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WO2020190757A1 - Improved processes for the preparation of semaglutide - Google Patents

Improved processes for the preparation of semaglutide Download PDF

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Publication number
WO2020190757A1
WO2020190757A1 PCT/US2020/022730 US2020022730W WO2020190757A1 WO 2020190757 A1 WO2020190757 A1 WO 2020190757A1 US 2020022730 W US2020022730 W US 2020022730W WO 2020190757 A1 WO2020190757 A1 WO 2020190757A1
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WO
WIPO (PCT)
Prior art keywords
semaglutide
gly
ser
tbu
impurity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2020/022730
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French (fr)
Inventor
Chaim Eidelman
Sharon PENIAS-NAVON
Luani NAVEH
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Teva Pharmaceutical Industries Ltd
Novetide Ltd
Teva Pharmaceuticals USA Inc
Original Assignee
Teva Pharmaceutical Industries Ltd
Novetide Ltd
Teva Pharmaceuticals USA Inc
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Publication of WO2020190757A1 publication Critical patent/WO2020190757A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Semaglutide is a glucagon-like peptide 1 (GLP-1) receptor agonist indicated to improve glycemic control in adults with type 2 diabetes mellitus, which has the following chemical name and formula:.
  • GLP-1 glucagon-like peptide 1
  • Semaglutide is described in US 8129343.
  • the amino acid backbone of semaglutide is prepared by standard sequential Fmoc-solid phase peptide synthesis followed by deprotection and then by coupling of the side chain fragment to the Lys 20 side chain.
  • the disclosed route has several disadvantages.
  • the sequential synthesis disclosed results in low purity.
  • the coupling of the side chain moiety to Lys side chain in the presence of additional amine group at the N-terminus is not selective enough and results in impurities due to wrong or double attachment of the side chain moiety. This results in the need of additional purification cycles and loss of the yield.
  • US 8129343 further discloses using Dde as protecting group on the Lys. This strategy is disadvantageous because hydrazine, which is a toxic and dangerous reagent, is required for the removal of the Dde group.
  • WO2007090496 discloses a method of synthesizing other GLP-1 peptide agonists, e.g. of formula:
  • WO 2013/098191 discloses use of Fmoc-His-Aib-OH for the preparation of a semaglutide wherein 3-31 amino acid backbone containing the side chain is acylated with Fmoc-His-Aib-OH to afford Semaglutide.
  • His-Aib to complete the synthesis allegedly provides a solution to racemization of His but requires the use of noncommercial and expensive starting materials that should be prepared separately.
  • US 8637647 discloses selective acylation of one amino acid group in a peptide which has two or more reactive nucleophilic functional groups wherein the acylation is performed in an aqueous media in alkaline pH.
  • the acylation step performed at this very late stage of the preparation of semaglutide requires the use of highly pure peptides. However, such use may cause the formation of many impurities which are difficult to remove due to lack of selectivity and the reaction conditions.
  • WO 2016/046753 discloses preparation of semaglutide by a process comprising coupling of fragment 1-4 with fragment 5-31.
  • WO 2016/046753 discloses that the Gly 4 and Gly 16 residues in Semaglutide enable convenient chemical ligation to form the peptide.
  • Such ligation to form the final peptide and peptide fragments and/or sub- fragments at Gly residues is described as advantageous where the final peptide contain a terminal His residue, because coupling reactions with His to form the final peptide, which have a tendency to result in racemization to produce D-His isomer impurity in the final peptide, can be reduced or avoided.
  • the D-His isomers are typically difficult to separate from the final peptide.
  • the convergent processes of the present invention in particular avoid final coupling reactions involving His.
  • CN104356224 discloses preparation of Lys(W) by a stepwise process in solution that entails isolation and purification of the intermediates formed in each step.
  • WO 2016/046753 discloses preparation of Lys(W) by solid phase synthesis wherein catalytic hydrogenation in the presence of metal catalysts was required for removal of the protecting group.
  • J. Med. Chem.2015, 58, 7370-7380 provides a very broad and standard analytical method for analysis of the purity of semaglutide, using C18 RP-HPLC with CAN/TFA as the eluent.
  • Other disclosures that relate to purity and purification methods of semaglutide, such as: CN104356224, CN105777872, CN105753964, CN108640985 and CN110540587 either include a very general analytical method that is not suitable for separation of semaglutide related peptides, or do not provide the analytical methods at all.
  • the present disclosure further provides pure intermediates that may be used for the preparation of highly pure semaglutide.
  • the disclosure provides highly pure semaglutide, wherein the purity of said semaglutide is at least 99.0% or at least 99.2% or at least 99.5% by weight, as measured by HPLC and wherein the content of each peptide-related impurities, is present in an amount of less than 0.3%, or less than 0.25%, or less than 0.20%, or less than 0.15%, or less than 0.10%, as measured by HPLC.
  • the disclosure provides a composition comprising semaglutide and at least one process-related impurity, wherein the purity of said semaglutide is at least 99.0% or at least 99.2% or at least 99.5%, as measured by HPLC; and wherein said process related impurities comprise impurities having epimerized amino acids, impurities characterized by a loss of one or more amino acids, impurities characterized by the presence of one or more additional amino acids, impurities characterized by the presence of one or more different amino acids, impurities characterized by N-terminal deletions, impurities characterized by beta-alanine insertions, structural isomers, and acetylated truncated sequences and impurities characterized by alkylation or acylation of amino acids, wherein each of said process-related impurities is present in an amount of less than 0.3%, or less than 0.25%, or less than 0.20%, or less than 0.15%, or less than 0.10%, as measured by HPLC.
  • the disclosure provides a composition comprising semaglutide and at least one process-related impurity, wherein the purity of said semaglutide is at least 99.5%, as measured by HPLC wherein each of said process-related impurities is present in an amount of less than 0.10%, as measured by HPLC.
  • the invention further comprises a process for purification of semaglutide wherein the process comprises:
  • step (b) comprises:
  • the process further comprises:
  • step (d) optionally subjecting the semaglutide fractions collected from step (c) to reversed phase HPLC on C8 or C18 silica for ion exchange;
  • the buffer is step (a) is selected from the group consisting of (i) an aqueous alkaline buffer solution comprising glycine; (ii) Tris buffer; and (iii) aqueous ammonium hydroxide; wherein the pH is adjusted during dissolution.
  • the disclosure further relates to the above processes wherein the purified semaglutide represents a semaglutide composition comprising semaglutide and at least one process-related impurity, wherein the purity of said semaglutide is at least 99.0% or at least 99.2% or at least 99.5%, as measured by HPLC; and wherein said peptide related impurities comprise impurities having epimerized amino acids, impurities characterized by a loss of one or more amino acids, impurities characterized by the presence of one or more additional amino acids, impurities characterized by the presence of one or more different amino acids, impurities characterized by N-terminal deletions, impurities characterized by beta-alanine insertions, impurity arising from deamidation of glutamine, structural isomers, and acetylated truncated sequences and impurities characterized by alkylation or acylation of amino acids, and wherein each of said process-related impurities is present in an amount of less than 0.3%, or
  • amino acid forming the semaglutide backbone are numbered consecutively from 1-31, starting from the terminal His residue as follows:
  • the present disclosure provides a process for preparing semaglutide which comprises coupling a peptide fragment containing amino acids (1-16) with a peptide fragment containing amino acids (17-31) which carries the W residue, to form Semaglutide.
  • Each one of the above fragments can be prepared on solid support.
  • the coupling process can be performed on solid support or in solution.
  • the process can include further deprotection and cleavage from the resin steps as required.
  • the disclosure further provides an alternative process for preparing semaglutide which comprises coupling a peptide fragment containing amino acids (2-16) with a peptide fragment containing amino acids (17-31) which carries the W residue, to form a third fragment (2-31) which is in turn coupled with protected His to afford Semaglutide.
  • the present disclosure also provides processes for purification of semaglutide.
  • the present disclosure provides a process for preparation of semaglutide comprising:
  • the N-terminus of His is optionally protected with a protecting group; in some embodiments, the protecting group is selected from the group consisting of Boc, Cbz or Fmoc, and - the Gly carboxylic acid group in Peptide 1 may be in the form of an activated carboxylic acid derivative;
  • W1 N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl
  • Peptide 2 is optionally conjugated to a solid support; (e.g., a CTC resin);
  • amino acid residues in Peptide 1 and Peptide 2 and W1 may be protected or unprotected, e.g., protected with acid-cleavable protecting groups;
  • the present disclosure provides a process for preparation of semaglutide comprising:
  • the N-terminal is optionally protected with a protecting group; in some embodiments, the protecting group is selected from the group consisting of, Cbz or Fmoc, and
  • the Gly carboxylic acid group in Peptide 1 may be in the form of an activated carboxylic acid derivative; with a peptide 2 having the sequence:
  • W1 N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl
  • Peptide 2 is optionally conjugated to a solid support; in one aspect, the solid support is a CTC resin);
  • amino acid residues in Peptide 1 and Peptide 2 and W1 may be protected or unprotected; in some aspects, the one or more of the amino acid residues in Peptide 1 and Peptide 2 and W1 are protected (e.g., with acid-cleavable protecting groups to afford peptide 3 having the sequence
  • any one of peptides 1, 1', and 2 are prepared by a sequential synthesis.
  • peptides 1, 1', and 2 are all prepared by sequential synthesis.
  • the present disclosure provides Semaglutide produced by the processes of the present disclosure and highly pure semaglutide obtained by the purification process disclosed herein.
  • Semaglutide and/or highly pure semaglutide produced by the processes of the present disclosure can be used in the preparation of pharmaceutical compositions of Semaglutide.
  • the present disclosure also encompasses the use of the Semaglutide and/or highly pure semaglutide prepared by the processes of the present disclosure for the preparation of pharmaceutical compositions of Semaglutide.
  • the present disclosure comprises processes for preparing the above mentioned pharmaceutical compositions.
  • the processes comprise combining the Semaglutide prepared by the processes of the present disclosure or salts thereof with at least one pharmaceutically acceptable excipient.
  • Semaglutide prepared by the processes of the present disclosure and the pharmaceutical compositions of Semaglutide prepared by the processes of the present disclosure can be used as medicaments, particularly to improve glycemic control in adults with type 2 diabetes mellitus
  • the present disclosure also provides methods to improve glycemic control in adults with type 2 diabetes mellitus, comprising administering a therapeutically effective amount of Semaglutide prepared by the processes of the present disclosure, or at least one of the above pharmaceutical compositions, to a subject in need of the treatment.
  • BRIEF DESCRIPTION OF THE FIGURES [0042]
  • Figure 1 is an HPLC chromatogram of W1 obtained according to to Example 3, step C.
  • Figure 3 is an HPLC chromatogram of semaglutide obtained according to Example 6.
  • Figure 4 is an HPLC chromatogram of semaglutide obtained according to Example 7.
  • Figure 5 is an HPLC chromatogram of semaglutide obtained according to Example 8.
  • Figure 6 is an HPLC chromatogram of semaglutide obtained according to Example 9. DETAILED DESCRIPTION
  • the present disclosure provides new processes for the preparation and purification of Semaglutide and semaglutide intermediates.
  • the process of the present invention provides highly pure semaglutide that is afforded in a process that combines use of highly pure W1 and Fmoc-Lys(W1)-OH intermediates and involves a novel and unique purification method of semaglutide.
  • the processes disclosed herein provide an efficient route which does not require the use of toxic or otherwise undesirable reagents and provides Semaglutide in high yield and quality, and that can be utilized in industrial scale.
  • the present disclosure provides the preparation of the highly pure intermediates W1 and Fmoc-Lys(W1)-OH and use thereof for preparation of highly pure semaglutide.
  • the disclosure also provides unique purification processes that comprises three purification steps that combine two purification steps with basic pH mobile phase. and that comprise one purification step using an acidic pH mobile phase and utilize phenyl- hexyl RP silica and C8 or C18 silica, thereby providing highly pure semaglutide
  • the semaglutide produced by the processes disclosed herein and purified by the processes disclosed herein displays high chemical purity of more than 99% and contains less than 0.20%, or less than 0.10% of any process related impurity, as measured by HPLC.
  • J. Med. Chem.2015, 58, 7370-7380 provides a very broad and standard analytical method for analysis of the purity of semaglutide, using C18 RP- HPLC with ACN/TFA as the eluent.
  • CN105753964, CN108640985 and CN110540587 either also include a very general analytical method that is not suitable for separation of semaglutide related peptides or does not provide the analytical methods at all.
  • the present disclosure provides improved analytical procedures for evaluating the purity of semaglutide samples. These analytical procedures allow detecting the presence of and/or measuring the relative amount of semaglutide related peptides.
  • EEDQ (2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline)
  • HAPyU 1-(1-pyrrolidinyl-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene) pyrrolidinmium hexafluorophosphate
  • PfPU pentafluorophenol-tetramethyluronium hexafluorophosphate PfTU pentafluorophenyl-1,1,3,3-bis(tetramethylene)uranium
  • the solid supports for the processes of the present invention can be resins that are cleavable using acid.
  • the acid used to cleaved the resin is trifluoroacetic acid.
  • the resin for use in the disclosed processes can be Wang resins and hyper- acid labile resins.
  • the hyper-acid labile resins can be a chlorotrityl based (CTC) resin, 4-methoxytrityl resin, or 4-methyl-trityl resin.
  • the hyper-acid labile resin is a CTC resin.
  • Hyper-acid labile resins such as CTC resins are cleavable under milder acidic conditions.
  • hyper-acid labile resins such as CTC resins can be removed using weak acid solutions, such as 2% trifluoroacetic acid.
  • the term “Wang resin” typically refers to a polyethylene-based resin, which can contain p- alkoxybenzyl alcohol or p-alkoxybenzyloxycarbonyhydrazide based resins, typically attached to a polyethylene glycol or polystyrene core (Wang, S., J. Am. Chem. Soc., 1973, 95(4), 1328-1333). Wang resins are typically removed under strong acid conditions, e.g.
  • trifluoroacetic acid solutions At least 50% trifluoroacetic acid solutions.
  • Wang and CTC resins suitable for the processes disclosed herein are those on a polystyrene support. These resins are commercially available.
  • H-Gly-Wang resin or H-Gly-CTC resin, or the free resins themselves are commercially available and are suitable starting materials for use in the processes disclosed herein.
  • the term "sequential synthesis” or“linear synthesis” refers to a process whereby the final product or an intermediate thereof is prepared by sequential transformations of a single starting material.
  • the final product is prepared by sequential condensation of single amino acids (pseudoproline dipeptide may also be used) to build the final peptide sequence.
  • the single amino acids are optionally side-chain protected as well as N-terminal protected with the usual protecting groups for peptide synthesis.
  • the N- terminal protecting groups are Fmoc, Boc, or Cbz.
  • the N-terminal protecting group is Fmoc or Boc.
  • the condensation(s) can be carried out as a solid phase synthesis (i.e. on a solid support, such as a resin) or in liquid phase (i.e. with the free peptide– i.e. a peptide that is not conjugated to a solid support/resin), or a combination of both.
  • the term“peptide” refers to a compound containing at least two amino acids in which the carboxyl group of one acid is linked to the amino group of the other (i.e. the two amino acids are linked by a peptide bond).
  • the term“peptide” as used herein encompasses amino acid sequences in which carboxyl and/or amino groups are protected or unprotected. Suitable protecting groups for the carboxyl groups of the amino acids include OtBu, OBzl, OFm. Suitable protecting groups for the amino groups of the amino acids include Fmoc, Boc, Mmt, Mtt, Cbz, Trt. Suitable protecting groups for the N- terminal amino acid include Fmoc, Boc and Cbz.
  • the amino acid or peptide fragment is coupled using Fmoc, Boc, or Cbz strategy which is well known in the art of peptide synthesis.
  • the typically side-chain protected amino acid or peptide fragment to be coupled onto another amino acid or peptide fragment is generally also N-terminal protected with Fmoc, Boc or Cbz to form a peptide or peptide fragment containing an N-terminal Fmoc, Boc or Cbz group.
  • the N-terminal protecting group is Fmoc or Boc.
  • the N-terminal protecting group is Fmoc.
  • the N-terminal protection of the peptide formed in the preceding coupling step is removed, for example by reaction with, e.g. a base such as piperidine in the case of Fmoc, or an acid, such as TFA (trifluoroacetic acid) in the case of Boc, before the next amino acid or peptide is coupled.
  • a base such as piperidine in the case of Fmoc
  • an acid such as TFA (trifluoroacetic acid) in the case of Boc
  • the coupling is carried out with Fmoc strategy using peptide fragments containing amino acid side chain protecting groups which are only acid-cleavable (i.e. are stable to the basic conditions that are generally employed to remove the base-cleavable N-terminal protecting groups), and the removal of the N-terminal protection (e.g. Fmoc) is conducted with a base.
  • the His residue of Peptide 1 contains an acid-labile N-terminal protecting group.
  • the N-terminal protecting group and the amino acid protecting groups in the protected semaglutide sequence can be removed (optionally along with any solid support, e.g. Wang resin) in one step.
  • the His residue of Peptide 1 can be Boc. If the His residue of Peptide 1 is Boc, the His N-terminal Boc group may be removed together with the acid-labile protecting groups and Wang resin by treatment with a cleavage cocktail (typically a cleavage cocktail comprising trifluoroacetic acid (TFA), and can be a mixture of TFA with dithiothreitol or other scavengers), thereby producing semaglutide.
  • a cleavage cocktail typically a cleavage cocktail comprising trifluoroacetic acid (TFA), and can be a mixture of TFA with dithiothreitol or other scavengers
  • semaglutide is prepared by liquid phase coupling, i.e. wherein a resin is not employed.
  • a resin e.g. CTC resin
  • the final coupling reaction of Peptide 1 with Peptide 2 is conducted in the liquid phase.
  • CTC resin is particularly suitable for such a process because this resin can be cleaved under mild conditions, such as dilute TFA solution (e.g. £ 10%, £ 5%, £ 2% vol/vol in a suitable organic solvent such as dichloromethane. These conditions leave most of the other acid cleavable amino acid protecting groups intact.
  • the coupling of Peptide 1 with Peptide 2 may be conducted as a solid phase synthesis, whereby Peptide 2 is conjugated to a solid support, which can be an acid cleavable resin.
  • the acid cleavable resin is a polystyrene-based resin.
  • the acid cleavable resin is a Wang resin or a CTC resin.
  • Peptide 2 is conjugated to a Wang resin or CTC resin.
  • the Gly carboxylic acid in Peptide 1 need not be preactivated by derivatisation into an activated carboxylic acid group (i.e. in the form of an isolated activated ester).
  • the coupling may be conducted in the presence of a coupling agent (i.e. in situ activation), such as those typically employed in peptide coupling reactions.
  • the coupling agents include BOP, AOP, PyBOP, PyAOP, HBTU, HATU, HCTU, HBPyU, HAPyU, TFFH, TBTU, BTFFH, EDC-HCl, PyBrop, DPPA, BOP-Cl, DCC, DIC, DEPC, EEDQ, IIDQ, CIP, PfTU, PfPU, BroP and CDI.
  • the coupling agent is TBTU and DIC (e.g. DIC/HOBt).
  • the term“segment” or“fragment” of semaglutide refers to a sequence of two or more amino acids present in semaglutide.
  • the amino acids in the segment or fragment may be protected or unprotected.
  • the amino acids in the fragments are protected.
  • the fragments are protected with acid-cleavable protecting groups.
  • the trifunctional amino acids namely: Thr, Ser, Asp, Tyr, Glu, Gln, Trp, His and Arg residues are protected with acid-cleavable protecting groups.
  • Suitable acid cleavable protecting groups are selected from the group consisting of: tBu, OtBu, Y Me,Me pro, Trt, and Pbf.
  • residues are protected as follows: Thr(tBu), Ser8(tBu), Ser8(Trt), Ser11(tBu), Ser11(Trt), Asp (OtBu), Ser12(YMe,Mepro), Ser12(Trt), Tyr(tBu), Glu(OtBu), Gln(Trt), Trp(Boc), His(Trt), His(Boc), and Arg(Pbf).
  • Glu 17 impurity of Semaglutide refers to semaglutide which contains a Glu residue at position 17 instead of Gln 17 i.e. H-His-Aib-Glu-Gly-Thr- Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Glu-Ala-Ala-Lys(W)-Glu-Phe-Ile-Ala- Trp-Leu-Val-Arg-Gly-Arg-Gly-Gly-OH (SEQ ID NO: 19).
  • b-Asp 9 impurity of Semaglutide refers to semaglutide which contains a b-Asp residue at position 9 instead of Asp 9 i.e. H-His-Aib-Glu-Gly-Thr- Phe-Thr-Ser-b-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Glu-Ala-Ala-Lys(W)-Glu-Phe-Ile- Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-Gly-OH (SEQ ID NO: 20).
  • the term "des-His impurity of Semaglutide” refers to a semaglutide which lacks the terminal His residue, i.e. H-Aib-Glu-Gly-Thr-Phe-Thr-Ser- Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(W)-Glu-Phe-Ile-Ala-Trp-Leu-Val- Arg-Gly-Arg-Gly-Gly-Gly-OH (SEQ ID NO: 21).
  • the term "des-His-Aib impurity of Semaglutide” refers to a semaglutide which lacks the terminal His-Aib residue, i.e. H-Glu-Gly-Thr-Phe-Thr-Ser- Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(W)-Glu-Phe-Ile-Ala-Trp-Leu-Val- Arg-Gly-Arg-Gly-Gly-Gly-OH (SEQ ID NO: 22).
  • peptide related impurities peptide related impurities comprise impurities having epimerized amino acids, impurities characterized by a loss of one or more amino acids, impurities characterized by the presence of one or more additional amino acids, impurities characterized by the presence of one or more different amino acids, impurities characterized by N-terminal deletions, impurities characterized by beta-alanine insertions, structural isomers, and acetylated truncated sequences and impurities characterized by alkylation or acylation of amino acids.
  • impurities may also form as a result of degradation processes.
  • acetylated truncated sequences refer to the products of N a acetylation.
  • HPLC analysis is carried out using a reversed phase silica gel column (e.g. Halo C8 column) using UV detection at 215 nm depending on the impurities.
  • the HPLC analytical method is designed to use UV absorption at a given wavelength for recording the presence and the amount of a compound in a sample passing the detector at any given point in time.
  • the primary output of any HPLC run with standard equipment will be an area percentage of the respective peak in the UV detection chromatogram.
  • residue“H-His” denotes that the terminal His residue (i.e. at amino acid position 1 of semaglutide) does not contain an N- terminal protecting group
  • “Boc-His” refers to a His residue which is protected at the N-terminal group with Boc.
  • “H- AA” refers to a terminal amino acid (AA) residue that does not contain an N-terminal protecting group.
  • residue“Gly-OH” denotes that the carboxylic acid group of the Gly residue is unsubstituted, and thus contains a free–OH group
  • “Gly-OtBu” refers to a Gly residue in which the carboxylic acid OH group is substituted to form OtBu
  • Gly-O-resin refers to a terminal Gly residue which is attached to a solid support (e.g. Gly-O-Wang resin, or Gly-O-CTC resin).
  • the term“AA-OH” may also be specified to refer to a terminal amino acid residue that is either optionally conjugated to a resin via the carboxylic acid terminal group or optionally the amino acid contains a carboxylic acid terminal group in activated form such as e.g. OSu.
  • a thing e.g., a reaction mixture
  • room temperature often abbreviated "RT.” This means that the temperature of the thing is close to, or the same as, that of the space, e.g., the room or fume hood, in which the thing is located.
  • room temperature is from about 20°C to about 30°C, or about 22°C to about 27°C, or about 25°C.
  • the processes or steps can be referred to herein as being carried out “overnight.” This refers to time intervals, e.g., for the processes or steps, that span the time during the night, when the processes or steps may not be actively observed.
  • the time intervals are from about 8 to about 20 hours, or about 10 to about 18 hours, or about 16 hours.
  • reduced pressure refers to a pressure of about 10 mbar to about 500 mbar, or about 50 mbar.
  • chlorinated solvent refers to a C1-C6 chlorinated hydrocarbon.
  • the chlorinated solvents are selected from the group consisting of, dichloromethane (CH 2 Cl 2 ), dichloroethane and chlorobenzene.
  • one pot process refers to continues process for preparing a desired product, in which penultimate product is converted to the desired product in the same vessel.
