(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2013/0310314 A1
`Wellings
`(43) Pub. Date:
`NOV. 21, 2013
`
`US 20l303l03l4Al
`
`(54) SOLID PHASE SYNTHESIS OF
`H(GLY2)GLP-2
`
`(75)
`
`Inventor: Don Wellings,Northwich (GB)
`
`(73) Assignee: TAKEDA GMBH,Konstanz (DE)
`
`(21) Appl. No.:
`
`13/820,082
`
`(22) PCT Filed:
`
`Aug.30, 2011
`
`(86) PCT No:
`§ 371 (C)(1),
`(2): (4) Date?
`
`PCT/EP2011/064877
`
`Jiii- 13: 2013
`
`(30)
`
`Foreign Aliliiicatioii Priority Data
`
`Aug. 30,
`
`................................ ..
`Publication Classification
`
`(51)
`
`Int. Cl.
`A61K 38/26
`
`(2006.01)
`
`(52) U.S. Cl.
`CPC .................................... .. A61K 38/26 (2013.01)
`USPC .......................................... .. 514/72; 530/324
`
`(57)
`
`ABSTRACT
`
`The present invention relates to a method of preparing a
`peptide comprising the amino acid sequence His-Gly-Asp-
`Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-
`Leu-Ala-Ala-Arg-Asp-Phe-I1e-Asn-Trp-Leu-Ile-Gln-Thr-
`Lys-Ile-Thr-Asp (SEQ ID NO:l). In particular, the method
`comprises the steps of providing a first peptide fragment
`having a first protection group, which peptide fragment is
`conjugated to a support; providing a second peptide fragment
`having a second protection group; removing the first protec-
`tion group from the first peptide fragment; and Coupling the
`second peptide fragment to the N-terminally deprotected,
`support-conjugated first peptide fragment. The present inven-
`tion further relates to a method of preparing a pharmaceutical
`composition containing said peptide.
`
`N PS EX. 2055
`CFAD V. N PS
`1PR20l5—OlO93
`
`Page 1
`
`Page 1
`
`NPS EX. 2055
`CFAD v. NPS
`IPR2015-01093
`
`

`
`Patent Application Publication
`
`Nov. 21, 2013 Sheet 1 of3
`
`US 2013/0310314 A1
`
`Teduglutide \
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`Page 2
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`Patent Application Publication
`
`Nov. 21, 2013 Sheet 2 of 3
`
`US 2013/0310314 A1
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`Patent Application Publication
`
`Nov. 21, 2013 Sheet 3 of 3
`
`US 2013/0310314 A1
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`Page 4
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`

`
`US 2013/0310314 A1
`
`Nov. 21,2013
`
`SOLID PHASE SYNTHESIS or
`H(GLY2)GLP-2
`
`SUBJECT OF THE INVENTION
`
`[0001] The present invention relates to a method of prepar-
`ing a peptide comprising the amino acid sequence His-Gly-
`Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-
`Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-
`Thr-Lys-Ile-Thr-Asp (SEQ ID NO: 1).
`In particular,
`the
`method comprises the steps of providing a first peptide frag-
`ment having a first protection group, which peptide fragment
`is conjugated to a support; providing a second peptide frag-
`ment having a second protection group; removing the first
`protection group from the first peptide fragment; and cou-
`pling the second peptide fragment to the N-terminally depro-
`tected,
`support-conjugated first peptide fragment. The
`present invention further relates to a method of preparing a
`pharmaceutical composition containing said peptide.
`
`BACKGROUND OF THE INVENTION
`
`[0002] Glucagon-like peptide-2 (GLP-2) is a 33 amino acid
`peptide having therapeutic applications in the treatment of
`diseases of the gastrointestinal tract. This naturally occurring
`hormone has been shown to regulate the growth, proliferation
`and maintenance of cells lining the gastrointestinal tract. In
`particular, it has been determined that GLP-2 and analogs
`thereof act as trophic agents to enhance and maintain the
`functioning of the gastrointestinal
`tract and to promote
`growth of intestinal tissue (see, e.g., European patent appli-
`cation EP 1 246 639 A2).
