A method of forming a conjugate of a sulfonamide and a polypeptide

BACKGROUND

Polypeptides are used in various application fields. Since polypeptides have several functional groups, for example, an NH2 group at the N terminus, a COOH group at the C terminus and normally one or more further functional groups directly bound to the polypeptide chain or to side chains, coupling with other compounds is complicated. Normally, byproducts are formed which reduce the yield of the desired product and which are hard to separate from the desired product.

Human insulin is a polypeptide of 51 amino acid residues, which are divided into 2 amino acid chains: the A chain having 21 amino acid residues and the B chain having 30 amino acid residues. The chains are connected to one another by means of 2 disulfide bridges. A third disulfide bridge exists between the cysteines at position 6 and 11 of the A chain. Some products in current use for the treatment of diabetes mellitus are insulin analogs, i.e. insulin variants whose sequence differs from that of human insulin by one or more amino acid substitutions in the A chain and/or in the B chain.

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

WO 2008/034881A1 (Novo Nordisk, Nielsen) discloses protease stabilized insulin analogs.

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 the attachment of the insulin to serum albumin by noncovalent, reversible binding. As a consequence, this insulin analog has a significantly prolonged time-action profile relative to human insulin (see e.g. Mayer et al. , Inc. Biopolymers (Pept Sci) 88: 687-713, 2007; or WO 2009/115469 A1 (Novo Nordisk,

Madsen)). Another conjugate comprising an insulin analog and a covalently attached functional group which allows the attachment of the insulin to serum albumin by noncovalent, reversible binding is disclosed in WO 2017/032798A1 (Novo Nordisk, Madsen): here, an acylated analogue of human insulin is described, which insulin analog is derivatized by

acylation of the epsilon amino group of the lysine residue at the A22 position with a group [Acyl]-[Linker]- wherein the Linker group is an amino acid chain composed of from 1 to 10 amino acid residues selected from gGlu (gamma glutamic acid residue) and/or OEG (residue of 8-amino-3,6-dioxaoctanoic acid).

One obstacle in synthesis of such conjugates, i.e. wherein an insulin analog is coupled with another molecule, which in turn allows the attachment of the insulin (analog) to serum albumin by noncovalent, reversible binding, is that the coupling step often results in poor yields and that a lot of work-up and purification steps are required which lower the yield further.

SUMMARY

Provided herein is a method of forming a conjugate of a sulfonamide and a polypeptide, which enables higher overall yields in conjugate synthesis, i.e. yields of more than 20 %, or more than 30 %, or more than 40 %, or more than 50 %, based on the polypeptide used. The method is described herein below in section A.

Further provided is a process for generating a conjugate of an albumin binder and an insulin polypeptide comprising: a) providing a proinsulin comprising from N- to C-terminus an insulin B-chain, a linker peptide and an insulin A-chain, b) cleaving the proinsulin provided in step a) with a first protease between the last amino acid of the insulin B-chain and the first amino acid of the linker peptide, thereby generating an insulin precursor, said insulin precursor comprising the insulin B-chain and an N-terminally extended A-chain comprising the linker peptide and the A-chain, c) contacting said insulin precursor with an albumin binder, thereby generating a conjugate of an albumin binder and the insulin precursor, d) cleaving the N-terminally extended A-chain of said insulin precursor comprised by the conjugate with a second protease between the last amino acid of the linker peptide and the first amino acid of the A-chain, thereby generating a conjugate of an albumin binder and a mature insulin. The process is described herein below in section B.

DETAILED DESCRIPTION

Section A: Method of forming a conjugate of a sulfonamide and a polypeptide

In order to increase the yield in conjugate synthesis, the number of steps required and the number as well as the ratio of byproducts plays a major role. Accordingly, there is a need for synthetic routes, which have a reduced number of steps and which at least have a more favorable ratio of desired product to undesired byproducts.

Surprisingly, it was found that using a specifically activated sulfonamide for the coupling reaction with the polypeptide helps to improve the ratio of desired product to undesired byproducts. Further, the use of a polypeptide precursor in combination with the use of specific enzymes enables a so called “one pot” reaction, i.e. coupling of the activated sulfonamide and cleavage of the polypeptide precursor to reach the final polypeptide can be done in one reaction vessel without the need for separation or intermediate purification steps. The use of additional protection groups for the sulfonamide can be avoided, this also reducing the need for intermediate separation or purification steps. The desired product, that is the conjugate of the sulfonamide and a polypeptide, can be obtained in yields of 50% or more.

Accordingly, provided herein is in a first aspect a method of forming a conjugate of a sulfonamide and a polypeptide, the method comprising:

a) Providing an activated sulfonamide, wherein the activated sulfonamide corresponds to Formula (I):

wherein

A is selected from the group consisting of oxygen atom, -CH2CH2- group, - OCH2- group and -CH2O- group;

E represents a -C6H3R- group with R being a hydrogen atom or a halogen atom, wherein the halogen atom is selected from the group consisting of fluorine, chlorine, bromine and iodine atom;

X represents a nitrogen atom or a -CH- group;

m is an integer in the range from 5 to 17;

n is zero or an integer in the range from 1 to 3;

p is zero or 1 ;

q is zero or 1 ;

r is an integer in the range from 1 to 6;

s is zero or 1 ;

t is zero or 1 ;

R1 represents at least one residue selected from the group of hydrogen atom, halogen atom, C1 to C3 alkyl group and halogenated C1 to C3 alkyl group;

R2 represents at least one residue selected from the group of hydrogen atom, halogen atom, C1 to C3 alkyl group and halogenated C1 to C3 alkyl group;

Rx represents an activation group;

wherein the combination of s being 1, p being zero, n being zero, A being an oxygen atom and t being 1 is excluded for Formula (I);

b) Providing an aqueous solution of a polypeptide, wherein the aqueous solution optionally comprises an alcohol;

c) Contacting the aqueous solution of b) with the activated sulfonamide of a); and d) Reacting the activated sulfonamide with the polypeptide, obtaining a solution comprising the conjugate of a sulfonamide and the active pharmaceutical ingredient or the diagnostic compound, wherein the sulfonamide is covalently bonded to the polypeptide.

In at least one embodiment, the activated sulfonamide of Formula (I) is covalently bound to the polypeptide in that the terminal carboxy group carrying the Rx group in the non-coupled state of the activated sulfonamide of Formula (I) is covalently bond to a suitable functional group of the polypeptide, for example to an amino group or a hydroxyl group of the polypeptide.

A “polypeptide” is a peptide which comprises at least 2 amino acid residues. In some embodiments, the peptide comprises at least 10 amino acid residues, or at least 20 amino acid residues. In some embodiments, the peptide comprises not more than 1000 amino acid residues, such as not more than 500 amino acid residues, for example not more than 100 amino acid residues.

As used herein, the term “polypeptide” includes any diagnostic chemical or biological polypeptide, pharmaceutically active chemical or biological polypeptide and any pharmaceutically acceptable salt of a diagnostic or pharmaceutically active polypeptide and any mixture thereof, that provides some diagnostic effect or some pharmacologic effect and is used for diagnosing, treating or preventing a condition.

The “polypeptide” is a mature polypeptide or a precursor thereof.