  • Protecting group refers to a grouping of atoms that when attached to a reactive functional group in a molecule masks, reduces or prevents reactivity of the functional group. Examples of protecting groups can be found in Greene and Wuts“Greene’s Protective Groups in Organic Synthesis”, 4th Edition, publ. Wiley, 2006 and Harrison et al., "Compendium of Synthetic Organic Methods", Vols.1-8 (John Wiley and Sons, 1971-1996).
  • Representative amine protecting groups include, but are not limited to, those where the amine group is converted to carbamate or amide such as Fmoc, cbz, benzyl, trityl, Boc, trifluoroacetyl derivative, phthalic anhydride, or succinic anhydride derivative.
  • Representative hydroxyl protecting groups include, but are not limited to, those where the hydroxyl group is converted to ethers, esters, carbonates, siloxanes, or acetals.
  • the amount of solvent employed in chemical processes, e.g., reactions or crystallizations, may be referred to herein as a number of "volumes” or “vol” or “V.”
  • a material may be referred to as being suspended in 10 volumes (or 10 vol or 10V) of a solvent.
  • this expression would be understood to mean milliliters of the solvent per gram of the material being suspended, such that suspending a 5 grams of a material in 10 volumes of a solvent means that the solvent is used in an amount of 10 milliliters of the solvent per gram of the material that is being suspended or, in this example, 50 mL of the solvent.
  • v/v can be used to indicate the number of volumes of a solvent that are added to a liquid mixture based on the volume of that mixture. For example, adding MTBE (1.5 v/v) to a 100 ml reaction mixture would indicate that 150 mL of MTBE was added.
  • the present disclosure provides new processes for the preparation and purification of Semaglutide and semaglutide intermediates.
  • the present disclosure further provides highly pure semaglutide and processes for preparation thereof.
  • the present disclosure further provides pure intermediates that may be used for the preparation of highly pure semaglutide.
  • the disclosure provides highly pure semaglutide, wherein the purity of said semaglutide is at least 99.0% or at least 99.2%, as measured by HPLC and wherein the content of each peptide-related impurities, is present in an amount of less than 0.3%, or less than 0.25%, or less than 0.20%, or less than 0.15%, or less than 0.10%, as measured by HPLC.
  • the disclosure provides a composition comprising semaglutide and at least one process-related impurity, wherein the purity of said semaglutide is at least 99.0% or at least 99.2% or at least 99.5%, as measured by HPLC; and wherein said peptide related impurities comprise impurities having epimerized amino acids, impurities characterized by a loss of one or more amino acids, impurities characterized by the presence of one or more additional amino acids, impurities characterized by the presence of one or more different amino acids, impurities characterized by N-terminal deletions, impurities characterized by beta-alanine insertions, structural isomers, and acetylated truncated sequences and impurities characterized by alkylation or acylation of amino acids, wherein each of said process-related impurities is present in an amount of less than 0.3%, or less than 0.25%, or less than 0.20%, or less than 0.15%, or less than 0.10%, as measured by HPLC
  • the disclosure relates to the above composition, wherein the at least one peptide-related impurity is selected from:
  • the disclosure is directed to a process for purification of semaglutide wherein the process comprises:
  • step (b) comprises:
  • the process further comprises:
  • step (d) optionally subjecting the semaglutide fractions collected from step (c) to reversed phase HPLC on C8 or C18 silica for ion exchange;
  • the buffer is step (a) is selected from the group consisting of (i) an aqueous alkaline buffer solution comprising glycine; (b) Tris buffer; and (iii) aqueous ammonium hydroxide solution; wherein the pH is adjusted during dissolution.
  • steps (b1) and (b2) can be carried out in the reversed order.
  • steps (b1) and (b2) and (c) is interchangeable.
  • step (b1) comprises elution using a mobile phase A which comprises water and optionally a chemical modifier and a mobile phase B which comprises a polar organic solvent selected from the group consisting of ethanol, isopropanol, acetonitrile, and mixtures thereof.
  • a mobile phase A which comprises water and optionally a chemical modifier
  • a mobile phase B which comprises a polar organic solvent selected from the group consisting of ethanol, isopropanol, acetonitrile, and mixtures thereof.
  • step (b2) comprises elution using a mobile phase C which comprises water and optionally a chemical modifier and a mobile phase D which comprises a polar organic solvent selected from the group consisting of ethanol, isopropanol, acetonitrile, and mixtures thereof.
  • a mobile phase C which comprises water and optionally a chemical modifier
  • a mobile phase D which comprises a polar organic solvent selected from the group consisting of ethanol, isopropanol, acetonitrile, and mixtures thereof.
  • step (c) comprises using a mobile phase A which comprises water and optionally a chemical modifier and a mobile phase B which comprises a polar organic solvent selected from the group consisting of ethanol, isopropanol, acetonitrile, and mixtures thereof.
  • a mobile phase A which comprises water and optionally a chemical modifier
  • a mobile phase B which comprises a polar organic solvent selected from the group consisting of ethanol, isopropanol, acetonitrile, and mixtures thereof.
  • the chemical modifier in mobile phase A is an ammonium salt or a sodium salt or a combination thereof.
  • the chemical modifier is selected from the group consisting of: ammonium chloride, ammonium acetate, ammonium bicarbonate, ammonium phosphate, ammonium sulfate, ammonium hydroxide, and combinations thereof.
  • the chemical modifier is ammonium chloride.
  • the chemical modifier is present in mobile phase A in a concentration of about 0.001 to about 1.0M. In some embodiments, the chemical modifier is present in mobile phase A in a concentration of about 0.002M to about 0.5M. In some embodiments, the chemical modifier is present in mobile phase A in a concentration of about 0.005M to about 0.1M. In some embodiments, the chemical modifier is present in mobile phase A in a concentration of about 0.01M to about 0.05M. In some embodiments, the chemical modifier is present in mobile phase A in a concentration of about 0.02M. [0118] In some embodiments, the pH of mobile phase A is about 5.5 to about 11.5. In some embodiments, the pH of mobile phase A is about 6.0 to about 11.0. In some embodiments, the pH of mobile phase A is about 7.0 to about 9.5. In some embodiments, the pH of mobile phase A is about 8.5.
  • the chemical modifier in mobile phase C is a strong acid.
  • the chemical modifier is TFA, HCl, H2SO4, perchloric acid, phosphoric acid, or a combination thereof. In some embodiments, the chemical modifier is TFA.
  • the chemical modifier in mobile phase C is present in a concentration of about 0.05% to about 0.3%. In some embodiments, the chemical modifier in mobile phase C is present in a concentration of about 0.1 to about 0.2%. In some embodiments, the chemical modifier in mobile phase C is present in a concentration of about 0.15%.
  • the chemical modifier in mobile phase E comprises acetic acid, ammonium hydroxide, or a volatile ammonium salt that is ammonium bicarbonate or ammonium acetate. In some embodiments, the chemical modifier in mobile phase E is ammonium hydroxide or an ammonium salt. In some embodiments, the chemical modifier in mobile phase E is ammonium bicarbonate, ammonium acetate, or acetic acid.
  • acetonitrile is the sole component of mobile phases B, D and F.
  • the purified semaglutide represents a semaglutide composition as defined above.
  • collected semaglutide fractions represent a semaglutide composition and are prepared by a process, comprising:
  • step (b1) subjecting the solution in step (a) to reversed-phase HPLC on a phenyl hexyl silica column using a gradient of about 0.02M ammonium chloride aqueous solution and acetonitrile, collecting the Semaglutide fractions; and optionally conducting additional purification cycles under the same conditions;
  • step (b2) subjecting the fractions collected in step (b) to reversed-phase HPLC on a phenyl hexyl silica using a gradient elution of about 0.15% TFA aqueous solution and acetonitrile, collecting the Semaglutide fractions; and optionally conducting additional purification cycles under the same conditions;
  • step (c) subjecting the fractions collected in step (c) to a C8 or C18 reversed phase HPLC column using a gradient of about 0.02M ammonium chloride aqueous solution and acetonitrile, collecting the Semaglutide fractions; and optionally conducting additional purification cycles under the same conditions;
  • step (d) subjecting the fractions collected in step (d) to a C8 or C18 reversed phase HPLC column for ion exchange and collecting the semaglutide fractions;
  • step (e) the salt is washed out and semaglutide is eluted with about 0.02M ammonium hydroxide aqueous solution and acetonitrile.
  • the combined semaglutide fractions can be concentrated in order to produce a purified semaglutide concentrate.
  • This purified semaglutide concentrate can be directly used to prepare a dry semaglutide product which is suitable for preparing a pharmaceutical composition.
  • the purified Semaglutide concentrate can be dried by any suitable process, especially processes which enable a rapid removal of water at low temperature, such as by spray drying, or lyophilization.
  • the drying step (f) comprises lyophilization.
  • the above described purification process for semaglutide is especially useful for purifying semaglutide obtained by chemical peptide synthesis techniques.
  • the crude semaglutide is obtained from a solid-phase or liquid phase peptide synthesis.
  • the crude semaglutide from such a synthesis can be treated before the HPLC steps, wherein the treatment comprises stirring the crude semaglutide with an aqueous alkaline buffer solution at a pH of about 8 to about 12.
  • the aqueous alkaline buffer solution has a pH of about 8 to about 11.
  • the aqueous alkaline buffer solution has a pH of about 8.5 to about 9.0.
  • ammonium hydroxide solution can be used for dissolution of semaglutide, while adjusting the pH to the above range.
  • a suitable aqueous alkaline buffer solution comprises aqueous glycine.
  • the buffer concentration can be about 1.5 M to about 0.01 M.
  • the buffer concentration can be about 1.3 M to about 0.1 M.
  • the buffer concentration can be about 1.2 M to about 0.8 M.
  • Tris buffer can also be used.
  • Tris buffer can be used at a concentration of about 0.01 to about 1 M.
  • Tris buffer can be used at a concentration of about 0.05 to about 0.5 M.
  • Tris buffer can be used at a concentration of about 0.1 to about 0.3 M.
  • the disclosure further relates to the above processes wherein the purified semaglutide represents a semaglutide composition comprising semaglutide and at least one process-related impurity, wherein the purity of said semaglutide is at least 99.0% or at least 99.2% or at least 99.5%, as measured by HPLC; and wherein said peptide related impurities comprise impurities having epimerized amino acids, impurities characterized by a loss of one or more amino acids, impurities characterized by the presence of one or more additional amino acids, impurities characterized by the presence of one or more different amino acids, impurities characterized by N-terminal deletions, impurities characterized by beta-alanine insertions, impurity arising from deamidation of glutamine, structural isomers, and acetylated truncated sequences and impurities characterized by alkylation or acylation of amino acids, and wherein each of the process-related impurities is present in an amount of less than 0.3%, or
  • kits for detecting and quantifying the presence of semaglutide process-related related impurities in a semaglutide sample can be used in order to determine whether a semaglutide sample meets FDA requirements with respect to the synthetic semaglutide purity.
  • the present disclosure provides a novel process for preparation of Semaglutide.
  • the present invention provides a process for preparing semaglutide which comprises coupling a peptide fragment containing amino acids (1-16) with a peptide fragment containing amino acids (17-31) which carries the W residue, to form Semaglutide.
  • each one of the above fragments can be prepared on solid support.
  • the coupling process can be performed on solid support or in solution.
  • the process can include further deprotection and cleavage from the resin steps as required.
  • the N-terminal of His is optionally protected with a protecting group (in some embodiments, the protecting group is selected from the group consisting of Boc, Cbz or Fmoc), and
  • the Gly carboxylic acid group in Peptide 1 may be in the form of an activated carboxylic acid derivative; with a peptide 2 having the sequence:
  • W1 N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl
  • Peptide 2 is optionally conjugated to a solid support; (e.g., a CTC resin);
  • amino acid residues in Peptide 1 and Peptide 2 and W1 may be protected or unprotected, e.g., protected with acid-cleavable protecting groups; (ii) optionally removing any protecting groups and/or cleaving the resin to form Semaglutide;
  • peptide 2 is not conjugated to the solid support and the coupling of peptide 2 with peptide 1 is performed in solution.
  • the present disclosure relates to a process for preparation of semaglutide comprising:
  • N-terminal is optionally protected with a protecting group (e.g., Boc, Cbz or Fmoc), and
  • the Gly carboxylic acid group in Peptide 1 may be in the form of an activated carboxylic acid derivative; with a peptide 2 having the sequence:
  • W1 N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl
  • Peptide 2 is optionally conjugated to a solid support; (e.g., a CTC resin);
  • amino acid residues in Peptide 1 and Peptide 2 and W1 can be protected or unprotected, e.g., with acid-cleavable protecting groups to afford peptide 3 having the sequence:
  • any one of peptides 1, 1', and 2 are prepared by a sequential synthesis.
  • peptides 1, 1', and 2 are all prepared by sequential synthesis.
  • Peptide 1 has the formula:
  • P1 represents a protecting group for the N-terminal of His, such as Boc, Fmoc, and Cbz.
  • P2 represents side chain protecting groups which can be the same or different and P2 is selected from: H, or a solid support (e.g., a CTC resin), or P2 represents an activated carboxylic ester of the Gly 4 residue (e.g., Su or Bt or Pfp).
  • Peptide 1 is selected from the group consisting of:
  • Peptide 1 is selected from the group consisting of:
  • Peptide 1' has the formula:
  • P1 represents a protecting group for the N-terminal, e.g., Boc, Fmoc, or Cbz; each P represents side chain protecting groups which may be the same or different and P2 is selected from: H, or a solid support (e.g., a CTC resin), or P2 represents an activated carboxylic ester of the Gly 16 residue (e.g., Su or Bt or Pfp).
  • peptide 1' is selected from the group consisting of:
  • Peptide 1' is selected from the group consisting of:
  • Peptide 2 has the formula:
  • W1 N-(17-carboxy(P)-1-oxoheptadecyl)-L-g-glutamyl(P)-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl ,
  • P1 represents H, or a protecting group for the N-terminal of Gln (e.g., Fmoc or Cbz), each P represents side chain protecting groups which may be the same or different, and P3 is selected from H, or a solid support, e.g., a CTC or Wang resin and P4 is selected from H or a protecting group.
  • peptide 2 has the formula:
  • P1 represents H, or a protecting group for the N-terminal of Gln (e.g., Fmoc or Cbz), each P represents side chain protecting groups which may be the same or different, and P3 is selected from H, or a solid support, e.g., a CTC or Wang resin.
  • a protecting group for the N-terminal of Gln e.g., Fmoc or Cbz
  • each P represents side chain protecting groups which may be the same or different
  • P3 is selected from H, or a solid support, e.g., a CTC or Wang resin.
  • peptide 2 has the formula:
  • W1 N-(17-carboxy(P)-1-oxoheptadecyl)-L-g-glutamyl(P)-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl
  • P1 represents H, or a protecting group for the N-terminal of Gln (e.g., Fmoc or Cbz).
  • a process for preparation of semaglutide comprising:
  • N-terminal of His is optionally protected with a protecting group selected from the group consisting of Boc, Cbz or Fmoc, and
  • the Gly carboxylic acid group in Peptide 1 may be in the form of an activated carboxylic acid derivative; with a peptide 2 having the sequence:
  • W1 N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl ,
  • Peptide 2 is optionally conjugated to a solid support; (e.g., a CTC resin);
  • amino acid residues in Peptide 1 and Peptide 2 and W1 may be protected or unprotected, e.g., protected with acid-cleavable protecting groups; (ii) optionally removing any protecting groups and/or cleaving the resin to form Semaglutide;
  • P1 represents a protecting group for the N-terminal of His
  • each P represents side chain protecting groups which may be the same or different and wherein P2 is selected from: H, or P2 represents an activated carboxylic ester of the Gly 16 residue, with a Peptide 2 having the formula:
  • W1 N-(17-carboxy(P)-1-oxoheptadecyl)-L-g-glutamyl(P)-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl,
  • P3 is selected from H, or a solid support, e.g., a CTC or Wang resin and wherein P4 is selected from H or a protecting group.
  • P2 is H or P2 represents an activated carboxylic ester of the Gly 16 residue.
  • Peptide 1 is selected from the group consisting of: Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(y Me,Me Pro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (SEQ ID NO: 24),
  • A7 The process according to any one of paragraphs A1-A3 wherein the coupling of peptide 2 with peptide 1 is conducted in the solid phase, in the presence of a coupling agent.
  • A8 The process according to paragraph A7 wherein coupling agent is selected from the list consisting of BOP, AOP, PyBOP, PyAOP, HBTU, HATU, HCTU, HBPyU, HAPyU, TFFH, TBTU, BTFFH, EDC-HCl, PyBrop, DPPA, BOP-Cl, DCC, DIC, DEPC, EEDQ, IIDQ, CIP, PfTU, PfPU, BroP and CDI.
  • coupling agent is selected from the list consisting of BOP, AOP, PyBOP, PyAOP, HBTU, HATU, HCTU, HBPyU, HAPyU, TFFH, TBTU, BTFFH, EDC-HCl, PyBrop, DPPA, BOP-Cl, DCC, DIC, DEPC, EEDQ, IIDQ, CIP, PfTU, PfPU, BroP and CDI.
  • a process for preparation of semaglutide comprising:
  • N-terminus is optionally protected with a protecting group (e.g., Boc, Cbz or Fmoc), and
  • the Gly carboxylic acid group in Peptide 1 may be in the form of an activated carboxylic acid derivative; with a peptide 2 having the sequence:
  • W1 N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl
  • Peptide 2 is optionally conjugated to a solid support; (e.g., a CTC resin);
  • amino acid residues in Peptide 1 and Peptide 2 and W1 may be protected or unprotected, e.g., protected with acid-cleavable protecting groups to afford peptide 3 having the sequence: Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala- Lys(W1)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (SEQ ID NO: 6),
  • P1 represents a protecting group for the N-terminal of Aib
  • each P represents side chain protecting groups which may be the same or different and wherein P2 is selected from: H, or P2 represents an activated carboxylic ester of the Gly 16 residue, with a Peptide 2 having the formula:
  • W1 N-(17-carboxy(P)-1-oxoheptadecyl)-L-g-glutamyl(P)-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl
  • P3 is selected from H, or a solid support, e.g., a CTC or Wang resin and wherein P4 is selected from H or a protecting group.
  • P1-Aib-Glu(P)-Gly-Thr(P)-Phe-Thr(P)-Ser(P)-Asp(P)-Val-Ser(P)-Ser(P)-Tyr(P)- Leu-Glu(P)-Gly-O-P2 (SEQ ID NO: 36), wherein P1 represents a protecting group for the N-terminal, e.g., Fmoc or Cbz; each P represents side chain protecting groups which may be the same or different and P2 is selected from: H, or P2 represents an activated carboxylic ester of the Gly 16 residue.
  • P1 represents a protecting group for the N-terminal, e.g., Fmoc and Cbz; each P represents side chain protecting groups which may be the same or different and P2 is selected from: H, or P2 represents an activated carboxylic ester of the Gly 16 residue (e.g., Su or Bt or Pfp)
  • Peptide 1' is selected from the group consisting of: Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(y Me,Me Pro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (SEQ ID NO: 37),
  • W1 N-(17-carboxy(P)-1-oxoheptadecyl)-L-g-glutamyl(P)-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl,
  • P1 represents H, or a protecting group for the N-terminal of Gln (e.g., Fmoc or Cbz), each P represents side chain protecting groups which may be the same or different, P3 is selected from H, or a solid support, e.g., a CTC or Wang resin and wherein P4 is selected from H or a protecting group.
  • a protecting group for the N-terminal of Gln e.g., Fmoc or Cbz
  • each P represents side chain protecting groups which may be the same or different
  • P3 is selected from H, or a solid support, e.g., a CTC or Wang resin and wherein P4 is selected from H or a protecting group.
  • W1 N-(17-carboxy(P)-1-oxoheptadecyl)-L-g-glutamyl(P)-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl,
  • P1 represents H, or a protecting group for the N-terminal of Gln (e.g., Fmoc or Cbz), each P represents side chain protecting groups which may be the same or different, and P3 is selected from H, or a solid support, e.g., a CTC or Wang resin.
  • a protecting group for the N-terminal of Gln e.g., Fmoc or Cbz
  • each P represents side chain protecting groups which may be the same or different
  • P3 is selected from H, or a solid support, e.g., a CTC or Wang resin.
  • W1 N-(17-carboxy(P)-1-oxoheptadecyl)-L-g-glutamyl(P)-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl
  • P1 represents H, or a protecting group for the N-terminal of Gln e.g., Fmoc or Cbz).
  • P1 represents a protecting group for the N-terminal of His, e.g., Boc, Fmoc, or Cbz; each P represents side chain protecting groups which may be the same or different; and
  • each P represents side chain protecting groups which may be the same or different.
  • P1 represents a protecting group for the N-terminal of His, e.g., Boc, Fmoc, or Cbz; each P represents side chain protecting groups which may be the same or different and represents an activated carboxylic ester of the (e.g., Su or Bt or Pfp); and g) removal of the protecting groups to obtain semaglutide.
  • each P represents side chain protecting groups which may be the same or different and wherein P2 represents an activated carboxylic ester Gly 16 .
  • P2 represents an activated carboxylic ester Gly 16 .
  • E5. The process according to any one of paragraphs D1-D3 and E1-E3, wherein Fmoc-Lys(W1)-OH used in step (c) is highly pure Fmoc-Lys(W1)-OH having a purity of more than 99.5% as measured by HPLC.
  • E6 The process according to any one of paragraphs D1-D3 and E1-E5, wherein Fmoc-Lys(W1)-OH used in step (c) is highly pure Fmoc-Lys(W1)-OH, wherein the content of each impurity is 0.30% or less as measured by HPLC.
  • E7 The process according to any one of paragraphs D1-D3 and E1-E6 wherein Fmoc-Lys(W1)-OH used in step (c) is highly pure Fmoc-Lys(W1)-OH, wherein the content of each impurity is 0.20% or less as measured by HPLC.
  • step d) coupling with Fmoc-Glu-OtBu and removing the Fmoc protecting group; d) coupling with octadecanedioic acid mono tert butyl ester; e) cleaving the product of step d) from the resin to obtain W1; and f) purifying W1.
  • G5. The highly pure W1 according to any one of paragraphs G1-G4, wherein the content of each impurity is 0.1% or less as measured by HPLC.
  • G6 A composition comprising W1 having a purity of more than 98% as measured by HPLC.
  • G8 The composition according to paragraph G6 or G7, wherein the content of each impurity is 0.3% or less as measured by HPLC.
  • G12 A process for preparation of highly pure W1 according to any one of paragraphs G1-G6 or a composition according to any one of paragraphs G6-G9 comprising:
  • step d) cleaving the product of step d) from the resin to obtain W1; and f) purifying W1 on preparative HPLC.
  • H6 A composition comprising Fmoc-Lys(W1)-OH wherein the Fmoc-Lys(W1)- OH has a purity of more than 99% as measured by HPLC and containing at least one impurity.
  • step a) contains a total amount of impurities of 1.0% or less as measured by HPLC.
  • step d) cleaving the product of step d) from the resin to obtain W1; f) purifying W1;
  • a method of purifying Semaglutide which can achieve a high purity product suitable for use in pharmaceutical formulations, and can meet the challenging requirements of the FDA draft guidelines.
  • the process employs a three stage HPLC purification using two different mobile phase systems and 2 reversed phase resins.
  • the present disclosure provides Semaglutide produced by the processes of the present disclosure.
  • Semaglutide produced by the processes of the present disclosure may be used in the preparation of pharmaceutical compositions of Semaglutide.
  • the present disclosure also encompasses the use of the Semaglutide prepared by the processes of the present disclosure for the preparation of pharmaceutical compositions of Semaglutide.
  • the present disclosure comprises processes for preparing the above mentioned pharmaceutical compositions.
  • the processes comprise combining the Semaglutide prepared by the processes of the present disclosure or salts thereof with at least one pharmaceutically acceptable excipient.
  • Semaglutide prepared by the processes of the present disclosure and the pharmaceutical compositions of Semaglutide prepared by the processes of the present disclosure can be used as medicaments, particularly to improve glycemic control in adults with type 2 diabetes mellitus
  • the present disclosure also provides methods to improve glycemic control in adults with type 2 diabetes mellitus, comprising administering a therapeutically effective amount of Semaglutide prepared by the processes of the present disclosure, or at least one of the above pharmaceutical compositions, to a subject in need of the treatment.