`[0003] Teduglutide, hereinafter also referred to as “h[Gly2]
`GLP-2”, is a single-chain and non-glycosylated 33-arnino
`acid peptide having the following sequence:
`
`Hisl — Glyz — Asp3 — Gly4 —Ser5 — Pheé —Ser7 —
`
`(SEQ ID NO5 1)
`
`Asps — Glug — Metlo — Asn“ — Thin — H213 — Len” —
`
`Aspls — Asnlé — Lew” — Alum — Ala” — Argzo —
`
`Aspzl — Phezz — H223 — Asn24 — Trpzs — Leuzé —
`
`H227 — Glnzs — Thrzg — Lys30 — H231 — Thr32 — Asp“
`
`[0004] This analog of GLP-2 differs from native GLP-2 by
`a change in one amino acid, i.e. alanine is replaced by glycine
`in position 2. This change has been determined to result in a
`peptide with a longer half-life. In particular, animal studies
`indicate that administration ofthis peptide produces a signifi-
`cant increase in both the mass and absorptive surface area of
`the epithelium lining the intestine, and moreover has a pro-
`nounced effect on reducing gut permeability.
`[0005] As many other therapeutic peptides, this GLP-2 ana-
`log can be manufactured recombinantly by expression in E.
`coli. However, in order to increase the production yield and to
`eliminate the need for some animal-derived raw materials in
`
`production, there was a need to provide alternative methods
`of preparing teduglutide.
`[0006]
`In the prior art, several solutions have been sought
`for chemically synthesizing peptides in general.
`[0007]
`Solid-phase peptide synthesis (SPPS) is a method
`introduced by Merrifield in 1963 (J. Amer. Chem. Soc. 1963,
`85: 2149-2154). Numerous peptides have been synthesized
`with this technique since then. A review of the chemical
`
`synthesis ofpeptides and proteins is provided by S. B. H. Kent
`(Armu. Rev. Biochem. 1988, 57: 957-989).
`[0008]
`In general, one strategy for the synthesis of peptide
`chains by solid-phase synthesis is the stepwise solid-phase
`synthesis. In stepwise SPPS, the C-terminal amino acid in the
`form of an N-[alpha]-protected, if necessary side-chain pro-
`tected reactive derivative is covalently coupled either directly
`or by means of a suitable linker to a solid support, e.g. a
`polymeric resin, which is swollen in an organic solvent. The
`N-[alpha]-protection group is removed, and the subsequent
`protected amino acids are added in a stepwise fashion. When
`the desired peptide chain length has been obtained, the side-
`chain protection groups are removed, and the peptide is
`cleaved from the support. Over the years, two major coupling
`strategies have been developed based on the use of different
`N-[alpha]-protection groups and matching side-chain protec-
`tion groups. Merrifield used t-butyloxycarbonyl (Boc) as the
`N-[alpha] protection group, while 9-fluorenylmethyloxy-car-
`bonyl (Fmoc) was introduced by Carpino and Han (J. Org.
`Chem. 1972, 37: 3404-3409).
`[0009] A general synthesis method for the preparation of
`GLP-2 molecules including teduglutide is described, e.g., in
`international patent applications WO 2006/ 1 17565 and WO
`2008/056155. According to these applications, peptides were
`synthesized batchwise in a polyethylene vessel equipped with
`a polypropylene filter for filtration using 9-fluorenylmethy-
`loxycarbonyl (Fmoc) as N-[alpha]-amino protection group
`and suitable common protection groups for side-chain func-
`tionalities. The amino acids were coupled as in situ generated
`N-hydroxybenzotriazole (HOBt) or 1-hydroxy-7-aza-benzo-
`triazole (HOAt) esters made from appropriate N-[alpha] -pro-
`tected amino acids and HOBt or HOAt by means of diisopro-
`pylcarbodiimide (DIC) in DMF. These substances can react
`with O-acylurea formed by the reaction of DIC and the car-
`boxylic acid group of the amino acid to form an active ester.
`Deprotection of the Fmoc group was performed by treatment
`with piperidine in DMF. Subsequently, the peptides were
`cleaved from the resins by treatment with 95% trifluoroacetic
`acid (TFA). The crude freeze-dried product was analyzed by
`high-performance liquid chromatography (HPLC) and iden-
`tified by mass spectrometry (MS).
`[0010] According to the prior art, GLP-2 molecules are
`being considered as candidates for standard chemical synthe-
`sis by the Fmoc-solid phase approach. It appeared to be a
`common understanding that GLP-2 molecules are probably
`best assembled in a linear fashion by solid phase chemistry
`due to the relative ease of assembly and the ultimate manu-
`facturing scale. However, numerous side reactions can occur
`during solidphase synthesis, some ofwhich are specific to the
`chemistries employed using Fmoc methodology.
