Process for the preparation of glucagon-like peptides

Abstract

The present invention relates to a method for preparing a glucagon-like peptide by precipitating the peptide or precursor peptide by mixing with an anti-solvent comprising diisopropyl ether and acetonitrile. Furthermore, the present invention relates to a peptide conjugated to a solid phase and a pharmaceutical composition comprising liraglutide obtainable according to the method of the present invention.

Description

[0001] The present invention relates to the field of industrial or laboratory scale peptide synthesis and discloses an improved process for the preparation of a glucagon-like peptide, in particular for the preparation of a glucagon-like peptide 1 analogue, such as Liraglutide. More specifically, the present invention relates to a process for the efficient preparation of a glucagon-like peptide, such as Liraglutide, as well as a process for the isolation of the crude peptide after synthesis.

[0002] As a preferred embodiment, the present invention relates to a process for the preparation of Liraglutide. The process comprises mixing Liraglutide or a precursor peptide with an anti-solvent comprising diisopropyl ether and acetonitrile to precipitate Liraglutide or the precursor peptide. Furthermore, the present invention relates to a Liraglutide peptide precursor bound to a solid phase, as well as a pharmaceutical composition comprising Liraglutide obtainable from the process according to the present invention.

[0003] The human GCG gene (HGNC: 4191) encodes a variety of related peptides, including glucagon, glucagon-like peptide 1 (GLP-1) and glucagon-like peptide 2 (GLP-2). They share considerable sequence homology (see Figure 1 ) and are involved in the control of glucose homeostasis, intestinal cell proliferation and satiety. The GLP-1 forms known to have biological activity are GLP-1 (7-37) and GLP-1 (7-36), which are amides, and exert their effects by activating the GLP-1 receptor. Among other physiological functions, GLP-1 is a glucose-dependent insulinotropic hormone that potently suppresses glucagon secretion, has a protective and proliferative effect on pancreatic beta cells, and suppresses gastrointestinal secretion and motility. GLP-1 dysfunction has been found to be associated with obesity, postprandial hypoglycemia and type 2 diabetes. Therefore, GLP-1 analogues have considerable significance in pharmaceutical research.

[0004] Variants and derivatives of the peptide hormone exendin-4 of the Gila monster (Heloderma suspectum) as well as variants and derivatives of the GLP-1 peptide itself are being intensively researched.

[0005] The drug compounds already on the market are Exenatide and Lixisenatide, both derived from the exendin-4 peptide, as well as Liraglutide, derived from GLP-1. Liraglutide (N-epsilon-(gamma-Glu(N-alpha-hexadecanoyl)))-Lys26Arg34-GLP-1(7-37), also known as NN2211, has been approved for the treatment of type 2 diabetes and for the treatment of obesity in adults with relevant comorbidities. This compound can be produced on an industrial scale by semi-synthetic techniques. EP-B 0944 648 describes the reaction of a recombinantly expressed precursor peptide with N alpha-hexadecanoyl-Glu (ONSu)-OtBu to obtain liraglutide (see its Example 35).

[0006] However, it is desirable to have a better process for large-scale preparation of a fully chemically synthesized glucagon-like peptide, such as liraglutide.

[0007] In general, the chemical synthesis of peptides is well known in the art (see the handbook "Solid Phase Peptide Synthesis Bachem - Pioneering Partner for Peptides", Global Marketing, Bachem group, published in June 2014). During synthesis, the formation of a peptide bond between the alpha amino group of a first amino acid and the alpha carboxyl group of a second amino acid should prevail over other side reactions. This is usually achieved by using "permanent" and "temporary" protecting groups. The former are used to block the reactivity of, for example, the side chain of a reactive amino acid and the C-terminal carboxyl group of the growing peptide chain and are removed only at the end of the entire synthesis. The latter are used to block, for example, the alpha amino group of a second amino acid during the coupling step, thus avoiding, for example, the formation of peptide bonds between multiple second amino acids. Chemical synthesis of peptides distinguishes between two standard methods, namely liquid phase peptide synthesis (LPPS) and solid phase peptide synthesis (SPPS).

[0008] LPPS, also known as solution peptide synthesis, is carried out in a homogeneous reaction medium. Sequential couplings yield the desired peptide. For liquid phase peptide synthesis, there is no standard synthesis protocol, which has to be carefully planned, choosing from a variety of possible combinations of protecting groups, coupling methods and solvents. LPPS usually involves isolation, characterization, and, if necessary, purification of intermediates after each coupling. Longer peptides are usually synthesized by convergent methods, i.e. several peptide fragments are first synthesized in parallel and then finally combined to yield the final product.

[0009] In standard SPPS, protected amino acids are added consecutively in the intended sequence, forming a peptide with the C-terminus anchored to an insoluble polymeric resin, i.e. the synthesis proceeds from the C- to the N-terminus of the peptide, with successive amino acid addition cycles, each cycle consisting of the following steps: a) cleavage of the N -protecting group from the resin-bound peptide, b) washing steps, c) coupling of a protected amino acid, and d) washing steps. Because the growing chain is bound to an insoluble support, excess reagents and soluble by-products can be removed by simple filtration. Washing steps with appropriate solvents can ensure complete removal of the cleaving agent after the deprotection step a) and eliminate excess reagents and soluble by-products resulting from the coupling step c). At the end of the synthesis, the peptide is cleaved from the resin and the protecting groups are removed (see the manual “Solid Phase Peptide Synthesis Bachem - Pioneering Partner for Peptides”, Global Marketing, Bachem group, published in June 2014). The so-called Fmoc SPPS is the method using 9-fluorenylmethyloxycarbonyl (Fmoc) as temporary amino protecting group, the most common form of SPPS. In contrast to LPPS, peptide purification can only be performed after the end of the synthesis, after cleavage from the resin. This is generally not favorable for the synthesis of large peptides, where various resin-bound by-products can accumulate in addition to by-products formed during deprotection or due to degradation. As a result, purification of the final product can be very challenging. Therefore, the purity of the crude peptide product has to be optimized when developing a SPPS method for industrial scale.

[0010] In addition to LPPS and SPPS, hybrid methods can be used, in which peptide fragments are first synthesized by one of the above techniques and then joined together using the other technique. This strategy is often used for large peptides with challenging sequences. Common to the above methods is the need to recover the final and / or intermediate peptide product from solution. Depending on the chosen synthesis strategy, other components of the peptide solution can vary, typically including cleavage composition containing TFA and scavengers.

[0011] The standard method for peptide isolation after TFA cleavage is the so-called cold ether work-up: the peptide solution is mixed with cold ether as antisolvent to cause precipitation of the peptide and the precipitate is collected by filtration and / or centrifugation.

[0012] Diethyl ether, diisopropyl ether or methyl tert-butyl ether. Methyl tert-butyl ether (MTBE) is commonly used for cold ether work-up. The method of adding an antisolvent to the peptide solution is called the “classical” precipitation protocol; the method of adding the peptide solution to the antisolvent is called the “reverse” precipitation protocol.

[0013] The properties of the precipitate often have some impact in the laboratory and in industrial manufacturing, as it influences the quality of the crude peptide as well as the further processing. Ideally, the precipitation is almost complete, the related impurities are reduced and the TFA content is low. Furthermore, an efficient filtration requires that the precipitate has a suitable consistency and particle size to avoid filter clogging or loss of material adhering to the vessel walls.

[0014] Although it has previously been recognized that a precipitation protocol can improve the purity and handling of the crude peptide (US 2005 / 0165216), surprisingly, the details of this step have received little attention.

[0015] The patent documents CN-A 103 275 209, CN-A 103 275 208, CN-A 104 045 706, CN-A 104 045 705, CN-A 103 145 828, CN-A 103 980 358 and EP-A 2 757 107 disclose SPPS or hybrid processes for the synthesis of liraglutide. In these patent documents, cold diethyl ether is used as antisolvent to precipitate crude liraglutide from a solution comprising TFA and scavenger.

[0016] WO2014 / 199397 and WO2016 / 005960 disclose SPPS and / or hybrid processes for the synthesis of liraglutide. Cold MTBE or diisopropyl ether is used as antisolvent to precipitate crude liraglutide from a solution comprising TFA and scavenger.

[0017] EP-B 1 987 052 discloses a SPPS process for the synthesis of glucagon-like peptides, wherein cold MTBE is used to precipitate the crude peptide from a solution comprising TFA and scavenger.

[0018] US-A 2005 / 0165216 discloses the use of alcohols having three or more carbon atoms for the precipitation of peptides.

[0019] WO2012 / 171984, WO2012 / 171982 and WO2012 / 171987 relate to problems encountered during precipitation when preparing peptides with LPPS. It is disclosed that direct precipitation from the polar aprotic solvent used in LPPS leads to the formation of a sticky, gel-like precipitate. A way to avoid this problem is to first extract the target peptide into 2-methyltetrahydrofuran or toluene and then precipitate the peptide from the extraction phase. Acetonitrile, diethyl ether, diisopropyl ether, n-heptane and toluene are considered suitable antisolvents to induce precipitation of the peptide from the extraction phase.

[0020] In individual cases, it has been reported that mixtures of acetonitrile and diisopropyl ether are used for precipitation when preparing short peptides. US 2010 / 0280221 describes the preparation of an octapeptide (length: 8 amino acids) by LPPS. The method comprises precipitation of the peptide from acetic acid / dioxane by acetonitrile / diisopropyl ether at ambient temperature. WO 2015 / 154031 describes a method for precipitating AMG 614, a highly polar artificial peptide compound consisting of seven D-amino acids and a single L-Cys bonded via a disulfide bond, by a mixture of acetonitrile and diisopropyl ether.

[0021] In view of the prior art, there is still a need for improvements in the preparation of larger size peptides, such as glucagon-like peptides, e.g. liraglutide. In particular, the synthesis and purification of the crude peptide product needs to be optimized to improve the purity and macroscopic properties of the crude peptide product obtained. The inventors have developed an improved method for the synthesis and precipitation of crude liraglutide, which can be used on an industrial scale.

[0022] Surprisingly, the present inventors have found that larger size peptides, such as glucagon-like peptides, e.g. liraglutide, are particularly well precipitated with an anti-solvent comprising diisopropyl ether and acetonitrile, in particular when the volume ratio of diisopropyl ether:acetonitrile is in the range of (3:1) to (10:1).

[0023] Thus, in general terms, one aspect of the present invention relates to a method for the preparation of a glucagon-like peptide or a salt thereof, comprising the steps of:

[0024] (i) providing a solution S containing a crude glucagon-like peptide.

[0025] (ii) mixing the peptide of step (i) with an anti-solvent comprising diisopropyl ether and acetonitrile, precipitating the peptide of step (i), wherein the volume ratio of diisopropyl ether:acetonitrile is in the range of (3:1) to (10:1); and

[0026] (iii) isolating the precipitate obtained in step (ii), preferably by filtration and / or centrifugation.

[0027] One embodiment of the present invention relates to a method for the preparation of liraglutide or a salt thereof, comprising:

[0028] (i) providing a solution S containing a peptide of formula I: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-B1-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Gly, wherein B1 is Lys(palmitoyl-Glu-OH) or Lys(H-Glu-OH);

[0029] (ii) precipitating the peptide of step (i) by mixing solution S with an anti-solvent comprising diisopropyl ether and acetonitrile, wherein the volume ratio of diisopropyl ether: acetonitrile is in the range of (3:1) to (10:1); and

[0030] (iii) isolating the precipitate obtained in step (ii), preferably by filtration and / or centrifugation.