  • METHODS HPLC Method 1 Chromatographic Conditions for semaglutide [0231] Column & packing: Halo C8150 ⁇ 4.6 mm, 2.7 ⁇ m, 90 ⁇ ;
  • HPLC Method 2 Chromatographic Conditions for W1 [0232] Column & packing: Halo C8150 ⁇ 4.6 mm, 2.7 ⁇ m, 90 ⁇ ;
  • HPLC Method 4 Chromatographic Conditions for Fmoc-Lys(W1)-OH [0234] Column & packing: Hypersil GOLD PFP 250 ⁇ 4.6 mm, 5 ⁇ m, 1750 ⁇ ;
  • Fmoc-Glu 15 -OH 4090 gr, 10mole
  • HOBt 490gr, 3mole
  • collidine 1277ml, 10mole
  • TBTU 2928 gr, 9moles
  • reaction mixture was agitated for about 10 min at 50C before being charged to the damp resin as a single aliquot.
  • the coupling reaction was allowed to proceed for 1.5 hour at room temperature.
  • the coupling of Boc-His(Boc)-OH was repeated with freshly prepared reaction mixture for 3 hours.
  • Fmoc-Glu 15 -OH (3067 gr, 7.5mole), HOBt (367gr, 2.25mole), collidine (958ml, 7mole) and TBTU (2196 gr, 6.75moles) were dissolved in NMP (15 liter) and stirred at room temperature for 10 min before being charged to the damp resin as a single aliquot.
  • NMP 15 liter
  • the coupling reaction was allowed to proceed for 3 hours at ambient temperature.
  • the coupling efficiency was tested by Kaiser test.
  • the resin was washed with DMF and NMP.
  • the resin was swollen in DCM for 15 min.
  • the first building block, Fmoc-Ethoxy-Ethoxy-OH, (2.88mole, 1109gr) and DIPEA (2940ml, 16.8mol) were introduced to the resin to start the loading step.
  • the Fmoc protecting group was removed by treatment with 25% piperidine in DMF.
  • the resin was washed with DMF and 5% HOBt in DMF.
  • octadecanedioic acid mono tert butyl ester (1068 gr, 2.88mole), HOBt (220gr, 1.44mole) and TBTU (924gr, 2.88mole) were dissolved in NMP (15 liter).
  • the reaction mixture was agitated for about 10 min before being charged to the damp resin as a single aliquot.
  • DIPEA (924ml, 5.28mol) was added in four portions to the reaction, the pH was monitored and additional DIPEA (42ml, 0.24mol) was added to pH»7.
  • the coupling reaction was allowed to proceed for 3 hours at ambient temperature followed by washing steps with DMF and DCM.
  • the W1 prepared as described above, was cleaved from the resin under mild cleavage conditions using the following cleavage cocktail: 1% TFA, 99% DCM (v/v) (50 liters).
  • the cleavage cocktail was added in 3 portions (50%, 25% and 25%) to the W1- resin each portion was stirred for 10 min at room temperature followed by 3 washes with DCM (15liter).
  • TFA was extracted by phase separation with DIPEA aqueous solution.
  • the DCM solution was evaporated until viscous oil was obtained.
  • octadecanedioic acid mono tert- butyl ester (534 gr, 1.44mole), HOBt (110gr, 0.72mole) and TBTU (462gr, 1.44mole) were dissolved in NMP (6 liter).
  • the reaction mixture was agitated for about 10 min before being charged to the damp resin as a single aliquot.
  • DIPEA (462ml, 2.64mol) was added in four portions to the reaction, the pH was monitored and additional DIPEA (42ml, 0.24mol) was added to pH»7.
  • the coupling reaction was allowed to proceed for 3 hours at ambient temperature followed by washing with DMF, IPA and DCM and dried under vacuum to obtain 2.546 Kg (103% yield, due to weight added).
  • Step B Cleavage of W1 from the resin:
  • Fmoc-Arg 30 (Pbf)-OH 102 g, 0.16mole
  • HOBt 8gr, 0.05mole
  • collidine 21ml, 0.16mole
  • TBTU 48 gr, 0.15mole
  • the coupling reaction was allowed to proceed for 1.5 hours at ambient temperature. Coupling efficiency was tested by Kaiser test. The resin was washed with NMP, and DMF. Coupling of the 3 rd amino acid was done as described for the 2 nd amino acid.
  • Fmoc-Arg 28 (Pbf)-OH (68 gr, 0.11mole) and HOBt (16gr, 0.11mole) were dissolved in NMP (750ml) and stirred at 50C for 10 min then DIC (33 ml, 0.22mole) was added to the reaction mixture.
  • the reaction mixture was agitated for about 10 min at 50C before being charged to the damp resin as a single aliquot.
  • the coupling reaction was allowed to proceed for 1.5 hours at ambient temperature coupling efficiency was tested by Kaiser test the resin was washed, followed by deblock reaction and additional washing step, as described above.
  • the 15 th coupling step the coupling of Boc-(1-16)-OH (prepared according to example 1, procedure A) was done by dissolving the protected fragment (303 gr, 0.13mole) and HOBt (19gr, 0.13mole) in NMP (750 ml). The solution was stirred at 50C for 10 min then DIC (39 ml, 0.26mole) was added to the reaction mixture. The reaction mixture was agitated for about 10 min at 50C before being charged to the damp resin as a single aliquot. The coupling reaction was allowed to proceed for 12 hours at ambient temperature. Coupling efficiency was tested by Kaiser test.
  • Fmoc- Arg 30 (Pbf)-OH 817 g, 1.26mole
  • HOBt 192gr, 1.26mole
  • collidine 168ml, 1.26mole
  • TBTU 384 gr, 1.2mole
  • Fmoc-Arg 28 (Pbf)-OH 408 gr, 0.63mole
  • HOBt 96gr, 0.44mole
  • DIC 195 ml, 1.26mole
  • the coupling reaction was allowed to proceed for 1.5 hours at ambient temperature coupling efficiency was tested by Kaiser test the resin was washed, followed by deblock reaction and additional washing step, as described above.
  • the 12 th amino acid Fmoc-Lys(W1) 25 -OH (602gr, 0.5mole, prepared according to example 2,) and OxymaPure (71gr, 0.5mole) were dissolved in NMP (6 liter) and stirred at 50C for 10min then DIC (156ml, 1moles) was added to the reaction mixture. The reaction mixture was then agitated for about 10 min at 50C before being charged to the damp resin as a single aliquot. The coupling reaction was allowed to proceed for 12 hours at ambient temperature.
  • the 15 th coupling step the coupling of Boc-(1-16)-OH (prepared according to example 1, procedure B) was done by dissolving the protected fragment (1211 gr, 0.5mole) and HOBt (77gr, 0.5mole) in NMP (6liter). The solution was stirred at 50C for 10 min then DIC (156 ml, 1mole) was added to the reaction mixture. The reaction mixture was agitated for about 10 min at 50C before being charged to the damp resin as a single aliquot. The coupling reaction was allowed to proceed for 12 hours at ambient temperature. Coupling efficiency was tested by Kaiser test.
  • Fmoc-(17-31)-CTC was carried out according to example 4, procedure B on 5 g Cl-CTC resin, loading of 0.35mmol/gr, 1.75mol.
  • the 15 th coupling step, the coupling of Fmoc-(2-16)-OH was done by dissolving the protected fragment (4.8gr, 2.1mmole) and HOBt (0.3gr, 1.2mmole) in NMP (25 ml). The solution was stirred at 50C for 10 min then DIC (1.3 ml, 2.4mmole) was added to the reaction mixture. The reaction mixture was agitated for about 10 min at 50C before being charged to the damp resin as a single aliquot. The coupling reaction proceeded for 16 hours at ambient temperature.
  • Semaglutide (16% yield, due to marker, 60.1% purity) were obtained.
  • Example 6 Purification of Semaglutide [0263] Dry Semaglutide crude after cleavage, was dissolved in 1.0 M buffer glycine pH 8.5-9.0, 15 gram crude /1L The pH of the solution was monitored and kept above 8.5 with NH 4 OH solution.
  • Comparative Example 7 Purification of Semaglutide [0266] Dry Semaglutide crude after cleavage was dissolved in Ammonium Acetate (0.06M, pH 7) containing 20% ACN, 20 gram crude/1L The pH of the solution was monitored and kept above 7.0 with NH4OH solution. Crude solution was loaded to RP HPLC column (packed with C18 silica, 15 ⁇ m, 100 ⁇ ), the Purification cycles were carried out by a gradient of 0.06M NH4Ac aqueous solution (pH-7) and ACN:EtOH 9:1.
  • Semaglutide was loaded to RP HPLC column (packed with C-18, 15 ⁇ m, 100 ⁇ ) and eluted in fast gradient of NH4OH aqueous solution and ACN.
  • the obtained peptide solution was concentrated under reduced vacuum and lyophilized to obtain Semaglutide powder (1.3 gr, Total purity 98.8%, 0.14% +Gly4, 0.22% D-Ser 12 and 0.23% of D-His 1 , according to analytical method 1.
  • the HPLC chromatogram is presented in Figure 4.
  • Comparative Example 8 Purification of Semaglutide [0269] Dry Semaglutide crude after cleavage was dissolved in Ammonium Chloride (0.02M, pH 8.5-9.0) contains 20% ACN, 10 gram crude/1L. The pH of the solution was monitored and kept above 8.5 with NH 4 OH solution. Crude solution was loaded to RP HPLC column (packed with C18 silica, 15 ⁇ m, 100 ⁇ , 280x2 inch), the Purification cycles were carried out by a gradient of 0.02M NH4Cl aqueous solution (pH-8.5) and ACN. The next purification stage was done using RP HPLC column (packed with C18 silica, 15 ⁇ m, 100 ⁇ , 280x2 inch).
  • Semaglutide was loaded to RP HPLC column (packed with C-18, 15 ⁇ m, 100 ⁇ , 280x2 inch) and eluted in fast gradient of NH 4 OH aqueous solution and ACN.
  • the obtained peptide solution was concentrated under reduced vacuum and lyophilized to obtain Semaglutide powder (2.9 gr, Total purity 98.8%,0.22% +Gly 4 , 0.18% D-Ser 12 , 0.15% Glu 17 , and 0.08% of D-His 1 according to analytical method 1.
  • the HPLC chromatogram is presented in Figure 5.
  • Example 9 Purification of Semaglutide [0270] Dry Semaglutide crude after cleavage was dissolved in glycine buffer (1.0 M, pH 8.5-9.0) 100 gram crude/1L. The pH of the solution was mixed, and the pH was monitored and kept above 8.5 with NH4OH solution. Crude solution was loaded to RP HPLC column (packed with Phenyl-hexyl silica, 15 ⁇ m, 100 ⁇ , 280x2 inch), the Purification cycles were carried out by a gradient of 0.02M NH4Cl aqueous solution (pH- 8.5) and ACN.
  • the next purification stage was done using RP HPLC column (packed with Phenyl-hexyl silica, 15 ⁇ m, 100 ⁇ , 280x2 inch). After loading the crude to the column, it was washed with 2% TFA aqueous solution containing 15% ACN and eluted with gradient of 0.15% TFA aqueous solution and ACN.
  • the Semaglutide was loaded to RP HPLC column (packed with C-8, 15 ⁇ m, 100 ⁇ , 280x2 inch), and eluted by gradient of 0.02M NH4Cl aqueous solution (pH-8.5) and ACN.
  • the Semaglutide was loaded to RP HPLC column (packed with C-8, 15 ⁇ m, 100 ⁇ , 280x2 inch) and eluted in fast gradient of NH4OH aqueous solution and ACN.
  • the obtained peptide solution was concentrated under reduced vacuum and lyophilized to obtain Semaglutide powder (12.2 gr, Total purity 99.7%, 0.07% +Gly4 and 0.07% D-Ser 12 according to analytical method 1.
  • the HPLC chromatogram is presented in Figure 6.

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Abstract

The present disclosure provides processes for the preparation and purification of semaglutide and intermediates thereof. The present disclosure further provides highly pure semaglutide and processes for preparation thereof.

Description

IMPROVED PROCESSES FOR THE PREPARATION OF SEMAGLUTIDE REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S. Provisional Application Nos. 62/819,031 filed March 15, 2019, and 62/819,826, filed March 18, 2019, each of which is hereby incorporated by reference herein in its entirety. REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB [0002] The content of the electronically submitted sequence listing (Name:
2873.301PC02_Seqlisting__ST25; Size: 46,931, and Date of Creation: March 12, 2020) is incorporated herein by reference in its entirety. FIELD OF THE DISCLOSURE [0003] The present disclosure provides procedures for the preparation of semaglutide that involve a convergent synthetic strategy. The disclosure further provides highly pure semaglutide and processes for purification of semaglutide. BACKGROUND OF THE DISCLOSURE [0004] Semaglutide is a glucagon-like peptide 1 (GLP-1) receptor agonist indicated to improve glycemic control in adults with type 2 diabetes mellitus, which has the following chemical name and formula:. Semaglutide, Glycine, L-histidyl-2-methylalanyl-L-a- glutamylglycyl-Lthreonyl-L-phenylalanyl-L-threonyl-L-seryl-L-a-aspartyl-L-valyl-L- seryl-L-seryl-Ltyrosyl-L-leucyl-L-a-glutamylglycyl-L-glutaminyl-L-alanyl-L-alanyl-N6- [N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl[2-(2-aminoethoxy)ethoxy]acetyl[2-(2- aminoethoxy)ethoxy]acetyl]-L-lysyl-L-a-glutamyl-L-phenylalanyl-L-isoleucyl-Lalanyl- L-tryptophyl-L-leucyl-L-valyl-L-arginylglycyl-L-arginyl-, has the following formula: wherein W = N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl (SEQ ID NO: 1).
[0005] Semaglutide is described in US 8129343. According to the disclosure of US 8129343, the amino acid backbone of semaglutide is prepared by standard sequential Fmoc-solid phase peptide synthesis followed by deprotection and then by coupling of the side chain fragment to the Lys20 side chain. The disclosed route has several disadvantages. For example, the sequential synthesis disclosed results in low purity. Additionally, the coupling of the side chain moiety to Lys side chain in the presence of additional amine group at the N-terminus is not selective enough and results in impurities due to wrong or double attachment of the side chain moiety. This results in the need of additional purification cycles and loss of the yield. US 8129343 further discloses using Dde as protecting group on the Lys. This strategy is disadvantageous because hydrazine, which is a toxic and dangerous reagent, is required for the removal of the Dde group.
[0006] WO2007090496 discloses a method of synthesizing other GLP-1 peptide agonists, e.g. of formula:
A-(R1)x-(R2)y-R3-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-R8-Gln- Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-R4-R5-(R6)w-(R7)z-B (SEQ ID NO: 2) by linear sequential synthesis, using an Fmoc-pseudoproline dipeptide unit at the relevant position in order to prepare the Val-Ser or Ser-Ser segment of the peptide chain. The remaining sequence is then prepared by stepwise sequential synthesis.
[0007] WO 2013/098191 discloses use of Fmoc-His-Aib-OH for the preparation of a semaglutide wherein 3-31 amino acid backbone containing the side chain is acylated with Fmoc-His-Aib-OH to afford Semaglutide. The use of His-Aib to complete the synthesis allegedly provides a solution to racemization of His but requires the use of noncommercial and expensive starting materials that should be prepared separately.
[0008] US 8637647 discloses selective acylation of one amino acid group in a peptide which has two or more reactive nucleophilic functional groups wherein the acylation is performed in an aqueous media in alkaline pH. The acylation step performed at this very late stage of the preparation of semaglutide requires the use of highly pure peptides. However, such use may cause the formation of many impurities which are difficult to remove due to lack of selectivity and the reaction conditions.
[0009] WO 2016/046753 discloses preparation of semaglutide by a process comprising coupling of fragment 1-4 with fragment 5-31. WO 2016/046753 discloses that the Gly4 and Gly16 residues in Semaglutide enable convenient chemical ligation to form the peptide. Such ligation to form the final peptide and peptide fragments and/or sub- fragments at Gly residues is described as advantageous where the final peptide contain a terminal His residue, because coupling reactions with His to form the final peptide, which have a tendency to result in racemization to produce D-His isomer impurity in the final peptide, can be reduced or avoided. The D-His isomers are typically difficult to separate from the final peptide. The convergent processes of the present invention in particular avoid final coupling reactions involving His.
[0010] CN104356224 discloses preparation of Lys(W) by a stepwise process in solution that entails isolation and purification of the intermediates formed in each step. WO 2016/046753 discloses preparation of Lys(W) by solid phase synthesis wherein catalytic hydrogenation in the presence of metal catalysts was required for removal of the protecting group.
[0011] In October 2017 the FDA published draft guidelines regarding ANDAs for highly purified synthetic peptides that refer to listed drug of rDNA origin. In this guideline, FDA recommends that the applicants apply sensitive and high resolution analytical procedures to detect and characterize process-related impurities in a proposed generic synthetic peptide in comparison to the Reference drug and that the applicant should identify each process-related impurity that is 0.10% of the drug substance or greater. This guide sets a very high challenge of producing a highly pure Semaglutide.
[0012] J. Med. Chem.2015, 58, 7370-7380 provides a very broad and standard analytical method for analysis of the purity of semaglutide, using C18 RP-HPLC with CAN/TFA as the eluent. Other disclosures that relate to purity and purification methods of semaglutide, such as: CN104356224, CN105777872, CN105753964, CN108640985 and CN110540587 either include a very general analytical method that is not suitable for separation of semaglutide related peptides, or do not provide the analytical methods at all.
[0013] In view of all of the above, there is still a need in the art to provide an efficient process for the preparation of highly pure semaglutide which should not require the use of toxic or otherwise undesirable reagents and would provide Semaglutide in high yield, quality and purity, and that can be utilized in industrial scale. BRIEF SUMMARY OF THE DISCLOSURE [0014] The present disclosure provides new processes for the preparation and purification of Semaglutide and semaglutide intermediates. The present disclosure further provides highly pure semaglutide and processes for preparation thereof.
[0015] The present disclosure further provides pure intermediates that may be used for the preparation of highly pure semaglutide.
[0016] In one aspect the disclosure provides highly pure semaglutide, wherein the purity of said semaglutide is at least 99.0% or at least 99.2% or at least 99.5% by weight, as measured by HPLC and wherein the content of each peptide-related impurities, is present in an amount of less than 0.3%, or less than 0.25%, or less than 0.20%, or less than 0.15%, or less than 0.10%, as measured by HPLC.
[0017] Alternatively, the disclosure provides a composition comprising semaglutide and at least one process-related impurity, wherein the purity of said semaglutide is at least 99.0% or at least 99.2% or at least 99.5%, as measured by HPLC; and wherein said process related impurities comprise impurities having epimerized amino acids, impurities characterized by a loss of one or more amino acids, impurities characterized by the presence of one or more additional amino acids, impurities characterized by the presence of one or more different amino acids, impurities characterized by N-terminal deletions, impurities characterized by beta-alanine insertions, structural isomers, and acetylated truncated sequences and impurities characterized by alkylation or acylation of amino acids, wherein each of said process-related impurities is present in an amount of less than 0.3%, or less than 0.25%, or less than 0.20%, or less than 0.15%, or less than 0.10%, as measured by HPLC.
[0018] In one embodiment the disclosure provides a composition comprising semaglutide and at least one process-related impurity, wherein the purity of said semaglutide is at least 99.5%, as measured by HPLC wherein each of said process-related impurities is present in an amount of less than 0.10%, as measured by HPLC.
[0019] In another aspect, the invention further comprises a process for purification of semaglutide wherein the process comprises:
(a) dissolving crude semaglutide in a solvent system comprising a buffer, having a pH of about 7 to about 12, about 8 to about 11, and about 8 to about 9; (b) subjecting semaglutide to one or more reversed phase HPLC separations on a phenyl hexyl silica column and collecting the semaglutide fractions;
[0020] In embodiments, step (b) comprises:
(b1) subjecting semaglutide to a reversed phase HPLC on a phenyl hexyl silica column under basic pH and collecting the semaglutide fractions; and
(b2) subjecting the collected semaglutide fractions to a reversed phase HPLC on a phenyl hexyl silica column under acidic pH and collecting the semaglutide fractions.
[0021] In embodiments the process further comprises:
(c) subjecting the collected semaglutide fractions to a reversed phase HPLC C8 or C18 silica column under basic pH and collecting the semaglutide fractions;
(d) optionally subjecting the semaglutide fractions collected from step (c) to reversed phase HPLC on C8 or C18 silica for ion exchange; and
(e) optionally concentrating the purified Semaglutide fractions to form a purified semaglutide concentrate; and
(f) optionally drying the purified semaglutide fractions or purified semaglutide concentrate.
[0022] In embodiments the buffer is step (a) is selected from the group consisting of (i) an aqueous alkaline buffer solution comprising glycine; (ii) Tris buffer; and (iii) aqueous ammonium hydroxide; wherein the pH is adjusted during dissolution.
[0023] The disclosure further relates to the above processes wherein the purified semaglutide represents a semaglutide composition comprising semaglutide and at least one process-related impurity, wherein the purity of said semaglutide is at least 99.0% or at least 99.2% or at least 99.5%, as measured by HPLC; and wherein said peptide related impurities comprise impurities having epimerized amino acids, impurities characterized by a loss of one or more amino acids, impurities characterized by the presence of one or more additional amino acids, impurities characterized by the presence of one or more different amino acids, impurities characterized by N-terminal deletions, impurities characterized by beta-alanine insertions, impurity arising from deamidation of glutamine, structural isomers, and acetylated truncated sequences and impurities characterized by alkylation or acylation of amino acids, and wherein each of said process-related impurities is present in an amount of less than 0.3%, or less than 0.25%, or less than 0.20%, or less than 0.15%, or less than 0.10%, as measured by HPLC. [0024] The present disclosure further provides a novel process for preparation of Semaglutide.
[0025] As used herein, the amino acid forming the semaglutide backbone are numbered consecutively from 1-31, starting from the terminal His residue as follows:
(SEQ ID NO: 1) wherein W = N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2- [2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl.
[0026] In one aspect, the present disclosure provides a process for preparing semaglutide which comprises coupling a peptide fragment containing amino acids (1-16) with a peptide fragment containing amino acids (17-31) which carries the W residue, to form Semaglutide.
[0027] Each one of the above fragments can be prepared on solid support.
[0028] The coupling process can be performed on solid support or in solution.
[0029] The process can include further deprotection and cleavage from the resin steps as required.
[0030] In another aspect, the disclosure further provides an alternative process for preparing semaglutide which comprises coupling a peptide fragment containing amino acids (2-16) with a peptide fragment containing amino acids (17-31) which carries the W residue, to form a third fragment (2-31) which is in turn coupled with protected His to afford Semaglutide.
[0031] The present disclosure also provides processes for purification of semaglutide.
[0032] In one embodiment the present disclosure provides a process for preparation of semaglutide comprising:
(i) Coupling a peptide 1 having the sequence:
His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly (SEQ ID NO: 3)
wherein:
- the N-terminus of His is optionally protected with a protecting group; in some embodiments, the protecting group is selected from the group consisting of Boc, Cbz or Fmoc, and - the Gly carboxylic acid group in Peptide 1 may be in the form of an activated carboxylic acid derivative;
with a peptide 2 having the sequence:
Gln-Ala-Ala-Lys(W1)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (SEQ ID NO: 4),
wherein W1 = N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl
and wherein:
Peptide 2 is optionally conjugated to a solid support; (e.g., a CTC resin);
and wherein one or more of the amino acid residues in Peptide 1 and Peptide 2 and W1 may be protected or unprotected, e.g., protected with acid-cleavable protecting groups;
(ii) optionally removing any protecting groups and/or cleaving the resin to form Semaglutide;
(iii) optionally purifying the Semaglutide formed; and
(iv) optionally isolating Semaglutide.