In at least one embodiment, the polypeptide is selected from the group consisting of antidiabetic polypeptide, antiobesity polypeptide, appetite regulating polypeptide,

antihypertensive polypeptide, polypeptide for the treatment and/or prevention of complications resulting from or associated with diabetes, polypeptides for the treatment and/or prevention of complications and disorders resulting from or associated with obesity, and a precursor of any one of those polypeptides.

In at least one embodiment of the method, the polypeptide is an antidiabetic polypeptide or a precursor thereof. In some embodiments, the polypeptide is GLP-1, GLP-1 analog, GLP-1 receptor agonist; dual GLP-1 receptor/glucagon receptor agonist; human FGF21, FGF21 analog, FGF21 derivative; insulin (for example human insulin), insulin analog, insulin derivative, or a precursor of any one of those polypeptides.

According to at least one embodiment of the method, the polypeptide is selected from the group consisting of insulin, insulin analog, GLP-1, and GLP-1 analog (for example GLP(-1) agonist) and a precursor of any one of those polypeptides.

As used herein, the terms ”GLP-1 analog” refer to a polypeptide which has a molecular structure which formally can be derived from the structure of a naturally occurring glucagon- like-peptide-1 (GLP-1), for example that of human GLP-1, by deleting and/or exchanging at least one amino acid residue occurring in the naturally occurring GLP-1 and/or adding at least one amino acid residue. The added and/or exchanged amino acid residue can either be codable amino acid residues or other naturally occurring residues or purely synthetic amino acid residues.

As used herein, the term “GLP(-1) receptor agonist” refers to analogs of GLP(-1), which activate the glucagon-like-peptide-1-rezeptor (GLP-1-rezeptor). Examples of GLP(-1) agonists include, but are not limited to, the following: lixisenatide, exenatide / exendin-4, semaglutide, taspoglutide, albiglutide, dulaglutide.

Lixisenatide has the following amino acid sequence (SEQ ID NO: 98):

Exenatide has the following amino acid sequence (SEQ ID NO: 99):

Semaglutide - albumin binder coupled to Lys(20) has the following amino acid sequence (SEQ ID NO: 100):

Dulaglutide (GLP1 (7-37) coupled via peptidic linker to an fc-fragment) has the following amino acid sequence (SEQ ID NO: 101):

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 “FGF-21 analog”) or a derivative of FGF-21 (referred to “FGF-21 derivative”).

According to at least one embodiment of the method, the polypeptide is insulin, an insulin analog, or a precursor of insulin or of insulin analog. The expression “insulin analog” as used herein refers to a peptide which has a molecular structure which formally can be derived from the structure of a naturally occurring insulin (herein also referred to as “parent insulin”, e.g. human insulin) by deleting and/or substituting at least one amino acid residue occurring in the naturally occurring insulin and/or adding at least one amino acid residue. The added and/or exchanged amino acid residue can either be codable amino acid residues or other naturally occurring residues or purely synthetic amino acid residues. The analog as referred to herein is capable of lowering blood glucose levels in vivo, such as in a human subject.

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 in the B chain.

In some embodiments, the insulin, insulin analog, or the precursor of insulin or of insulin analog has an epsilon amino group of a lysine present in the insulin or insulin analog or precursor of insulin or of insulin analog or is the N-terminal amino group of the B chain of the insulin, the insulin analog, the precursor of insulin or the precursor of insulin analog. For example, the insulin or insulin analog or precursor thereof has one lysine in the A chain and/or B chain. In some embodiments, the insulin, insulin analog, precursor of insulin or precursor of insulin analog has one lysine in the A and in the B chain. In some embodiments, the activated sulfonamide of Formula (I) is covalently bond to lysine, for example to the epsilon amino group of the lysine of the polypeptide in that the terminal carboxy group

carrying the Rx group in the pre-coupled state of the activated sulfonamide of Formula (I) forms an amide bond with the amino group.

In some embodiments, the insulin analog provided herein comprises two peptide chains, an A-chain and a B-chain. Typically, the two chains are connected by disulfide bridges between cysteine residues. For example, in some embodiments, insulin analogs provided herein comprise three disulfide bridges: one disulfide bridge between the cysteines at position A6 and A11, one disulfide bridge between the cysteine at position A7 of the A-chain and the cysteine at position B7 of the B-chain, and one between the cysteine at position A20 of the A-chain and the cysteine at position B19 of the B-chain. Accordingly, insulin analogs provided herein may comprise cysteine residues at positions A6, A7, A11, A20, B7 and B19.

Mutations of insulin, i.e. mutations of a parent insulin, are indicated herein by referring to the chain, i.e. either the A-chain or the B-chain of the analog, the position of the mutated amino acid residue in the A- or B-chain (such as A14, B16 and B25), and the three letter code for the amino acid substituting the native amino acid in the parent insulin. The term “desB30” refers to an analog lacking the B30 amino acid from the parent insulin (i.e. the amino acid at position B30 is absent). For example, Glu(A14)lle(B16)desB30 human insulin, is an analog of human insulin in which the amino acid residue at position 14 of the A-chain (A14) of human insulin is substituted with glutamic acid, the amino acid residue at position 16 of the B-chain (B16) is substituted with isoleucine, and the amino acid at position 30 of the B chain is deleted (i.e. is absent).

Insulin analogs that can be used in the method described herein comprise at least one mutation (substitution, deletion, or addition of an amino acid) relative to parent insulin. The term “at least one”, as used herein means one, or more than one, such as “at least two”, “at least three”, “at least four”, ”at least five”, etc. In some embodiments, the insulin analogs provided herein comprise at least one mutation in the B-chain and at least one mutation in the A-chain. In a further embodiment, the insulin analogs provided herein comprise at least two mutations in the B-chain and at least one mutation in the A-chain.

In some embodiments, the insulin analog comprises an A chain and a B chain, wherein the A chain comprises at least one mutation relative to the A chain of the parent insulin (such as human insulin) and/or wherein the B chain comprises at least one mutation relative to the parent insulin (such as human insulin). For example, the at least one mutation relative to 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, the mutation relative to the B chain of human insulin may be a substitution at position B16, such as a substitution with an amino acid selected from the group consisting of valine (Val), isoleucine (lie), leucine (Leu), alanine (Ala) or histidine (His), a substitution at position B25, such as a substitution with valine (Val), isoleucine (lie), leucine (Leu), alanine (Ala) or histidine (His), and/or a deletion at position B30.

In some embodiments, the insulin analog comprises a deletion at position B30. In some embodiments, the insulin analog may comprise a substitution at position B16, a deletion at position B30 and a substitution at position A14. In some embodiments, the insulin analog may comprise a substitution at position B25, a deletion at position B30 and a substitution at position A14. In some embodiments, the insulin analog may comprise a substitution at position B16, a substitution at position B25, a deletion at position B30 and a substitution at position A14.

The insulin analogs provided herein may comprise mutations in addition to the mutations above. In some embodiments, the number of mutations does not exceed a certain number.