[0031] Those skilled in the art will appreciate that formula I also encompasses salts of the above polypeptide chains. Those skilled in the art will also appreciate that the peptides used in the present application can optionally bear any counterion known in the art, such as an anion or a cation, for example chloride, acetate, carbonate, bicarbonate, sodium, potassium, magnesium, any ion of the cleavage solution (e.g., TFA ion, bromide, perchlorate, ammonium) and / or a cation or anion of a protecting group residue. Furthermore, the peptides can optionally be covalently or non-covalently associated with trace amounts of one or more scavengers, such as triisopropylsilane (TIPS), dithiothreitol (DTT), dithioerythritol (DTE), anisole, thioanisole or 1,2-ethanedithiol.

[0032] The process of the present application can be advantageously used to improve the preparation, yield and / or purity of crude glucagon-like peptide (in particular liraglutide), in particular for its large scale preparation. Of particular interest is a process comprising performing a solid state synthesis (SPPS) of a glucagon-like peptide precursor, cleaving said precursor from the resin to obtain crude glucagon-like peptide in a cleavage composition, and separating the cleaved crude glucagon-like peptide from the cleavage composition.

[0033] Conventionally, several abbreviations and definitions are used in the present application:

[0034] Abbreviations:

[0035] Boc tert.butyl ester

[0036] CLEAR cross-linked ethoxylate acrylate

[0037] DBU diazabicyclo[5.4.0]undec-7-ene

[0038] DEPBT 3-(diethoxy-phosphoryloxy)-3H-benzo[d][l,2,3]triazin-4-one

[0039] DIC diisopropylcarbodiimide

[0040] DIPEA diisopropylethylamine

[0041] Dmb 2,4-dimethoxybenzyl

[0042] DMF N,N-dimethylformamide

[0043] DTE 1,4-dithioerythriol

[0044] DTT 1,4-dithiothreitol

[0045] EDT 1,2-ethanedithiol

[0046] Fmoc 9-fluorenylmethyloxycarbonyl

[0047] Hmb 2-hydroxy-4-methoxybenzyl

[0048] HOBt hydroxybenzotriazole

[0049] HPLC High Performance Liquid Chromatography

[0050] IPE diisopropyl ether

[0051] LPPS Liquid Phase Peptide Synthesis

[0052] MALDI-MS Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

[0053] MTBE methyl tert.butyl ether

[0054] MTT 4-methyltrityl

[0055] NMP N-methylpyrrolidone

[0056] OMpe 3-methylpent-3-yl ester

[0057] OtBu tert.butyl ester

[0058] ONSu=OSu N-hydroxysuccinimide

[0059] cyano-hydroxyimino-acetic acid ethyl ester

[0060] PEG polyethylene glycol

[0061] PEGA acrylamide-PEG co-polymer

[0062] Pbf 2,2,4,6,7-Pentamethyldihydrobenzofurane-5-sulfonyl

[0063] RT room temperature

[0064] SPPS Solid Phase Peptide Synthesis

[0065] tBu tert.butyl

[0066] TBTU benzotriazolyl tetramethyluronium tetrafluoroborate

[0067] TES triethylsilane

[0068] THF tetrahydrofuran

[0069] TIPS triisopropylsilane

[0070] Trt trityl

[0071] TFA trifluoroacetic acid

[0072] UHPLC Ultra High Performance Liquid Chromatography

[0073] Unless otherwise indicated, liquid mixtures are defined by volume percent and volume ratios. In the present invention, the terms "peptide" and "polypeptide" are interchangeable.

[0074] Unless otherwise indicated, peptide sequences in the present invention start at the N-terminus (left) and end at the C-terminus (right).

[0075] Table 1 illustrates different notations which are equivalent and can be used interchangeably in the present specification.

[0076] Amino acid notation can be by its full name (e.g. alanine), or by the 3-letter code (e.g. Ala) or the single letter code (e.g. A) according to WIPO Standard ST.25, which are interchangeable. In the absence of explicit indication, the enantiomeric form is typically the L-amino acid.

[0077] It is understood, however, that the present invention can also be practiced using D-amino acids and other stereoisomers.

[0078] Table 1. Notations for peptides

[0079]

[0080] The present invention employs the following widely accepted notation for amino acid derivatives: substituents on the alpha amino group (Nα) are denoted by symbols on the left side of the amino acid symbol and separated by a hyphen, substituents on the alpha carboxy group are denoted by symbols on the right side of the amino acid symbol and separated by a hyphen, and substituents on the side chain are denoted in parentheses on the right side of the amino acid symbol. For unmodified alpha-amino acids, the substituent on the alpha amino group (Nα) is a proton (H-), and the substituent on the alpha carboxy group is a hydroxyl group (-OH).

[0081] For branched dipeptides, the notation follows a nested form. For example, Fmoc-Lys(Boc-Glu-OtBu)-OH refers to a Lys derivative having a Fmoc-protected alpha amino group and a free alpha carboxyl group, whose side chain is substituted with a glutamyl group having a Boc-protected alpha amino group and an OtBu-protected carboxyl group. The glutamyl group forms an amide bond with the Lys side chain through its gamma-carboxyl group.

[0082] A similar notation is used for substituted amino acids that are part of a peptide. For example, Aaa1-Aaa2-Lys(Boc-Glu-OtBu)-Aaa4-Aaa5 refers to a branched pentapeptide in which the Lys side chain at position 3 is substituted with a glutamyl group via an amide bond, the glutamyl group having a Boc-protected alpha amino group and an OtBu-protected carboxyl group. Thus, the amide bond is between the epsilon-amino group of Lys and the gamma-carboxyl group of Glu.

[0083] As another example of expressing a substituted amino acid in a peptide, Lys(Palmitoyl-Glu-OH) refers to a Lys residue incorporated into a peptide and its epsilon amino group (Ne), i.e., side chain, is bound to a Palmitoyl-Glu moiety through its gamma carboxyl group (Cy), thereby forming an amide bond between the Ne of Lys and the Cy of Glu. In the Palmitoyl-Glu moiety, the Palmitoyl residue is bound to the alpha amino group (Na) of Glu. The Lys(Palmitoyl-Glu-OH) moiety can also be referred to as Lys(Hexadecanoyl-Glu-OH), Lys(N-epsilon-(gamma-Glu-(N alpha-Hexadecanoyl))), or Lys(Ne-(gamma-)glutamyl-(N alpha-hexadecanoyl))).

[0084] As yet another example, the Lys(H-Glu-OH) moiety, when expressing a peptide sequence, refers to a Lys residue whose epsilon amino group (Ne) is bound to an unprotected Glu residue through its gamma carboxyl group (Cy). Thus, an amide bond is formed between the Ne of Lys and the Cy of Glu. In the Glu residue, the alpha amino group (Na) is free. The Lys(H-Glu-OH) moiety can also be referred to as Lys(N-epsilon-(gamma-Glu(H)-OH), Lys(N-epsilon-(gamma-Glu-OH), or Lys(Ne-)(gamma glutamyl-OH).

[0085] The skilled person will immediately understand that the peptide of formula I is a derivative of the common liraglutide, written in single letter code:

[0086] HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 4),

[0087] wherein the lysyl moiety (Lys 20 , K 20) is modified. More specifically, Lys 20 The ε-amino group is bonded to the γ-carboxyl residue (γ-Glu, γ-E) of the glutamyl moiety via an amide bond. This glutamyl moiety typically has a free α-carboxyl group. The glutamyl moiety can be bonded to the palmitic acid (hexadecanoic acid) moiety via its amino group, or it can have a free -NH2 (α-amino, Nα) group.

[0088] Preferably, the peptide of formula I contains (substantially) no protecting groups, and except for Lys 20 Apart from the amino acid side chains, there are no other modifications. Therefore, the peptide of Formula I is preferably a completely unprotected peptide, and preferably without further modification.

[0089] Alternatively, the N-terminus of peptide I can be modified (e.g., acylation (e.g., acetylation)). Occasionally, the C-terminus can be modified (e.g., amidation).

[0090] Occasionally, one or more amino acid residues on the side chain may bind to a fluorophore. Optionally, the peptide of formula I may also be radiolabeled (e.g., by means of radiolabeling). 3 H, 32 P, 35 S, 14 C, 99m Tc or lanthanides (e.g., 64 Gd can be labeled with spin markers, such as one or more heavy isotopes, for example, 13 C can be detected by nuclear magnetic resonance (NMR).

[0091] The glucagon-like peptide present in the solution of step (i) is typically composed of natural L-amino acids. However, alternatively, the peptide may also contain one or more non-natural amino acids, such as D-amino acids, β-amino acids, methylated amino acids (e.g., N-methylated amino acids), or may even consist entirely of these non-natural amino acids.

[0092] Those skilled in the art will understand that in polar environments, particularly in aqueous environments, the peptide chain of a type I peptide or any other glucagon-like peptide can form salts, for example, by binding protons or other cations and / or anions to the terminal and / or some amino acid side chains, or by releasing protons or other cations and / or anions.

[0093] The term "glucagon-like peptide" or GLP as used in this invention refers to homologous peptides derived from the GCG gene (HGNC: 4191), venom exopeptides and their analogues, and any derivatives of the aforementioned substances. Figure 1 This shows sequence alignment between the prototype glucagon-like peptides.

[0094] The terms "glucagon-like peptide 1 analogue" and "GLP-1 analogue" are used interchangeably in the present invention and refer to a peptide which is capable of binding to a GLP-1 receptor. Derivatives and analogues of GLP-1 (7-37) and exendin-4 (1-39) such as exenatide, liraglutide and lixisenatide are preferred GLP-1 analogues. Exemplarily, a GLP-1 analogue can comprise a polypeptide chain which is at least 80% homologous to SEQ ID NO: 4, more preferably a polypeptide chain which is at least 90% homologous to SEQ ID NO: 4, in particular a polypeptide chain which is at least 95% homologous to SEQ ID NO: 4. Optionally, there is also a modification on the lysine which is homologous to Lys20 of SEQ ID NO: 4. In the present invention homology preferably is sequence homology determined over the entire length of SEQ ID NO: 4.

[0095] In the present invention sequence homology can refer to any definition of sequence homology known in the art. In particular, sequence homology can be understood as sequence homology determinable by the BLAST (Basic Local Alignment Search Tool) provided by the National Center for Biotechnology Information (NCBI) in the version available at the date of filing of the present application.

[0096] The term "analog" or "analogue" as used in the present invention refers to a peptide which is derived from a first peptide sequence by substitution of up to 50% of the amino acid residues in the first peptide, and / or by deletion of up to 10% of the amino acid residues from the first peptide, and / or by addition of up to 10 amino acid residues to the first peptide. Preferred analogues are derived from a first peptide by substitution of up to 20% of the amino acid residues, and / or by deletion of up to 10% of the amino acid residues, and / or by addition of up to 10 amino acid residues.

[0097] The term "derived" or "derivative" as used herein refers to a compound which is obtainable from a first compound by a chemical reaction. The result of the derivation can be distinguished from the first compound by the presence or absence of a substituent. For example, amino acid derivatives used in SPPS are typically different from the amino acids from which they are derived at least because of the presence of an amino protecting group.