`[0011]
`In particular, it has been found that one particular
`problem in the synthesis of teduglutide by Fmoc-solid phase
`chemistry involves rearrangement of the -Asp-Gly-bond at
`position 3-4 in the molecule resulting in the formation of the
`[beta]-Asp analogue (so-called “aspartimide by-product for-
`mation”). The [beta]-isomerization of -Asp-Gly- bonds
`involves the carboxy side-chain group from the aspartic acid
`forming a peptide bond with the [alpha]-amino group of the
`adjacent glycine via a succinimide intermediate. The main
`cause of this reaction is the treatment ofthe teduglutide-solid
`phase with piperidine, or other bases during the Fmoc
`removal stage. This reaction resulting in the undesired by-
`product is about 10% per N-terminal deprotection cycle but
`can be significantly higher.
`
`Page 5
`
`Page 5
`
`

`
`US 2013/0310314 A1
`
`Nov. 21,2013
`
`In particular, when teduglutide is assembled at a
`[0012]
`laboratory scale the piperidine treatment to remove the N-ter-
`minal Fmoc protection group usually takes a maximum of
`~10 minutes. However, at a large scale addition, filtration and
`removal of piperidine from the peptide-polymer takes much
`longer. The slower addition and mixing ofreagents at process
`scale results in an extended exposure of the peptide polymer
`to piperidine much longer which even may exaggerate the
`problem of aspartimide by-product formation.
`[0013] Accordingly, there is a need for alternative synthesis
`methods for preparing teduglutide, wherein aspartimide by-
`product formation can be reduced or even avoided. In particu-
`lar embodiments, such a route of synthesizing teduglutide
`should also be easy to accomplish and inexpensive. Further-
`more, such route of synthesizing teduglutide should be suited
`for industrial scale.
`
`[0014] Administration oftherapeutic peptides such as tedu-
`glutide further requires compositions that remain stable dur-
`ing storage. Because of their size and the resulting difficulty
`in crossing biological membranes and because of their sus-
`ceptibility to digestion, peptides are frequently administered
`parenterally. However, peptides can be particularly difficult
`to formulate because of their tendency to degrade over time
`and/or undergo aggregation and precipitation. Degradation,
`aggregation, and precipitation are all indicative of an unstable
`composition which may not be commercially viable. Com-
`position variables which affect the degradation of peptides
`during storage include pH, the quantity of salts present, and
`the type and quantity of excipients.
`[0015] Hence, there is also a need in the art for commer-
`cially suitable compositions of teduglutide which can be pre-
`pared using a commercially acceptable process.
`
`OBJECT AND SUMMARY OF THE INVENTION
`
`It is an object of the present invention to provide a
`[0016]
`method ofpreparing teduglutide, wherein aspartimide forma-
`tion can be reduced or even avoided. Accordingly, it is a
`further object of the present invention to provide a method of
`preparing teduglutide, which method is easy to accomplish
`and inexpensive. Furthermore, such method of preparing
`teduglutide should be suited for industrial scale. It is a further
`object of the present invention to provide a commercially
`acceptable method of preparing compositions of teduglutide.
`[0017] According to an embodiment of the present inven-
`tion, a method of preparing teduglutide is provided which
`breaks the assembly into two fragments.
`[0018]
`It has been found that the method according to the
`present invention may offer a series of advantages compared
`to the known synthesis method. In particular,
`it has been
`found that the level ofrearrangement at the Asp-Gly positions
`3-4 can be reduced by preparing the peptide via a fragment
`based assembly. This means that
`the peptide can be
`assembled up to the 5 position by solid phase assembly. The
`tetrapeptide corresponding to positions 1-4 of teduglutide
`(His-Gly-Asp-Gly) can be assembled separately, and then
`optionally be purified to remove the [beta]-Asp analogue
`before coupling to the 5-33 fragment on the solid phase.
`Accordingly, the peptide is not subjected to e.g. piperidine
`following coupling of this tetrapeptide fragment. Surpris-
`ingly, the method according to the present invention not only
`increases the purity of the peptide, but also the overall yield.
`[0019] These objectives as well as others which will
`become apparent from the ensuing description are attained by
`the subject matter of the independent claims. Some of the
`
`embodiments of the present invention are defined by the
`subject matter of the dependent claims.
