of a solution containing either a placebo or one of the respective insulin conjugates. Insulin conjugates 1–4 and insulin conjugate 5 (described in WO2018109162A1 [Novo Nordisk, Norrman]) were tested. Blood was collected from K-EDTA plasma via a pre-implanted central venous catheter to determine blood glucose, pharmacokinetic biomarkers, and additional biomarkers. Blood sampling began before administration of the test substance (baseline) and was repeated 1 to 4 times per day until the end of the study. During the study period, animals were fed after the last blood sampling of the day. All animals were handled uniformly, and clinical signs were recorded at least twice on the treatment day and once per day for the remainder of the study period. Animals were carefully monitored for all clinical signs of hypoglycemia, including behavior, skin condition, urine and fecal excretion, condition of body orifices, and any signs of disease. In cases of severe hypoglycemia, researchers were available to provide food, or, if food intake was not possible, to administer intravenous (iv) glucose solution. After the last blood sampling, animals were returned to the animal housing facility.

[0307] A) Effects on blood glucose levels during fasting The results are also shown in Figure 1.

[0308] [Table 10]

[0309] B) Measurement of pharmacokinetic parameters The results are also shown in Figure 2.

[0310] [Table 11]

[0311] C) Conclusion A single dose of insulin conjugate 4 (Ile(B25)) at a 30 nM / kg dose showed a flat profile up to 152 hours and exhibited a low to mild glucose-lowering effect. Insulin conjugate 3, containing mutant Val(B16) and Val(B25), also showed a flat profile up to 152 hours and exhibited a mild to moderate glucose-lowering effect. Furthermore, both insulin conjugates 1 and 2, containing mutant Val(B25), provided a stable glucose-lowering effect at a 30 nM / kg dose without inducing hypoglycemia. In contrast, insulin conjugate 5 (described in WO2018109162A1) was found to exhibit a stronger glucose-lowering effect at a dose of only 18 nM / kg, with a less flat time-action profile compared to insulin conjugates 1-4. The compounds may carry a higher risk of hypoglycemia. According to pharmacokinetic parameters, insulin conjugates 1-4 have a C25 effect up to 50 hours. max Along with the plateau, earlier T is within the range of 8-20 hours. max These indicate a long terminal phase t in the range of 39-45 hours. 1 / 2 This results in a flat pharmacokinetic profile, which is desirable with once-weekly dosing due to the potential reduction in the risk of hypoglycemic events.

Claims

1. A method for forming a conjugate of a sulfonamide and a polypeptide: a) A step of preparing an activated sulfonamide, wherein the activated sulfonamide is of formula (I): 【Chemistry 1】 [In the formula, A is an oxygen atom, -CH 2 CH 2 - base, - OCH 2 - group, and -CH 2 Selected from the group consisting of O-groups; E is -C 6 H 3 R represents a group, where R is a hydrogen atom or a halogen atom, where the halogen atom is selected from the group consisting of fluorine, chlorine, bromine, and iodine atoms; X represents a nitrogen atom or a -CH- group; m is an integer in the range of 5 to 17; n is an integer between 0 and 3; p is either 0 or 1; q is either 0 or 1; r is an integer in the range of 1 to 6; s is either 0 or 1; t is either 0 or 1; R 1 These include hydrogen atoms, halogen atoms, C1-C3 alkyl groups, and halogenated C1-C3 alkyl groups. Represents at least one residue selected from the group of three alkyl groups; R 2 These include hydrogen atoms, halogen atoms, C1-C3 alkyl groups, and halogenated C1-C3 alkyl groups. Represents at least one residue selected from the group of three alkyl groups; R x is 7-azabenzotriazole, 4-nitrobenzene, and N-succinimide Represents an activating group selected from the group consisting of zyl groups; Here, the combination where s is 1, p is 0, n is 0, A is an oxygen atom, and t is 1 is excluded from equation (I). The process corresponding to; b) A step of preparing an aqueous solution of a polypeptide having free amino groups, wherein the aqueous solution may contain alcohol; c) The step of contacting the aqueous solution of b) with the activated sulfonamide of a); and d) A step of reacting the activated sulfonamide with the polypeptide having a free amino group to obtain a solution containing the conjugate of the sulfonamide and the polypeptide, wherein the sulfonamide is covalently bonded to the polypeptide. The method comprising the above.