[0098] The term "providing a solution S comprising a peptide of formula I" can be understood in the broadest sense as obtaining any liquid composition containing a peptide of formula I. The peptide of formula I can be provided by any method known in the art. Exemplarily, it can be obtained from solid phase peptide synthesis (SPPS) or liquid phase peptide synthesis (LPPS) or a combination thereof. Alternatively, the polypeptide chain can also be obtained from a biotechnological method, followed by a chemical / synthetic method to modify the obtained polypeptide chain. Preferably, the peptide of formula I is obtained from SPPS, LPPS or a combination thereof. More preferably, the peptide of formula I is obtained from a method consisting of or comprising SPPS.

[0099] Generally, peptide synthesis (based on SPPS and LPPS) involves the use of various protecting groups and activated esters. Thus, during the synthesis of a peptide of formula I various protecting groups and activated esters can be used.

[0100] The term "protecting group" as used in the present invention can be understood in the broadest sense as a group introduced into a molecule by chemical modification of a functional group which prevents said functional group from participating in a reaction in a subsequent process step, e.g. to prevent side reactions of the amino acid side chains. Examples of amino protecting groups are Boc and Fmoc groups, examples of carboxylic acid protecting groups are non-reactive esters such as methyl, benzyl or tert-butyl esters.

[0101] In the present invention the term "activated ester" can be understood in the broadest sense as an ester which is suitable for spontaneous reaction with an amino group. Examples of activated esters are p-nitrophenyl, pentafluorophenyl and succinimidyl esters.

[0102] While other methods, in particular other synthetic methods, for obtaining a peptide of formula I are also comprised within the scope of the present invention, the peptide of formula I is preferably obtained by solid phase peptide synthesis (SPPS). On the other hand, the SPPS method disclosed in the present invention can also be combined with different peptide isolation protocols for the production of liraglutide.

[0103] In SPPS, the precursor peptide of a peptide of formula I is synthesized on a resin (here step (i-a)), i.e. on a "solid phase", which is usually a bead-like structure which can easily be separated from the liquid phase by means of filtration. After the synthesis of the peptide on the resin is completed, the peptide is released from the resin and the protecting groups are removed. Thus, step (i) of the present invention can comprise the following steps

[0104] (i-a) providing a solid phase-bound glucagon-like peptide, wherein at least the side chains of Glu, Asp and Lys carry a protecting group,

[0105] (i-b) cleaving the peptide from the resin and optionally removing the protecting groups.

[0106] Thus, in a preferred embodiment, step (i) of the process of the present invention comprises:

[0107] (i-a) providing a solid phase-bound glucagon-like peptide of the formula H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-B 2 -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-[resin], wherein B 2 is Lys(palmitoyl-Glu-OR1) or B2 is Lys(R2-Glu-OR1), R1 is a carboxylic acid protecting group, R2 is an amino protecting group; wherein at least the side chains of Glu, Asp and Lys carry a protecting group; and

[0108] (i-b) cleaving the precursor peptide from the resin.

[0109] In a preferred embodiment, B 2 is Lys(Palmitoyl-Glu-OtBu) or Lys(Boc-Glu-OtBu). The Lys(Palmitoyl-Glu-OtBu) moiety can also be referred to as Lys(Hexadecanoyl-Glu-OtBu).

[0110] In the present invention, the terms "resin" and "[resin]" can be understood in the broadest sense as a bead-like structure that can be used for SPPS. The terms "resin", "solid phase" and "support" are used interchangeably in the invention.

[0111] SPPS is typically performed on a gel phase rather than a solid phase support. Suitable resins can be based on polystyrene, polystyrene-PEG composites, PEG, PEGA, cross-linked ethoxylated acrylate (CLEAR), polyamide, polydimethylacrylamide, or any other support having the desired physical and chemical properties. Resins based on bead polystyrene with 1% divinylbenzene are the routinely used supports, typically having a size distribution of 200-400 mesh or 100-200 mesh. Polystyrene-based 4-alkyloxybenzyl alcohol (Wang) resins, diphenyl diazomethane (PDDM) resins, 4-(2',4'-dimethoxyphenyl-Fmoc- aminomethyl)-phenoxy-methyl-polystyrene (Rink) resins, 2-methoxy-4-alkyloxybenzyl alcohol (Sasrin) resins, especially 2-chlorotrityl chloride (CTC) resins, are all particularly suitable resins for use in the methods of the invention and are commercially available from suppliers such as Sigma-Aldrich, Bachem and EMD Millipore. Of course, any other resin suitable for SPPS can be used.

[0112] As an alternative to immobilization via the C-terminal carboxyl group, the peptide can also be bound to the resin via the side chain of an (preferably terminal) amino acid.

[0113] As a further alternative, the peptide can also be bound to the resin via the N-terminus and synthesized from the N-terminus to the C-terminus (i.e. reverse peptide synthesis).

[0114] The skilled person will be aware of a variety of alternative SPPS methods. In general, any type of SPPS can be used in the methods of the application. Various types of equipment can be employed for SPPS. There are manual, semi-automated and fully automated synthesizers, SPPS can be performed in batch or continuous operation. For any given equipment, the appropriate resin can be chosen to meet the mechanical requirements of the equipment.

[0115] The skilled person will be fully aware that resin loading can affect the efficiency of SPPS, particularly in industrial SPPS. This effect can be particularly large for long, aggregation-prone peptide sequences (e.g. glucagon-like peptide): on the one hand, process efficiency increases with increasing resin loading; on the other hand, precipitation on the resin has to be reduced by reducing resin loading. Therefore, for any given SPPS protocol, the optimal resin loading needs to be established by routine experimentation to meet its delicate balance. Methods to vary resin loading are, for example, by using commercially available resins of different degrees of substitution, or by coupling the second amino acid in a molar excess relative to the first amino acid, followed by acetylation, i.e. blocking of unreacted first amino acid. Likewise, the first amino acid can be coupled to the resin in a molar excess, followed by a blocking step. In preferred embodiments of the application, a resin loading of about 0.2 mmol / g to about 0.9 mmol / g is used, e.g. about 0.2 mmol / g, 0.3 mmol / g, 0.4 mmol / g, 0.5 mol / g, 0.6 mmol / g, 0.7 mmol / g, 0.8 mmol / g, or 0.9 mmol / g. In this context, it can be noted that swelling and shrinking of the resin in the various solvents used during SPPS can lead to significant fluctuations in the volume of the resin, and thus to significant fluctuations in the concentration of the peptide on the resin.

[0116] In a preferred embodiment of the application, the glucagon-like peptide is prepared by Fmoc-SPPS. Suitable protected amino acid derivatives (i.e. amino acid moieties conjugated with one or more protecting groups), such as Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Mtt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OMpe)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Gly-OH, Fmoc-Gln(Mtt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-His(l-Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Val-OH, are commercially available from various vendors. It should be noted that the use of non-natural amino acid derivatives such as Aib (alpha-aminoisobutyric acid), Nle (norleucine) or Orn (ornithine) in the synthesis of glucagon-like peptides is also included within the scope of the present application. In a preferred embodiment, the amino acid derivatives Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OMpe)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Boc-His(Boc)-OH, Fmoc-His(l-Trt)-OH Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Val-OH are employed. The use of Fmoc-Trp(Boc)-OH together with Fmoc-Arg(Pbf)-OH can suppress the sulfonyl modification of the tryptophan moiety by cations upon cleavage of the peptide from the resin. However, since the cleavage of TFA from the resin can lead to the formation of side products such as carbamates, it can be advantageous to perform a decarboxylation reaction on the crude peptide.This can, exemplarily, be achieved by subjecting the crude peptide to a high pH treatment, e.g. a pH of at least 7.2, at least 8.0, at least 8.5, at least 9, at least 9.5, at least 10, at least 10.5, at least 11, or at least 11.5. Occasionally, the decarboxylation reaction can be performed under mild acidic conditions, e.g. a pH of 6.0 to 7.0, a pH of 5.5 to 6.5, or a pH of 5.0 to 6.0. Optionally, the treatment can be accompanied by a heat treatment, e.g. at a temperature of 30-70 °C, e.g. at 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, or 70 °C.

[0117] SPPS methods based on Fmoc synthesis protocols are well known to the skilled person. Each cycle of adding an amino acid to the resin typically starts with Fmoc cleavage, i.e. removal of the Fmoc protecting group from the resin-bound peptide chain. This is achieved by incubating the peptide resin with a base in a solvent capable of swelling the resin and dissolving the reagent. Commonly used bases for this purpose include, e.g., secondary amines such as piperidine and 4-methylpiperidine. Suitable solvents include, e.g., DMF, NMP, dimethylsulfoxide, dichloromethane, tetrahydrofuran, acetonitrile, toluene and mixtures thereof. The reaction is typically performed at ambient temperature, e.g. in the temperature range of 15-30 °C. Typically, the base-labile and acid-stable Fmoc is cleaved off by a short treatment (2 to 15 minutes, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 minutes) in DMF containing 5-50% (v / v), preferably 20%, piperidine.

[0118] If necessary, the above treatment can be repeated and / or slightly prolonged (7 to 30 minutes, e.g. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 minutes). For the synthesis of large peptides with segments that are difficult to cleave, the duration of the Fmoc cleavage and the number of repetitions can be gradually increased. For example, the cleavage time can be 15-75 minutes, e.g. 15, 30, 45, 60 or 75 minutes, and the cleavage can be repeated up to 8 times, e.g. 2, 3, 4, 5, 6, or 8 times. Also, the temperature can be increased, e.g. between 30 °C and 45 °C. Under these conditions, complete cleavage can be achieved in most cases. Occasionally, the cleavage reagent for Fmoc can be changed.

[0119] It was found that even slight changes in the reagents can significantly accelerate the cleavage, for example, using DMF containing 1-5% DBU, DMF containing 20% piperidine and 1-5% DBU, NMP containing 20% piperidine, or DMF containing 20% piperidine at 45°C. In addition, acceleration of the cleavage reaction can be achieved by microwave treatment. On the other hand, the nature of the peptide can make it more advantageous to use milder treatments. In particular, the mild cleavage conditions are 0.1 M HOBt and 20% piperidine in DMF, 50% morpholine in DMF, and 2% HOBt, 2% hexamethyl eneimine and 50% DMSO (containing 25% N-methylpyrrolidine) in NMP. The person of ordinary skill will routinely optimize and control the Fmoc cleavage conditions at each step of the synthesis.

[0120] In a preferred embodiment, the reagents used to cleave the Fmoc protecting group from the growing peptide chain conjugated to the solid phase are a mixture of the following solutions: 5-50% piperidine or 4-methylpiperidine in dimethylformamide (DMF), 5-50% piperidine or 4-methylpiperidine in N-methylpyrrolidone (NMP), 1-5% diazabicyclo[5.4.0]undec-7-ene (DBU) in DMF, and 50% morpholine in DMF, all percentages being volume percentages.

[0121] After Fmoc removal, the cleavage reagent is usually carefully washed off. DMF and optionally IPA can be used to wash the product to neutral pH. To ensure complete removal of the base, it can be advantageous to add a small amount of HOBt in a later wash cycle.

[0122] The coupling of an amino acid derivative to the resin-bound peptide, i.e. the elongation step, is one of the core steps of the SPPS cycle.