[0033] In another embodiment the present disclosure provides a process for preparation of semaglutide comprising:
(i) Coupling a peptide 1' having the sequence:
Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly (SEQ ID NO: 5),
Wherein:
- the N-terminal is optionally protected with a protecting group; in some embodiments, the protecting group is selected from the group consisting of, Cbz or Fmoc, and
- the Gly carboxylic acid group in Peptide 1 may be in the form of an activated carboxylic acid derivative; with a peptide 2 having the sequence:
Gln-Ala-Ala-Lys(W1)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (SEQ ID NO: 4),
wherein W1 = N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl
and wherein: Peptide 2 is optionally conjugated to a solid support; in one aspect, the solid support is a CTC resin);
and wherein one or more of the amino acid residues in Peptide 1 and Peptide 2 and W1 may be protected or unprotected; in some aspects, the one or more of the amino acid residues in Peptide 1 and Peptide 2 and W1 are protected (e.g., with acid-cleavable protecting groups to afford peptide 3 having the sequence
Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala- Lys(W1)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (SEQ ID NO: 6)),
(ii) coupling of protected His on the resin to peptide 3;
(iii) optionally removing any protecting groups and/or cleaving the resin to form Semaglutide;
(iv) optionally purifying the Semaglutide and;
(v) optionally isolating semaglutide.
[0034] In some embodiments, any one of peptides 1, 1', and 2 are prepared by a sequential synthesis.
[0035] In some embodiments, peptides 1, 1', and 2 are all prepared by sequential synthesis.
[0036] In another aspect, the present disclosure provides Semaglutide produced by the processes of the present disclosure and highly pure semaglutide obtained by the purification process disclosed herein.
[0037] Semaglutide and/or highly pure semaglutide produced by the processes of the present disclosure can be used in the preparation of pharmaceutical compositions of Semaglutide.
[0038] The present disclosure also encompasses the use of the Semaglutide and/or highly pure semaglutide prepared by the processes of the present disclosure for the preparation of pharmaceutical compositions of Semaglutide.
[0039] The present disclosure comprises processes for preparing the above mentioned pharmaceutical compositions. The processes comprise combining the Semaglutide prepared by the processes of the present disclosure or salts thereof with at least one pharmaceutically acceptable excipient.
[0040] Semaglutide prepared by the processes of the present disclosure and the pharmaceutical compositions of Semaglutide prepared by the processes of the present disclosure can be used as medicaments, particularly to improve glycemic control in adults with type 2 diabetes mellitus
[0041] The present disclosure also provides methods to improve glycemic control in adults with type 2 diabetes mellitus, comprising administering a therapeutically effective amount of Semaglutide prepared by the processes of the present disclosure, or at least one of the above pharmaceutical compositions, to a subject in need of the treatment. BRIEF DESCRIPTION OF THE FIGURES [0042] Figure 1 is an HPLC chromatogram of W1 obtained according to to Example 3, step C.
[0043] Figure 2 HPLC chromatogram of Fmoc-Lys(W1)-OH obtained according to Example 3, step E.
[0044] Figure 3 is an HPLC chromatogram of semaglutide obtained according to Example 6.
[0045] Figure 4 is an HPLC chromatogram of semaglutide obtained according to Example 7.
[0046] Figure 5 is an HPLC chromatogram of semaglutide obtained according to Example 8.
[0047] Figure 6 is an HPLC chromatogram of semaglutide obtained according to Example 9. DETAILED DESCRIPTION
[0048] The present disclosure provides new processes for the preparation and purification of Semaglutide and semaglutide intermediates.
[0049] As discussed earlier, the processes described in the literature have significant disadvantages. In contrast to the prior art processes, the process of the present invention uses only two fragments and thus, is a simpler process offering lower production costs.
[0050] Further, while the processes described in the literature do not lead to highly pure semaglutide, the process of the present invention provides highly pure semaglutide that is afforded in a process that combines use of highly pure W1 and Fmoc-Lys(W1)-OH intermediates and involves a novel and unique purification method of semaglutide. [0051] The processes disclosed herein provide an efficient route which does not require the use of toxic or otherwise undesirable reagents and provides Semaglutide in high yield and quality, and that can be utilized in industrial scale.
[0052] The present disclosure provides the preparation of the highly pure intermediates W1 and Fmoc-Lys(W1)-OH and use thereof for preparation of highly pure semaglutide.
[0053] The disclosure also provides unique purification processes that comprises three purification steps that combine two purification steps with basic pH mobile phase. and that comprise one purification step using an acidic pH mobile phase and utilize phenyl- hexyl RP silica and C8 or C18 silica, thereby providing highly pure semaglutide
[0054] The semaglutide produced by the processes disclosed herein and purified by the processes disclosed herein displays high chemical purity of more than 99% and contains less than 0.20%, or less than 0.10% of any process related impurity, as measured by HPLC.
[0055] As discussed above, J. Med. Chem.2015, 58, 7370-7380 provides a very broad and standard analytical method for analysis of the purity of semaglutide, using C18 RP- HPLC with ACN/TFA as the eluent. Other disclosures that relate to purity and
purification methods of semaglutide, such as: CN104356224, CN105777872,
CN105753964, CN108640985 and CN110540587 either also include a very general analytical method that is not suitable for separation of semaglutide related peptides or does not provide the analytical methods at all.
[0056] In contrast, the present disclosure provides improved analytical procedures for evaluating the purity of semaglutide samples. These analytical procedures allow detecting the presence of and/or measuring the relative amount of semaglutide related peptides.
[0057] For the purpose of clarity and as an aid in the understanding of the invention, as disclosed and claimed herein, the following terms and abbreviations are defined below: AOP (7-azabenzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate
Boc or t-Boc t-butyloxycarbonyl
BOP benzotriazol-1-yl-oxytris-(dimethylamino)-phosphonium
hexafluorophosphate
BOP-Cl bis(2-oxo-3-oxazolidinyl)phosphonic chloride
BroP bromo-tris(dimethylamino)phosphonium hexafluorophosphate Cbz carboxybenzyl CDI 1,1’-Carbonyl-diimidazole
CIP 2-Chloro-1,3-dimethylimidazolidinium hexafluorophosphate DCC N,N’-dicyclohexyl carbodiimide
DCHA dicyclohexylamine
DCM dichloromethane
DIC N,N’-diisopropylcarbodiimide
DMF dimethylformamide
DTT dithiothreitol
EDC-HCl 1-ethyl-3-(3’-dimethyl-aminopropyl)carbodiimide hydrochloride EEDQ (2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline)
Fmoc 9-fluorenylmethoxycarbonyl
HAPyU 1-(1-pyrrolidinyl-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene) pyrrolidinmium hexafluorophosphate
HATU 2-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1- ylmethylene]-Nmethylmethanaminium hexafluorophosphate HBTU 2-(1H-benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium
hexafluorophosphate
HCTU 2-(6-Chloro-1H-benzotriazol-1-yl)-N,N,N’,N’ tetramethyluronium hexafluorophosphate
HBPyU 1-[(1H-benzotryazole-1-yl-oxy)(pyrrolidine-1-yl)methylidene] pyrrolidinium hexafluorophosphate
HOBt N-hydroxybenzotriazole
HPLC High Performance Liquid Chromatography
IIDQ N-isobutoxycarbonyl-2-isobutoxy-1,2-dihydroquinoline
Mmt monomethoxytrityl [(4-methoxyphenyl)diphenylmethyl]
MTBE methyl-t-butyl ether
Mtt 4-methyltrityl
NMP N-methylpyrrolidone
OAt hydroxy-7-azabenzotriazole
OBt O-benzotriazole
OBzl O-benzyl
Oct ethyl 1-hydroxy-1H-1,2,3-triazole-4-carboxylate
ODhbt 1-oxo-2-hydroxydihydrobenzotriazine ODNP dinitrophenol
Fm 9-fluorenylmethyl
ONB N-hydroxy-5-norbornene-endo-2,3-dicarboxyimide
OPfp O-pentafluorophenyl
OPht N-hydroxy-phthalimide
OPNP p-nitrophenol
OSu succinimide ester
Ot N-hydroxytetrazole
OtBu tert-butyl ester
OTCP trichlorophenol
TFFH 1,1,3,3-tetramethylfluoroformamidinium hexafluorophosphate Pal palmitoyl
Pbf 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl
PfPU pentafluorophenol-tetramethyluronium hexafluorophosphate: PfTU pentafluorophenyl-1,1,3,3-bis(tetramethylene)uranium
hexafluorophosphate:
Figure imgf000013_0001
PyAOP (7-azabenzotriazol-1-yloxy)tris(pyrrolidino)phosphonium
Hexafluorophosphate
Figure imgf000013_0002
PyBOP benzotriazol-1-yl-oxy-tris-pyrrolidinophosphonium
hexafluorophosphate
PyBrop bromo-tris-pyrrolidino-phosphonium hexafluorophosphate SPPS solid phase peptide synthesis
LPPS liquid phase peptide synthesis TBTU 2-(1H-benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium
tetrafluoroborate
tBu tert-butyl
TFA trifluoroacetic acid
Trt trityl
[0058] As used herein, unless stated otherwise, percentages relate to weight percent. The solid supports for the processes of the present invention can be resins that are cleavable using acid. In some aspects, the acid used to cleaved the resin is trifluoroacetic acid. In some aspects, the resin for use in the disclosed processes can be Wang resins and hyper- acid labile resins. In some aspects, the hyper-acid labile resins can be a chlorotrityl based (CTC) resin, 4-methoxytrityl resin, or 4-methyl-trityl resin. In one aspect, the hyper-acid labile resin is a CTC resin. Hyper-acid labile resins such as CTC resins are cleavable under milder acidic conditions. For example, hyper-acid labile resins such as CTC resins can be removed using weak acid solutions, such as 2% trifluoroacetic acid. The term “Wang resin” typically refers to a polyethylene-based resin, which can contain p- alkoxybenzyl alcohol or p-alkoxybenzyloxycarbonyhydrazide based resins, typically attached to a polyethylene glycol or polystyrene core (Wang, S., J. Am. Chem. Soc., 1973, 95(4), 1328-1333). Wang resins are typically removed under strong acid conditions, e.g. at least 50% trifluoroacetic acid solutions. Wang and CTC resins suitable for the processes disclosed herein are those on a polystyrene support. These resins are commercially available. For example, H-Gly-Wang resin or H-Gly-CTC resin, or the free resins themselves are commercially available and are suitable starting materials for use in the processes disclosed herein.
[0059] As used herein, the term "sequential synthesis" or“linear synthesis” refers to a process whereby the final product or an intermediate thereof is prepared by sequential transformations of a single starting material. Typically, in a sequential or linear synthesis, the final product is prepared by sequential condensation of single amino acids (pseudoproline dipeptide may also be used) to build the final peptide sequence. Typically the single amino acids are optionally side-chain protected as well as N-terminal protected with the usual protecting groups for peptide synthesis. In some embodiments, the N- terminal protecting groups are Fmoc, Boc, or Cbz. In some embodiments, the N-terminal protecting group is Fmoc or Boc. The condensation(s) can be carried out as a solid phase synthesis (i.e. on a solid support, such as a resin) or in liquid phase (i.e. with the free peptide– i.e. a peptide that is not conjugated to a solid support/resin), or a combination of both.
[0060] As used herein, the term“peptide” refers to a compound containing at least two amino acids in which the carboxyl group of one acid is linked to the amino group of the other (i.e. the two amino acids are linked by a peptide bond). The term“peptide” as used herein encompasses amino acid sequences in which carboxyl and/or amino groups are protected or unprotected. Suitable protecting groups for the carboxyl groups of the amino acids include OtBu, OBzl, OFm. Suitable protecting groups for the amino groups of the amino acids include Fmoc, Boc, Mmt, Mtt, Cbz, Trt. Suitable protecting groups for the N- terminal amino acid include Fmoc, Boc and Cbz.
[0061] In the coupling reactions of any embodiment of the present disclosure, the amino acid or peptide fragment is coupled using Fmoc, Boc, or Cbz strategy which is well known in the art of peptide synthesis. Thus, the typically side-chain protected amino acid or peptide fragment to be coupled onto another amino acid or peptide fragment is generally also N-terminal protected with Fmoc, Boc or Cbz to form a peptide or peptide fragment containing an N-terminal Fmoc, Boc or Cbz group. In some embodiments, the N-terminal protecting group is Fmoc or Boc. In some embodiments, the N-terminal protecting group is Fmoc.
[0062] In any subsequent coupling step, the N-terminal protection of the peptide formed in the preceding coupling step is removed, for example by reaction with, e.g. a base such as piperidine in the case of Fmoc, or an acid, such as TFA (trifluoroacetic acid) in the case of Boc, before the next amino acid or peptide is coupled.
[0063] In the case of solid phase synthesis according to the present disclosure, the coupling is carried out with Fmoc strategy using peptide fragments containing amino acid side chain protecting groups which are only acid-cleavable (i.e. are stable to the basic conditions that are generally employed to remove the base-cleavable N-terminal protecting groups), and the removal of the N-terminal protection (e.g. Fmoc) is conducted with a base. The coupling of Peptide 1 with Peptide 2 to form semaglutide which typically contains protected amino acid residues. In some embodiments, the His residue of Peptide 1 contains an acid-labile N-terminal protecting group. In some embodiments, the N-terminal protecting group and the amino acid protecting groups in the protected semaglutide sequence can be removed (optionally along with any solid support, e.g. Wang resin) in one step. In some embodiments, the His residue of Peptide 1, can be Boc. If the His residue of Peptide 1 is Boc, the His N-terminal Boc group may be removed together with the acid-labile protecting groups and Wang resin by treatment with a cleavage cocktail (typically a cleavage cocktail comprising trifluoroacetic acid (TFA), and can be a mixture of TFA with dithiothreitol or other scavengers), thereby producing semaglutide.
[0064] In one of the preferred embodiments of the present invention, semaglutide is prepared by liquid phase coupling, i.e. wherein a resin is not employed. In this embodiment, although the intermediate peptide fragments may be prepared on a resin (e.g. CTC resin), the final coupling reaction of Peptide 1 with Peptide 2 is conducted in the liquid phase. CTC resin is particularly suitable for such a process because this resin can be cleaved under mild conditions, such as dilute TFA solution (e.g. £ 10%, £ 5%, £ 2% vol/vol in a suitable organic solvent such as dichloromethane. These conditions leave most of the other acid cleavable amino acid protecting groups intact.
[0065] The coupling of Peptide 1 with Peptide 2 may be conducted as a solid phase synthesis, whereby Peptide 2 is conjugated to a solid support, which can be an acid cleavable resin. In some embodiments, the acid cleavable resin is a polystyrene-based resin. In some embodiments, the acid cleavable resin is a Wang resin or a CTC resin.
[0066] Thus, in one aspect, where the coupling of Peptide 1 with Peptide 2 is conducted in the solid phase, Peptide 2 is conjugated to a Wang resin or CTC resin.
[0067] When the coupling of Peptide 1 with Peptide 2 is conducted in the solid phase, such as on a Wang resin, the Gly carboxylic acid in Peptide 1 need not be preactivated by derivatisation into an activated carboxylic acid group (i.e. in the form of an isolated activated ester). However, the coupling may be conducted in the presence of a coupling agent (i.e. in situ activation), such as those typically employed in peptide coupling reactions. In some aspects, the coupling agents include BOP, AOP, PyBOP, PyAOP, HBTU, HATU, HCTU, HBPyU, HAPyU, TFFH, TBTU, BTFFH, EDC-HCl, PyBrop, DPPA, BOP-Cl, DCC, DIC, DEPC, EEDQ, IIDQ, CIP, PfTU, PfPU, BroP and CDI. In some aspects, the coupling agent is TBTU and DIC (e.g. DIC/HOBt).
[0068] As used herein, the term“segment” or“fragment” of semaglutide refers to a sequence of two or more amino acids present in semaglutide. The amino acids in the segment or fragment may be protected or unprotected.
[0069] In any embodiment disclosed herein, the amino acids in the fragments are protected. In some embodiments, the fragments are protected with acid-cleavable protecting groups. In particular, the trifunctional amino acids, namely: Thr, Ser, Asp, Tyr, Glu, Gln, Trp, His and Arg residues are protected with acid-cleavable protecting groups. Suitable acid cleavable protecting groups are selected from the group consisting of: tBu, OtBu, YMe,Mepro, Trt, and Pbf. In particular, the residues are protected as follows: Thr(tBu), Ser8(tBu), Ser8(Trt), Ser11(tBu), Ser11(Trt), Asp (OtBu), Ser12(YMe,Mepro), Ser12(Trt), Tyr(tBu), Glu(OtBu), Gln(Trt), Trp(Boc), His(Trt), His(Boc), and Arg(Pbf).
[0070] As used herein, the term "D-His impurity of Semaglutide" refers to H-D-His-Aib- Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(W)-Glu- Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH {W = N-(17-carboxy-1-oxoheptadecyl)- L-g-glutamyl-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2- aminoethoxy)ethoxy]acetyl}(SEQ ID NO: 1).
[0071] As used herein, the term“[+Gly4] impurity of semaglutide” refers to semaglutide which contains an extra Gly residue at position 4 (i.e. the Gly residue at position 4 is replaced by Gly- Gly), i.e.: the H-His-Aib-Glu-Gly-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser- Ser-Tyr-Leu- Glu-Gly-Gln-Ala-Ala-Lys(W)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg- Gly-OH {W = N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl}(SEQ ID NO: 7).
[0072] As used herein, the term "[+Gly16] impurity of semaglutide" refers to semaglutide which contains an extra Gly residue at position 16 (i.e. the Gly residue at position 16 is replaced by Gly-Gly), i.e.: H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr- Leu-Glu-Gly-Gly-Gln-Ala-Ala-Lys(W)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly- OH {W = N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl}(SEQ ID NO: 8).
[0073] As used herein, the term "[+Gly29] impurity of semaglutide" refers to semaglutide which contains an extra Gly residue at position 29 (i.e. the Gly residue at position 29 is replaced by Gly-Gly), i.e.: H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr- Leu-Glu-Gly-Gln-Ala-Ala-Lys(W)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Gly-Arg-Gly- OH {W = N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl}(SEQ ID NO: 9).
[0074] As used herein, the term“[+Gly31] impurity of semaglutide” refers to semaglutide which contains an extra terminal Gly residue, i.e. H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser- Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(W)-Glu-Phe-Ile-Ala-Trp-Leu-Val- Arg-Gly-Arg-Gly-Gly-OH {W = N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl}(SEQ ID NO: 10).
[0075] As used herein, the term“[+Ala18] impurity of semaglutide” refers to semaglutide which contains an extra Ala residue at position 18 i.e. the Ala residue at position 18 is replaced by Ala-Ala : H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu- Glu-Gly-Gln-Ala-Ala-Ala-Lys(W)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH {W = N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl} (SEQ ID NO: 11).
[0076] As used herein, the term "D-Ser8 impurity of Semaglutide" refers to H-His-Aib- Glu-Gly-Thr-Phe-Thr-D-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(W)- Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH {W = N-(17-carboxy-1- oxoheptadecyl)-L-g-glutamyl-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2- aminoethoxy)ethoxy]acetyl} (SEQ ID NO: 12).
[0077] As used herein, the term "D-Ser11 impurity of Semaglutide" refers to H-His-Aib- Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-D-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(W)- Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH {W = N-(17-carboxy-1- oxoheptadecyl)-L-g-glutamyl-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2- aminoethoxy)ethoxy]acetyl} (SEQ ID NO: 13).
[0078] As used herein, the term "D-Ser12 impurity of Semaglutide" refers to H-His-Aib- Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-D-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(W)- Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH {W = N-(17-carboxy-1- oxoheptadecyl)-L-g-glutamyl-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2- aminoethoxy)ethoxy]acetyl} (SEQ ID NO: 14).
[0079] As used herein, the term“[D-Ala18] impurity of semaglutide” refers to: H-His-Aib- Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-D-Ala-Ala-Lys(W)- Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH {W = N-(17-carboxy-1- oxoheptadecyl)-L-g-glutamyl-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2- aminoethoxy)ethoxy]acetyl} (SEQ ID NO: 15).
[0080] As used herein, the term“[D-Ala19] impurity of semaglutide” refers to: H-His-Aib- Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-D-Ala-Lys(W)- Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH {W = N-(17-carboxy-1- oxoheptadecyl)-L-g-glutamyl-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2- aminoethoxy)ethoxy]acetyl (SEQ ID NO: 16). [0081] As used herein, the term“[D-Glu21] impurity of semaglutide” refers to: H-His- Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(W)- D-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH {W = N-(17-carboxy-1- oxoheptadecyl)-L-g-glutamyl-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2- aminoethoxy)ethoxy]acetyl (SEQ ID NO: 17).
[0082] As used herein, the term“[D-Arg30] impurity of semaglutide” refers to: H-His- Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(W)- Glu-Phe-Ile-Ala-Trp-Leu-Val-D-Arg-Gly-Arg-Gly-OH {W = N-(17-carboxy-1- oxoheptadecyl)-L-g-glutamyl-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2- aminoethoxy)ethoxy]acetyl (SEQ ID NO: 18).
[0083] As used herein, the term "Glu17 impurity of Semaglutide" refers to semaglutide which contains a Glu residue at position 17 instead of Gln17 i.e. H-His-Aib-Glu-Gly-Thr- Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Glu-Ala-Ala-Lys(W)-Glu-Phe-Ile-Ala- Trp-Leu-Val-Arg-Gly-Arg-Gly-Gly-OH (SEQ ID NO: 19).
[0084] As used herein, the term "b-Asp9 impurity of Semaglutide" refers to semaglutide which contains a b-Asp residue at position 9 instead of Asp9 i.e. H-His-Aib-Glu-Gly-Thr- Phe-Thr-Ser-b-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Glu-Ala-Ala-Lys(W)-Glu-Phe-Ile- Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-Gly-OH (SEQ ID NO: 20).
[0085] As used herein, the term "des-His impurity of Semaglutide" refers to a semaglutide which lacks the terminal His residue, i.e. H-Aib-Glu-Gly-Thr-Phe-Thr-Ser- Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(W)-Glu-Phe-Ile-Ala-Trp-Leu-Val- Arg-Gly-Arg-Gly-Gly-OH (SEQ ID NO: 21).
[0086] As used herein, the term "des-His-Aib impurity of Semaglutide" refers to a semaglutide which lacks the terminal His-Aib residue, i.e. H-Glu-Gly-Thr-Phe-Thr-Ser- Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(W)-Glu-Phe-Ile-Ala-Trp-Leu-Val- Arg-Gly-Arg-Gly-Gly-OH (SEQ ID NO: 22).
[0087] As used herein the term peptide related impurities peptide related impurities comprise impurities having epimerized amino acids, impurities characterized by a loss of one or more amino acids, impurities characterized by the presence of one or more additional amino acids, impurities characterized by the presence of one or more different amino acids, impurities characterized by N-terminal deletions, impurities characterized by beta-alanine insertions, structural isomers, and acetylated truncated sequences and impurities characterized by alkylation or acylation of amino acids. Several of the above mentioned impurities may also form as a result of degradation processes.
[0088] As used herein, unless stated otherwise, the term acetylated truncated sequences refer to the products of Na acetylation.
[0089] As used herein, unless stated otherwise, chemical purity (area %) can be measured by HPLC analysis. The HPLC analysis is carried out using a reversed phase silica gel column (e.g. Halo C8 column) using UV detection at 215 nm depending on the impurities. The HPLC analytical method is designed to use UV absorption at a given wavelength for recording the presence and the amount of a compound in a sample passing the detector at any given point in time. For example, the primary output of any HPLC run with standard equipment will be an area percentage of the respective peak in the UV detection chromatogram.
[0090] Unless otherwise indicated, the reference to the residue“H-His”– denotes that the terminal His residue (i.e. at amino acid position 1 of semaglutide) does not contain an N- terminal protecting group, whereas, for example,“Boc-His” refers to a His residue which is protected at the N-terminal group with Boc. Similarly, unless otherwise indicated,“H- AA” refers to a terminal amino acid (AA) residue that does not contain an N-terminal protecting group.
[0091] Unless otherwise indicated, the reference to the residue“Gly-OH” denotes that the carboxylic acid group of the Gly residue is unsubstituted, and thus contains a free–OH group, whereas, for example,“Gly-OtBu” refers to a Gly residue in which the carboxylic acid OH group is substituted to form OtBu, and Gly-O-resin refers to a terminal Gly residue which is attached to a solid support (e.g. Gly-O-Wang resin, or Gly-O-CTC resin). In some instances, the term“AA-OH” may also be specified to refer to a terminal amino acid residue that is either optionally conjugated to a resin via the carboxylic acid terminal group or optionally the amino acid contains a carboxylic acid terminal group in activated form such as e.g. OSu.