In some embodiments, the insulin analogs comprise less than twelve mutations (i.e. deletions, substitution, additions) relative to the parent insulin. In another embodiment, the analog comprises less than ten mutations relative to the parent insulin. In another embodiment, the analog comprises less than eight mutations relative to the parent insulin. In another embodiment, the analog comprises less than seven mutations relative to the parent insulin. In another embodiment, the analog comprises less than six mutations relative to the parent insulin. In another embodiment, the analog comprises less than five mutations relative to the parent insulin. In another embodiment, the analog comprises less than four mutations relative to the parent insulin. In another embodiment, the analog comprises less than three mutations relative to the parent insulin.

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

In some embodiments, the parent insulin is human insulin. The sequence of human insulin is well known in the art and shown in Table 1 in the Example section. Human insulin comprises an A chain having an amino acid sequence as shown in SEQ ID NO: 1

and a B chain having an amino acid sequence as shown in SEQ ID NO: 2

In another embodiment, the parent insulin is bovine insulin. The sequence of bovine insulin is well known in the art. Bovine insulin comprises an A chain having an amino acid sequence as shown in SEQ ID NO: 81
and a B chain having an amino acid sequence as shown in SEQ ID NO: 82

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 an amino acid sequence as shown in SEQ ID NO: 83
and a B chain having an amino acid sequence as shown in SEQ ID NO: 84

Human, bovine, and porcine insulin comprises three disulfide bridges: one disulfide bridge between the cysteines at position A6 and A11, one disulfide bridge between the cysteine at position A7 of the A-chain and the cysteine at position B7 of the B-chain, and one between the cysteine at position A20 of the A-chain and the cysteine at position B19 of the B-chain.

The term “mature insulin” as referred to herein shall include parent insulin, such as human insulin, and insulin analogs. In some embodiments, the mature insulin is an insulin analog, such as an insulin analog listed in Table 1 of the Examples section. For example, the insulin analog may be insulin analog 24 of Table.

The insulin analogs provided herein typically have an insulin receptor binding affinity which is reduced as compared to the insulin receptor binding affinity of the corresponding parent insulin, e.g. of human insulin.

The insulin receptor can be any mammalian insulin receptor, such as a bovine, porcine or human insulin receptor. In some embodiments, the insulin receptor is a human insulin receptor, e.g. human insulin receptor isoform A or human insulin receptor isoform B (which was used in the Examples section).

Advantageously, the human insulin analogs provided herein have a significantly reduced binding affinity to the human insulin receptor as compared to the binding affinity of human insulin to the human insulin receptor (see Examples). Thus, the insulin analogs have a very low clearance rate, i.e. a very low insulin-receptor-mediated clearance rate.

In some embodiments, the insulin analogs that are used in the method of the present invention have, i.e. exhibit, less than 20 % of the binding affinity to the corresponding insulin receptor compared to its parent insulin. In another embodiment, the insulin analogs provided herein have less than 10 % of the binding affinity to the corresponding insulin receptor compared to its parent insulin. In another embodiment, the insulin analogs provided herein have less than 5 % of the binding affinity to the corresponding insulin receptor compared to its parent insulin, such as less than 3 % of the binding affinity compared to its parent insulin. For example, the insulin analogs provided herein may have between 0.1% to 10 %, such as between 0.3 % to 5 % of the of the binding affinity to the corresponding insulin receptor compared to its parent insulin. Also, the insulin analogs provided herein may have between 0.5% to 3 %, such as between 0.5 % to 2 % of the of the binding affinity to the corresponding insulin receptor compared to its parent insulin.

Methods for determining the binding affinity of an insulin analog to an insulin receptor are well known in the art. For example, the insulin receptor binding affinity can be determined by a scintillation proximity assay which is based on the assessment of competitive binding between [125l]-labelled parent insulin, such as [125l]-labelled human insulin, and the (unlabeled) insulin analog to the insulin receptor. The insulin receptor can be present in a membrane of a cell, e.g. of CHO (Chinese Hamster Ovary) cell, which overexpresses a recombinant insulin receptor. In an embodiment, the insulin receptor binding affinity is determined as described in the Examples section.

Binding of a naturally occurring insulin or an insulin analog to the insulin receptor activates the insulin signaling pathway. The insulin receptor has tyrosine kinase activity. Binding of insulin to its receptor induces a conformational change that stimulates the autophosphorylation of the receptor on tyrosine residues. The autophosphorylation of the insulin receptor stimulates the receptor’s tyrosine kinase activity toward intracellular substrates involved in the transduction of the signal. The autophosphorylation of the insulin receptor by an insulin analog is therefore considered as a measure for signal transduction caused by said analog.

Thus, the insulin analogs that can be used in the method of the present invention may have a low binding activity, and consequently a lower receptor-mediated clearance rate, but are nevertheless capable of causing a relatively high signal transduction. Therefore, the insulin analogs provided herein could be used as long-acting insulins. In some embodiments, the insulin analog provided herein are capable of inducing 1 to 10 %, such as 2 to 8 %, insulin receptor autophosphorylation relative to the parent insulin (such as human insulin). Further, in some embodiments, the insulin analogs provided herein are capable of inducing 3 to 7 %, such as 5 to 7% insulin receptor autophosphorylation relative to the parent insulin (such as human insulin). The insulin receptor autophosphorylation relative to a parent insulin can be determined as described in the Examples section.

As disclosed above, the activated sulfonamide of Formula (I) is covalently bond to the polypeptide in that the terminal carboxy group carrying the Rx group in the non-coupled state of the activated sulfonamide of Formula (I) is covalently bound to a suitable functional group of the polypeptide, for example to an amino group or a hydroxyl group of the polypeptide.

According to at least one embodiment of the method, an amino group of the polypeptide to which the activated sulfonamide of formula (I) is covalently bound is an epsilon amino group of a lysine present at position B26 to B29, for example B29, of the B chain of human insulin, human insulin analog, precursor of human insulin or precursor of human insulin analog, for example of human insulin analog or a precursor of human insulin analog. In some embodiments, the polypeptide and the activated sulfonamide of formula (I) are connected by an amide bond, formed between the terminal carboxy group carrying the Rx group in the pre-coupled state of the activated sulfonamide of formula (I) and an amino group of the polypeptide, for example the epsilon amino group of a lysine present at position B26 to B29, for example B29, of the B chain of human insulin, human insulin analog, precursor of human insulin or precursor of human insulin analog, for example of human insulin analog or precursor of human insulin analog. It goes without saying that in case of an amide bond, the carboxyl group carrying the Rx group in the pre-coupled state is present in the conjugate formed as carbonyl group -C(=O)-, i.e. the amide bond is -C(=O)-NH- as shown below, wherein all residues E, A, R1, R2, X, as well as the indices m, s, p, n, t, r and q have the meaning as indicated above for formula (I) and the NH---- group is already the part remaining from the peptide’s amino group :

In an exemplary embodiment of the first aspect, a method of forming a conjugate of a sulfonamide and a polypeptide is provided, the method comprising:

a) Providing an activated sulfonamide, wherein the activated sulfonamide corresponds to Formula (I):

wherein

wherein

A is selected from the group consisting of oxygen atom, -CH2CH2- group, -OCH2- group and -CH2O- group;

E represents a -C6H3R- group with R being a hydrogen atom or a halogen

atom, wherein the halogen atom is selected from the group consisting of fluorine, chlorine, bromine and iodine atom;