[0123] The rate and yield of the coupling reaction can be influenced by various parameters, such as the choice of solvent, steric hindrance and reactivity of the activated carboxylic acid. The solvent can not only determine the swelling of the precursor peptide-resin, and thus the accessibility of the reaction site; it can also directly influence the kinetics of the coupling reaction. Suitable solvents are able to swell the resin and dissolve the reagents, including, for example, DMF, NMP, dimethyl sulfoxide, dichloromethane, tetrahydrofuran, acetonitrile, toluene, and mixtures thereof. The steric hindrance is determined by the nature of the amino acid side chain and its protecting group. The reactivity of the activated carboxylic acid determines the rate of acylation, as well as the extent of side reactions, such as racemization. Depending on the chosen synthesis strategy, peptide derivatives, such as pseudoproline dipeptide derivatives, di- or tripeptide derivatives or branched dipeptide derivatives, can be used instead of individual amino acid derivatives.

[0124] In certain embodiments of the application, amino acid activation is performed in DMF as the solvent, i.e., the amino acid or peptide derivative, coupling agent, and optional additive are dissolved in DMF and mixed. DIC can be used as the coupling agent with HOBt as the additive. Alternatively, Fmoc amino acids can be converted to active OBt or ODhbt esters using TBTU or DEPBT in the presence of a base, preferably DIPEA. The selected amino acid derivative can be pre-activated by incubation with the reagents described above for 1-30 minutes, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,..., 28, 29, or 30 minutes, prior to addition to the resin. The coupling reaction can be performed for 1 to 74 hours, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,..., 71, 72, 73, or 74 hours. The amount of amino acid derivative used can be 0.4-3 molar equivalents relative to the amount of amine groups on the resin, e.g., 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 molar equivalents. Addition of a second portion of the activating agent or base to the reaction mixture after a period of time, e.g., 10, 20, 30, 40, or 60 minutes, can be beneficial in achieving complete coupling. The pre-activation and coupling steps are typically performed at room temperature, but can be performed at other temperatures. One or more repeated coupling steps can be performed to achieve near complete conversion of the amino groups.

[0125] In other embodiments of the application, amino acid activation is performed in a solvent consisting of NMP, dimethylsulfoxide, dichloromethane, tetrahydrofuran, acetonitrile, toluene, and mixtures thereof, and DMF can optionally be added.

[0126] In a preferred embodiment, step (i-a) of the application comprises Fmoc-based solid phase peptide synthesis (SPPS) using appropriately protected amino acid derivatives or dipeptide derivatives, wherein the protected amino acid derivatives or dipeptide derivatives are activated by one or more coupling agent / additive mixtures

[0127] (A) TBTU / DIPEA;

[0128] (B) DIC / OximaPure (cyano-hydroxy imino-ethyl acetate);

[0129] (C) DEPBT / DIPEA; and

[0130] (d) DIC / HOBt.

[0131] Coupling of histidine (His) derivatives is preferably performed to avoid racemization. This side reaction can be reduced by three different approaches: 1) blocking the N3 of the imidazole ring, 2) blocking the N1 of the imidazole ring by an electron withdrawing group such as Boc or Tos, and 3) using Fmoc-His(1-Trit)-OH to optimize the coupling conditions. Thus, when introducing an N-terminal His, it is recommended to use Fmoc-His(1-Trit)-OH, Boc-His(1-Trt)-OH, or Boc-His(Boc)-OH, most preferably Boc-His(Boc)-OH, and to combine it with DEPBT / DIPEA.

[0132] Thus, in a preferred embodiment, an N-terminal histidine is introduced into a precursor peptide conjugated to the solid phase using a histidine derivative selected from the group consisting of Boc-His(Boc)-OH, Boc-His(1-Trt)-OH, and Fmoc-His(1-Trt)-OH and DEPBT / DIPEA as coupling agent / additive mixture.

[0133] Capping can be performed to block unreacted amines from forming peptide bonds in subsequent synthesis steps, i.e. to avoid the formation of deletion variants of the sequence to be synthesized. This can be achieved by treating the peptide resin with a large excess of a highly reactive, unhindered acid derivative and a base for a short time, wherein the unhindered acid derivative can be selected from N-hydroxysuccinimide, acetic anhydride or benzoyl chloride; the base can be selected from e.g. pyridine, collidine or DIPEA. Optionally, this treatment can be performed in the presence of an additive such as OxymaPure or HOBt. Capping will typically result in a truncated sequence, which is typically significantly different from the final peptide and can easily be separated off. Preferably, capping is performed systematically after double coupling. At the end of the capping step, the reagents are typically filtered off and the resin is carefully washed, e.g. using DMF and optionally IPA, before the next deprotection step is performed.

[0134] Preferably, aggregation of glucagon-like peptides, such as liraglutide, on the resin can be prevented during SPPS.

[0135] Glucagon-like peptides like liraglutide tend to aggregate on their own, which constitutes an additional challenge for their efficient synthesis. Once aggregation of resin-bound peptides occurs, the steps of Fmoc-SPPS can become difficult or even fail.

[0136] There are a number of possible options available to mitigate this aggregation, such as controlling the resin loading by controlling the coupling density, controlling the resin shrinkage in the washing steps, adding solvents such as DMSO, adding chaotropic salts, adding non-ionic detergents and ethylene carbonate, performing the coupling reaction under high temperature / microwave treatment, and sonicating the coupling reaction mixture.

[0137] Further possible measures also include the introduction of at least one O-isoacyl peptide bond into the serine or threonine, the introduction of at least one pseudoproline dipeptide, or the N-alkylation of at least one peptide bond by 2-hydroxy-4-methoxybenzyl (Hmb) or 2,4-dimethoxybenzyl (Dmb). Thus, at least one dipeptide derivative comprising an O-isoacyl peptide bond such as Boc-Ser(Val-Fmoc)-OH or Boc-Thr(Gly-Fmoc)-OH can be used for this purpose. Occasionally, at least one dipeptide with an N-alkylated peptide bond can be used, such as Fmoc-Glu(OtBu)-(Dmb)Gly-OH and Fmoc-Ala-(Dmb)Ala-OH.

[0138] Surprisingly, it was found that the use of at least one Fmoc pseudoproline dipeptide advantageously inhibits the aggregation of the peptide and thus the formation of by-products due to inefficient synthesis. Preferably, the position of the introduction of the pseudoproline dipeptide corresponds or is identical to the position of Gly 4 -Thr 5 , Phe 6 -Thr 7 , Thr 7 -Ser 8 , Val 10 -Ser 11 or Ser 11 -Ser 12 of the peptide of the formula I.

[0139] Thus, the introduction of a pseudoproline residue provides a solid phase conjugated glucagon-like peptide, preferably a solid phase conjugated liraglutide, which is characterized by a reduced percentage of relevant impurities. A variety of pseudoproline dipeptide derivatives are known to the person skilled in the art, see “Pseudoproline Dipeptides Bachem-Pioneering Partner for Peptides”, Global Marketing, November 2015, published by the Bachem Group. Thus, in a preferred embodiment, at least one dipeptide derivative comprising a pseudoproline residue can be used in the position of Gly 4 -Thr 5 , Phe 6 -Thr 7 , Thr 7-Ser 8 , Val 10 -Ser 11 or Ser 11 -Ser 12 one or more pseudoproline dipeptides are introduced at the position.

[0140] In a particularly preferred embodiment one or more pseudoproline dipeptide derivatives selected from the group of Fmoc-Gly-Thr(Psi(Me,Me)pro)-OH, Fmoc-Phe-Thr(Psi(Me,Me)pro)-OH, Fmoc-Thr(tBu)-Ser(Psi(Me,Me)pro)-OH, Fmoc-Val-Ser(Psi(Me,Me)pro)-OH, and Fmoc-Ser(tBu)-Ser(Psi(Me,Me)pro)-OH are employed.

[0141] According to a preferred embodiment one or more pseudoproline dipeptides are introduced at the position corresponding to Gly 4 -Thr 5 , Phe 6 -Thr 7 or Thr 7 -Ser 8 of the peptide of formula I. Preferably a single pseudoproline dipeptide is introduced at the position corresponding or identical to Thr7-Ser 8 of the peptide of formula I. In another preferred embodiment a single pseudoproline dipeptide is introduced at the position corresponding or identical to Phe 6 -Thr 7 of the peptide of formula I. In a particularly preferred embodiment one or more pseudoproline dipeptides selected from the group of Fmoc-Gly-Thr(Psi(Me,Me)pro)-OH, Fmoc-Phe-Thr(Psi(Me,Me)pro)-OH, and Fmoc-Thr(tBu)-Ser(Psi(Me,Me)pro)-OH are employed.

[0142] According to a preferred embodiment one or more pseudoproline dipeptides are introduced at the position corresponding to Gly 4 -Thr 5 , Phe 6 -Thr 7 or Thr 7 -Ser 8one or more pseudoproline dipeptides at the same or a corresponding position. In a particularly preferred embodiment, all introduced pseudoproline dipeptides are selected from Fmoc-Gly-Thr(Psi(Me,Me)pro)-OH, Fmoc-Phe-Thr(Psi(Me,Me)pro)-OH, or Fmoc-Thr(tBu)-Ser(Psi(Me,Me)pro)-OH.

[0143] According to a preferred embodiment, only one single pseudoproline dipeptide is introduced at the same or a corresponding position of Gly 4 -Thr 5 , Phe 6 -Thr 7 , Thr 7 -Ser 8 , Val 10 -Ser 11 or Ser 11 -Ser 12 , wherein the preferred positions are Gly 4 -Thr 5 , Phe 6 -Thr 7 and Thr 7 -Ser 8 . In a particularly preferred embodiment, the single pseudoproline dipeptide is selected from Fmoc-Gly-Thr(Psi(Me,Me)pro)-OH, Fmoc-Phe-Thr(Psi(Me,Me)pro)-OH, or Fmoc-Thr(tBu)-Ser(Psi(Me,Me)pro)-OH.

[0144] The term "pseudoproline dipeptide" as used herein refers to a temporary proline mimic which is readily accessible from Ser and Thr via formation of an oxazolidine and from Cys via formation of a thiazolidine. This pseudoproline dipeptide is well known to the person skilled in the art. These dipeptides are a possible option to reduce aggregation on the resin during SPPS. Since 2,2-dimethyloxazolidine is smoothly cleavable by TFA, it is particularly suitable for Fmoc-SPPS. Thus, Fmoc-Phe-Thr(Psi(Me,Me)pro)-OH, Fmoc-Gly-Thr(Psi(Me,Me)pro)-OH, Fmoc-Thr(tBu)-Ser(Psi(Me,Me)pro)-OH, Fmoc-Val-Ser(Psi(Me,Me)pro)-OH, or Fmoc-Ser(tBu)-Ser(Psi(Me,Me)pro)-OH can be particularly selected.

[0145] The term "position corresponding to the position of Gly 4 -Thr 5 , Phe 6 -Thr 7 , Thr 7 -Ser 8 , Val 10 -Ser 11 or Ser 11 -Ser 12 of the peptide of formula I" as used herein refers to a position within the primary sequence of a glucagon-like peptide which, based on a sequence alignment of said glucagon-like peptide with the peptide of formula I, is considered to be homologous to the position of Gly 4 -Thr 5 , Phe 6 -Thr 7 , Thr 7 -Ser 8 , Val 10 -Ser 11 or Ser 11 -Ser 12 of the peptide of formula I. Typically, in such an alignment display, positions which are superimposed on each other are considered to be homologous, i.e. corresponding to each other. Typical sequence alignment tools like BLAST or ClustalW are well known to the person skilled in the art.

[0146] As mentioned above, liraglutide can comprise palmitic acid conjugated to the epsilon amino group of the lysinyl residue at amino acid position 20 (Lys20), i.e. Lys(Palmitoyl-Glu-OH). This can be introduced in any way.