[0092] As used herein, and unless indicated otherwise, the term "isolated" in reference to the intermediates of the present disclosure, their salts or solid state forms thereof corresponds to compounds that are physically separated from the reaction mixture in which they are formed. [0093] A thing, e.g., a reaction mixture, may be characterized herein as being at, or allowed to come to "room temperature", often abbreviated "RT." This means that the temperature of the thing is close to, or the same as, that of the space, e.g., the room or fume hood, in which the thing is located. Typically, room temperature is from about 20°C to about 30°C, or about 22°C to about 27°C, or about 25°C.
[0094] The processes or steps can be referred to herein as being carried out "overnight." This refers to time intervals, e.g., for the processes or steps, that span the time during the night, when the processes or steps may not be actively observed. The time intervals are from about 8 to about 20 hours, or about 10 to about 18 hours, or about 16 hours.
[0095] As used herein, and unless indicated otherwise, the term "reduced pressure" refers to a pressure of about 10 mbar to about 500 mbar, or about 50 mbar.
[0096] As used herein, and unless indicated otherwise, the term "chlorinated solvent" refers to a C1-C6 chlorinated hydrocarbon. In some embodiments, the chlorinated solvents are selected from the group consisting of, dichloromethane (CH2Cl2), dichloroethane and chlorobenzene.
[0097] As used herein, and unless indicated otherwise, the term "one pot process" refers to continues process for preparing a desired product, in which penultimate product is converted to the desired product in the same vessel.
[0098] As used herein, and unless indicated otherwise, the term "Protecting group" refers to a grouping of atoms that when attached to a reactive functional group in a molecule masks, reduces or prevents reactivity of the functional group. Examples of protecting groups can be found in Greene and Wuts“Greene’s Protective Groups in Organic Synthesis”, 4th Edition, publ. Wiley, 2006 and Harrison et al., "Compendium of Synthetic Organic Methods", Vols.1-8 (John Wiley and Sons, 1971-1996).
[0099] Representative amine protecting groups include, but are not limited to, those where the amine group is converted to carbamate or amide such as Fmoc, cbz, benzyl, trityl, Boc, trifluoroacetyl derivative, phthalic anhydride, or succinic anhydride derivative.
[0100] Representative hydroxyl protecting groups include, but are not limited to, those where the hydroxyl group is converted to ethers, esters, carbonates, siloxanes, or acetals.
[0101] The amount of solvent employed in chemical processes, e.g., reactions or crystallizations, may be referred to herein as a number of "volumes" or "vol" or "V." For example, a material may be referred to as being suspended in 10 volumes (or 10 vol or 10V) of a solvent. In this context, this expression would be understood to mean milliliters of the solvent per gram of the material being suspended, such that suspending a 5 grams of a material in 10 volumes of a solvent means that the solvent is used in an amount of 10 milliliters of the solvent per gram of the material that is being suspended or, in this example, 50 mL of the solvent. In another context, the term "v/v" can be used to indicate the number of volumes of a solvent that are added to a liquid mixture based on the volume of that mixture. For example, adding MTBE (1.5 v/v) to a 100 ml reaction mixture would indicate that 150 mL of MTBE was added.
[0102] In one aspect, the present disclosure provides new processes for the preparation and purification of Semaglutide and semaglutide intermediates. The present disclosure further provides highly pure semaglutide and processes for preparation thereof.
[0103] The present disclosure further provides pure intermediates that may be used for the preparation of highly pure semaglutide.
[0104] In one aspect the disclosure provides highly pure semaglutide, wherein the purity of said semaglutide is at least 99.0% or at least 99.2%, as measured by HPLC and wherein the content of each peptide-related impurities, is present in an amount of less than 0.3%, or less than 0.25%, or less than 0.20%, or less than 0.15%, or less than 0.10%, as measured by HPLC.
[0105] Alternatively, the disclosure provides a composition comprising semaglutide and at least one process-related impurity, wherein the purity of said semaglutide is at least 99.0% or at least 99.2% or at least 99.5%, as measured by HPLC; and wherein said peptide related impurities comprise impurities having epimerized amino acids, impurities characterized by a loss of one or more amino acids, impurities characterized by the presence of one or more additional amino acids, impurities characterized by the presence of one or more different amino acids, impurities characterized by N-terminal deletions, impurities characterized by beta-alanine insertions, structural isomers, and acetylated truncated sequences and impurities characterized by alkylation or acylation of amino acids, wherein each of said process-related impurities is present in an amount of less than 0.3%, or less than 0.25%, or less than 0.20%, or less than 0.15%, or less than 0.10%, as measured by HPLC.
[0106] In some embodiments, the disclosure relates to the above composition, wherein the at least one peptide-related impurity is selected from:
D-His1 impurity of semaglutide;
[+Gly4] impurity of semaglutide; [+Gly16] impurity of semaglutide;
[+Gly29] impurity of semaglutide;
[+Gly31] impurity of semaglutide;
[+Ala18] impurity of semaglutide;
[D-Ser8] impurity of semaglutide;
[D-Ser11] impurity of semaglutide;
[D-Ser12] impurity of semaglutide;
[D-Ala18] impurity of semaglutide;
[D-Ala19] impurity of semaglutide;
[D-Glu21] impurity of semaglutide;
[D-Arg30] impurity of semaglutide;
b-Asp9 impurity of semaglutide;
des-His impurity of semaglutide; and
combinations thereof.
[0107] In another aspect, the disclosure is directed to a process for purification of semaglutide wherein the process comprises:
(a) dissolving crude semaglutide in a solvent system comprising a buffer, having a pH of about 7 to about 12, about 8 to about 11, or about 8 to about 9;
(b) subjecting semaglutide to one or more reversed phase HPLC separations on a phenyl hexyl silica column and collecting the semaglutide fractions;
[0108] In some embodiments, step (b) comprises:
(b1) subjecting semaglutide to a reversed phase HPLC on a phenyl hexyl silica column under basic pH and collecting the semaglutide fractions; and
(b2) subjecting the collected semaglutide fractions to a reversed phase HPLC on a phenyl hexyl silica column under acidic pH and collecting the semaglutide fractions.
[0109] In some embodiments, the process further comprises:
(c) subjecting the collected semaglutide fractions to a reversed phase HPLC C8 or C18 silica column under basic pH and collecting the semaglutide fractions;
(d) optionally subjecting the semaglutide fractions collected from step (c) to reversed phase HPLC on C8 or C18 silica for ion exchange; and
(e) optionally concentrating the purified Semaglutide fractions to form a purified semaglutide concentrate; and (f) optionally drying the purified semaglutide fractions or purified semaglutide concentrate.
[0110] In some embodiments, the buffer is step (a) is selected from the group consisting of (i) an aqueous alkaline buffer solution comprising glycine; (b) Tris buffer; and (iii) aqueous ammonium hydroxide solution; wherein the pH is adjusted during dissolution.
[0111] In some embodiments, steps (b1) and (b2) can be carried out in the reversed order.
[0112] In some embodiments, the order of steps (b1) and (b2) and (c) is interchangeable.
[0113] In some embodiments, step (b1) comprises elution using a mobile phase A which comprises water and optionally a chemical modifier and a mobile phase B which comprises a polar organic solvent selected from the group consisting of ethanol, isopropanol, acetonitrile, and mixtures thereof.
[0114] In some embodiments, step (b2) comprises elution using a mobile phase C which comprises water and optionally a chemical modifier and a mobile phase D which comprises a polar organic solvent selected from the group consisting of ethanol, isopropanol, acetonitrile, and mixtures thereof.
[0115] In some embodiments, step (c) comprises using a mobile phase A which comprises water and optionally a chemical modifier and a mobile phase B which comprises a polar organic solvent selected from the group consisting of ethanol, isopropanol, acetonitrile, and mixtures thereof.
[0116] In some embodiments, the chemical modifier in mobile phase A is an ammonium salt or a sodium salt or a combination thereof. In some embodiments, the chemical modifier is selected from the group consisting of: ammonium chloride, ammonium acetate, ammonium bicarbonate, ammonium phosphate, ammonium sulfate, ammonium hydroxide, and combinations thereof. In some embodiments, the chemical modifier is ammonium chloride.
[0117] In some embodiments, the chemical modifier is present in mobile phase A in a concentration of about 0.001 to about 1.0M. In some embodiments, the chemical modifier is present in mobile phase A in a concentration of about 0.002M to about 0.5M. In some embodiments, the chemical modifier is present in mobile phase A in a concentration of about 0.005M to about 0.1M. In some embodiments, the chemical modifier is present in mobile phase A in a concentration of about 0.01M to about 0.05M. In some embodiments, the chemical modifier is present in mobile phase A in a concentration of about 0.02M. [0118] In some embodiments, the pH of mobile phase A is about 5.5 to about 11.5. In some embodiments, the pH of mobile phase A is about 6.0 to about 11.0. In some embodiments, the pH of mobile phase A is about 7.0 to about 9.5. In some embodiments, the pH of mobile phase A is about 8.5.
[0119] In some embodiments, the chemical modifier in mobile phase C is a strong acid.
In some embodiments, the chemical modifier is TFA, HCl, H2SO4, perchloric acid, phosphoric acid, or a combination thereof. In some embodiments, the chemical modifier is TFA.
[0120] In some embodiments, the chemical modifier in mobile phase C is present in a concentration of about 0.05% to about 0.3%. In some embodiments, the chemical modifier in mobile phase C is present in a concentration of about 0.1 to about 0.2%. In some embodiments, the chemical modifier in mobile phase C is present in a concentration of about 0.15%.
[0121] In some embodiments, the chemical modifier in mobile phase E comprises acetic acid, ammonium hydroxide, or a volatile ammonium salt that is ammonium bicarbonate or ammonium acetate. In some embodiments, the chemical modifier in mobile phase E is ammonium hydroxide or an ammonium salt. In some embodiments, the chemical modifier in mobile phase E is ammonium bicarbonate, ammonium acetate, or acetic acid.
[0122] In some embodiments, acetonitrile is the sole component of mobile phases B, D and F.
[0123] In some embodiments, the purified semaglutide represents a semaglutide composition as defined above.
[0124] In some embodiments, collected semaglutide fractions represent a semaglutide composition and are prepared by a process, comprising:
(a) dissolving crude semaglutide in a solution selected from the group consisting of (i) an aqueous alkaline buffer solution comprising glycine; (ii) a Tris buffer having a pH of about 8 to about 9; and (iii) an aqueous ammonium hydroxide solution; wherein the pH is adjusted to about 8 to about 9 during dissolution;
(b1) subjecting the solution in step (a) to reversed-phase HPLC on a phenyl hexyl silica column using a gradient of about 0.02M ammonium chloride aqueous solution and acetonitrile, collecting the Semaglutide fractions; and optionally conducting additional purification cycles under the same conditions; (b2) subjecting the fractions collected in step (b) to reversed-phase HPLC on a phenyl hexyl silica using a gradient elution of about 0.15% TFA aqueous solution and acetonitrile, collecting the Semaglutide fractions; and optionally conducting additional purification cycles under the same conditions;
(c) subjecting the fractions collected in step (c) to a C8 or C18 reversed phase HPLC column using a gradient of about 0.02M ammonium chloride aqueous solution and acetonitrile, collecting the Semaglutide fractions; and optionally conducting additional purification cycles under the same conditions;
(d) subjecting the fractions collected in step (d) to a C8 or C18 reversed phase HPLC column for ion exchange and collecting the semaglutide fractions;
(e) optionally concentrating the purified Semaglutide fractions to form a purified semaglutide concentrate; and
(f) optionally drying the purified semaglutide fractions or purified semaglutide.
[0125] In embodiments in step (e) the salt is washed out and semaglutide is eluted with about 0.02M ammonium hydroxide aqueous solution and acetonitrile.
[0126] Following the final HPLC run, the combined semaglutide fractions can be concentrated in order to produce a purified semaglutide concentrate. This purified semaglutide concentrate can be directly used to prepare a dry semaglutide product which is suitable for preparing a pharmaceutical composition.
[0127] The purified Semaglutide concentrate can be dried by any suitable process, especially processes which enable a rapid removal of water at low temperature, such as by spray drying, or lyophilization. In some embodiments, the drying step (f) comprises lyophilization.
[0128] The above described purification process for semaglutide is especially useful for purifying semaglutide obtained by chemical peptide synthesis techniques. In some embodiments, the crude semaglutide is obtained from a solid-phase or liquid phase peptide synthesis.
[0129] The crude semaglutide from such a synthesis can be treated before the HPLC steps, wherein the treatment comprises stirring the crude semaglutide with an aqueous alkaline buffer solution at a pH of about 8 to about 12. In some embodiments, the aqueous alkaline buffer solution has a pH of about 8 to about 11. In some embodiments, the aqueous alkaline buffer solution has a pH of about 8.5 to about 9.0. In some embodiments, ammonium hydroxide solution can be used for dissolution of semaglutide, while adjusting the pH to the above range.
[0130] A suitable aqueous alkaline buffer solution comprises aqueous glycine. In some embodiments, the buffer concentration can be about 1.5 M to about 0.01 M. In some embodiments, the buffer concentration can be about 1.3 M to about 0.1 M. In some embodiments, the buffer concentration can be about 1.2 M to about 0.8 M. In some embodiments, Tris buffer can also be used. In some embodiments, Tris buffer can be used at a concentration of about 0.01 to about 1 M. In some embodiments, Tris buffer can be used at a concentration of about 0.05 to about 0.5 M. In some embodiments, Tris buffer can be used at a concentration of about 0.1 to about 0.3 M.
[0131] The disclosure further relates to the above processes wherein the purified semaglutide represents a semaglutide composition comprising semaglutide and at least one process-related impurity, wherein the purity of said semaglutide is at least 99.0% or at least 99.2% or at least 99.5%, as measured by HPLC; and wherein said peptide related impurities comprise impurities having epimerized amino acids, impurities characterized by a loss of one or more amino acids, impurities characterized by the presence of one or more additional amino acids, impurities characterized by the presence of one or more different amino acids, impurities characterized by N-terminal deletions, impurities characterized by beta-alanine insertions, impurity arising from deamidation of glutamine, structural isomers, and acetylated truncated sequences and impurities characterized by alkylation or acylation of amino acids, and wherein each of the process-related impurities is present in an amount of less than 0.3%, or less than 0.25%, or less than 0.20%, or less than 0.15%, or less than 0.10%, as measured by HPLC.
[0132] Provided herein are methods of detecting and quantifying the presence of semaglutide process-related related impurities in a semaglutide sample. The methods provided herein can be used in order to determine whether a semaglutide sample meets FDA requirements with respect to the synthetic semaglutide purity.
[0133] The present disclosure provides a novel process for preparation of Semaglutide.
[0134] As used herein, the amino acid forming the semaglutide backbone are numbered consecutively from 1-31, starting from the terminal His residue as follows: wherein W = N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl (SEQ ID NO: 1).
[0135] In one aspect, the present invention provides a process for preparing semaglutide which comprises coupling a peptide fragment containing amino acids (1-16) with a peptide fragment containing amino acids (17-31) which carries the W residue, to form Semaglutide.
[0136] In some embodiments, each one of the above fragments can be prepared on solid support.
[0137] In some embodiments, the coupling process can be performed on solid support or in solution.
[0138] In some embodiments, the process can include further deprotection and cleavage from the resin steps as required.
[0139] Also disclosed herein is a process for preparation of semaglutide comprising:
(i) Coupling a peptide 1 having the sequence:
His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly (SEQ ID NO: 3),
Wherein:
- the N-terminal of His is optionally protected with a protecting group (in some embodiments, the protecting group is selected from the group consisting of Boc, Cbz or Fmoc), and
- the Gly carboxylic acid group in Peptide 1 may be in the form of an activated carboxylic acid derivative; with a peptide 2 having the sequence:
Gln-Ala-Ala-Lys(W1)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (SEQ ID NO: 4),
wherein W1 = N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl
wherein:
Peptide 2 is optionally conjugated to a solid support; (e.g., a CTC resin);
and wherein one or more of the amino acid residues in Peptide 1 and Peptide 2 and W1 may be protected or unprotected, e.g., protected with acid-cleavable protecting groups; (ii) optionally removing any protecting groups and/or cleaving the resin to form Semaglutide;
(iii) optionally purifying the Semaglutide; and
(iv) optionally isolating semaglutide.
[0140] In embodiments peptide 2 is not conjugated to the solid support and the coupling of peptide 2 with peptide 1 is performed in solution.
[0141] In another embodiment, the present disclosure relates to a process for preparation of semaglutide comprising:
(i) Coupling a peptide 1' having the sequence:
Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly (SEQ ID NO: 5),
wherein:
- the N-terminal is optionally protected with a protecting group (e.g., Boc, Cbz or Fmoc), and
- the Gly carboxylic acid group in Peptide 1 may be in the form of an activated carboxylic acid derivative; with a peptide 2 having the sequence:
Gln-Ala-Ala-Lys(W1)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (SEQ ID NO: 4),
wherein W1 = N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl
and wherein:
Peptide 2 is optionally conjugated to a solid support; (e.g., a CTC resin);
and wherein one or more of the amino acid residues in Peptide 1 and Peptide 2 and W1 can be protected or unprotected, e.g., with acid-cleavable protecting groups to afford peptide 3 having the sequence:
Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala- Lys(W1)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (SEQ ID NO: 6),
(ii) coupling of Protected His on the resin to peptide 3
(iii) optionally removing any protecting groups and/or cleaving the resin to form Semaglutide;
(iv) optionally purifying the Semaglutide; and
(v) optionally isolating semaglutide. [0142] In some embodiments, any one of peptides 1, 1', and 2 are prepared by a sequential synthesis.
[0143] In some embodiments, peptides 1, 1', and 2 are all prepared by sequential synthesis.
[0144] The coupling of Peptide 1 with Peptide 2 forms an optionally protected Semaglutide sequence which (when Peptide 2 is attached to a solid support) is optionally attached to a resin at the Gly31 residue. Subsequent removal of any protecting groups and resin, and optional purification enables the Semaglutide to be obtained in high yield and high purity.
[0145] In some embodiments, Peptide 1 has the formula:
P1-His(Boc)-Aib-Glu(P)-Gly-Thr(P)-Phe-Thr(P)-Ser(P)-Asp(P)-Val-Ser(P)- Ser(P)-Tyr(P)-Leu-Glu(P)-Gly-O-P2 (SEQ ID NO: 23),
wherein P1 represents a protecting group for the N-terminal of His, such as Boc, Fmoc, and Cbz. Each P represents side chain protecting groups which can be the same or different and P2 is selected from: H, or a solid support (e.g., a CTC resin), or P2 represents an activated carboxylic ester of the Gly4 residue (e.g., Su or Bt or Pfp).
[0146] In some embodiments, Peptide 1 is selected from the group consisting of:
Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (SEQ ID NO: 24),
Fmoc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (SEQ ID NO: 25),
Cbz-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (SEQ ID NO: 26),
Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OSu (SEQ ID NO: 27),
Fmoc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OSu (SEQ ID NO: 28),
Cbz-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OSu (SEQ ID NO: 29),
Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OBt (SEQ ID NO: 30), Fmoc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OBt (SEQ ID NO: 31),
Cbz-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OBt (SEQ ID NO: 32),
Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OPfp (SEQ ID NO: 33), Fmoc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OPfp (SEQ ID NO: 34); and Cbz-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OPfp (SEQ ID NO: 35).
[0147] In some embodiments, Peptide 1 is selected from the group consisting of:
Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (SEQ ID NO: 24),
Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OSu (SEQ ID NO: 27),
Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OBt (SEQ ID NO: 30); and Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OPfp (SEQ ID NO: 33).
[0148] In some embodiments Peptide 1' has the formula:
P1-Aib-Glu(P)-Gly-Thr(P)-Phe-Thr(P)-Ser(P)-Asp(P)-Val-Ser(P)-Ser(P)-Tyr(P)- Leu-Glu(P)-Gly-O-P2 (SEQ ID NO: 36),
wherein P1 represents a protecting group for the N-terminal, e.g., Boc, Fmoc, or Cbz; each P represents side chain protecting groups which may be the same or different and P2 is selected from: H, or a solid support (e.g., a CTC resin), or P2 represents an activated carboxylic ester of the Gly16 residue (e.g., Su or Bt or Pfp).
[0149] In some embodiments, peptide 1' is selected from the group consisting of:
Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (SEQ ID NO: 37),
Cbz-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (SEQ ID NO: 38), Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OSu (SEQ ID NO: 44),
Cbz-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OSu (SEQ ID NO: 39),
Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OBt (SEQ ID NO: 40),
Cbz-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OBt (SEQ ID NO: 41),
Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OPfp (SEQ ID NO: 42); and
Cbz-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OPfp (SEQ ID NO: 43).
[0150] In some embodiments, Peptide 1' is selected from the group consisting of:
Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (SEQ ID NO: 37),
Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OSu (SEQ ID NO: 44),
Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OBt (SEQ ID NO: 40); and
Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OPfp (SEQ ID NO: 42).
[0151] In some embodiments, Peptide 2 has the formula:
P1-Gln(P)-Ala-Ala-Lys(W1)-Glu(P)-Phe-Ile-Ala-Trp(P4)-Leu-Val-Arg(P)-Gly- Arg(P)-Gly-O-P3(SEQ ID NO: 45),
wherein W1 = N-(17-carboxy(P)-1-oxoheptadecyl)-L-g-glutamyl(P)-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl ,
wherein P1 represents H, or a protecting group for the N-terminal of Gln (e.g., Fmoc or Cbz), each P represents side chain protecting groups which may be the same or different, and P3 is selected from H, or a solid support, e.g., a CTC or Wang resin and P4 is selected from H or a protecting group. In embodiments peptide 2 has the formula:
P1-Gln(P)-Ala-Ala-Lys(W1)-Glu(P)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(P)-Gly- Arg(P)-Gly-O-P3(SEQ ID NO: 46), wherein W1 = N-(17-carboxy(P)-1-oxoheptadecyl)-L-g-glutamyl(P)-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl,
wherein P1 represents H, or a protecting group for the N-terminal of Gln (e.g., Fmoc or Cbz), each P represents side chain protecting groups which may be the same or different, and P3 is selected from H, or a solid support, e.g., a CTC or Wang resin.
[0152] In some embodiments, peptide 2 has the formula:
P1-Gln(Trt)-Ala-Ala-Lys(W1)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val- Arg(Pbf)-Gly-Arg(Pbf)-Gly-CTC (SEQ ID NO: 47),
wherein W1 = N-(17-carboxy(P)-1-oxoheptadecyl)-L-g-glutamyl(P)-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl wherein P1 represents H, or a protecting group for the N-terminal of Gln (e.g., Fmoc or Cbz).
[0153] Further aspects and embodiments are set forth in the following numbered paragraphs: Preparation of Semaglutide:
[0154] A1. A process for preparation of semaglutide comprising:
(i) Coupling a peptide 1 having the sequence:
His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly (SEQ ID NO: 3).
Wherein:
- the N-terminal of His is optionally protected with a protecting group selected from the group consisting of Boc, Cbz or Fmoc, and
- the Gly carboxylic acid group in Peptide 1 may be in the form of an activated carboxylic acid derivative; with a peptide 2 having the sequence:
Gln-Ala-Ala-Lys(W1)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly wherein W1 = N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl ,
wherein:
Peptide 2 is optionally conjugated to a solid support; (e.g., a CTC resin);
and wherein one or more of the amino acid residues in Peptide 1 and Peptide 2 and W1 may be protected or unprotected, e.g., protected with acid-cleavable protecting groups; (ii) optionally removing any protecting groups and/or cleaving the resin to form Semaglutide;
(iii) optionally purifying the Semaglutide; and
(iv) optionally isolating semaglutide.
[0155] A2. The process according to paragraph A1 wherein the process comprises:
(i) coupling the peptide 1 having the formula P1-His(Boc)-Aib-Glu(P)-Gly- Thr(P)-Phe-Thr(P)-Ser(P)-Asp(P)-Val-Ser(P)-Ser(P)-Tyr(P)-Leu-Glu(P)-Gly-O-P2 (SEQ ID NO: 23),
wherein P1 represents a protecting group for the N-terminal of His, each P represents side chain protecting groups which may be the same or different and wherein P2 is selected from: H, or P2 represents an activated carboxylic ester of the Gly16 residue, with a Peptide 2 having the formula:
H-Gln(P)-Ala-Ala-Lys(W1)-Glu(P)-Phe-Ile-Ala-Trp(P4)-Leu-Val-Arg(P)-Gly- Arg(P)-Gly-O-P3 (SEQ ID NO: 48),
wherein W1 = N-(17-carboxy(P)-1-oxoheptadecyl)-L-g-glutamyl(P)-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl,
and wherein P3 is selected from H, or a solid support, e.g., a CTC or Wang resin and wherein P4 is selected from H or a protecting group.