X represents a nitrogen atom or a -CH- group;

m is an integer in the range from 5 to 17;

n is zero or an integer in the range from 1 to 3;

p is zero or 1 ;

q is zero or 1 ;

r is an integer in the range from 1 to 6;

s is zero or 1 ;

t is zero or 1 ;

R1 represents at least one residue selected from the group of hydrogen atom, halogen atom, C1 to C3 alkyl group and halogenated C1 to C3 alkyl group;

R2 represents at least one residue selected from the group of hydrogen atom, halogen atom, C1 to C3 alkyl group and halogenated C1 to C3 alkyl group;

Rx represents an activation group;

wherein the combination of s being 1, p being zero, n being zero, A being an oxygen atom and t being 1 is excluded for Formula (I);

b) Providing an aqueous solution of a polypeptide having a free amino group, wherein the aqueous solution optionally comprises 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, obtaining a solution comprising the conjugate of a sulfonamide and a polypeptide, wherein the sulfonamide is covalently bonded to the polypeptide,

wherein the polypeptide is an insulin polypeptide.

In some embodiments, the activated sulfonamide is an activated albumin binder, for example, if the method is a method of forming a conjugate of a sulfonamide and an insulin polypeptide.

Regarding the activated sulfonamide of formula (I): In some embodiments, s is zero, wherein the remaining residues and indices have the meaning as indicated above for formula (I).

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

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

In at least one embodiment of the method, contacting the aqueous solution of b) with the activated sulfonamide of a) according to step c) is done in that the activated sulfonamide of a) is added as a solution of the activated sulfonamide to the aqueous solution of b). In some embodiments, the solution of the activated sulfonamide is an organic solution, such as a solution comprising the activated sulfonamide and a polar aprotic organic solvent. In some embodiments, the polar aprotic organic solvent has an octanol-water-partition coefficient (Kow) in the range of from 1 to 5 at standard conditions (T: 20-25 °C, p: 1013 mbar). In some embodiments, the polar aprotic organic solvent has an octanol-water-partition coefficient (Kow) in the range of from 2 to 4 at standard conditions (T: 20-25 °C, p: 1013 mbar). In some embodiments, the polar aprotic organic solvent is selected from the group consisting of tetrahydrofuran, acetonitrile, dimethylformamide, and mixtures of two or more thereof. In some embodiments, the polar aprotic organic solvent is selected from the group of tetrahydrofuran, acetonitrile and mixtures of tetrahydrofuran and acetonitrile.

In at least one embodiment of the method, contacting the aqueous solution of b) with the activated sulfonamide of a) according to step c) is done in that the activated sulfonamide of a) is added in solid form to the aqueous solution of b). n some embodiments, the activated sulfonamide of a) is added in at least partially in crystalline form. In some embodiments, the activated sulfonamide of a) is added so that at least 90 weight-% thereof are in crystalline form.

In at least one embodiment of the method, step d) comprises:

d.1) Reacting the activated sulfonamide with a precursor of a mature polypeptide having a free amino group at a pH in the range from 9 to 12, obtaining a pre-conjugate comprising the sulfonamide and the precursor of the mature polypeptide, wherein the sulfonamide is covalently bonded to the precursor of the mature polypeptide 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 precursor of the mature polypeptide;

d.2) Enzymatic digestion of the precursor of the mature insulin of the pre-conjugate obtained according to d.1), obtaining a solution comprising the conjugate of the sulfonamide and the mature polypeptide.

In some embodiments, reacting the activated sulfonamide with a precursor of the mature polypeptide having a free amino group according to d.1) is done at a pH in the range from

9.5 to 11.5. In some embodiments, reacting the activated sulfonamide with a precursor of the mature polypeptide having a free amino group according to d.1) is done at a pH in the range from 10 to 11. In some embodiments, the enzymatic digestion according to d.2) is done at a pH in the range below 9. In some embodiments, the enzymatic digestion according to d.2) is done at a pH in the range of 7 to 9.

In at least one embodiment, the method further comprises:

e) Isolating the conjugate of the sulfonamide and the mature polypeptide from the solution obtained in d) or d.2).

According to at least one embodiment, the activated sulfonamide has the formula (1-1)

wherein:

E represents a -C6H3R- group with R being a hydrogen atom or a halogen atom, wherein the halogen atom is selected from the group consisting of fluorine, chlorine, bromine and iodine atom and is for example a fluorine atom;

X represents a nitrogen atom or a -CH- group;

p is zero or 1 ;

q is zero or 1 ;

r is an integer in the range from 1 to 6;

R1 represents at least one residue selected from the group of hydrogen atom and halogen atom, wherein the halogen atom is for example a fluorine or chlorine atom;

R2 represents at least one residue selected from the group of hydrogen atom, C1 to C3 alkyl group and halogenated C1 to C3 alkyl group, wherein the C1 to C3 alkyl group is for example a methyl group and the halogenated C1 to C3 alkyl group is for example perhalogenated such as a trifluoromethyl group;

Rx represents an activation group;

with m being an integer in the range from 5 to 15 if p is zero, or m being an integer in the range from 7 to 15 if p is 1.

In some embodiments, the residues R1 and R2 of the activated sulfonamide are hydrogen atoms. In some embodiments, the residue X of the activated sulfonamide represents a nitrogen atom. In some embodiments, the HOOC-(CH2)m-(O)s-(E)p-(CH2)n-(A)t- group of formula (I) or the HOOC-(CH2)m-(E)p-O- group of formula (1-1) of the activated sulfonamide is situated in meta or para position on phenyl ring Ph with respect to the -S(O)2- group. In some embodiments, if p is 1, the HOOC-(CH2)m-(O)s- group and the -(CH2)n-(A)t- group are situated in meta or para position on (E)p of formula (I) of the activated sulfonamide or the HOOC-(CH2)m- group and the -O- are situated in meta or para position on (E)p of formula (1-1). In some embodiments, the index q of the activated sulfonamide is zero.

In some embodiments, the activated sulfonamide has the formula (1-1-2)

wherein X is a nitrogen atom or a -CH- group, for example a nitrogen atom; m is an integer in the range from 5 to 15; r is an integer in the range from 1 to 6; q is zero or 1, for example zero;

Rx is an activation group; and the HOOC-(CH2)m-O- group is situated in meta or para position on phenyl ring Ph with respect to the -S(O)2- group.

According to some embodiments, the activated sulfonamide has the formula (1-1-2a)

wherein Rx is an activation group.

In at least one embodiment of the method, the activation group Rx of the activated

sulfonamide of Formula (I) is selected from the group consisting of 7-azabenzotriazole, 4- nitro benzene and N-succinimidyl-group. The 7-azabenzotriazole may be derived from HATU (1-[bis(dimethylamino)methylene]-1 H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) or HBTU (3-[bis(dimethylamino)methyliumyl]-3H-benzotriazol-1-oxide hexafluorophosphate). In some embodiments, Rx is a N-succinimidyl-group.

In at least one embodiment of the method, the aqueous solution of the polypeptide having a free amino group according to b) comprises an alcohol which is 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 which is 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 which is selected from the group consisting of ethanol, propan-2-ol, propan-1-ol, and mixtures of two or more thereof.