[0147] Various options are available for introducing the (N-epsilon-(gamma-glutamyl (N alpha-fatty acid ester))) substituted Lys side chain (see, e.g., US-B 6,451,974, EP-A 2 757 107, WO 2013 / 171 135). In the present application, the group is introduced at least partially during SPPS, preferably using a branched dipeptide building block of formula 2 below, wherein R3 is an amino protecting group, R1 is a carboxylic acid protecting group, R2 is an amino protecting group or a fatty acid residue, in particular a palmitoyl [CH3(CH2) 14 CO-] group.

[0148] Formula 2:

[0149]

[0150] It is particularly preferred that the SPPS is carried out using Fmoc-Lys(palmitoyl-Glu-OtBu)-OH building blocks (commercially available from Iris Biotech GmbH or Peptides International) or Fmoc-Lys(Boc-Glu-OtBu)-OH building blocks (commercially available from Active Peptide).

[0151] In case the latter is used, the resulting peptide can be subjected to N-palmitoylation at the alpha amino function of the glutamyl moiety. Various activated palmitates, such as p-nitrophenyl and succinimidyl esters can be used for this purpose. In a preferred embodiment, N-succinimidyl palmitate is used.

[0152] Thus, in a preferred embodiment, the method of the present application comprises a step of reacting the peptide with an activated palmitate, preferably N-succinimidyl palmitate, which typically results in a peptide of formula I.

[0153] In a more preferred embodiment, the method comprises a step of reacting an activated palmitate, preferably N-succinimidyl palmitate, with Lys(H-Glu-OH) or Lys(H-Glu-OR1) of a peptide of formula I. The peptide of formula I is obtained from the precursor peptide provided in step (i-a) by cleavage of the amino protecting group R2.

[0154] Even more preferably, the method comprises a step of reacting an activated palmitate, preferably N-succinimidyl palmitate, with Lys(H-Glu-OR1) obtainable from the precursor peptide provided in step (i-a) by cleavage of the amino protecting group R2, in particular, the precursor peptide provided in step (i-a) carries protecting groups at least on the side chains of Glu, Asp and Lys.

[0155] Optionally, the progress of the SPPS reaction can be monitored by process control to ensure efficient Fmoc removal, coupling and / or capping steps. On the other hand, Fmoc assay and free amine assay can provide complementary information. In summary, these methods can enable efficient monitoring of each step of the SPPS process. Some commonly used monitoring methods that can be used in the present application are exemplified below.

[0156] Optionally, the amount of Fmoc cleaved from the resin-bound peptide can be easily quantified, for example by spectroscopic assays. The Fmoc cleavage reagent can be collected from the resin effluent and the Fmoc concentration therein determined by measuring the absorbance at 301 nm. Based on the amount of Fmoc cleaved, the resin loading, i.e. the original amount of Fmoc peptide on the resin, can be calculated. Furthermore, to assess the completeness of Fmoc removal, a small sample of the resin after Fmoc deprotection can be subjected to an additional rigorous Fmoc cleavage treatment to determine the amount of residual Fmoc removed by this treatment. Alternatively, a small scale cleavage test can be performed on the peptide from the resin sample to assess the thoroughness of Fmoc removal. The resulting peptide can be analyzed using standard gradient analytical RP-HPLC, where Fmoc protected and free peptide sequences will typically be well separated. Occasionally, the peptide sample can be analyzed by mass spectrometry, for example, using LC-MS or MALDI-MS. Also, thin layer chromatography can detect traces of Fmoc peptide.

[0157] The amount of free amine on the resin can be assessed by various assays, including the colorimetric Kaiser (i.e. ninhydrin), TNBS, chloroquinone and bromophenol blue test. This is well known to the person skilled in the art.

[0158] These tests can advantageously be used to assess the production of free amino functional groups after Fmoc cleavage, and their disappearance after coupling of Fmoc protected amino acid derivatives and / or capping steps. Preferably, at least two colorimetric tests can be performed in parallel, for example the Kaiser and TNBS tests. The Kaiser test is based on the reaction of ninhydrin with amines. This is a very sensitive test for primary amines, with a strong blue color easily observed, but slightly less suitable for secondary amines, which produce a reddish-brown color. The color typically occurs mainly in the beads, partly in the supernatant. When spectroscopic quantification of the amount of unreacted amino groups is intended, the color can be transferred completely into the solution. The intensity of the color depends on the nature of the amino terminus to be detected. With N-terminal side chain protected Asp, Asn, Cys, Ser and Thr a non-specific color tone is obtained, and with N-terminal Pro a reddish-brown bead is produced. As the resin sample is typically heated, "hidden" NH2 groups can become more accessible and thus detectable.

[0159] However, long heating or overheating should be avoided, as this can lead to cleavage of Lys(Boc) or removal of Fmoc (via pyridine). The TNBS (2,4,6-trinitrobenzenesulfonic acid) test is almost as sensitive as the Kaiser test, but can only be used to detect primary amino groups. The beads will only turn orange-red, and the intensity of the color is independent of the nature of the N-terminal amino acid. As the slight orange staining in the bead core cannot be detected by simple visual inspection, it is recommended to use a more sensitive detection, for example, by inspecting the beads under a microscope.

[0160] When the synthesis of the precursor peptide is finished by SPPS, it is still conjugated to the resin. Thus, it is a solid phase conjugate and at least partially side chain protected. In order to obtain the peptide of formula I, e.g. liraglutide or any other glucagon-like peptide of the present application, the peptide has to be cleaved from the resin. This is indicated in the present specification by step (i-b), i.e. cleavage of the precursor peptide from the resin.

[0161] Most preferably, during this step, most or all side chain protecting groups are cleaved from the peptide at the same time, i.e. the peptide is deprotected, thereby providing the peptide of formula I.

[0162] Thus, preferably, deprotection of the resin and cleavage (step (i-b)) are performed simultaneously by incubation with a cleavage composition comprising TFA and one or more scavengers.

[0163] For cleaving the peptide from the resin, any composition suitable for this purpose can be used. Preferably, the cleavage composition for cleaving and deprotection contains more than 50% TFA, more preferably more than 75% TFA, in particular at least 80% (v / v) or even at least 90% TFA. Higher compositions are possible. The composition can also contain water and / or one or more scavengers. Preferably, the composition comprises TFA, water and one or more scavengers. Particularly advantageous scavengers are thiol scavengers, such as EDT, and / or silane scavengers, such as TIPS. The cleavage composition can comprise at least 80% TFA, preferably at least 90% TFA, and EDT. The cleavage composition can comprise at least 80% TFA, preferably at least 90% TFA, water and EDT. The cleavage composition can comprise at least 80% TFA, preferably at least 90% TFA, water and TIPS. The cleavage composition can comprise at least 80% TFA, preferably at least 90% TFA, water, TIPS and EDT. Exemplarily, the cleavage composition for use in the present application can be selected from the group consisting of TFA / water / TIPS (90:5:5), TFA / water / phenol (90:5:5), TFA / water / EDT / TIPS (90:5:2.5:2.5), TFA / water / EDT / TIPS (90:4:3:3), TFA / water / EDT (90:5:5), TFA / anisole / anisidine / EDT (90:5:3:2) and TFA / anisole / water / phenol / EDT (82.5:5:5:5:2.5). The above mentioned percentages and numbers ratios are volume percentages and volume ratios, respectively.

[0164] The step of cleaving the precursor peptide from the resin (step (ib)) can be carried out under any conditions suitable for this purpose. Preferably, the washed resin is incubated with the cleavage composition (preferably under an inert gas) for about 1 to 4 hours and / or cleaved at a temperature of 0 to 32°C.

[0165] For example, during pyrolysis, the washed resin can be incubated with the pyrolysis composition (preferably under an inert gas) for 1, 1.5, 2, 2.5, 3, 3.5, 4 hours or more, at temperatures approximately 0-4°C, 4-10°C, 10-15°C, 15-25°C, or 25-35°C. Alternatively, during pyrolysis, the washed resin can be incubated with the pyrolysis composition (preferably under an inert gas) for 1, 1.5, 2, 2.5, 3, 3.5, 4 hours or more, at temperatures approximately 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32°C.

[0166] Alternatively, another cleavage composition can be used. For example, when cleaving protected peptide fragments from 2-chlorotriphenylmethyl resin, it can be achieved by using TFE / AcOH / DCM (1:1:3), 0.5% TFA / DCM, or HFIP / DCM.

[0167] Those skilled in the art will routinely optimize the cleavage composition suitable for the present invention based on the amino acid composition of the given peptide, and the optional use of one or more scavengers, such as DTE, EDT, TES, TIPS, 2-mercaptoethanol, ethyl methyl sulfide, m- or p-cresol, 2-methylindole, Ac-Trp-OMe, or tryptamine, is conceivable.

[0168] In this invention, the term "scavenger" refers to a compound added to the reaction mixture to inhibit side reactions during the cleavage of the post-SPPS peptide from the resin and / or the removal of protecting groups. Typical scavengers used for cleavage compositions are "thiol scavengers" (e.g., EDT, DTE, DTT, and β-mercaptoethanol) and "silane scavengers" (e.g., TES and TIPS). Other commonly used scavengers include ethyl methyl sulfide, anisole, anisole, m- or p-cresol, 2-methylindole, Ac-Trp-OMe, or tryptamine. Those skilled in the art will readily recognize the various scavengers available.

[0169] According to the above description, the solution S obtained from step (i) also preferably contains TFA (typically at least 80% (volume ratio) or at least 90% (volume ratio)), residues of protecting groups cleaved from the peptide, and, more preferably, water and one or more scavenging agents.

[0170] The resin is then typically separated from the solution S obtained in step (i), i.e., the step of cleaving the peptide from the resin, by filtration. In one embodiment of the invention, the solution S obtained from step (i) comprises liraglutide and reagents for cleavage (e.g., TFA, water, a scavenging agent, and residues of the protecting group cleaved from the peptide). Optionally, the resin is rinsed after filtration, for example, with concentrated TFA or concentrated TFA plus a scavenging agent.

[0171] Optionally, the rinsing solution may also be part of the solution S obtained in step (i), i.e., it may be combined with the solution obtained directly from the cleavage of the peptide on the resin.

[0172] Therefore, in a preferred embodiment, the solution S obtained from step (i) further comprises trifluoroacetic acid (TFA), water, and one or more scavenging agents.

[0173] In a more preferred embodiment, the solution S obtained from step (i) comprises trifluoroacetic acid (TFA), water, and one or more scavengers selected from thiol scavengers and / or silane scavengers.

[0174] Scavengers can be used to prevent adverse side reactions with sensitive amino acids such as Cys, Met, Ser, Thr, Trp, and Tyr. Without wishing to be bound by any theory, it is believed that inhibition of these side reactions is achieved by capturing highly reactive carbocations generated during the cleavage reaction.

[0175] Those skilled in the art will know that it is generally desirable to separate the peptide of formula I from solution S to obtain a crude peptide (typically present as a TFA salt). In a particularly preferred embodiment of the invention, this is achieved by precipitating the peptide of step (i) with an antisolvent containing diisopropyl ether (IPE) and acetonitrile (ACN), wherein the volume ratio of IPE to ACN is in the range of (3:1) to (10:1); and then separating the resulting precipitate.