(ii) optionally removing any protecting groups and/or cleaving the resin to form Semaglutide;
(iii) optionally purifying the Semaglutide; and
(iv) optionally isolating semaglutide.
[0156] A3. The process according to any of claims A1-A2 wherein peptide 1 has the formula: Boc-His(Boc)-Aib-Glu(P)-Gly-Thr(P)-Phe-Thr(P)-Ser(P)-Asp(P)-Val-Ser(P)- Ser(P)-Tyr(P)-Leu-Glu(P)-Gly-O-P2 (SEQ ID NO: 49),
wherein P2 is H or P2 represents an activated carboxylic ester of the Gly16 residue.
[0157] A4. The process according to any one of paragraphs A1-A3 wherein the coupling of peptide 2 with peptide 1 is performed in solution and the Gly16 in peptide 1 is preactivated by derivatization into an activated carboxylic acid group.
[0158] A5. The process according to any one of paragraphs A2-A4 wherein Peptide 1 is selected from the group consisting of: Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (SEQ ID NO: 24),
Fmoc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (SEQ ID NO: 25),
Cbz-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (SEQ ID NO: 26),
Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OSu (SEQ ID NO: 27),
Fmoc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OSu (SEQ ID NO: 28),
Cbz-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OSu (SEQ ID NO: 29),
Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OBt (SEQ ID NO: 30),
Fmoc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(t
Figure imgf000035_0001
Tyr(tBu)-Leu-Glu(OtBu)-Gly-OBt (SEQ ID NO: 31),
Cbz-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(t
Figure imgf000035_0002
Tyr(tBu)-Leu-Glu(OtBu)-Gly-OBt (SEQ ID NO: 32),
Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OPfp (SEQ ID NO: 33), Fmoc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OPfp (SEQ ID NO: 34); and Cbz-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OPfp (SEQ ID NO: 35).
[0159] A6. The process according to any one of paragraphs A2-A5 wherein Peptide 1 is selected from the group consisting of:
Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (SEQ ID NO: 24),
Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OSu (SEQ ID NO: 27),
Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OBt (SEQ ID NO: 30); and Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OPfp (SEQ ID NO: 33).
[0160] A7. The process according to any one of paragraphs A1-A3 wherein the coupling of peptide 2 with peptide 1 is conducted in the solid phase, in the presence of a coupling agent.
[0161] A8. The process according to paragraph A7 wherein coupling agent is selected from the list consisting of BOP, AOP, PyBOP, PyAOP, HBTU, HATU, HCTU, HBPyU, HAPyU, TFFH, TBTU, BTFFH, EDC-HCl, PyBrop, DPPA, BOP-Cl, DCC, DIC, DEPC, EEDQ, IIDQ, CIP, PfTU, PfPU, BroP and CDI.
[0162] A9. The process according to paragraph A8 wherein coupling agent is selected from TBTU and DIC.
[0163] A10. The process according to any one of paragraphs A1-A9 wherein both peptide 1 and peptide 2 are prepared by sequential synthesis.
[0164] B1. A process for preparation of semaglutide comprising:
(i) Coupling a peptide 1' having the sequence:
Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly (SEQ ID NO: 5),
wherein:
- the N-terminus is optionally protected with a protecting group (e.g., Boc, Cbz or Fmoc), and
- the Gly carboxylic acid group in Peptide 1 may be in the form of an activated carboxylic acid derivative; with a peptide 2 having the sequence:
Gln-Ala-Ala-Lys(W1)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (SEQ ID NO: 4),
wherein W1 = N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl
and wherein:
Peptide 2 is optionally conjugated to a solid support; (e.g., a CTC resin);
and wherein one or more of the amino acid residues in Peptide 1 and Peptide 2 and W1 may be protected or unprotected, e.g., protected with acid-cleavable protecting groups to afford peptide 3 having the sequence: Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala- Lys(W1)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (SEQ ID NO: 6),
(ii) coupling of optionally protected His on the resin to peptide 3; (iii) optionally removing any protecting groups and/or cleaving the resin to form Semaglutide;
(iv) optionally purifying the Semaglutide; and
(v) optionally isolating semaglutide.
[0165] B2. The process according to paragraph B1 wherein the process comprises:
(i) coupling the peptide 1' having the formula P1-Aib-Glu(P)-Gly-Thr(P)- Phe-Thr(P)-Ser(P)-Asp(P)-Val-Ser(P)-Ser(P)-Tyr(P)-Leu-Glu(P)-Gly-O-P2 (SEQ ID NO: 36),
wherein P1 represents a protecting group for the N-terminal of Aib, each P represents side chain protecting groups which may be the same or different and wherein P2 is selected from: H, or P2 represents an activated carboxylic ester of the Gly16 residue, with a Peptide 2 having the formula:
H-Gln(P)-Ala-Ala-Lys(W1)-Glu(P)-Phe-Ile-Ala-Trp(P4)-Leu-Val-Arg(P)-Gly- Arg(P)-Gly-O-P3 (SEQ ID NO: 48),
wherein W1 = N-(17-carboxy(P)-1-oxoheptadecyl)-L-g-glutamyl(P)-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl
and wherein P3 is selected from H, or a solid support, e.g., a CTC or Wang resin and wherein P4 is selected from H or a protecting group.
(ii) coupling of P5-His(P)-OH, wherein P5 and P represent protecting groups which may be the same or different, on the resin to peptide 3
(iii) optionally removing any protecting groups and/or cleaving the resin to form Semaglutide;
(iv) optionally purifying the Semaglutide; and
(v) optionally isolating semaglutide.
[0166] B3. The process according to any one of paragraphs B1 or B2 wherein Peptide 1' has the formula:
P1-Aib-Glu(P)-Gly-Thr(P)-Phe-Thr(P)-Ser(P)-Asp(P)-Val-Ser(P)-Ser(P)-Tyr(P)- Leu-Glu(P)-Gly-O-P2 (SEQ ID NO: 36), wherein P1 represents a protecting group for the N-terminal, e.g., Fmoc or Cbz; each P represents side chain protecting groups which may be the same or different and P2 is selected from: H, or P2 represents an activated carboxylic ester of the Gly16 residue.
[0167] B5. The process according to any one of paragraphs B2–B4 wherein Peptide 1' has the formula:
P1-Aib-Glu(P)-Gly-Thr(P)-Phe-Thr(P)-Ser(P)-Asp(P)-Val-Ser(P)-Ser(P)-Tyr(P)- Leu-Glu(P)-Gly-O-P2 (SEQ ID NO: 36),
wherein P1 represents a protecting group for the N-terminal, e.g., Fmoc and Cbz; each P represents side chain protecting groups which may be the same or different and P2 is selected from: H, or P2 represents an activated carboxylic ester of the Gly16 residue (e.g., Su or Bt or Pfp)
[0168] B6. The process according to any one of paragraphs B2–B5 wherein Peptide 1’ is selected from the group consisting of:
Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (SEQ ID NO: 37),
Cbz-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (SEQ ID NO: 38),
Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OSu (SEQ ID NO: 44),
Cbz-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OSu (SEQ ID NO: 39),
Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OBt (SEQ ID NO: 40),
Cbz-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OBt (SEQ ID NO: 41),
Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OPfp (SEQ ID NO: 42); and
Cbz-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OPfp (SEQ ID NO: 43).
[0169] B7. The process according to any one of paragraphs B2–B6 wherein Peptide 1' is selected from the group consisting of: Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (SEQ ID NO: 37),
Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OSu (SEQ ID NO: 44),
Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OBt (SEQ ID NO: 40); and
Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OPfp (SEQ ID NO: 42).
[0170] B8. The process according to any one of paragraphs B1-B7 wherein the coupling of peptide 2 with peptide 1' is conducted in the solid phase, in the presence of a coupling agent.
[0171] B9. The process according to paragraph B8, wherein the coupling agent is selected from the list consisting of BOP, AOP, PyBOP, PyAOP, HBTU, HATU, HCTU, HBPyU, HAPyU, TFFH, TBTU, BTFFH, EDC-HCl, PyBrop, DPPA, BOP-Cl, DCC, DIC, DEPC, EEDQ, IIDQ, CIP, PfTU, PfPU, BroP and CDI.
[0172] B10. The process according to paragraph B9 wherein the coupling agent is selected from TBTU and DIC.
[0173] B11. The process according to any one of paragraphs B1–B10 wherein both peptide 1' and peptide 2 are prepared by sequential synthesis.
[0174] C1. The processes according to any one of paragraphs A1-A10 or B1-B11 wherein Peptide 2 has the formula:
P1-Gln(P)-Ala-Ala-Lys(W1)-Glu(P)-Phe-Ile-Ala-Trp(P4)-Leu-Val-Arg(P)-Gly- Arg(P)-Gly-O-P3 (SEQ ID NO: 45),
wherein W1 = N-(17-carboxy(P)-1-oxoheptadecyl)-L-g-glutamyl(P)-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl,
wherein P1 represents H, or a protecting group for the N-terminal of Gln (e.g., Fmoc or Cbz), each P represents side chain protecting groups which may be the same or different, P3 is selected from H, or a solid support, e.g., a CTC or Wang resin and wherein P4 is selected from H or a protecting group.
[0175] C2. The processes according to any one of paragraphs A1-A10 or B1-B12 and C1 wherein Peptide 2 has the formula: P1-Gln(P)-Ala-Ala-Lys(W1)-Glu(P)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(P)-Gly- Arg(P)-Gly-O-P3 (SEQ ID NO: 46),
wherein W1 = N-(17-carboxy(P)-1-oxoheptadecyl)-L-g-glutamyl(P)-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl,
wherein P1 represents H, or a protecting group for the N-terminal of Gln (e.g., Fmoc or Cbz), each P represents side chain protecting groups which may be the same or different, and P3 is selected from H, or a solid support, e.g., a CTC or Wang resin.
[0176] C3. The processes according to any one of paragraphs A1-A10 or B1-B11 and C1- C2 wherein Peptide 2 has the formula:
P1-Gln(Trt)-Ala-Ala-Lys(W1)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val- Arg(Pbf)-Gly-Arg(Pbf)-Gly-CTC (SEQ ID NO: 47),
wherein W1 = N-(17-carboxy(P)-1-oxoheptadecyl)-L-g-glutamyl(P)-2-[2-(2- aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl wherein P1 represents H, or a protecting group for the N-terminal of Gln e.g., Fmoc or Cbz).
[0177] D1. A process for preparing semaglutide:
(SEQ ID NO: 1) wherein W = N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2- [2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl,
wherein the process comprises:
a) coupling P1-Gly-OH to a resin, e.g., a Wang or CTC resin, and removing P1;
b) sequentially coupling N-terminal P1-protected amino acids Arg, Gly, Arg, Val, Leu, Trp, Ala, Ile, Phe and Glu which may be optionally side-chain protected, and removing P1 to form a peptide-resin comprising amino acid sequence 21-31 of semaglutide;
c) coupling of Fmoc-Lys(W1)-OH wherein W1 = N-(17-carboxy(OtBu)-1- oxoheptadecyl)-L-g-glutamyl(OtBu)-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2- aminoethoxy)ethoxy]acetyl to the Glu21 residue, and removing Fmoc;
d) sequentially coupling N-terminal P1-protected amino acids: Ala, Ala, Gln, which may be optionally side-chain protected, and removing P1 to form a peptide resin comprising amino acid sequence 17-31 of semaglutide; e) coupling with a Peptide 1 containing the sequence P1-His(Boc)-Aib- Glu(P)-Gly-Thr(P)-Phe-Thr(P)-Ser(P)-Asp(P)-Val-Ser(P)-Ser(P)-Tyr(P)-Leu-Glu(P)-Gly- OH (SEQ ID NO: 50),
wherein P1 represents a protecting group for the N-terminal of His, e.g., Boc, Fmoc, or Cbz; each P represents side chain protecting groups which may be the same or different; and
f) cleavage from the resin and removal of protecting groups.
[0178] D2. The process according to paragraph D1 wherein peptide 1 has the formula:
Boc-His(Boc)-Aib-Glu(P)-Gly-Thr(P)-Phe-Thr(P)-Ser(P)-Asp(P)-Val-Ser(P)- Ser(P)-Tyr(P)-Leu-Glu(P)-Gly-OH (SEQ ID NO: 24),
wherein each P represents side chain protecting groups which may be the same or different.
[0179] D3. The process according to any one of claims D1 and D2 wherein peptide 1 has the formula:
Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)- Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (SEQ ID NO: 24). [0180] E1. A process for preparation of semaglutide:
(SEQ ID NO: 1) wherein W = N-(17-carboxy-1-oxoheptadecyl)-L-g-glutamyl-2- [2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl wherein the process comprises:
a) coupling P1-Gly-OH to a resin, e.g., a Wang or CTC resin, and removing P1;
b) sequentially coupling N-terminal P1-protected amino acids Arg, Gly, Arg, Val, Leu, Trp, Ala, Ile, Phe and Glu which may be optionally side-chain protected, and removing P1 to form a peptide-resin comprising amino acid sequence 21-31 of semaglutide;
c) coupling of Fmoc-Lys(W1)-OH wherein W1 = N-(17-carboxy(OtBu)-1- oxoheptadecyl)-L-g-glutamyl(OtBu)-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2- aminoethoxy)ethoxy]acetyl to the Glu21 residue, and removing Fmoc; d) sequentially coupling N-terminal P1-protected amino acids: Ala, Ala, Gln, which may be optionally side-chain protected, and removing P1 to form a peptide resin comprising amino acid sequence 17-31 of semaglutide;
e) cleavage of amino acid sequence 17-31 of semaglutide of the resin; and f) reacting with the activated form of Peptide 1 containing the sequence P1- His(Boc)-Aib-Glu(P)-Gly-Thr(P)-Phe-Thr(P)-Ser(P)-Asp(P)-Val-Ser(P)-Ser(P)-Tyr(P)- Leu-Glu(P)-Gly-O-P2 (SEQ ID NO: 23),
wherein P1 represents a protecting group for the N-terminal of His, e.g., Boc, Fmoc, or Cbz; each P represents side chain protecting groups which may be the same or different and represents an activated carboxylic ester of the (e.g., Su or Bt or Pfp); and g) removal of the protecting groups to obtain semaglutide.
[0181] E2. The process according to paragraph E1 wherein peptide 1 has the formula:
Boc-His(Boc)-Aib-Glu(P)-Gly-Thr(P)-Phe-Thr(P)-Ser(P)-Asp(P)-Val-Ser(P)- Ser(P)-Tyr(P)-Leu-Glu(P)-Gly-O-P2 (SEQ ID NO: 49),
wherein each P represents side chain protecting groups which may be the same or different and wherein P2 represents an activated carboxylic ester Gly16.
[0182] E3. The process according to any one of claims E1 and E2 wherein peptide 1 has the formula:
Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(yMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OP2 (SEQ ID NO: 51),
wherein P2 represents an activated carboxylic ester Gly16.
[0183] E4. The process according to any one of paragraphs D1-D3 and E1-E3 wherein Fmoc-Lys(W1)-OH used in step (c) is highly pure Fmoc-Lys(W1)-OH having a purity of more than 99% as measured by HPLC.
[0184] E5. The process according to any one of paragraphs D1-D3 and E1-E3, wherein Fmoc-Lys(W1)-OH used in step (c) is highly pure Fmoc-Lys(W1)-OH having a purity of more than 99.5% as measured by HPLC.
[0185] E6. The process according to any one of paragraphs D1-D3 and E1-E5, wherein Fmoc-Lys(W1)-OH used in step (c) is highly pure Fmoc-Lys(W1)-OH, wherein the content of each impurity is 0.30% or less as measured by HPLC. [0186] E7. The process according to any one of paragraphs D1-D3 and E1-E6 wherein Fmoc-Lys(W1)-OH used in step (c) is highly pure Fmoc-Lys(W1)-OH, wherein the content of each impurity is 0.20% or less as measured by HPLC.
[0187] E8. The process according to any one of paragraphs D1-D3 and E1-E7 wherein Fmoc-Lys(W1)-OH used in step (c) is highly pure Fmoc-Lys(W1)-OH, wherein the content of each impurity is 0.10% or less as measured by HPLC.
[0188] F1. The processes according to any one of paragraphs D1-D3 and E1-E8 wherein highly pure Fmoc-Lys(W1)-OH is prepared by a process comprising:
a) providing highly pure W1 wherein W1 is not connected to a resin and wherein W1 has a purity of more than 98.0% as measured by HPLC;
b) optionally activating the carboxylic acid of W1 and coupling with Fmoc- Lys-OH or its HCl salt to form Fmoc-Lys{N-(17-carboxy-1-oxoheptadecyl)-L-g- glutamyl[2-(2-aminoethoxy) ethoxy]acetyl[2-(2-aminoethoxy)ethoxy]acetic acid; and c) purifying the obtained Fmoc-Lys(W1)-OH to obtain highly pure Fmoc- Lys(W1)-OH.
[0189] F2. The process according to paragraph F1, wherein the highly pure W1 provided in step a) has a purity of more than 99.0% as measured by HPLC.
[0190] F3. The process according to paragraph F1 or F2, wherein the highly pure W1 provided in step a) has a content of each impurity of 0.3% or less as measured by HPLC.
[0191] F4. The process according to any one of paragraphs F1-F3, wherein the highly pure W1 provided in step a) has a content of each impurity of 0.20% or less as measured by HPLC.
[0192] F5. The process according to any one of paragraphs F1-F3, wherein the highly pure W1 provided in step a) has a content of each impurity of 0.10% or less as measured by HPLC.
[0193] F6. The process according to any one of paragraphs F1-F5 wherein highly pure W1 is prepared by a process comprising:
a) loading Fmoc-Ethoxy-Ethoxy-OH to the resin, e.g., CTC resin;
b) removing the Fmoc protecting group and coupling the second Fmoc- Ethoxy-Ethoxy-OH and removing the Fmoc protecting group;
c) coupling with Fmoc-Glu-OtBu and removing the Fmoc protecting group; d) coupling with octadecanedioic acid mono tert butyl ester; e) cleaving the product of step d) from the resin to obtain W1; and f) purifying W1.
[0194] G1. Highly pure W1, wherein W1 has a purity of more than 98.0% as measured by HPLC.
[0195] G2. The highly pure W1 according to paragraph G1, wherein W1 has a purity of more than 99.0% as measured by HPLC.
[0196] G3. The highly pure W1 according to paragraph G1 or G2, wherein the content of each impurity is 0.3% or less as measured by HPLC.
[0197] G4. The highly pure W1 according to any one of paragraphs G1-G3, wherein the content of each impurity is 0.2% or less as measured by HPLC.
[0198] G5. The highly pure W1 according to any one of paragraphs G1-G4, wherein the content of each impurity is 0.1% or less as measured by HPLC.
[0199] G6. A composition comprising W1 having a purity of more than 98% as measured by HPLC.
[0200] G7. The composition according to paragraph G6, wherein the W1 has a purity of more than 99% as measured by HPLC.
[0201] G8. The composition according to paragraph G6 or G7, wherein the content of each impurity is 0.3% or less as measured by HPLC.
[0202] G9. The composition according to any one of paragraphs G6-G8, wherein the content of each impurity is 0.2% or less as measured by HPLC.
[0203] G10. The composition according to any one of paragraphs G6-G9, wherein the content of each impurity is 0.1% or less as measured by HPLC.
[0204] G11. Use of W1 according to any one of paragraphs G1-G6 or a composition according to any one of paragraphs G6-G9 for the preparation of Fmoc-Lys(W1)-OH and/or for the preparation of semaglutide.
[0205] G12. A process for preparation of highly pure W1 according to any one of paragraphs G1-G6 or a composition according to any one of paragraphs G6-G9 comprising:
a) loading Fmoc-Ethoxy-Ethoxy-OH to the resin, e.g., CTC resin;
b) removing the Fmoc protecting group and coupling the second Fmoc- Ethoxy-Ethoxy-OH and removing the Fmoc protecting group;
c) coupling with Fmoc-Glu-OtBu and removing the Fmoc protecting group; d) coupling with octadecanedioic acid mono tert butyl ester;
e) cleaving the product of step d) from the resin to obtain W1; and f) purifying W1 on preparative HPLC.
[0206] H1. Highly pure Fmoc-Lys(W1)-OH, wherein the Fmoc-Lys(W1)-OH has a purity of more than 99% as measured by HPLC.
[0207] H2. The highly pure Fmoc-Lys(W1)-OH according to paragraph H1, wherein the Fmoc-Lys(W1)-OH has a purity of more than 99.5% as measured by HPLC.
[0208] H3. The highly pure Fmoc-Lys(W1)-OH according to paragraph H1 or H2, wherein the content of each impurity is 0.3% or less as measured by HPLC.
[0209] H4. The highly pure Fmoc-Lys(W1)-OH according to any one of paragraphs H1- H3, wherein the content of each impurity is 0.2% or less as measured by HPLC.
[0210] H5. The highly pure Fmoc-Lys(W1)-OH according to any one of paragraphs H1- H4, wherein the content of each impurity is 0.1% or less as measured by HPLC.
[0211] H6. A composition comprising Fmoc-Lys(W1)-OH wherein the Fmoc-Lys(W1)- OH has a purity of more than 99% as measured by HPLC and containing at least one impurity.
[0212] H7. The composition according to paragraph H6, wherein the Fmoc-Lys(W1)-OH has a purity of more than 99.5% as measured by HPLC and containing at least one impurity.
[0213] H8. The composition according to paragraph H6 or H7, wherein the content of each impurity is 0.3% or less as measured by HPLC.
[0214] H9. The composition according to any one of paragraphs H6-H8, wherein the content of each impurity is 0.2% or less as measured by HPLC.
[0215] H10. The composition according to any one of paragraphs H6-H9, wherein the content of each impurity is 0.1% or less as measured by HPLC.
[0216] H11. Use of Fmoc-Lys(W1)-OH according to any one of paragraph H1-H5 or the composition according to any one of paragraphs H6-H10 for the preparation of semaglutide.
[0217] H12. A process for preparation of highly pure Fmoc-Lys(W1)-OH according to any one of paragraphs H1-H5 or the composition according to any one of paragraphs H6- H10 comprising:
a) providing highly pure W1, wherein the W1 is not connected to a resin and wherein the W1 contains a total amount of impurities of 2.0% or less as measured by HPLC; b) optionally activating the carboxylic acid of W1 and coupling with Fmoc- Lys-OH or its HCl salt to form Fmoc-Lys{N-(17-carboxy-1-oxoheptadecyl)-L-g- glutamyl[2-(2-aminoethoxy) ethoxy]acetyl[2-(2-aminoethoxy)ethoxy]acetic acid; and c) purifying the obtained Fmoc-Lys(W1)-OH on preparative HPLC to obtain highly pure Fmoc-Lys(W1)-OH.
[0218] H13. The process according to paragraph H12, wherein the highly pure W1
provided in step a) contains a total amount of impurities of 1.0% or less as measured by HPLC.
[0219] H14. The process according to paragraph H12 or H13, wherein the content of each impurity in the highly pure W1 provided in step a) is 0.3% or less as measured by HPLC.
[0220] H15. The process according to any one of paragraphs H12-H14, wherein the content of each impurity in the highly pure W1 provided in step a) is 0.2% or less as measured by HPLC.
[0221] H16. The process according to any one of paragraphs H12-H15, wherein the content of each impurity in the highly pure W1 provided in step a) is 0.1% or less as measured by HPLC.