In at least one embodiment of the method, the aqueous solution according to b) comprises an alcohol, wherein the alcohol is present in the aqueous solution in an amount in the range from 0.0001 to 35 volume-%, based on the total volume of water and alcohol. In some embodiments, the aqueous solution according to b) comprises an alcohol, wherein the alcohol is present in the aqueous solution in an amount in the range from 0.001 to 30 volume-%, based on the total volume of water and alcohol. In some embodiments, the aqueous solution according to b) comprises an alcohol, wherein the alcohol is present in the aqueous solution in an amount in the range from 0.01 to 25 volume-%, based on the total volume of water and alcohol. In some embodiments, the aqueous solution according to b) comprises an alcohol, wherein the alcohol is present in the aqueous solution in an amount in the range from 0.1 to 20 volume-%, based on the total volume of water and alcohol.

In at least one embodiment of the method, the enzymatic digestion according to d.2) comprises use of at least one enzyme selected from the group consisting of trypsin, a TEV protease (Tobacco Etch Virus protease) and mixtures of two or more thereof.

In at least one embodiment of the method, the mature polypeptide is a mature insulin, which comprises an A chain and a B chain, wherein the A chain comprises at least one mutation relative to the A chain of human insulin and/or the B chain comprises at least mutation relative to human insulin. In some embodiments, the at least one mutation relative to 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). In some embodiments, the mutation relative to the B chain of human insulin is a substitution at position B16, such as a substitution with an amino acid selected from the group consisting of valine (Val), isoleucine (lie), leucine (Leu), alanine (Ala) or histidine (His), a substitution at position B25, such as a substitution with valine (Val), isoleucine (lie), leucine (Leu), alanine (Ala) or histidine (His), and/or a deletion at position B30.

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

In another embodiment, the insulin analog comprises 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.

In another embodiment, insulin analog comprises a mutation at position B16 which is substituted with a hydrophobic amino acid and a mutation at position B25 which is substituted with a hydrophobic amino acid. The hydrophobic amino acid may be any hydrophobic amino acid. For example, the hydrophobic amino acid may be an aliphatic amino acid such as a branched-chain amino acid.

In some embodiments, the hydrophobic amino acid used for the substitution at position B16 and/or B25 is isoleucine, valine, leucine, alanine, tryptophan, methionine, proline, glycine, phenylalanine or tyrosine. In some embodiments, the hydrophobic amino acid used for the substitution at position B16 and/or B25 is isoleucine, valine, leucine, such as valine. Further, it is envisaged that the amino acid at position B16 and/or at position B25 is substituted with a histidine.

The insulin analog may comprise further mutations. For example, the insulin analog may further comprise a mutation at position A14. Such mutations are known to increase protease stability (see e.g. WO 2008/034881 A1 [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).

Further, the insulin analog may comprise a mutation at position B30. In some embodiment, the mutation at position B30 is the deletion of threonine at position B30 of the parent insulin (also referred to as Des(B30)-mutation).

Further, the insulin analog of the present invention may further comprise a mutation at position B3 which is substituted with a glutamic acid (Glu), and/or a mutation at position A21 which is substituted with glycine (Gly).

In some embodiments, the insulin analog comprises a substitution at position A14, a substitution a position B25, and a deletion at position B30 (i.e. the amino acid at position B30 is absent).

In some embodiments, the A chain of the insulin analog comprises or consists of the following sequence:

(SEQ ID NO: 109),

and the B-chain of the insulin analog comprises or consists of the following sequence:

(SEQ ID NO: 110),

wherein Xaa9 is glutamic acid (Glu), aspartic acid (Asp) or histidine (His)

wherein Xaa10 is tyrosine (Tyr), valine (Val), isoleucine (lie), leucine (Leu), alanine (Ala) or histidine (His), and/or

wherein Xaa11 is phenylalanine (Phe), valine (Val), isoleucine (lie), leucine (Leu), alanine (Ala) or histidine (His).

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

In some embodiments, the mature insulin is selected from the group consisting of Leu(B16)-human insulin,

Val(B16)-human insulin,

lle(B16)-human insulin,

Leu(B16)Des(B30)-human insulin,

Val(B16)Des(B30)-human insulin,

lle(B16)Des(B30)-human insulin,

Leu(B25)-human insulin,

Val(B25)-human insulin,

lle(B25)-human insulin,

Leu(B25)Des(B30)-human insulin,

Val(B25)Des(B30)-human insulin,

lle(B25)Des(B30)-human insulin,

Glu(A14)Leu(B16)Des(B30)-human insulin, Glu(A14)lle(B16)Des(B30)-human insulin, Glu(A14)Val(B16)Des(B30)-human insulin,

Glu(A14)Leu(B16)-human insulin,

Glu(A14)lle(B16)-human insulin,

Glu(A14)Val(B16)-human insulin,

Glu(A14)Leu(B25)Des(B30)-human insulin, Glu(A14)lle(B25)Des(B30)-human insulin, Glu(A14)Val(B25)Des(B30)-human insulin,

Glu(A14)Leu(B25)-human insulin,

Glu(A14)lle(B25)-human insulin,

Glu(A14)Val(B25)-human insulin,

Glu(A14)Gly(A21)Glu(B3)Val(B25)Des(B30)-human insulin, Glu(A14)lle(B16)lle(B25)Des(B30)-human insulin, Glu(A14)Glu(B3)lle(B16)lle(B25)Des(B30)-human insulin, Glu(A14)lle(B16)Val(B25)Des(B30)-human insulin, Glu(A14)Gly(A21)Glu(B3)lle(B16)Val(B25)Des(B30)-human insulin, Glu(A14)Val(B16)lle(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)lle(B16)lle(B25)-human insulin, Glu(A14)Glu(B3)lle(B16)lle(B25)-human insulin, Glu(A14)lle(B16)Val(B25)-human insulin, Glu(A14)Gly(A21)Glu(B3)lle(B16)Val(B25)-human insulin, Glu(A14)Val(B16)lle(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

Glu (A14)His(B16)His(B25) Des(B30) human insulin.

In some embodiments, the amino acid residues referred to herein are L-amino acid residues (such as L-isoleucine, L-valine, or L-leucine). Accordingly, the amino acid residues (or the derivatives thereof) used for e.g. the substitution at position B16, B25, A14 and/or A21 are typically L-amino acid residues.

In another embodiment, the insulin analog is Leu(B25)Des(B30)-lnsulin (such as Leu(B25)Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 11).

In another embodiment, the insulin analog is Val(B25)Des(B30)-lnsulin (such as Val(B25)Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 12).

In another embodiment, the insulin analog is Glu(A14)lle(B25)Des(B30)-lnsulin (such as Glu(A14)lle(B25)Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 22).

In another embodiment, the insulin analog is Glu(A14)Val(B25)Des(B30)-lnsulin (such as Glu(A14)Val(B25)Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 24).

In another embodiment, the insulin analog is Glu(A14)Gly(A21)Glu(B3)Val(B25)Des(B30)-Insulin (such as Glu(A14)Gly(A21)Glu(B3) Val(B25)Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 25).