[0176] Here, a mixture of IPE and ACN is used as an antisolvent. This term is well understood by those skilled in the art. As used herein, the term "antisolvent" can be understood in the broadest sense as any agent that induces peptide precipitation when mixed with a peptide solution. Exemplary antisolvents include diethyl ether, IPE, MTBE, and mixtures of IPE and ACN. According to the invention, the antisolvent comprises IPE and ACN selected from a volume ratio of (3:1) to (10:1). More preferably, the antisolvent comprises IPE and ACN, wherein the IPE:ACN volume ratio is in the range of (3:1) to (5:1). Surprisingly, it has been found that the recovery of crude peptides from cleavage compositions is particularly effective when using an antisolvent comprising IPE and ACN in a volume ratio selected from (3:1) to (10:1), preferably in a volume ratio of (3:1) to (5:1), such as in ratios of (3:1), (3,5:1), (4:1), (4,5:1), or (5:1).

[0177] The present invention has found that using the aforementioned antisolvent can improve the purity and physical properties of the precipitate, thereby enabling the efficient separation of peptide products from the cleavage composition.

[0178] Advantageously, the present invention enables rapid recovery of crude peptides by forming a non-sticky precipitate with a size distribution that facilitates rapid filtration. Further advantageous are the purity of the precipitate and its low TFA content.

[0179] In a preferred embodiment, the antisolvent used in step (ii) comprises at least 50% (by volume) of a mixture M consisting of diisopropyl ether and acetonitrile. Preferably, the antisolvent comprises at least 75% (by volume) of said mixture M, and more preferably, the antisolvent is substantially composed of a mixture of diisopropyl ether and acetonitrile.

[0180] In a particularly preferred embodiment, the antisolvent is essentially a mixture of diisopropyl ether and acetonitrile, wherein the volume ratio of diisopropyl ether to acetonitrile is in the range of (3:1) to (5:1).

[0181] The antisolvent can be mixed with solution S by any suitable method. In a preferred embodiment, step (ii) includes mixing the antisolvent with solution S from step (i) in the following manner:

[0182] (ii-a) Premix diisopropyl ether and acetonitrile, and then mix them with the solution S obtained in step (i);

[0183] (ii-b) First, diisopropyl ether is mixed with solution S obtained in step (i), and then acetonitrile is mixed with the mixture containing solution S and diisopropyl ether; or

[0184] (ii-c) First, mix acetonitrile with the solution S obtained in step (i), and then mix diisopropyl ether with the mixture containing solution S and acetonitrile.

[0185] In other words, the antisolvent can be separated into an acetonitrile-containing fraction and a diisopropyl ether-containing fraction, which are then contacted with a solution containing the peptide. Alternatively, the antisolvent can be combined into a single liquid mixture.

[0186] In summary, the (IPE:ACN) volume ratio range of this invention was ultimately obtained. Those skilled in the art will understand that acetonitrile and diisopropyl ether can also be mixed with solution S in batches, as long as the final volume ratio range required by this invention is achieved.

[0187] Preferably, the volume ratio of solution S to antisolvent is 1:5 to 1:15, such as 1:5, 1:8, 1:10, 1:12, or 1:15. In other words, one volume of solution S is mixed with a certain amount of antisolvent, which is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 volumes.

[0188] The step of precipitating peptide of formula I from solution S (step (ii)) can be performed at any temperature and at any time interval suitable for this purpose. Exemplarily, step (ii) is performed at -5°C to 25°C, for example, about -5 to 0°C, 0 to 10°C, 10 to 20°C, or 20 to 25°C.

[0189] For the purposes of this application, the term "about" is used to indicate a deviation from any given value that may be as high as 10%.

[0190] In a preferred embodiment, the precipitation of the peptide of Formula I (step (ii)) is carried out at a temperature of -5°C to 10°C, preferably 0°C to 10°C. Suitable temperature ranges may be -5 to 0°C, -2.5 to 2.5°C, 0 to 5°C, 2.5 to 7.5°C, or 5 to 10°C.

[0191] For example, step (ii) is performed for a period of approximately 30 to 360 minutes, such as approximately 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, or 360 minutes.

[0192] Therefore, the preferred precipitation conditions are characterized by a temperature in the range of about -5°C to 25°C, for example, about -5 to 0°C, 0 to 10°C, 10 to 20°C or 20 to 25°C, and a reaction time of about 30 to 360 minutes, for example, about 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330 or 360 minutes.

[0193] In a particularly preferred embodiment, a method for preparing liraglutide or a salt thereof includes:

[0194] (i) Provide a solution S containing a peptide of formula I, wherein formula I is:

[0195] His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-B 1 -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly,

[0196] Among them B 1 It is Lys(palmitoyl-Glu-OH) or Lys(H-Glu-OH);

[0197] The steps for providing solution S include:

[0198] (ia) Provide precursor peptides for solid-phase conjugation:

[0199] H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-B 2 -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-[resin],

[0200] Among them B 2 It is Lys(palmitoyl-Glu-OtBu) or Lys(Boc-Glu-OtBu), wherein at least the side chains of Glu, Asp, and Lys have protecting groups; and

[0201] (ib) The precursor peptide is cleaved from the resin using a cleavage composition containing trifluoroacetic acid (TFA).

[0202] The solution S obtained from step (i) comprises trifluoroacetic acid (TFA), water, and one or more scavengers selected from thiol scavengers and / or silane scavengers;

[0203] (ii) Mixing solution S with an antisolvent consisting of diisopropyl ether and acetonitrile to precipitate the peptide from step (i), wherein the volume ratio of diisopropyl ether to acetonitrile is in the range of (3:1) to (5:1); and

[0204] (iii) Separate the precipitate obtained in step (ii), preferably by filtration and / or centrifugation.

[0205] The solution S obtained in step (i) and the antisolvent can be mixed by any method known in the art. The solution S obtained in step (i) and the antisolvent can be mixed in any order.

[0206] According to the preferred embodiment, step (ii) is performed using a classic precipitation method, i.e., the antisolvent is added to the solution S obtained from step (i). This can be achieved by adding it all at once, by adding it dropwise, and / or by adding it slowly.

[0207] According to another preferred embodiment, step (ii) is performed using a reverse precipitation scheme, i.e., the solution S obtained in step (i) is added to the antisolvent. This can be achieved by adding it all at once, by adding it dropwise, and / or by adding it slowly. Those skilled in the art are well aware of how to perform such a precipitation step.

[0208] A suspension containing liraglutide is then formed in the precipitate and antisolvent, the suspension also containing reagents for cleaving the composition (e.g., TFA, water, and scavenging agents) and residues of protecting groups cleaved from the peptide. The precipitate consisting of (or substantially consisting of) liraglutide can then be separated from the crude suspension by any method known in the art for this purpose.

[0209] Therefore, the present invention also includes a separation step (iii) to separate the precipitate obtained from step (ii).

[0210] According to the preferred embodiment, step (iii) is performed by filtration and / or centrifugation.

[0211] For the purposes of this invention, the term "separation" can be understood in the broadest sense as any means used to obtain the desired product, for example, obtaining a crude peptide precipitate from a more complex composition. The crude peptide precipitate may contain at least 30%, preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, particularly at least 95%, or even 100% liraglutide in a dry state. Typically, the crude peptide precipitate contains 40-70% liraglutide in a dry state. All percentages mentioned above are volume percentages.

[0212] The filtration can be any filtration method known in the art, such as dead-end filtration or cross-flow filtration. As used herein, the terms "cross-flow filtration," "tangential filtration," "tangential flow filtration," or "tangential filtration" are used interchangeably. The filter can be any material known in the art of filtration, such as plastics (e.g., nylon, polystyrene), metals, alloys, glass, ceramics, cellophane, cellulose, or composite materials. The filter can be hydrophobic or hydrophilic. The filter surface can be neutral, positively charged, or negatively charged.

[0213] Centrifugation can be understood in the broadest sense as any method that accelerates the settling of suspended precipitates. Exemplarily, usable centrifugal forces can be 100xg, at least 100xg, at least 1,000xg, at least 2,500xg, at least 5,000xg, at least 7,500xg, at least 10,000xg, at least 15,000xg, at least 25,000xg, or at least 50,000xg. Centrifugation forms a filter cake containing (or substantially containing only) crude liraglutide. Optionally, the filter cake can be resuspended in an antisolvent, which may be the same as or different from the antisolvent described above. Optionally, centrifugation and resuspending the precipitate in the antisolvent can be repeated several times, which can further improve the purity of the crude liraglutide.

[0214] Preferably, the crude peptide obtained by the method of the present invention can be further purified by one or more preparation steps. The purification and separation methods optionally used in the present invention include one or more electrophoretic methods (e.g., gel electrophoresis or capillary (CE) electrophoresis), one or more additional precipitation-based methods (e.g., salting or salting out), one or more dialysis methods (dialysis), and / or one or more chromatographic methods, such as gel permeation chromatography (GPC), size exclusion chromatography, ion exchange chromatography (IEC), high performance liquid chromatography (HPLC), reversed-phase HPLC (RP-HPLC), rapid protein liquid chromatography (FPLC), rapid chromatography (flash), rapid fluidized liquid chromatography (RRLC), rapid separation liquid chromatography (RSLC), ultra-fast liquid chromatography (UFLC), reversed-phase UFLC (RP-UFLC), ultra-high performance liquid chromatography (UPLC), or reversed-phase UPLC (RP-UPLC). Preferably, the crude peptide is first subjected to three-dimensional reversed-phase HPLC, followed by size exclusion chromatography, ion exchange chromatography, or ultrafiltration.

[0215] Those skilled in the art will understand that the present invention also includes crude liraglutide precipitate with improved purity, which is particularly easy to handle due to its improved particle size and consistency.

[0216] Therefore, another aspect of the present invention relates to liraglutide precipitates obtainable by the method of the present invention.

[0217] In a preferred embodiment, the liraglutide precipitate obtainable from the method according to the invention is characterized by having an average particle size greater than 280 micrometers, greater than 300 micrometers, greater than 350 micrometers, or greater than 400 micrometers.

[0218] Furthermore, the liraglutide precipitate that can be obtained by the method according to the invention is characterized by having a peptide purity of at least 50%, preferably at least 55%, and more preferably at least 60%.

[0219] In a preferred embodiment, the liraglutide precipitate obtainable according to the method of the invention is characterized by containing truncated N-terminal and / or C-terminal liraglutide variants. In some embodiments, the liraglutide precipitate obtainable according to the method of the invention is characterized by containing truncated variant liraglutide 21-31-OH, which may be acylated, for example, acetylated at the N-terminus. In other embodiments, the liraglutide precipitate obtainable according to the method of the invention is characterized by containing truncated variant liraglutide 1-30.

[0220] As used in this invention, the term "truncated variant" refers to a continuous segment of the peptide, i.e., a gapless subsequence, which lacks one or more amino acids at the N-terminus or C-terminus. N-terminal truncated variants may be acylated, for example, acetylated.

[0221] In a further preferred embodiment, the liraglutide precipitate obtainable by the present invention is characterized by reduced or even non-viscous properties and / or a TFA content of less than 20% (by weight). In a particularly preferred embodiment, the liraglutide precipitate obtainable by the present invention is characterized by an average particle size greater than 350 micrometers and a purity of at least 55%. In another preferred embodiment, the liraglutide precipitate obtainable by the present invention is characterized by an average particle size greater than 400 micrometers and a purity of at least 55%. In another preferred embodiment, the liraglutide precipitate obtainable by the present invention is characterized by an average particle size greater than 350 micrometers and a purity of at least 60%. In a particularly preferred embodiment, the liraglutide precipitate obtainable by the present invention is characterized by an average particle size greater than 350 micrometers, a purity of at least 55%, and non-viscous properties. In another preferred embodiment, the liraglutide precipitate obtainable by the present invention is characterized by an average particle size greater than 350 micrometers, a purity of at least 55%, and a TFA content of less than 20% (by weight).