[0222] H17. A process for preparation of highly pure Fmoc-Lys(W1)-OH according to any one of paragraphs H1-5 or the composition according to any one of paragraphs H6- H10 comprising:
a) loading Fmoc-Ethoxy-Ethoxy-OH to the resin;
b) removing the Fmoc protecting group and coupling the second Fmoc- Ethoxy-Ethoxy-OH and removing the Fmoc protecting group;
c) coupling with Fmoc-Glu-OtBu and removing the Fmoc protecting group; d) coupling with octadecanedioic acid mono tert butyl ester;
e) cleaving the product of step d) from the resin to obtain W1; f) purifying W1;
g) optionally activating the carboxylic acid of W1 and coupling with Fmoc- Lys-OH or HCl salt thereof to form Fmoc-Lys{N-(17-carboxy-1-oxoheptadecyl)-L-g- glutamyl[2-(2-aminoethoxy) ethoxy]acetyl[2-(2-aminoethoxy)ethoxy]acetic acid; and h) purifying W1 Fmoc-Lys(W1)-OH. [0223] H18. Highly pure Fmoc-Lys(W1)-OH according to any one of paragraphs H1-H5 or according to the composition according to any one of paragraphs H6-H10 produced by the processes according to any one paragraphs H12-H17. Semaglutide purification
[0224] In a further aspect of the present invention, there is provided a method of purifying Semaglutide which can achieve a high purity product suitable for use in pharmaceutical formulations, and can meet the challenging requirements of the FDA draft guidelines. The process employs a three stage HPLC purification using two different mobile phase systems and 2 reversed phase resins.
[0225] In another aspect the present disclosure provides Semaglutide produced by the processes of the present disclosure.
[0226] Semaglutide produced by the processes of the present disclosure may be used in the preparation of pharmaceutical compositions of Semaglutide.
[0227] The present disclosure also encompasses the use of the Semaglutide prepared by the processes of the present disclosure for the preparation of pharmaceutical compositions of Semaglutide.
[0228] The present disclosure comprises processes for preparing the above mentioned pharmaceutical compositions. The processes comprise combining the Semaglutide prepared by the processes of the present disclosure or salts thereof with at least one pharmaceutically acceptable excipient.
[0229] Semaglutide prepared by the processes of the present disclosure and the pharmaceutical compositions of Semaglutide prepared by the processes of the present disclosure can be used as medicaments, particularly to improve glycemic control in adults with type 2 diabetes mellitus
[0230] The present disclosure also provides methods to improve glycemic control in adults with type 2 diabetes mellitus, comprising administering a therapeutically effective amount of Semaglutide prepared by the processes of the present disclosure, or at least one of the above pharmaceutical compositions, to a subject in need of the treatment. METHODS HPLC Method 1: Chromatographic Conditions for semaglutide [0231] Column & packing: Halo C8150×4.6 mm, 2.7µm, 90Å;
Eluent A: 94% {0.05M Sodium 1-Heptanesulfonate (CH3(CH2)6SO3Na)]: 6% ACN} pH 2.7±0.1 (v/v);
Eluent B: 94% ACN : 6% 1-propanol (v/v);
Flow: 0.7 mL/min, Detector: 215nm;
Column temp: 60°C;
Auto sampler temp: 5°C;
Diluent: 60% (0.02M NH4Cl pH 8.5): 40% acetonitrile;
Program:
Figure imgf000048_0001
HPLC Method 2: Chromatographic Conditions for W1 [0232] Column & packing: Halo C8150×4.6 mm, 2.7µm, 90Å;
Eluent A: 94% [0.05% H3PO4 in water pH 2.9]: 6% ACN (v/v);
Eluent B: ACN;
Flow: 0.7 mL/min, Detector: 220nm;
Column temp: 25±3°C;
Diluent: 75% ACN: 25% water;
Program:
Figure imgf000048_0002
Figure imgf000049_0001
HPLC Method 3: Chromatographic Conditions for W1 and Fmoc-Lys(W1)-OH [0233] Column & packing: Hypersil GOLD PFP 250×4.6 mm, 5µm, 1750Å
Eluent A: 0.05% TFA in H2O;
Eluent B: 0.05% TFA in ACN;
Flow: 1 mL/min, Detector: 220nm;
Column temp: 55°C;
Diluent: 95% ACN 5% H2O;
Program:
Figure imgf000049_0002
HPLC Method 4: Chromatographic Conditions for Fmoc-Lys(W1)-OH [0234] Column & packing: Hypersil GOLD PFP 250×4.6 mm, 5µm, 1750Å;
Eluent A: 0.05% TFA in H2O;
Eluent B: 0.05% TFA in ACN;
Flow: 1 mL/min, Detector: 220nm;
Column temp: 55°C;
Diluent: NMP;
Program:
Figure imgf000049_0003
Figure imgf000050_0001
EXAMPLES
Example 1: Synthesis of protected fragment Boc(1-16)-OH Procedure A
[0235] Synthesis of the peptide was carried out by a stepwise Fmoc SPPS procedure starting from CTC resin (4 Kg Cl-CTC resin, loading of 0.8mmol/gr, 3.2mol). The resin was swollen in DCM for 30 min. The first amino acid, Fmoc-Gly-OH, (3.84mole, 1236gr) and DIPEA (3920ml, 22mol) was introduced to the resin to start the loading step. The Fmoc protecting group was removed by treatment with 25% piperidine in DCM for 5min and 25% piperidine in DMF for 15 min. The resin was washed with DMF, IPA, and NMP. For introducing the 2nd amino acid, Fmoc-Glu15-OH (4090 gr, 10mole), HOBt (490gr, 3mole), collidine (1277ml, 10mole) and TBTU (2928 gr, 9moles) were dissolved in NMP (20 liter) and stirred at room temperature for 10 min before being charged to the damp resin as a single aliquot. The coupling reaction was allowed to proceed for 3 hours at ambient temperature. The coupling efficiency was tested by Kaiser test. The resin was washed with NMP, IPA and DMF. For introducing the 3rd amino acid, Fmoc-Leu-OH (1694 gr, 5mole), and HOBt (734gr, 5mole) were dissolved in NMP (20 liter) and stirred at 5⁰C for 10 min then DIC (1488 ml, 10mole) was added to the reaction mixture. The reaction mixture was agitated for about 10 min at 5⁰C before being charged to the damp resin as a single aliquot. The coupling reaction was allowed to proceed for 3 hours at ambient temperature followed by washing step, deblock and additional washing step as described above. These steps were repeated each time with the suitable protected amino acid according to the peptide sequence, except for the following: Fmoc-Ser(tBu)- Ser(Y(Me,Me)Pro)-OH - (1958 gr, 4mole, the 5th &6th amino acids) and HOBt (588gr, 4mole) were dissolved in NMP (20 liter) and stirred at 5⁰C for 10 min then DIC (1190 ml, 8mole) was added to the reaction mixture. The reaction mixture was agitated for about 10 min at 5⁰C before being charged to the damp resin as a single aliquot. Fmoc-Gly4-OH (1901 gr, 6mole), HOBt (490gr, 3mole), collidine (851ml, 6mole) and TBTU (1952 gr, 6mole) were dissolved in NMP (20 liter) and added to the reaction mixture. The mixture was agitated for about 10 min before being charged to the damp resin. The 16th amino acid, Boc-His(Boc)1-OH DCHA (1717 gr, 3 mole), was dissolved in DCM (12 liter), after full dissolution, HOBt (490gr, 3mole) and NMP (12 liter) were added. The mixture was stirred at 5⁰C for 10 min then DIC (992 ml, 6mole) was added to the reaction mixture. The reaction mixture was agitated for about 10 min at 5⁰C before being charged to the damp resin as a single aliquot. The coupling reaction was allowed to proceed for 1.5 hour at room temperature. The coupling of Boc-His(Boc)-OH was repeated with freshly prepared reaction mixture for 3 hours.
[0236] At the end of the synthesis the peptide-resin was washed with NMP, IPA & DCM, and dried under vacuum to obtain 12.1 Kg (103% yield, due to weight added).
[0237] The peptide, prepared as described above, was cleaved from the resin under mild cleavage conditions using the following cleave cocktail: 1% TFA, 99% DCM (v/v). 43.2 liters of premixed cleavage cocktail was prepared and cooled to 10⁰C. The cleavage cocktail was added in 3 portions (50%, 25%, and 25%) to the peptide-resin (3.6kg) each portion was stirred for 10 min at room temperature. The TFA was extracted by phase separation with DIPEA aqueous solution. The DCM was evaporated and precipitated in Heptane (97 liter), the mixture was agitated for further 10 min and the protected peptide was filtered. The obtained solid was washed with heptane.
[0238] 2.47 kg (63.5% yield, due to marker, 86.7% purity with 0.5% of D-His1 according to HPLC analysis) were obtained. Procedure B
[0239] Synthesis of the peptide was carried out by a stepwise Fmoc SPPS procedure starting from CTC resin ( 3 Kg CTC resin, loading of 0.8mmol/gr, 2.4mol). The resin was swollen in DCM for 30 min. The first amino acid, Fmoc-Gly-OH, (2.88mole, 927gr) and DIPEA (2940ml, 16.5mol) were introduced to the resin to start the loading step. The Fmoc protecting group removal for all synthetic stages was done by treatment with 25% piperidine in DMF at the end of deblocking stages. The resin was washed with DMF, 5% HOBt in DMF and NMP. For introducing the 2nd amino acid, Fmoc-Glu15-OH (3067 gr, 7.5mole), HOBt (367gr, 2.25mole), collidine (958ml, 7mole) and TBTU (2196 gr, 6.75moles) were dissolved in NMP (15 liter) and stirred at room temperature for 10 min before being charged to the damp resin as a single aliquot. The coupling reaction was allowed to proceed for 3 hours at ambient temperature. The coupling efficiency was tested by Kaiser test. The resin was washed with DMF and NMP. For introducing the 3rd amino acid, Fmoc-Leu-OH (1271 gr, 3.75mole), and HOBt (551gr, 3.75mole) were dissolved in NMP (15 liter) and stirred at 5⁰C for 10 min then DIC (1116 ml, 7.5mole) was added to the reaction mixture. The reaction mixture was agitated for about 10 min at 5⁰C before being charged to the damp resin as a single aliquot. The coupling reaction was allowed to proceed for 3 hours at ambient temperature followed by washing step, deblock and additional washing step as described above. These steps were repeated each time with the suitable protected amino acid according to the peptide sequence, except for the following: Fmoc-Ser(tBu)-Ser(Y(Me,Me)Pro)-OH - (1469 gr, 3mole, the 5th &6th amino acids) and HOBt (441gr, 3mole) were dissolved in NMP (15 liter) and stirred at 5⁰C for 10 min then DIC (893 ml, 6mole) was added to the reaction mixture. The reaction mixture was agitated for about 10 min at 5⁰C before being charged to the damp resin as a single aliquot. Fmoc-Gly4-OH (1426 gr, 4.8mole), HOBt (367gr, 4.8mole), collidine (638ml, 4.8mole) and TBTU (1464 gr, 4.6mole) were dissolved in NMP (15 liter) and added to the reaction mixture. The mixture was agitated for about 10 min before being charged to the damp resin. The 16th amino acid, Boc-His(Boc)1-OH DCHA (1288 gr, 2.25 mole), was dissolved in DCM (7.5 liter), after full dissolution, HOBt (367gr, 2.25mole) and NMP (7.5 liter) were added. The mixture was stirred at 5⁰C for 10 min then DIC (744 ml, 4.5mole) was added to the reaction mixture. The reaction mixture was agitated for about 10 min at 5⁰C before being charged to the damp resin as a single aliquot. The coupling reaction was allowed to proceed for 1.5 hour at room temperature. The coupling of Boc-His(Boc)-OH was repeated with freshly prepared reaction mixture for 9 hours.
[0240] At the end of the synthesis the peptide-resin was washed with NMP and DCM, and dried under vacuum to obtain 9.5 Kg (93% yield, due to weight added).
[0241] The peptide, prepared as described above, was cleaved from the resin under mild cleavage conditions using the following cleave cocktail: 1% TFA, 99% DCM (v/v). 57 liters of premixed cleavage cocktail was prepared and cooled to 10⁰C. The cleavage cocktail was added in 3 portions (50%, 25%, and 25%) to the peptide-resin (4.75kg) each portion was stirred for 10 min at room temperature. The TFA was extracted by phase separation with DIPEA aqueous solution. The DCM was evaporated and the protected peptide was precipitated in Heptane (160 liter), the mixture was agitated for further 10 min and the protected peptide was filtered. The obtained solid was washed 3 times with heptane (48 liter).
[0242] 1.95 kg (67.6% yield, due to weight added, 79.6% purity with 0.25% of D-His1 according to HPLC analysis) were obtained. Procedure C
[0243] The same procedure on 15 g Cl-CTC resin, loading of 0.8mmol/gr, 12mmol.
Coupling solvent was DMF, washing steps after deblock were done with DMF, 1% HOBt in DMF and DMF, and the washing steps before deblock were done with DMF. At the end of the synthesis the peptide-resin was washed with DMF and DCM, and dried under vacuum to obtain 36.5g (53.3% yield due to marker, 88.8% purity 0.16% of D-His1 according to HPLC analysis) were obtained. Example 2: Synthesis of Fmoc-Lys(W1)-OH [0244] Synthesis of the peptide was carried out by a stepwise Fmoc SPPS procedure starting from CTC resin (3 Kg CTC resin, loading of 0.8mmol/gr, 2.4mol). The resin was swollen in DCM for 15 min. The first building block, Fmoc-Ethoxy-Ethoxy-OH, (2.88mole, 1109gr) and DIPEA (2940ml, 16.8mol) were introduced to the resin to start the loading step. The Fmoc protecting group was removed by treatment with 25% piperidine in DMF. The resin was washed with DMF and 5% HOBt in DMF. For introducing the 2nd building block, Fmoc-Ethoxy-Ethoxy-OH (1386 gr, 3.6mole), HOBt (551gr, 3.6mole) and DIC (1116ml, 7.2mole) were dissolved in NMP (15 liter) and stirred at room temperature for 10 min before being charged to the damp resin as a single aliquot. The coupling reaction was allowed to proceed for 1.5 hours at ambient temperature. The coupling efficiency was tested by Kaiser test. The resin was washed with DMF. For introducing the 3rd amino acid, Fmoc-Glu-OtBu (1530 gr, 3.6mole), and HOBt (551gr, 3.6mole) were dissolved in NMP (15 liter) and stirred at RT then DIC (1116 ml, 7.2mole) was added to the reaction mixture. The reaction mixture was agitated for about 10 min before being charged to the damp resin as a single aliquot. The coupling reaction was allowed to proceed for 3 hours at ambient temperature followed by washing step, deblock and additional washing step as described above. For introducing the 4th building block, octadecanedioic acid mono tert butyl ester (1068 gr, 2.88mole), HOBt (220gr, 1.44mole) and TBTU (924gr, 2.88mole) were dissolved in NMP (15 liter). The reaction mixture was agitated for about 10 min before being charged to the damp resin as a single aliquot. DIPEA (924ml, 5.28mol) was added in four portions to the reaction, the pH was monitored and additional DIPEA (42ml, 0.24mol) was added to pH»7. The coupling reaction was allowed to proceed for 3 hours at ambient temperature followed by washing steps with DMF and DCM.
[0245] The W1, prepared as described above, was cleaved from the resin under mild cleavage conditions using the following cleavage cocktail: 1% TFA, 99% DCM (v/v) (50 liters). The cleavage cocktail was added in 3 portions (50%, 25% and 25%) to the W1- resin each portion was stirred for 10 min at room temperature followed by 3 washes with DCM (15liter).
[0246] The TFA was extracted by phase separation with DIPEA aqueous solution. The DCM solution was evaporated until viscous oil was obtained.
[0247] The obtained W1 was dissolved in DCM (12 liter), HOSu (551gr, 4.8mole) and DIC (550ml, 3.6mole) were added. The reaction was allowed to proceed for 1.5 hours at ambient temperature. Activation efficiency was tested by HPLC. After full activation of the W1, Fmoc-Lys-OH (1058gr, 2.87mmole) was added to the solution (Fmoc-Lys- OH*HCl can also be used). The reaction was allowed to proceed for 12 hours at ambient temperature.700 grams of Fmoc-Lys(W1)-OH were obtained (33.4% yield, 93% purity). The reaction solution was evaporated until viscous oil was obtained. The obtained protected Fmoc-Lys(W1)-OH was purified using ACN and H2O on C18 RP preparative column. The obtained pure Fmoc-Lys(W1)-OH solution was evaporated to obtain 0.6 kg (29.5% yield, due to marker, 98.1% purity, according to analytical method 4) of viscous oil. Example 3: Synthesis of highly pure Fmoc-Lys(W1)-OH Step A: Synthesis of protected W1
[0248] Synthesis was carried out by a stepwise Fmoc SPPS procedure starting from CTC resin (1.5 Kg CTC resin, loading of 0.8mmol/gr, 1.2mol). The resin was swollen in DCM for 15 min. The first building block, Fmoc-Ethoxy-Ethoxy-OH, (1.44mole, 554gr) and DIPEA (1470ml, 8.4mol) were introduced to the resin to start the loading step. The Fmoc protecting group was removed by treatment with 25% piperidine in DMF. The resin was washed with DMF and IPA. For introducing the 2nd building block, Fmoc-Ethoxy- Ethoxy-OH (693 gr, 1.8mole), HOBt (275gr, 1.8mole) and DIC (558ml, 3.6mole) were dissolved in NMP (6 liter) and stirred at room temperature for 10 min before being charged to the damp resin as a single aliquot. The coupling reaction was allowed to proceed for 1.5 hours at ambient temperature. The coupling efficiency was tested by Kaiser test. The resin was washed with DMF and IPA. For introducing the 3rd amino acid, Fmoc-Glu-OtBu (1020 gr, 2.4mole), and HOBt (367gr, 2.4mole) were dissolved in NMP (6 liter) and stirred at RT then DIC (744 ml, 4.8mole) was added to the reaction mixture. The reaction mixture was agitated for about 10 min before being charged to the damp resin as a single aliquot. The coupling reaction was allowed to proceed for 3 hours at ambient temperature followed by washing step, deblock and additional washing step as described above. For introducing the 4th building block, octadecanedioic acid mono tert- butyl ester (534 gr, 1.44mole), HOBt (110gr, 0.72mole) and TBTU (462gr, 1.44mole) were dissolved in NMP (6 liter). The reaction mixture was agitated for about 10 min before being charged to the damp resin as a single aliquot. DIPEA (462ml, 2.64mol) was added in four portions to the reaction, the pH was monitored and additional DIPEA (42ml, 0.24mol) was added to pH»7. The coupling reaction was allowed to proceed for 3 hours at ambient temperature followed by washing with DMF, IPA and DCM and dried under vacuum to obtain 2.546 Kg (103% yield, due to weight added). Step B: Cleavage of W1 from the resin:
[0249] 840 grams of W1-resin were cleaved from the resin under mild cleavage conditions using the following cleavage cocktail: 10% HFIP, 90% DCM (v/v). 8.4 liter of the premixed cleave cocktail was prepared. The cleavage cocktail was added in 2 portions (70% and 30%) to the W1-resin the first portion was stirred for 1.5 hour and the second for 0.5 hour at room temperature followed by DCM (2.1liter). The DCM solution was evaporated. The obtained protected W1 was purified using ACN and H2O on C18 RP preparative column. The obtained solution was evaporated to obtain 233gr (69.5% yield, due to marker, 97.5% purity, analyzed by method 3) of viscous oil. Step C: Purification of W1:
[0250] 13 grams of W1 (92.3% purity), was purified using ACN and H2O on C18 RP (15 µm, 100 Å) preparative column (2inch*X mm), up to 2.3 gram of W1 per 100 gram silica were loaded. The obtained solution fractions containing W1 were collected and re- purified in the same condition. The pure W1 solution was evaporated to obtain 8.5gr (42.5% yield, due to marker, 99.9% purity, 0.07% of +44 on Ethoxy2, analyzed by method 2) of viscous oil. The HPLC chromatogram is presented in Figure 1. Step D: Synthesis of protected Fmoc-Lys(W1)-OH
Procedure 1:
[0251] 233 gram of W1 was dissolved in DCM (2000 ml), HOSu (69.7gr, 610mmole) and DIC (68.3ml, 440mmole) were added, the reaction was allowed to proceed for 1.5 hours at ambient temperature. Activation efficiency was tested by HPLC. After full activation of the W1, Fmoc-Lys-OH (131gr, 358mole) was added to the solution. The reaction was allowed to proceed for 12 hours at ambient temperature. 275 grams of Fmoc-Lys(W1)-OH were obtained (58% yield, 93.6% purity, according to analytical method 3). The reaction solution was evaporated until a viscous oil was obtained. The obtained protected Fmoc-Lys(W1)-OH was purified using ACN and H2O on C18 RP preparative column. The obtained solution was evaporated to obtain 239 gr (50.2% total reaction yield, due to marker, 96.8% purity, according to analytical method 3) of viscous oil. Procedure 2: Fmoc-Lys(W1)-OH preparation from highly pure W1, with Fmoc-Lys- OH*HCl
[0252] 8.5 gram of highly pure W1, obtained according to example 3, step C, were dissolved in 4:1 (v/v) DCM: ACN solution (50 ml), HOSu (2.5gr, 2.2mmole) and DIC (2.9ml, 18.5mmole) were added, the reaction was allowed to proceed for 1.5 hours at ambient temperature. After activation completion of W1, Acetic acid (0.84ml, 14mmole) was added to the solution. The reaction was allowed to proceed to 1hr at ambient temperature. The solution was filtered followed by addition of DMF (10ml), Fmoc-Lys- OH*HCl (5.2gr, 13mmole) and collidine (3.4ml, 26mmole) were added to the solution. The reaction was allowed to proceed for 3hr at ambient temperature. The reaction solution was evaporated until viscous oil of Fmoc-Lys(W1)-OH, 7.7 grams (26.8% yield, 96.83% purity, according to analytical method 3) were obtained. Step E: Purification of Fmoc-Lys(W1)-OH
[0253] 7.7 gram of Fmoc-Lys(W1)-OH, prepared by procedure 2 of step D, (96.8% purity) were purified using ACN and H2O on C18 RP (15mm, 100 Å) preparative column (280x2 inch). Up to 2 gram of Fmoc-Lys(W1)-OH per 100 gram silica were loaded, in each purification cycle. The fraction that contain Fmoc-Lys(W1)-OH were collected. Additional purification cycles were done to obtain highly pure Fmoc-Lys(W1)-OH. The highly pure Fmoc-Lys(W1)-OH solution was evaporated to obtain 5.6 gr (19.1% yield, due to marker, 99.96% purity according to analytical method 3) of viscous oil. The HPLC chromatogram is presented in Figure 2. Example 4: Synthesis of Semaglutide (coupling of peptide 1 and peptide 2) Procedure A
[0254] Synthesis of the peptide was carried out by a stepwise Fmoc procedure starting from CTC resin (150 g CTC resin, loading of 0.35mmol/gr, 52.5mmol). The resin was swollen using DCM for 15 min. The first amino acid Fmoc-Gly31-OH (23 gr, 80mmol) and DIPEA (201ml, 1.16mol) were introduced to the resin to start the loading step. The Fmoc protecting group was removed by treatment with 25% piperidine in DCM for 5min and 25% piperidine in DMF for 15 min. The resin was washed with DMF, IPA, and NMP. For introducing the 2nd amino acid, Fmoc-Arg30(Pbf)-OH (102 g, 0.16mole), HOBt (8gr, 0.05mole), collidine (21ml, 0.16mole) and TBTU (48 gr, 0.15mole) were dissolved in NMP (750ml) and stirred at room temperature for 10 min before being charged to the damp resin as a single aliquot. The coupling reaction was allowed to proceed for 1.5 hours at ambient temperature. Coupling efficiency was tested by Kaiser test. The resin was washed with NMP, and DMF. Coupling of the 3rd amino acid was done as described for the 2nd amino acid. For introducing the 4th amino acid, Fmoc-Arg28(Pbf)-OH (68 gr, 0.11mole) and HOBt (16gr, 0.11mole) were dissolved in NMP (750ml) and stirred at 5⁰C for 10 min then DIC (33 ml, 0.22mole) was added to the reaction mixture. The reaction mixture was agitated for about 10 min at 5⁰C before being charged to the damp resin as a single aliquot. The coupling reaction was allowed to proceed for 1.5 hours at ambient temperature coupling efficiency was tested by Kaiser test the resin was washed, followed by deblock reaction and additional washing step, as described above. These steps were repeated each time with the suitable protected amino acid according to the peptide sequence, except for the following: The 8th amino acid, Fmoc-Trp25(Boc)-OH (83gr, 0.16mole) and OxymaPure (22gr, 0.16mole) were dissolved in NMP (750ml) and stirred at 5⁰C for 10 min then DIC (49 ml, 0.32mole) was added to the reaction mixture. The reaction mixture was agitated for about 10 min at 5⁰C before being charged to the damp resin as a single aliquot. From the 10th amino acid the Fmoc deprotection was carried out using 4% DBU and 5% piperidine in DMF. The 12th amino acid Fmoc-Lys(W1)25-OH (75gr, 0.06mole, prepared according to example 3, step D, procedure 1) and OxymaPure (9gr, 0.06mole) were dissolved in NMP (750 ml) and stirred at 5⁰C for 10min then DIC (20ml, 0.12moles) was added to the reaction mixture. The reaction mixture was then agitated for about 10 min at 5⁰C before being charged to the damp resin as a single aliquot. The coupling reaction was allowed to proceed for 12 hours at ambient temperature. The 15th coupling step, the coupling of Boc-(1-16)-OH (prepared according to example 1, procedure A) was done by dissolving the protected fragment (303 gr, 0.13mole) and HOBt (19gr, 0.13mole) in NMP (750 ml). The solution was stirred at 5⁰C for 10 min then DIC (39 ml, 0.26mole) was added to the reaction mixture. The reaction mixture was agitated for about 10 min at 5⁰C before being charged to the damp resin as a single aliquot. The coupling reaction was allowed to proceed for 12 hours at ambient temperature. Coupling efficiency was tested by Kaiser test.