In another embodiment, the insulin analog is Glu(A14)lle(B16)lle(B25)Des(B30)-lnsulin(such as Glu(A14)lle(B16)lle(B25)Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 29).

In another embodiment, the insulin analog is Glu(A14)Glu(B3)lle(B16)lle(B25)Des(B30)-lnsulin(such as Glu(A14)Glu(B3)lle(B16) lle(B25)Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 30).

(FVEQHLCGSHLVEALILVCGERGFIYTPK).

In another embodiment, the insulin analog is Glu(A14)lle(B16)Val(B25)Des(B30)-lnsulin (such as Glu(A14)lle(B16)Val(B25)Des(B30)-human insulin) The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 32).

In another embodiment, the insulin analog is Glu(A14)Gly(A21)Glu(B3) lle(B16)Val(B25)Des(B30)-lnsulin (such as Glu(A14)Gly(A21)Glu(B3)lle(B16)Val(B25) Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 33).

In another embodiment, the insulin analog is Glu(A14)Val(B16)lle(B25)Des(B30)-lnsulin (such as Glu(A14)Val(B16)lle(B25)Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 35).

In another embodiment, the insulin analog is Glu(A14)Val(B16)Val(B25)Des(B30)-lnsulin (such as Glu(A14)Val(B16)Val(B25) Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 38).

In another embodiment, the insulin analog is Glu(A14)Glu(B3)Val(B16)Val(B25)Des(B30)-Insulin (such as Glu(A14)Glu(B3)Val(B16) Val(B25)Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 39).

In another embodiment, the insulin analog is

Glu(A14)Gly(A21)Glu(B3)Val(B16)Val(B25)Des(B30)-lnsulin (such as Glu(A14)Gly(A21) Glu(B3)Val(B16)Val(B25)Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 40).

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

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

In at least one embodiment of the method, the precursor of the mature polypeptide is a precursor of a mature insulin, which comprises a sequence as listed in detail herein above for the mature insulin, comprising an A chain and a B chain, and an additional linker peptide, which has a length of at least two amino acid residues. Optionally, the linker peptide has a length in the range from 2 to 30 amino acid residues, for example a length in the range from 4 to 9 amino acid residues. In some embodiments, the precursor is a precursor as defined in section B of the present application.

In at least one embodiment of the 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 a valine residue, 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, phenylalanine residue, a glutamine residue, a glutamic acid residue, an asparagine residue or an aspartic acid residue. At least these amino acid residues for the first amino acid of the linker peptide are amino acid residues having an amino group at the N-terminus which has a low nucleophilicity. This reduces the reactivity regarding a reaction with the activated carboxyl group -COORx of the sulfonamide.

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

In at least one embodiment of the method, the linker peptide comprises or consists of the following sequence

(SEQ ID NO: 106)

wherein Xaa1 to Xaa8 may be as follows:

Xaa1 may 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 may 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 is absent.

Xaa3 may 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 is absent.

Xaa4 may 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 is absent.

Xaa5 may 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 is absent.

Xaa6 may 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 is absent.

Xaa7 may 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 is absent.

Xaa8 may 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 is absent.

In at least one embodiment of the method, the linker peptide has the sequence TEGR (SEQ ID NO: 112). A pre-conjugate comprising an exemplary sulfonamide and a precursor of a mature polypeptide, wherein the linker peptide has the sequence TEGR (SEQ ID NO: 112), and an exemplary sulfonamide is shown in Fig. 8.

In at least one embodiment of the method, the linker peptide is a linker peptide as defined in section B of the present application.

In at least one embodiment of the method, the sulfonamide is covalently bonded to the mature polypeptide and the precursor thereof respectively by an amide bond C(=O)-NH-formed between the -C(=O)-O(Rx) of the (activated) sulfonamide of Formula (I) and the free amino group of the mature polypeptide and the precursor thereof respectively. In some embodiments, the free amino group of the polypeptide is the amino group of a lysine comprised in the mature polypeptide and the precursor thereof respectively. In some embodiments, the free amino group of the polypeptide is the amino group of a terminal lysine. In some embodiments, the free amino group of the polypeptide is the amino group of a lysine present at a C terminus of the mature polypeptide and the precursor thereof

respectively. In some embodiments, the free amino group of the polypeptide is the amino group of a lysine present at the C terminus of the B-chain.

The (cleavable) linker peptide, especially the linker peptide TEGR (SEQ ID NO: 112), protects the N-terminus of the A-chain from coupling with the activated sulfonamide of Formula (I). The peptide TEGR (SEQ ID NO: 112) does not or only to a minor extend below 1% react with the activated carboxyl group -COORx of the sulfonamide, which lowers the excess of sulfonamide required. The cleavage of TEGR (SEQ ID NO: 112) after the coupling with the sulfonamide can be achieved in one pot by adjusting the pH to a value in the range below 9, followed by the addition of trypsin, a 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, the A1-acylated byproduct is not produced. The

separation of the desired compound is thus simplified, because the A1-acylated byproduct shows similar retention times as the most desired product, wherein only the lysine at B29 is coupled to the sulfonamide conjugate.

Exemplary conjugates of the sulfonamide of formula (I) and polypeptides, for example,

insulin analogs, which are obtained or obtainable by the method described herein above are disclosed in section B; exemplary conjugates are shown in Figures 3 to 6.

In at least one embodiment of the method, the activated sulfonamide of Formula (I) is obtained or obtainable from a protected activated sulfonamide of Formula (0)

wherein A, E, X, m, n, p, q, r, s, t, R1, R2and Rx have the meaning as defined herein above, wherein the protected activated sulfonamide of Formula (0) is optionally deprotected by addition of one or more acids, for example by addition of at least trifluoroacetic acid.

In a second aspect, a conjugate obtained or obtainable from the method of any one of embodiments as described herein above is provided.

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

In a fourth aspect, a procedure for crystallizing an activated sulfonamide corresponding to Formula (I) is provided

wherein A, E, X, m, n, p, q, r, s, t, R1, R2and Rx have the meaning as defined above with respect to the method for preparing a conjugate, comprising

A) Providing a solution comprising the activated sulfonamide and an organic solvent;

B) Removing the organic solvent at least partially, for example by distillation, obtaining a phase of the activated sulfonamide having a reduced amount of the organic solvent compared to the solution provided in A);

C) Adding organic solvent to the phase obtained in B) obtaining a solution of the activated sulfonamide; and

D) Repeating step B) with the solution obtained in C) obtaining a phase of the activated sulfonamide having a reduced amount of the organic solvent compared to the solution obtained in C);

E) Optionally repeating steps C) and D) at least one further time.

The “phase of the activated sulfonamide having a reduced amount of the organic solvent compared to the solution provided in A)” comprises solutions (i.e. liquid phases) of the activated sulfonamide having a reduced amount of the organic solvent compared to the solution provided in A), oily phases of the activated sulfonamide and solid phases of the activated sulfonamide. In at least one embodiment, the procedure for crystallizing an activated sulfonamide corresponding to Formula (I) comprises an additional step:

F) Adding organic solvent to the phase of the activated sulfonamide obtained in D) and/or to the phase of the activated sulfonamide obtained in E) and keeping the resulting solution at a temperature in the range from 5 to 40 °C for at least one hour, thus obtaining a precipitate comprising the activated sulfonamide in solid form.