[0222] The particle size of the precipitate can be measured by any method in the art, for example, by the focused beam reflectance measurement (FBRM) technique described below.

[0223] Unless otherwise stated, peptide purity in this specification is referred to as "HPLC purity," which is the relative peak area observed in analytical reversed-phase high-performance liquid chromatography (RP-HPLC), obtained by detection at 205 and 230 nm UV wavelengths, i.e., the wavelengths of maximum absorption of the peptide bond. In other words, this value is determined as the area percentage obtained by dividing a given peak area by the sum of the peak areas observed at 205 and 230 nm UV wavelengths in the chromatogram. This method is routine in the art, and those skilled in the art routinely design RP-HPLC assay protocols for a given product and quantify according to established guidelines specified in the United States Pharmacopeia. Peak purity determination by LC-MS routinely assesses whether a given RP-HPLC assay protocol can be used to detect peptide purity. Assuming that all peptide components have the same absorption due to their similar structures, RP-HPLC purity can represent weight percentage purity.

[0224] All definitions listed above apply to the liraglutide precipitation of this invention. Those skilled in the art will note that the liraglutide in the precipitate may be present in the form of a salt, particularly a TFA salt.

[0225] Optionally, one or more amino acid residues of the liraglutide of the present invention may be esterified, phosphorylated, sulfated, cyclized, oxidized, reduced, decarboxylated, acetylated, acylated, amidated, deamidated, biotinylated, or combined with one or more other small molecules and / or terpenes after obtaining the peptide. Optionally, the liraglutide of the present invention can be labeled, and available labels include one or more small molecule dyes (e.g., Cy dyes such as Cy3, Cy5, Cy5.5, Cy7), Alexa dyes (e.g., Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 647, Alexa Fluor 680, Alexa Fluor 750), VisEn dyes (e.g., VivoTag680, VivoTag750), S dyes (e.g., SO387), DyLight fluorophores (e.g., DyLight 750, DyLight 800), IRDyes (e.g., IRDye 680, IRDye 800), fluorescein dyes (e.g., fluorescein, carboxyfluorescein, fluorescein isothiocyanate (FITC)), rhodamine dyes (e.g., rhodamine, tetramethylrhodamine (TAMRA)), or HOECHST dyes, or one or more quantum dots.

[0226] As described above, according to a preferred embodiment of the method of the present invention, liraglutide is prepared on a resin by SPPS.

[0227] Therefore, another aspect of the present invention relates to precursor peptides conjugated with resin:

[0228] His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-B 2 -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-[resin],

[0229] Among them B 2 Is it Lys (palmitoyl-Glu-OR1) or B? 2 It is Lys(R2-Glu-OR1), where R1 is a carboxylic acid protecting group and R2 is an amino protecting group.

[0230] At least Glu, Asp, and Lys have protecting groups on their side chains, and

[0231] At least one pseudoproline dipeptide is present in Gly 4 -Thr 5 Phe 6 -Thr 7 Thr 7 -Ser 8 Val 10 -Ser 11 or Ser 11 -Ser 12 The location.

[0232] All the definitions listed above also apply to precursor peptides. Those skilled in the art will understand that the invention also includes salts of said precursor peptides, particularly pharmaceutically acceptable salts. In a preferred embodiment, at least one pseudoproline is present in the Gly of the precursor peptide. 4 -Thr 5 Phe 6 -Thr 7 or Thr 7 -Ser 8 The position of [the residue]. Exemplarily, it can be a pseudoproline residue as described in this invention.

[0233] For the purposes of this invention, the term "precursor peptide" can be understood in the broadest sense as a compound that can be converted into the peptide described in this invention, i.e., a glucagon-like peptide, particularly liraglutide. Typically, such a precursor is a product of SPPS with its side chains partially or completely protected and conjugated to a resin, i.e., solid-phase conjugation.

[0234] Furthermore, the present invention provides solid-phase conjugated glucagon-like precursor peptides (particularly liraglutide precursor peptides) wherein at least the side chains of Glu, Asp, and Lys have protecting groups, and wherein at least one O-isoacyl peptide bond is linked to a serine or threonine, and / or at least one pseudoproline is present in Gly 4 -Thr 5 Phe 6 -Thr 7 Thr 7 -Ser 8 Val 10 -Ser 11 or Ser 11 -Ser 12 The position, and / or at least one peptide bond is N-alkylated by 2-hydroxy-4-methoxybenzyl (Hmb) or 2,4-dimethoxybenzyl (Dmb).

[0235] Particularly preferably, the precursor peptide comprises at least one pseudoproline dipeptide selected from Fmoc-Gly-Thr(Psi(Me,Me)pro)-OH, Fmoc-Thr(tBu)-Ser(Psi(Me,Me)pro)-OH, Fmoc-Val-Ser(Psi(Me,Me)pro)-OH, Fmoc-Ser(tBu)-Ser(Psi(Me,Me)pro)-OH, and Fmoc-Phe-Thr(Psi(Me,Me)pro)-OH.

[0236] The present invention further relates to a method for preparing a type I peptide, wherein the peptide is obtained from the above-described resin-conjugated precursor peptide (i.e., a solid-bound, side-chain-protected liraglutide derivative) by incubation with a cleavage composition containing TFA. This incubation can be performed as described above.

[0237] The present invention also relates to liraglutide, which can be obtained from a precursor peptide cleaved from a resin as described above.

[0238] Furthermore, the present invention provides pharmaceutical compositions comprising glucagon-like peptides produced according to the method of the present invention, or glucagon-like peptides derived from the cleavage of solid-phase conjugated glucagon-like peptides of the present invention.

[0239] Therefore, another aspect of the present invention relates to a pharmaceutical composition comprising:

[0240] (A) Liraglutide that can be obtained from the method according to the invention or liraglutide that can be obtained from the precursor peptide of the invention by cleaving its solid phase.

[0241] (B) Pharmaceutically acceptable carriers.

[0242] Pharmaceutically acceptable carriers can be any additive or additive composition known for this purpose. Such additives can be, exemplarily, non-toxic solvents such as water, dimethyl sulfoxide (DMSO), ethanol, vegetable oils, paraffin oils, or combinations thereof. Furthermore, the carrier may contain one or more detergents, one or more foaming agents (e.g., sodium lauryl sulfate (SLS) / sodium diethyl sulfate (SDS)), one or more colorants (e.g., TiO2, food coloring), one or more additional vitamins, one or more salts (e.g., sodium, potassium, calcium, zinc salts), one or more humectants (e.g., sorbitol, glycerin, mannitol, propylene glycol, polydextrose), one or more enzymes, one or more preservatives (e.g., benzoic acid, methyl terephthalic acid), one or more deformants (e.g., carboxymethyl cellulose (CMC), polyethylene glycol (PEG), sorbitol), one or more emulsifiers, one or more fillers, and one or more varnishes. The ingredients include: one or more separating agents, one or more antioxidants, one or more herbal and plant extracts, one or more stabilizers, one or more polymers (e.g., hydroxypropyl methacrylamide (HPMA), polyethyleneimine (PEI), carboxymethyl cellulose (CMC), polyethylene glycol (PEG)), one or more uptake mediators (e.g., polyethyleneimine (PEI), dimethyl sulfoxide (DMSO), cell-penetrating peptides (CPP), protein transduction domains (PTD), antimicrobial peptides, etc.), one or more antibodies, one or more sweeteners (e.g., sucrose, sodium saccharin, stevia)), one or more counterstaining dyes (e.g., fluorescein, fluorescein derivatives, Cy dyes, Alexa Fluor dyes, S dyes, rhodamine, quantum dots, etc.), one or more homeopathic ingredients, one or more taste substances, and / or one or more flavorings.

[0243] The accompanying drawings and embodiments, including the experiments conducted and the results obtained, as well as the claims provided below, are used to describe the present invention. Brief description of the attached diagram

[0245] Figure 1 The sequence alignment of the selected glucagon-like peptide is shown. Parts identical to the GLP-1 sequence are indicated in bold.

[0246] Figure 2The particle size distribution of the peptide precipitate, measured by FBRM technology, is shown (performed on a ParticleTrack G600L, Mettler Toledo, setup: Macro V1.1.11, cubeweight, normalized, mean (cubeweight)). The chord length is the distance per particle calculated based on the scan rate and the number and duration of different pulses of backscattered light. The percentages given are relative to the number of detected particles and are normalized so that the maximum number of particles of a certain size within a single scan is 100%. Here, the precipitate EOP26a (antisolvent: diethyl ether; mean particle size: 261 μm) of Example 5 is compared with the precipitate EOP22a (antisolvent: IPE / ACN; mean particle size: 422 μm) of Example 2. Example

[0247] Example 1:

[0248] Liraglutide was subjected to stepwise Fmoc-SPPS on 100-200 or 200-400 mesh H-Gly-2-chlorotriphenylmethyl resin (Bachem no. 4092098 or 4026823) using the following standard Fmoc amino acid derivatives: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OMpe)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Boc-His(Boc)-OH, and Fmoc-His(1-Trt)-OH. Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Val-OH, as well as the previously mentioned structural unit Fmoc-Lys(Nε-(γ-glutamyl(OtBu)-(Nα-hexadecanoyl))) (i.e. Fmoc-Lys(palmitoyl- -Glu-OtBu)-OH (see WO2013 / 171135), plus at least one pseudoproline dipeptide selected from Fmoc-Gly-Thr(Psi(Me,Me)pro)-OH, Fmoc-Phe-Thr(Psi(Me,Me)pro)-OH, Fmoc-Thr(tBu)-Ser(Psi(Me,Me)pro)-OH, Fmoc-Val-Ser(Psi(Me,Me)pro)-OH, and Fmoc-Ser(tBu)-Ser(Psi(Me,Me)pro)-OH. The coupling reaction employs... TBTU / DIPEA or DEPBT / DIPEA, couple for an appropriate time (1.5-24.0 hours) and then deprotect Fmoc (0.5-4.0 hours).

[0249] When necessary, NMP containing 20% ​​piperidine was used instead of DMF containing 20% ​​piperidine to improve deprotection. Acetylation was optionally performed using acetic anhydride after the coupling step. DMF and IPA were used as solvents for the washing step after acetylation or Fmoc deprotection. Crude liraglutide was typically obtained after lysis and precipitation, with a U-HPLC purity of approximately 50%.

[0250] Alternatively, as described below, a stepwise synthesis protocol can be used, including the use of Fmoc-Lys(Boc-Glu-OtBu)-OH as a structural unit and palmitoylation of the resulting purified peptide.

[0251] Stepwise Fmoc SPPS a fully protected peptide with a primary amino acid sequence:

[0252] His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Boc-Glu-OtBu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-[Resin]

[0253] The synthesis was carried out using H-Gly-2-chlorotriphenylmethyl resin in an automated synthesizer. Various corresponding standard Fmoc amino acid derivatives were used, with… Alternatively, TBTU / DIPEA can be used as the coupling agent and DMF as the solvent for the coupling reaction. As an exception, the coupling of Boc-His(Boc)-OH is carried out using DEPBT / DIPEA in DMF. The coupling times vary, ranging from 1.5 hours to 4 hours. The introduction of the Fmoc-Lys(Boc-Glu-OtBu)-OH structural unit is performed manually. The coupling time was 4 hours, while the remaining synthesis was performed using fully automated peptide synthesis. Fmoc deprotection was carried out using a 20% piperidine DMF solution (v / v). Deprotection time varied between 15 and 90 minutes. After each coupling, a systematic capping step was performed using acetic anhydride.