[0255] At the end of the synthesis the peptide-resin was washed with NMP, IPA & DCM, and dried under vacuum to obtain 380 gr (84.7% yield, due to weight added).
[0256] The peptide, prepared as described above, was cleaved from the resin using the following cleave cocktail: 91% TFA, 5% DTT, 3% H2O and 1% TIS (v/v). 1200 ml of premixed cleavage cocktail was prepared and cooled to 15⁰C. The peptide-resin (120gr) was added to the cleavage cocktail for 2 hours at 15⁰C. The TFA was evaporated to 60ml and the peptide was precipitated in MTBE (450 ml), the mixture was agitated for further 10 min and the peptide was filtered. The obtained solid was with MTBE and dried under atmospheric pressure at RT. 20 gr (27.8% yield, due to marker, 59% purity according to HPLC analysis) were obtained. Procedure B
[0257] Synthesis of the peptide was carried out by a stepwise Fmoc procedure starting from CTC resin (1.2 kg CTC resin, loading of 0.35mmol/gr, 0.42mmol). The resin was swollen using DCM for 15 min. The first amino acid Fmoc-Gly31-OH (187 gr, 0.63mmol) and DIPEA (511ml, 2.94mol) were introduced to the resin to start the loading step. The Fmoc protecting group was removed by treatment with 25% piperidine in DMF. The resin was washed with DMF and 5% HOBt in DMF. For introducing the 2nd amino acid, Fmoc- Arg30(Pbf)-OH (817 g, 1.26mole), HOBt (192gr, 1.26mole), collidine (168ml, 1.26mole) and TBTU (384 gr, 1.2mole) were dissolved in DMF (6 liter) and stirred at room temperature for 10 min before being charged to the damp resin as a single aliquot. The coupling reaction was allowed to proceed for 1.5 hours at ambient temperature. Coupling efficiency was tested by Kaiser test. The resin was washed with DMF. Coupling of the 3rd amino acid was done as described for the 2nd amino acid. For introducing the 4th amino acid, Fmoc-Arg28(Pbf)-OH (408 gr, 0.63mole) and HOBt (96gr, 0.44mole) were dissolved in DMF (6liter) and stirred at 5⁰C for 10 min then DIC (195 ml, 1.26mole) was added to the reaction mixture. The reaction mixture was agitated for about 10 min at 5⁰C before being charged to the damp resin as a single aliquot. The coupling reaction was allowed to proceed for 1.5 hours at ambient temperature coupling efficiency was tested by Kaiser test the resin was washed, followed by deblock reaction and additional washing step, as described above. These steps were repeated each time with the suitable protected amino acid according to the peptide sequence, except for the following: The 8th amino acid, Fmoc-Trp25(Boc)-OH (331gr, 0.63mole) and OxymaPure (89gr, 0.63mole) were dissolved in DMF (6 liter) and stirred at 5⁰C for 10 min then DIC (195 ml, 1.26mole) was added to the reaction mixture. The reaction mixture was agitated for about 10 min at 5⁰C before being charged to the damp resin as a single aliquot. From the 10th amino acid the Fmoc deprotection was carried out using 4% DBU and 5% piperidine in DMF. The 12th amino acid Fmoc-Lys(W1)25-OH (602gr, 0.5mole, prepared according to example 2,) and OxymaPure (71gr, 0.5mole) were dissolved in NMP (6 liter) and stirred at 5⁰C for 10min then DIC (156ml, 1moles) was added to the reaction mixture. The reaction mixture was then agitated for about 10 min at 5⁰C before being charged to the damp resin as a single aliquot. The coupling reaction was allowed to proceed for 12 hours at ambient temperature.
[0258] The 15th coupling step, the coupling of Boc-(1-16)-OH (prepared according to example 1, procedure B) was done by dissolving the protected fragment (1211 gr, 0.5mole) and HOBt (77gr, 0.5mole) in NMP (6liter). The solution was stirred at 5⁰C for 10 min then DIC (156 ml, 1mole) was added to the reaction mixture. The reaction mixture was agitated for about 10 min at 5⁰C before being charged to the damp resin as a single aliquot. The coupling reaction was allowed to proceed for 12 hours at ambient temperature. Coupling efficiency was tested by Kaiser test.
[0259] At the end of the synthesis the peptide-resin was washed with NMP and DCM and dried under vacuum to obtain 2410 gr (66.5% yield, due to weight added).
[0260] The peptide, prepared as described above, was cleaved from the resin using the following cleave cocktail: 91% TFA, 5% DTT, 3% H2O and 1% TIS (v/v). 35 liter of premixed cleavage cocktail was prepared and cooled to 15⁰C. The peptide-resin (3.5Kg) was added to the cleavage cocktail for 2 hours at 15⁰C. The TFA was evaporated and the peptide was precipitated in MTBE (240 liter), the mixture was agitated for further 10 min and the peptide was filtered. The obtained solid was washed with MTBE and dried under nitrogen stream at RT. 314 gr (18% yield, due to marker, 37.6% purity according to HPLC analysis) were obtained. Example 5: Synthesis of Semaglutide (coupling of peptide 1' and peptide 2 to afford peptide 3 followed by coupling with protected His) [0261] Synthesis and cleavage of (2-16) was done according to example 1, procedure A.
The synthesis of Fmoc-(17-31)-CTC was carried out according to example 4, procedure B on 5 g Cl-CTC resin, loading of 0.35mmol/gr, 1.75mol. The 15th coupling step, the coupling of Fmoc-(2-16)-OH was done by dissolving the protected fragment (4.8gr, 2.1mmole) and HOBt (0.3gr, 1.2mmole) in NMP (25 ml). The solution was stirred at 5⁰C for 10 min then DIC (1.3 ml, 2.4mmole) was added to the reaction mixture. The reaction mixture was agitated for about 10 min at 5⁰C before being charged to the damp resin as a single aliquot. The coupling reaction proceeded for 16 hours at ambient temperature. Coupling efficiency was tested by Kaiser test. For the 16th coupling stage, Boc-His(Boc)1- OH*DCHA (1.88 gr, 3.5 mole), was dissolved in DCM (12.5 ml), HOBt (0.5gr, 3.5mmole) and NMP (12.5 ml) were added. The mixture was stirred at 5⁰C for 10 min then DIC (1.1 ml, 7mmole) was added to the reaction mixture. The reaction mixture was agitated for about 10 min at 5⁰C before being charged to the damp resin as a single aliquot. The coupling reaction proceeded for 3 hour at room temperature.
[0262] At the end of the synthesis the peptide-resin was washed and dried under vacuum.
After cleavage, 10.8 g Semaglutide (16% yield, due to marker, 60.1% purity) were obtained. Example 6: Purification of Semaglutide [0263] Dry Semaglutide crude after cleavage, was dissolved in 1.0 M buffer glycine pH 8.5-9.0, 15 gram crude /1L The pH of the solution was monitored and kept above 8.5 with NH4OH solution.
[0264] The purification of Semaglutide, was done by 3 purification systems on preparative HPLC column (280x2 inch):
1. A gradient of 0.02M NH4Cl pH 8.5 and ACN on phenyl hexyl silica (15 µm, 100 Å). Up to 8 gram per 100 gram of silica.
2. A gradient of 0.02M NH4Cl pH 8.5 and ACN on C8-RP silica (15 µm, 100 Å)Up to 8 gram per 100 gram of silica
3. A gradient of 0.05% TFA aqueous solution, pH < 2.0 and 0.05% TFA in ACN, on C8-RP silica (15 µm, 100 Å). Up to 8 gram per 100 gram of silica.
[0265] The obtained pure Semaglutide was loaded to C-18 RP (15 µm, 100 Å) preparative column (280x2inch). The Semaglutide was washed with solution of 0.2% of 29%NH4OH:ACN, 98: 2 v/v then it was kicked from the column with 0.1% of 29%NH4OH:ACN, 55: 45 v/v. The obtained solution was evaporated. The free base Semaglutide was dried by Lyophilization. 24.5 grams of Semaglutide, 98.9.0% purity with, 0.14% +Gly4, 0.07% D-Ser12, 0.08% Glu17, and 0.08% of D-His1 according to analytical method 1, were obtained. The HPLC chromatogram is presented in Figure 3. Comparative Example 7: Purification of Semaglutide [0266] Dry Semaglutide crude after cleavage was dissolved in Ammonium Acetate (0.06M, pH 7) containing 20% ACN, 20 gram crude/1L The pH of the solution was monitored and kept above 7.0 with NH4OH solution. Crude solution was loaded to RP HPLC column (packed with C18 silica, 15 µm, 100 Å), the Purification cycles were carried out by a gradient of 0.06M NH4Ac aqueous solution (pH-7) and ACN:EtOH 9:1.
[0267] The next purification stage was done using RP HPLC column (packed with C18 silica, 15 µm, 100 Å, 280x2 inch) with a gradient of 0.01M NH4Cl aqueous solution adjusted to pH=1.5 and ACN.
[0268] For ion exchange, the Semaglutide was loaded to RP HPLC column (packed with C-18, 15 µm, 100 Å) and eluted in fast gradient of NH4OH aqueous solution and ACN. The obtained peptide solution was concentrated under reduced vacuum and lyophilized to obtain Semaglutide powder (1.3 gr, Total purity 98.8%, 0.14% +Gly4, 0.22% D-Ser12 and 0.23% of D-His1, according to analytical method 1. The HPLC chromatogram is presented in Figure 4. Comparative Example 8: Purification of Semaglutide [0269] Dry Semaglutide crude after cleavage was dissolved in Ammonium Chloride (0.02M, pH 8.5-9.0) contains 20% ACN, 10 gram crude/1L. The pH of the solution was monitored and kept above 8.5 with NH4OH solution. Crude solution was loaded to RP HPLC column (packed with C18 silica, 15 µm, 100 Å, 280x2 inch), the Purification cycles were carried out by a gradient of 0.02M NH4Cl aqueous solution (pH-8.5) and ACN. The next purification stage was done using RP HPLC column (packed with C18 silica, 15 µm, 100 Å, 280x2 inch). After loading the crude to the column, it was eluted with gradient of 0.02M NH4Cl aqueous solution adjusted to pH=1.5 and ACN. For ion exchange, the Semaglutide was loaded to RP HPLC column (packed with C-18, 15 µm, 100 Å, 280x2 inch) and eluted in fast gradient of NH4OH aqueous solution and ACN. The obtained peptide solution was concentrated under reduced vacuum and lyophilized to obtain Semaglutide powder (2.9 gr, Total purity 98.8%,0.22% +Gly4, 0.18% D-Ser12, 0.15% Glu17, and 0.08% of D-His1 according to analytical method 1. The HPLC chromatogram is presented in Figure 5. Example 9: Purification of Semaglutide [0270] Dry Semaglutide crude after cleavage was dissolved in glycine buffer (1.0 M, pH 8.5-9.0) 100 gram crude/1L. The pH of the solution was mixed, and the pH was monitored and kept above 8.5 with NH4OH solution. Crude solution was loaded to RP HPLC column (packed with Phenyl-hexyl silica, 15 µm, 100 Å, 280x2 inch), the Purification cycles were carried out by a gradient of 0.02M NH4Cl aqueous solution (pH- 8.5) and ACN.
[0271] The next purification stage was done using RP HPLC column (packed with Phenyl-hexyl silica, 15 µm, 100 Å, 280x2 inch). After loading the crude to the column, it was washed with 2% TFA aqueous solution containing 15% ACN and eluted with gradient of 0.15% TFA aqueous solution and ACN.
[0272] At the final purification stage, the Semaglutide was loaded to RP HPLC column (packed with C-8, 15 µm, 100 Å, 280x2 inch), and eluted by gradient of 0.02M NH4Cl aqueous solution (pH-8.5) and ACN. For ion exchange, the Semaglutide was loaded to RP HPLC column (packed with C-8, 15 µm, 100 Å, 280x2 inch) and eluted in fast gradient of NH4OH aqueous solution and ACN. The obtained peptide solution was concentrated under reduced vacuum and lyophilized to obtain Semaglutide powder (12.2 gr, Total purity 99.7%, 0.07% +Gly4 and 0.07% D-Ser12 according to analytical method 1. The HPLC chromatogram is presented in Figure 6.

Claims

WHAT IS CLAIMED: 1. A composition comprising highly pure semaglutide, wherein the purity of said semaglutide is at least 99.0%, or at least 99.2%, or at least 99.5%, as measured by HPLC.
2. The composition of claim 1, further comprising at least one process-realated impurity selected from the group consisting of:
(a) an impurity having epimerized amino acids;
(b) an impurity characterized by a loss of one or more amino acids;
(c) an impurity characterized by the presence of one or more additional amino acids; (d) an impurity characterized by the presence of one or more different amino acids;
(e) an impurity characterized by N-terminal deletions;
(f) an impurity characterized by beta-alanine insertions, structural isomers, and/or acetylated truncated sequences;
(g) an impurity characterized by alkylation or acylation of amino acids; and
(h) combinations thereof. 3. The composition of claim 3, wherein each process-realated impurity is present in an amount of less than 0.
3%, or less than 0.25%, or less than 0.20%, or less than 0.15%, or less than 0.10%, as measured by HPLC.
4. The composition according to any one of claims 1-3, wherein the purity of semaglutide is at least 99.5% and the at least one process-realated impurity is present in an amount of less than 0.10%, as measured by HPLC.
5. The composition according to any one of claims 1 to 4, wherein the at least one process- realated impurity is selected from the group consisting of: D-His1 impurity of semaglutide; [+Gly4] impurity of semaglutide; [+Gly16] impurity of semaglutide; [+Gly29] impurity of semaglutide; [+Gly31] impurity of semaglutide; [+Ala18] impurity of semaglutide; [D-Ser8] impurity of semaglutide; [D-Ser11] impurity of semaglutide; [D-Ser12] impurity of semaglutide; [D-Ala18] impurity of semaglutide; [D-Ala19] impurity of semaglutide; [D-Glu21] impurity of semaglutide; [D-Arg30] impurity of semaglutide; b-Asp9 impurity of semaglutide; des-His impurity of semaglutide; and combinations thereof.
6. The composition according to any one of claims 1 to 5, wherein the semaglutide has a purity of at least 99.2% and the composition contains less than 0.10% of each process-realated impurity.
7. Use of the composition according to any one of claims 1 to 6 for preparing a pharmaceutical composition.
8. A pharmaceutical composition comprising the composition according to any one of claims 1 to 6.
9. The composition according to any one of claims 1 to 6, or the pharmaceutical composition according to claim 8, for use as a medicament.
10. The composition according to any one of claims 1 to 6, or the pharmaceutical composition according to claim 8, for use in improving glycemic control in adults with type 2 diabetes mellitus.
11. A process for the purification of semaglutide comprising: (a) dissolving crude semaglutide; and (b) subjecting semaglutide to one or more reversed phase HPLC separations on a phenyl hexyl silica column and collecting the semaglutide fractions.
12. The process according to claim 11 wherein in step (a) semaglutide is dissolved in a solvent system having a pH of about 7 to about 12, or about 8 to about 11, or about 8 to about 9.
13. The process according to claim 11 or 12 wherein in step (a) semaglutide is dissolved in (i) an aqueous alkaline buffer solution comprising glycine; (ii) Tris buffer; or (iii) an aqueous ammonium hydroxide solution; wherein the pH is adjusted during dissolution.
14. The process according to any one of claims 11-13, wherein step (b) comprises: (b1) subjecting semaglutide to a reversed phase HPLC on a phenyl hexyl silica column under basic pH and collecting the semaglutide fractions; and (b2) subjecting the collected semaglutide fractions to a reversed phase HPLC on a phenyl hexyl silica column under acidic pH and collecting the semaglutide fractions.
15. The process according to claim 14, wherein steps (b1) and (b2) are carried out in reverse order.
16. The process according to claim 14 or 15, wherein the process further comprises: (c) subjecting the collected semaglutide fractions to a reversed phase HPLC C8 or C18 silica column under basic pH and collecting the semaglutide fractions; (d) optionally subjecting the semaglutide fractions collected from step (c) to reversed phase HPLC on C8 or C18 silica for ion exchange; (e) optionally concentrating the purified Semaglutide fractions to form a purified semaglutide concentrate; and (f) optionally drying the purified semaglutide fractions or purified semaglutide concentrate.
17. The process according to claim 16, wherein the order of steps (b1), (b2) and (c) is interchangeable.
18. The process according to any one of claims 14-17, wherein any one of steps (b1) (b2) and (c) may be repeated.
19. The process according to any one of claims 14-18, wherein step (b1) comprises elution using a mobile phase A which comprises water and optionally a chemical modifier, and a mobile phase B which comprises a polar organic solvent selected from the group consisting of ethanol, isopropanol, acetonitrile, and mixtures thereof.
20. The processes according to any one of claims 14-19, wherein step (b2) comprises elution using a mobile phase C, which comprises water and optionally a chemical modifier, and a mobile phase D, which comprises a polar organic solvent selected from the group consisting of ethanol, isopropanol, acetonitrile, and mixtures thereof.
21. The process according to any one of claims 14-20, wherein step (c) comprises using a mobile phase A which comprises water and optionally a chemical modifier, and a mobile phase B which comprises a polar organic solvent selected from the group consisting of ethanol, isopropanol, acetonitrile, and mixtures thereof.
22. The process according to any one of claims 14-21, wherein step (d) comprises using a mobile phase E, which comprises water and optionally a chemical modifier, and a mobile phase F, which comprises a polar organic solvent selected from the group consisting of ethanol, isopropanol, acetonitrile, and mixtures thereof.
23. The process according to any one of claims 19-22, wherein mobile phase A comprises a chemical modifier that is an ammonium salt or a sodium salt or a combination thereof.
24. The process according to claim 23, wherein the chemical modifier in mobile phase A is selected from the group consisting of: ammonium chloride, ammonium acetate, ammonium bicarbonate, ammonium phosphate, ammonium sulfate, ammonium hydroxide, and combinations thereof.
25. The process according to claim 24, wherein the chemical modifier in mobile phase A is ammonium chloride.
26. The process according to any one of claims 23-25, wherein the chemical modifier is present in mobile phase A in a concentration of about 0.001 to about 1.0M, about 0.002M to about 0.5M, about 0.005M to about 0.1M, about 0.01M to about 0.05M or about 0.02M.
27. The process according to any one of claims 19-26, wherein the pH of mobile phase A is about 5.5 to about 11.5, about 6.0 to about 11.0, about 7.0 to about 9.5, or about 8.5.
28. The process according to any one of claims 20-27, wherein mobile phase C comprises a chemical modifier that is a strong acid.
29. The process according to claim 28, wherein the chemical modifier is selected from the group consisting of TFA, HCl, H2SO4, perchloric acid, phosphoric acid, and a combination thereof.
30. The process according to claim 29, wherein the chemical modifier is TFA.
31. The process according to any one of claims 28-30, wherein the chemical modifier in mobile phase C is present in a concentration of about 0.05% to about 0.3%, about 0.1 to about 0.2%, or about 0.15%.
32. The process according to claim 22, wherein mobile phase E comprises acetic acid, ammonium hydroxide, or a volatile ammonium salt that is ammonium bicarbonate or ammonium acetate.
33. The process according to any one of claims 19-32, wherein acetonitrile is the sole component of mobile phases B, D and F.
34. The process according to any one of claims 14-33, wherein any one of steps (b1), (b2) and (c) is carried out by gradient elution.
35. The process according to any one of claims 14-34, wherein the collected semaglutide fractions represent a semaglutide composition as defined in any one of claims 1-6.
36. A process according to any one of claims 14-35, wherein the collected semaglutide fractions represent a semaglutide composition as defined in any one of claims 1-6, the process comprising: (a) dissolving crude semaglutide in a solution selected from a group consisting of (i) aqueous alkaline buffer solution comprising glycine; (ii) in Tris buffer having a pH of about 8 to about 9; and (iii) an aqueous ammonium hydroxide solution; wherein the pH is adjusted to about 8 to about 9; (b1) subjecting the solution in step (a) to reversed-phase HPLC on a phenyl hexyl silica column using a gradient of about 0.02M ammonium chloride aqueous solution and acetonitrile, collecting the Semaglutide fractions; and optionally conducting additional purification cycles under the same conditions; (b2) subjecting the fractions collected in step (b1) to reversed-phase HPLC on a phenyl hexyl silica using a gradient elution of about 0.15% TFA aqueous solution and acetonitrile, collecting the Semaglutide fractions; and optionally conducting additional purification cycles under the same conditions; (c) subjecting the fractions collected in step (b2) to a C8 or C18 reversed phase HPLC column using a gradient of about 0.02M ammonium chloride aqueous solution and acetonitrile, collecting the Semaglutide fractions; and optionally conducting additional purification cycles under the same conditions; (d) subjecting the fractions collected in step (c) to a C8 or C18 reversed phase HPLC column for ion exchange and collecting the semaglutide fractions; (e) optionally concentrating the purified semaglutide fractions to form a purified semaglutide concentrate; and (f) optionally drying the purified semaglutide fractions or purified semaglutide.
37. The process according to claim 36, wherein in step (e) a salt is washed out and semaglutide is eluted with about 0.02M ammonium hydroxide aqueous solution and acetonitrile.
38. The process according to claim 36, wherein in step (e) a salt is washed out and acetic acid or ammonium acetate is used for elution of semaglutide acetate.
39. Semaglutide according to any one of claims 1-6 prepared according to any one of claims 11-38.
40. A composition comprising Fmoc-Lys(W1)-OH having a purity of more than 98% or more than 99%, and containing at least one impurity, wherein each impurity is present in an amount of less than 0.3%, less than 0.2%, or less than 0.10%, as measured by HPLC.
41. Use of the composition according to claim 40 for the preparation of semaglutide.
42. A composition comprising W1 having a purity of more than 98% or more than 99%, and containing at least one impurity, wherein each impurity is present in an amount of less than 0.3%, less than 0.2%, or less than 0.10%, as measured by HPLC.
43. Use of the composition according to claim 42 for the preparation of semaglutide.
44. An analytical method for analyzing the presence and quantity of impurities present in a semaglutide sample comprising: subjecting the sample to reversed phase HPLC silica gel chromatography.
45. The analytical method according to claim 44, wherein the sample is subjected to reversed phase HPLC silica gel chromatography which comprises a Halo C8 column (150X4.6 mm, 2.7 um, 90A), and eluted with a mobile phase gradient wherein: Eluent A: 94% {[0.05M Sodium 1-Heptanesulfonate (CH3(CH2)6SO3Na)]: 6% ACN}, pH 2.7±0.1 (v/v); Eluent B: 94% ACN : 6% 1-propanol (v/v); optionally wherein Flow: 0.7 mL/min; Column temp: 60°C;
Auto sampler temp: 5°C;
Diluent: 60% (0.02M NH4Cl pH 8.5): 40% acetonitrile; and wherein the HPLC employs UV detection at 215 nm.
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