Said precipitate obtained according to (F) can be separated from the solution by means known in the art, for example, by filtration.

In some embodiments, the resulting solution in F) is kept at a temperature in the range from 10 to 35 °C. In some embodiments, the resulting solution in F) is kept at a temperature in the range from 20 to 30 °C. In some embodiments, the resulting solution in F) is kept at the respective temperature for 1 to 72 hours. In some embodiments, the resulting solution in F) is kept at the respective temperature for 10 to 48 hours. In some embodiments, the resulting solution in F) is kept at the respective temperature for 15 to 30 hours. In some embodiments, the precipitate obtained in F) comprises the activated sulfonamide at least partially in crystalline form. In some embodiments, the precipitate obtained in F) comprises the activated sulfonamide at least 90 weight-% in crystalline form.

In at least one embodiment of the procedure for crystallizing an activated sulfonamide corresponding to Formula (I), the solution comprising the activated sulfonamide and an organic solvent the organic solvent provided 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 aceotropic mixture with trifluoroacetic acid. In at least one embodiment of the procedure for crystallizing an 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 (Kow) in the range from 1 to 5 at standard conditions (temperature: 20-25 °C, pressure: 1013 mbar). In some embodiments, the polar aprotic organic solvent has an octanol-water-partition coefficient (Kow) in the range from 2 to 4, at standard conditions (temperature: 20-25 °C, pressure: 1013 mbar). In some embodiments, the organic solvent is selected from the group of acetonitrile, tetrahydrofuran and mixtures of acetonitrile and tetrahydrofuran. In some embodiments, the organic solvent at least comprises acetonitrile.

Due to, for example, synthesis reasons, the activated sulfonamide corresponding to Formula (I) comprises minor amounts of trifluoroacetic acid (less than 5 weight-% based on the weight of the activated sulfonamide). However, these minor amounts disturb the solidification and crystallisation respectively of the activated sulfonamide, which results in the activated sulfonamide being present in oil form. The repeated addition of organic solvent and the off-distillation thereof enables an aceotropic removal of trifluoroacetic acid, which in turn results in solidification / crystallisation of the activated sulfonamide after reduction and removal respectively of the trifluoroacetic acid amount. According to the Dortmunder Datenbank, acetonitrile has an octanol-water-partition coefficient (Kow) of 2, tetrahydrofuran (THF) has a Kow of 4, dimethylformamide has a Kow of 4.

The organic solvent used for providing 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 the same or different and are independently selected from the group of organic solvents capable of forming an aceotropic mixture with trifluoroacetic acid and/or from the group of polar aprotic organic solvents, which may have a Kow in the range of from 1-5. In some embodiments, the polar aprotic organic solvents has a Kow in the range from 2-4. In some embodiments, the same organic solvent is used for providing the solution in A), in C), optionally in E) and in F).

In a fifth aspect, a solid form of the activated sulfonamide corresponding to Formula (I) is provided

wherein A, E, X, m, n, p, q, r, s, t, R1, R2and Rx have the meaning as defined herein above.

In some embodiments, the activated binder is crystalline.

In one embodiment, an exemplary sulfonamide of Formula (I) is prepared by coupling of two building blocks A and B as shown in schema 1 below, wherein the coupling of building blocks A and B gives the exemplary sulfonamide, which is called Pyrimidine-bis-OEG-acid:

Schema 1

Schema 2 shows the synthesis of building block A, and schema 3 shows the synthesis of building block B:

Schema 2

Schema 3

The definitions and explanations provided herein above in Section A shall apply mutatis mutandis to the embodiments described herein below in section B.

Section B

As set forth in section A, the presence of one or more additional amino acid residues at the N-terminus of the A-chain, for example the presence of a 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.

Accordingly, the A1-acylated byproduct is not produced when preparing a conjugate of an albumin binder and an insulin precursor comprising an N-terminally extended A-chain and a B-chain. After conjugation, the one or more additional peptides at the N-terminus of the A-chain can be advantageously removed by proteolytic cleavage, e.g. with trypsin, thereby generating the conjugate of a mature insulin (such as an insulin analog) and an albumin binder. The separation of the desired conjugated is simplified, because the A1-acylated byproduct shows similar retention times as the desired product, wherein only the lysine at B29 is coupled to the albumin binder conjugate.

Further, the amount of binder used for the conjugation might be reduced, if the first amino acid of the N-terminally extended A-chain has a lower nucleophilicity than the A1 amino acid. This reduces the reactivity regarding a reaction with the activated carboxyl group -COORx of the albumin binder. For example, threonine has a lower nucleophilicity than glycine which is frequently found at the A1 position of insulin analogs.

An insulin precursor comprising an N-terminally extended A-chain and a B-chain can be generated by cleaving a proinsulin comprising from N- to C-terminus an insulin B-chain, a linker peptide and an insulin A-chain with a protease between the last amino acid of the insulin B-chain and the first amino acid of the linker peptide. The generated N-terminally extended A-chain comprises, from N- to C-terminus, the linker peptide and the A-chain. The linker peptide then protects the A1 amino acid of the A-chain in the subsequent conjugation step.

Accordingly, the present invention relates to a process for generating a conjugate of an albumin binder and a mature insulin, said process comprising

a) Providing a proinsulin comprising from N- to C-terminus an insulin B-chain, a linker peptide and an insulin A-chain,

b) Cleaving the proinsulin provided in step a) with a first protease between the last amino acid of the insulin B-chain and the first amino acid of the linker peptide, thereby generating an insulin precursor, said insulin precursor comprising the insulin B-chain and an N-terminally extended A-chain comprising the linker peptide and the A-chain, c) Contacting said insulin precursor with an albumin binder, wherein the albumin binder comprises a functional group capable of binding to albumin;

thereby generating a conjugate of an albumin binder and the insulin precursor, d) Cleaving the N-terminally extended A-chain of said insulin precursor comprised by the conjugate with a second protease between the last amino acid of the linker peptide and the first amino acid of the A-chain, thereby generating a conjugate of an albumin binder and a mature insulin.

An ’’albumin binder” is a compound capable of binding non-covalently to an albumin, for example, human albumin, for example in a blood sample.

Optionally, the albumin binder is an activated albumin binder. The activated albumin binder may comprise an activated carboxyl group -COORx, wherein Rx is an activation group. The activation group Rx is in at least one embodiment selected from the group consisting of 7-azabenzotriazole, for example derived from HATU or HBTU, 4-nitro benzene and N-succinimidyl-group. Optionally, Rx is a N-succinimidyl-group.