[0254] Following Fmoc deprotection and acetylation, a washing step was performed using DMF and / or IPA as solvents. The peptide was then cleaved from the solid support for 2.5 hours using a lysis composition of TFA / H2O / EDT (90:5:5, v / v), removing all side-chain protecting groups, followed by precipitation. The crude material was subjected to two-dimensional preparative reversed-phase HPLC using C8-bonded silica as the stationary phase, TEA as the first-dimensional eluent system, and TFA as the second-dimensional eluent system. The purified precursor peptide typically had a U-HPLC purity of 95% and was reacted with N-succinimide palmitate in an aqueous solution containing 66% THF. The product was precipitated by solvent evaporation, dissolved in a 10% ACN / H2O solution of 50% AcOH, and filtered.

[0255] Example 2: Precipitation of glucagon-like peptides from lysed compositions

[0256] General procedure:

[0257] The peptide resin (5 g) was suspended in 50 mL of the lysis composition and stirred at room temperature for 3 hours. The resin was then filtered off and washed with TFA (2 × 3.5 mL). The lysis composition and wash stage were combined and 570 mL of the appropriate antisolvent was added to initiate precipitation. The resulting suspension was stirred for another 2 hours, and the peptide precipitate was separated using a filter funnel or a standard pressure filtration apparatus (“bag filter”). The filtration time and the overall yield based on the peptide resin were determined. The precipitate was washed with the appropriate ether (3 × 15 mL) and dried under vacuum at room temperature to obtain the crude peptide.

[0258] Alternatively, 3 g of peptide resin was suspended in 30 mL of the lysis composition and stirred at room temperature for 3 hours. The resin was then filtered off and washed with TFA (2 × 2.1 mL). The lysis composition and wash fraction were combined and added to 342 mL of antisolvent to initiate precipitation. The resulting suspension was stirred for another 2 hours, and the peptide precipitate was separated using a filter funnel or a standard pressure filtration apparatus (“bag filter”). The filtration time and the overall yield based on the peptide resin were determined. The precipitate was washed with the appropriate ether (3 × 18 mL) and dried under vacuum at room temperature to obtain the crude peptide.

[0259] result:

[0260] The purity and TFA content of the dried peptide precipitate were analyzed by analytical reversed-phase UHPLC, and the aggregate content was determined by analytical size exclusion UHPLC. The appearance and viscosity of the precipitate were assessed by visual inspection.

[0261] As shown in Table 2, precipitation with a mixture of IPE / ACN in the range of 1:3 to 1:10 not only yielded acceptable yields and filtration times, but also improved the purity of the precipitate compared to pure ether antisolvent (compare rows 5-7 and 1-3).

[0262] When the antisolvent is not premixed, but instead the ACN and IPE fractions are separated and subsequently contacted separately with the peptide solution, the purity can be further improved to 63.1% (data not shown). To facilitate further comparison of various precipitation methods, the performance score P is calculated as follows: P = P0 滤波时间 x P 纯度 x P 产量 x P 外观 x P 聚集物含量 Since each item has a score range of 1-3, the maximum possible score is 243, and the minimum score is 1. This quantitative assessment revealed that the IPE / ACN mixture was significantly superior to other tested antisolvents.

[0263] Table 2. Liraglutide precipitation from TFA / water / EDT / TIPS cleavage compositions

[0264]

[0265] Example 3: Composition of the peptide solution

[0266] Further experiments were conducted according to Example 2 above, except that the cleavage composition for preparing liraglutide disclosed in EP-A 2 757 107, with the composition of anisole / anisole / EDT (90:5:3:2, volume ratio), was used. The observed performance score was 108 (3×2×3×3×2), indicating that the beneficial effect of the IPE / ACN antisolvent is not limited to a specific peptide-containing solution.

[0267] Example 4: Precipitation Temperature

[0268] Further experiments were conducted according to Example 2 above to analyze the preferred temperature range for precipitation.

[0269] Table 3. Settling Temperature

[0270]

[0271] Example 5: Comparative Example

[0272] Liraglutide was cleaved from the resin by incubation with TFA / anisole / anisole / EDT (90:5:3:2, volume unspecified) according to the method described in EP-A 2 757 107, and precipitated with ice-cold diethyl ether. The precipitate was analyzed as described in Example 2 above.

[0273] Table 4. Performance comparison of precipitation schemes

[0274] Experiment No. Performance score Yield [%] Filtration time [min] HPLC purity [%] Appearance EOP26a 18 (2x1x3x3x1) 38 22：30 44.89 Fine precipitate

[0275] Representative suspensions obtained from Examples 5 (Experiment EOP26a) and 2 (Experiment EOP22a) were analyzed using a ParticleTrack G600L device via focused beam reflectance measurement (FBRM) technology. FBRM technology can determine the particle size distribution after precipitation based on the detection of backscattered laser light.

[0276] like Figure 2 As shown, the precipitate of the present invention is characterized by a particle size distribution that varies towards larger sizes. The precipitate prepared according to the present invention has an average particle size of 422 μm, while the precipitate prepared according to the scheme of EP-A 2 757 107 has an average particle size of 261 μm. This results in a significant difference in filtration characteristics. The precipitate obtained by the present invention has a much faster filtration rate. Therefore, it can be concluded that the precipitate obtained according to the present invention differs from the precipitates disclosed in the prior art not only in its improved purity and macroscopic properties, but also in its particle size distribution.

[0277] Example 6: Composition of peptide precipitation

[0278] The crude liraglutide precipitate obtained in Example 2 above was analyzed by analytical reversed-phase UHPLC and mass spectrometry to detect truncated liraglutide variants. N-terminal truncated liraglutide variants, particularly acylated liraglutide, were detected in the crude liraglutide precipitate [21-31]. Furthermore, C-terminal truncated variant liraglutide was observed [1-30].

Claims

1. A process for the preparation of liraglutide or a salt thereof, comprising: (i) providing a solution S containing a peptide of formula I: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln- Ala-Ala-B 1 -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly, wherein B 1 is Lys(palmitoyl-Glu-OH) or Lys(H-Glu-OH); (ii) precipitating the peptide of step (i) by mixing solution S with an anti-solvent containing diisopropyl ether and acetonitrile, wherein the anti-solvent containing diisopropyl ether and acetonitrile is a mixture M of 100 vol-% diisopropyl ether and acetonitrile, and the volume ratio of diisopropyl ether: acetonitrile in the mixture M is in the range of 3:1 to 5:1; and (iii) isolating the precipitate obtained in step (ii).

2. The process according to claim 1, wherein step (i) comprises: (i-a) providing a precursor peptide conjugated to a solid phase: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-B 2 -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-[resin], wherein B 2 is Lys (palmitoyl-Glu-OR1) or Lys (R2-Glu-OR1), R1 is a carboxylic acid protecting group, R2 is an amino protecting group; wherein at least the side chains of Glu, Asp, and Lys bear protecting groups; and (i-b) cleaving the precursor peptide from the resin.

3. The process according to claim 2, wherein step (i-a) comprises Fmoc-based solid phase peptide synthesis using a protected amino acid derivative selected from the group consisting of Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OMpe)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Boc-His(Boc)-OH, Fmoc-His(l-Trt)-OH Fmoc-lle-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Val-OH, wherein the protected amino acid derivative is activated by a mixture of one or more coupling agents / additives, which mixture of coupling agents / additives is independently for each step selected from the group consisting of: (A) (Benzotriazolyl)tetramethyluronium tetrafluoroborate (TBTU) / diisopropylethylamine (DIPEA) (B) diisopropylcarbodiimide (DIC) / cyano-hydroxy imino-acetic acid ethyl ester; (C) 3-(Diethoxy-phosphoryloxy)-3H-benzo[d][l,2,3]triazine-4-one (DEPBT) / DIPEA; and (D) DIC / hydroxybenzotriazole (HOBt).

4. The method according to claim 2, wherein one or more pseudoproline dipeptide derivatives are introduced into the position of Gly 4 -Thr 5 , Phe 6 -Thr 7 , Thr 7 -Ser 8 , Val 10 -Ser 11 or Ser 11 -Ser 12 of the peptide of formula I, wherein the one or more pseudoproline dipeptide derivatives used are selected from the group of Fmoc-Gly-Thr(Psi(Me,Me)pro)-OH, Fmoc-Phe-Thr(Psi(Me,Me)pro)-OH, Fmoc-Thr(tBu)-Ser(Psi(Me,Me)pro)-OH, Fmoc-Val-Ser(Psi(Me,Me)pro)-OH, and Fmoc-Ser(tBu)-Ser(Psi(Me,Me)pro)-OH.​​​​​​​​​​ 5. The process according to claim 2, wherein the N-terminal histidine residue is introduced into the precursor peptide conjugated to the solid phase using an amino acid derivative selected from the group consisting of Boc-His(Boc)-OH, Boc-His(l-Trt)-OH, and Fmoc-His(l-Trt)-OH, and employing the coupling agent / additive mixture DEPBT / DIPEA.

6. The method according to claim 3, wherein the mixture selected from the group consisting of N,N-dimethylformamide (DMF) containing 5-50 vol.% piperidine or 4-methylpiperidine, N-methylpyrrolidone (NMP) containing 5-50 vol.% piperidine or 4-methylpiperidine, DMF containing 1-5 vol.% diazabicyclo[5.4.0]undec-7-ene, and DMF containing 50 vol.% morpholine is used to cleave the Fmoc protecting group from the growing peptide chain conjugated to the solid phase.

7. The method according to claim 2, further comprising a step of reacting the activated palmitate with one Lys (H-Glu-OH) of the peptide of formula I or with Lys (H-Glu-OR1) obtainable by cleaving the amino protecting group R2 from the precursor peptide provided in step (i-a).

8. The method according to claim 1 or 2, wherein the solution S obtained from step (i) further comprises trifluoroacetic acid (TFA) and one or more scavengers.

9. The method according to claim 1 or 2, wherein step (ii) comprises mixing an antisolvent with the solution S obtained from step (i) by: (ii-a) pre-mixing diisopropyl ether and acetonitrile and then mixing the pre-mix with the solution S obtained from step (i); (ii-b) first mixing diisopropyl ether with the solution S obtained from step (i) and then mixing acetonitrile with the mixture containing the solution S and diisopropyl ether; or (ii-c) first mixing acetonitrile with the solution S obtained from step (i) and then mixing diisopropyl ether with the mixture comprising the solution S and acetonitrile.

10. The method according to claim 1 or 2, wherein step (ii) is carried out at a temperature of -5 °C to 10 °C and / or step (ii) is carried out using a classical or reverse precipitation protocol.

11. The method according to claim 1 or 2, wherein the separation step of step (iii) is carried out by filtration and / or centrifugation.

12. The method according to claim 7, wherein the activated palmitate is N-succinimidyl palmitate.

13. The method according to claim 8, wherein the scavenger is selected from a thiol scavenger and / or a silane scavenger.

14. The method according to claim 10, wherein step (ii) is carried out at a temperature of 0 °C to 10 °C.

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