2. The method according to claim 1, comprising forming a conjugate of a sulfonamide and an insulin polypeptide, wherein the activated sulfonamide is an activated albumin binder.

3. Contacting the aqueous solution of b) with the activated sulfonamide of a) according to step c) is carried out by adding the activated sulfonamide of a) to the aqueous solution of b) as a solution of the activated sulfonamide, and / or The method according to claim 1 or 2, wherein contacting the aqueous solution of b) with the activated sulfonamide of a) according to step c) is carried out by adding the activated sulfonamide of a) to the aqueous solution of b) in solid form, or at least partially in crystalline form, or at least 90% by weight in crystalline form.

4. Preferably, step d) is: d. 1) A step of reacting the activated sulfonamide with a polypeptide precursor having a free amino group at a pH in the range of 9 to 12, or in the range of 9.5 to 11.5, or in the range of 10 to 11, to obtain a preconjugate comprising the sulfonamide and the polypeptide precursor, wherein the sulfonamide is covalently bonded to the polypeptide precursor by an amide bond C(=O)-NH- formed between the -C(=O)-O(R) of the (activated) sulfonamide of formula (I) and the amino group of the polypeptide precursor; d. 2) A step of obtaining a solution containing the conjugate of the sulfonamide and the polypeptide by enzymatic digestion of the precursor of the preconjugate obtained according to d. 1) at a pH in the range of less than 9 or a pH in the range of 7 to 9. The method according to any one of claims 1 to 3, including the method described in any one of claims 1 to 3.

5. The method according to claim 4, wherein the precursor polypeptide is an insulin precursor.

6. A conjugate of a sulfonamide and a polypeptide, wherein the sulfonamide is covalently bonded to the polypeptide, The sulfonamide in the conjugate is given by formula (I): 【Chemistry 2】 [In the formula, A is an oxygen atom, -CH 2 CH 2 - base, - OCH 2 - group, and -CH 2 Selected from the group consisting of O-groups; E is -C 6 H 3 R represents a group, where R is a hydrogen atom or a halogen atom, where the halogen atom is selected from the group consisting of fluorine, chlorine, bromine, and iodine atoms; X represents a nitrogen atom or a -CH- group; m is an integer in the range of 5 to 17; n is an integer between 0 and 3; p is either 0 or 1; q is either 0 or 1; r is an integer in the range of 1 to 6; s is either 0 or 1; t is either 0 or 1; R 1 These include hydrogen atoms, halogen atoms, C1-C3 alkyl groups, and halogenated C1-C3 alkyl groups. Represents at least one residue selected from the group of three alkyl groups; R 2 These include hydrogen atoms, halogen atoms, C1-C3 alkyl groups, and halogenated C1-C3 alkyl groups. Represents at least one residue selected from the group of three alkyl groups; R x These are 7-azabenzotriazole, 4-nitrobenzene, and N-succinimi Represents an activating group selected from the group consisting of zyl groups; Here, the combination where s is 1, p is 0, n is 0, A is an oxygen atom, and t is 1 is excluded from equation (I). Formed from the activated sulfonamide, The conjugate wherein the sulfonamide in the conjugate is covalently bonded to the amino group of the polypeptide via its terminal carboxyl group.

7. The conjugate according to claim 6, wherein the polypeptide in the conjugate is an insulin polypeptide.

8. Activated sulfonamide corresponding to formula (I) 【Transformation 3】 (In the formula, A, E, X, m, n, p, q, r, s, t, R 1 , R 2 , and R x Claim 1 A method for crystallizing (having the meaning defined in the above), A) A step of preparing a solution containing the activated sulfonamide and an organic solvent; B) Remove the organic solvent at least partially and compare it with the solution prepared in A). A step of obtaining the activated sulfonamide phase having the organic solvent in a reduced amount; C) Adding an organic solvent to the phase obtained in B) to obtain a solution of the activated sulfonamide; and D) A step of repeating step B) with the solution obtained in C) to obtain a phase of the activated sulfonamide having a reduced amount of the organic solvent compared to the solution obtained in C); E) A process in which steps C) and D) are repeated at least one more time, depending on the circumstances. The method comprising the above.