In at least one embodiment, the albumin binder comprises a functional group capable of binding to albumin, such as human serum albumin. Optionally, the functional group capable of binding to albumin is a carboxyl group or a bioisostere of a carboxyl group. Optionally, the functional group capable of binding to albumin is selected from the group consisting of a carboxyl group, hydroxamic acid group, hydroxamic ester group, phosphonic acid group, phosphinic acid group, sulfonic acid group, sulfinic acid group, sulfonamide group, acyl sulfonamide group, sulfonyl urea group, acyl urea group, tetrazole group, thiazolinine dione group, oxazolindine dione group, oxadiazol-5(4H)- one group, thiadiazol-5(4H)-one group, oxathiadiazole-2-oxide group, oxadiazole-5(4H)-thione group, isoxazole group, tetramic acid group, cyclopentane 1,3-diones, cyclopentane 1,2-diones, squaric acid derivatives, substituted phenols, -CO-Asp, -CO-Glu, -CO-Gly, -CO-Sar (-CO-sarcosine), -CH(COOH)2, and -N(CH2COOH)2. Optionally, the functional group capable of binding to albumin is selected from the group consisting of a carboxyl group, -CO-Asp, -CO-Glu, -CO-Gly, -CO-Sar, -CH(COOH)2,-N(CH2COOH)2, sulfonic acid group (-SO3H) and phosphonic acid group (-PO3H).

CLAIMS

1. A method of forming a conjugate of a sulfonamide and a polypeptide, the method comprising:

a) Providing an activated sulfonamide, wherein the activated sulfonamide

corresponds to Formula (I):

wherein

A is selected from the group consisting of oxygen atom, -CH2CH2- group, - OCH2- group and -CH2O- group;

E represents a -C6H3R- group with R being a hydrogen atom or a halogen atom, wherein the halogen atom is selected from the group consisting of fluorine, chlorine, bromine and iodine atom;

X represents a nitrogen atom or a -CH- group;

m is an integer in the range from 5 to 17;

n is zero or an integer in the range from 1 to 3;

p is zero or 1 ;

q is zero or 1 ;

r is an integer in the range from 1 to 6;

s is zero or 1 ;

t is zero or 1 ;

R1 represents at least one residue selected from the group of hydrogen atom, halogen atom, C1 to C3 alkyl group and halogenated C1 to C3 alkyl group;

R2 represents at least one residue selected from the group of hydrogen atom, halogen atom, C1 to C3 alkyl group and halogenated C1 to C3 alkyl group;

Rx represents an activation group;

wherein the combination of s being 1, p being zero, n being zero, A being an oxygen atom and t being 1 is excluded for Formula (I);

b) Providing an aqueous solution of a polypeptide having a free amino group, wherein the aqueous solution optionally comprises 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, obtaining a solution comprising the conjugate of a sulfonamide and a polypeptide, wherein the sulfonamide is covalently bonded to the polypeptide.

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

3. The method of claim 1 or 2, wherein contacting the aqueous solution of b) with the activated sulfonamide of a) according to step c) is done in that the activated sulfonamide of a) is added as a solution of the activated sulfonamide to the aqueous solution of b).

4. The method of any one of claims 1 to 3, wherein contacting the aqueous solution of b) with the activated sulfonamide of a) according to step c) is done in that the activated sulfonamide of a) is added in solid form to the aqueous solution of b), or at least partially in crystalline form, or at least 90 weight-% in crystalline form;

preferably wherein step d) comprises:

d.1) Reacting the activated sulfonamide with a precursor of the polypeptide having a free amino group at a pH in the range from 9 to 12, or in the range from 9.5 to 11.5 or in the range from 10 to 11, obtaining a pre-conjugate comprising the sulfonamide and the precursor of the polypeptide, wherein the sulfonamide is covalently bonded to the precursor of the polypeptide 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 precursor of the polypeptide;

d.2) Enzymatic digestion optionally at a pH in the range below 9, or at a pH in the range of 7 to 9, of the precursor of the polypeptide of the pre-conjugate obtained according to d.1), obtaining a solution comprising the conjugate of the sulfonamide and the polypeptide.

5. A conjugate obtained or obtainable from the method of any one of claims 1 to 4.

6. An N-terminally extended insulin A-chain comprising from N- to C-terminus:

(a) a linker peptide, and

(b) an insulin A-chain,

wherein said N-terminally extended insulin A-chain comprises a cleavage site for trypsin between the last amino acid of the linker peptide and the first amino acid of the

A-chain.

7. An insulin precursor comprising the N-terminally extended insulin A-chain of claim 6

and an insulin B-chain.

8. A procedure for crystallizing an activated sulfonamide corresponding to Formula (I)

wherein A, E, X, m, n, p, q, r, s, t, R1, R2and Rx have the meaning as defined in claim

1 , comprising

A) Providing a solution comprising the activated sulfonamide and an organic solvent;

B) Removing the organic solvent at least partially, obtaining a phase of the

activated sulfonamide having a reduced amount of the organic solvent compared to the solution provided in A);

C) Adding organic solvent to the phase obtained in B) obtaining a solution of the activated sulfonamide; and

D) Repeating step B) with the solution obtained in C) obtaining a phase of the activated sulfonamide having a reduced amount of the organic solvent compared to the solution obtained in C);

E) Optionally repeating steps C) and D) at least one further time.

9. A solid, optionally crystalline, form of the activated sulfonamide corresponding to

Formula (I)

wherein A, E, X, m, n, p, q, r, s, t, R1 , R2 and Rx have the meaning as defined in claim 1.

10. A process for generating a conjugate of an albumin binder and a mature insulin, said process comprising

a) Providing a proinsulin comprising from N- to C-terminus an insulin B-chain, a linker peptide and an insulin A-chain,

b) Cleaving the proinsulin provided in step a) with a first protease between the last amino acid of the insulin B-chain and the first amino acid of the linker peptide, thereby generating an insulin precursor, said insulin precursor comprising the insulin B-chain and an N-terminally extended A-chain comprising the linker peptide and the A-chain,

c) contacting said insulin precursor with an albumin binder, wherein the albumin binder comprises a functional group capable of binding to albumin, thereby generating a conjugate of an albumin binder and the insulin precursor,

d) Cleaving the N-terminally extended A-chain of said insulin precursor comprised by the conjugate with a second protease between the last amino acid of the linker peptide and the first amino acid of the A-chain, thereby generating a conjugate of a sulfonamide and a mature insulin.

11. The process of claim 10, wherein the last amino acid of the insulin B-chain is a lysine residue.

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

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

14. A proinsulin comprising from N- to C-terminus:

a) an insulin B-chain,

b) a linker peptide, and

c) an insulin A-chain,

wherein said proinsulin comprises a cleavage site for endoproteinase 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.

15. The process of any one of claims 10 to 13, or the proinsulin of claim 14, wherein the linker peptide comprises the following sequence

(SEQ ID NO: 106)

wherein

Xaa1 is any naturally occurring amino acid residue,

Xaa2 is any naturally occurring amino acid residue, or wherein Xaa2 is absent, Xaa3 is any naturally occurring amino acid residue or wherein Xaa3 is absent,

Xaa4 is any naturally occurring amino acid residue, or wherein Xaa4 is absent,

Xaa5 is any naturally occurring amino acid residue, or wherein Xaa5 is absent,

Xaa6 is any naturally occurring amino acid residue, or wherein Xaa6 is absent,

Xaa7 is any naturally occurring amino acid residue, or wherein Xaa7 is absent, and Xaa8 is any naturally occurring amino acid residue, or wherein Xaa8 is absent.

16. The process of any one of claims 10 to 13 and 15, or the proinsulin of claim 14, wherein the linker peptide has the sequence TEGR (SEQ ID NO: 112).