9. Activated sulfonamides corresponding to formula (I), in solid form, and sometimes in crystalline form. 【Chemistry 4】 (In the formula, A, E, X, m, n, p, q, r, s, t, R 1 , R 2 , and R x Claim 1 (Having the meaning defined in [the relevant context]).

10. A method for producing a conjugate of albumin-binding agent and mature insulin, a) A step of preparing proinsulin containing an insulin B chain, a linker peptide, and an insulin A chain from the N-terminus to the C-terminus, b) A step of cleaving the proinsulin prepared in step a) between the last amino acid of the insulin B chain and the first amino acid of the linker peptide using a first protease, thereby generating an insulin precursor, wherein the insulin precursor includes the insulin B chain, the linker peptide and an N-terminally extended A chain including the A chain, c) A step of contacting the insulin precursor with an activated albumin binder, wherein the activated albumin binder is an activated sulfonamide according to any one of claims 1 to 4 and includes a functional group capable of binding to albumin, thereby generating a conjugate of the albumin binder and the insulin precursor. d) The step of cleaving the N-terminally extended A chain of the insulin precursor contained in the conjugate between the last amino acid of the linker peptide and the first amino acid of the A chain using a second protease, thereby producing a conjugate of sulfonamide and mature insulin. The method comprising the above.

11. The method according to claim 10, wherein the last amino acid of the insulin B chain is a lysine residue.

12. The method according to claim 10 or 11, wherein the linker peptide has a length of at least two amino acid residues, for example, the linker peptide has a length of 2 to 30 amino acid residues, such as a length of 4 to 9 amino acid residues.

13. The method according to any one of claims 10 to 12, wherein the first amino acid of the linker peptide is a threonine residue, a phenylalanine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue, and / or the last amino acid of the linker peptide is an arginine residue.

14. Insulin polypeptides are transported from the N-terminus to the C-terminus: a) Insulin B chain, b) Linker peptide, and c) Insulin A chain Formed from proinsulin containing, The conjugate according to claim 7, comprising an endoproteinase Lys-C cleavage site between the last amino acid of the insulin B chain and the first amino acid of the linker peptide, and a trypsin cleavage site between the last amino acid of the linker peptide and the first amino acid of the insulin A chain.

15. The linker peptide has the following sequence Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Arg (SEQ ID NO: 106) (In the formula, Xaa1 is any naturally occurring amino acid residue, Xaa2 is any naturally occurring amino acid residue, or, in this case, Xaa2 is not present. Xaa3 is any naturally occurring amino acid residue, or, in this case, Xaa3 is not present. Xaa4 is any naturally occurring amino acid residue, or, in this case, Xaa4 is not present. Xaa5 is any naturally occurring amino acid residue, or, in this case, Xaa5 is not present. Xaa6 is any naturally occurring amino acid residue, or, in this case, Xaa6 is not present. Xaa7 is any naturally occurring amino acid residue, or, in this case, Xaa7 is not present. Xaa8 is either any naturally occurring amino acid residue, or Xaa8 does not exist in this case. The conjugate according to claim 14, including the following:

16. The linker peptide has the sequence TEGR (Sequence ID: 112), as described in claim 14.

17. The linker peptide has the following sequence Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Arg (SEQ ID NO: 106) (In the formula, Xaa1 is any naturally occurring amino acid residue, Xaa2 is any naturally occurring amino acid residue, or, in this case, Xaa2 is not present. Xaa3 is any naturally occurring amino acid residue, or, in this case, Xaa3 is not present. Xaa4 is any naturally occurring amino acid residue, or, in this case, Xaa4 is not present. Xaa5 is any naturally occurring amino acid residue, or, in this case, Xaa5 is not present. Xaa6 is any naturally occurring amino acid residue, or, in this case, Xaa6 is not present. Xaa7 is any naturally occurring amino acid residue, or here Xaa7 is It does not exist, Xaa8 is either any naturally occurring amino acid residue, or Xaa8 does not exist in this case. The method according to any one of claims 10 to 13, including the method described in any one of claims 10 to 13.

18. The method according to any one of claims 10 to 13, wherein the linker peptide has the sequence TEGR (Sequence ID: 112).

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