`[0020]
`In one embodiment, the present invention relates to
`a method of preparing a peptide comprising the amino acid
`sequence His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-
`Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-
`Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp (SEQ ID NO:l), the
`method comprising the steps of:
`(a) providing a first peptide fragment comprising the amino
`acid sequence X-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-
`Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-
`Ile-Gln-Thr-Lys-Ile-Thr-Asp (SEQ ID NO:2), wherein X is a
`first protection group and the C-terminal residue of the first
`peptide fragment is conjugated to a support;
`(b) providing a second peptide fragment comprising the
`amino acid sequence Y-His-Gly-Asp-Gly (SEQ ID NO:3),
`whereinY is a second protection group;
`(c) removing the first protection group from the first peptide
`fragment; and
`(d) coupling the second peptide fragment to the N-terminally
`deprotected, support-conjugated first peptide fragment.
`[0021]
`In one embodiment, the first peptide fragment and/
`or the second peptide fragment are prepared by solid phase
`peptide synthesis. For example, the first peptide fragment
`and/or the second peptide fragment are provided by conju-
`gating the C-terminal amino acid residue to a support and
`sequentially adding appropriately protected amino acids to
`the N-terminus ofthe C-terminal, support-conjugated residue
`(5)
`In particular, amino acids to be sequentially added
`[0022]
`to the N-terminus of the C-terminal, support-conjugated resi-
`due(s) of the first peptide fragment and/or the second peptide
`fragment can each be protected by a protection group selected
`from the group consisting of Boc and Fmoc.
`[0023]
`In another embodiment of the present invention, the
`first protection group is Fmoc.
`[0024]
`In another embodiment of the present invention, the
`second protection group is an acid-labile protection group,
`optionally selected from the group consisting of Boc and
`benzyloxycarbonyl (Z).
`[0025] Typically, the histidine residue ofthe secondpeptide
`fragment can be protected at the side chain with a protection
`group which is e.g. selected from the group consisting of
`trityl, Boc, Bom and Bum.
`[0026] Also, the aspartic acid residue of the second peptide
`fragment can be protected at the side chain, e.g. with a tert-
`butyl ester protection group.
`[0027]
`In another embodiment, prior to coupling the sec-
`ond peptide fragment to the N-terminally deprotected, sup-
`port-conjugated first peptide fragment the second peptide
`fragment is cleaved from the support.
`[0028]
`In some embodiments, prior to coupling the second
`peptide fragment to the N-terminally deprotected, support-
`conjugated first peptide fragment the cleaved second peptide
`fragment is purified, optionally by chromatography and/or
`crystallization.
`[0029] According to one embodiment, the Fmoc protection
`group can be removed from the first peptide fragment by
`adding a secondary amine selected from the group consisting
`of piperidine, piperazine, morpholine and dicyclohexy-
`lamine.
`
`[0030] According to a further embodiment, the inventive
`method further comprises cleaving the first peptide fragment
`coupled to the second peptide fragment from the support.
`
`Page 6
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`

`
`US 2013/0310314 A1
`
`Nov. 21,2013
`
`In another embodiment, the method according to the
`[003 1]
`present invention further comprises purifying the cleaved first
`peptide fragment coupled to the second peptide fragment,
`optionally by chromatography.
`the present invention
`[0032]
`In a specific embodiment,
`relates to a method of preparing a peptide comprising the
`amino acid sequence His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-
`Glu-Met-Asn-Thr-lle-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-
`Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp (SEQ ID
`NO: 1), the method comprising the steps of:
`(a) providing a first peptide fragment comprising the amino
`acid sequence X-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-
`Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-
`Ile-Gln-Thr-Lys-Ile-Thr-Asp (SEQ ID NO:2) by peptide syn-
`thesis, wherein X is a Fmoc protection group and the
`C-terminal residue ofthe first peptide fragment is conjugated
`to a support;
`(b) providing a second peptide fragment comprising the
`amino acid sequence Y-His-Gly-Asp-Gly (SEQ ID NO:3) by
`peptide synthesis, wherein Y is an acid-labile protection
`group, optionally selected from the group consisting of Boc
`and benzyloxycarbonyl, and the C-terminal residue of the
`second peptide fragment is conjugated to a support;
`(c) cleaving the second peptide fragment from the support;
`(d) purifying the cleaved second peptide fragment, optionally
`by reversed-phase high-pressure liquid chromatography;
`(e) removing the Fmoc protection group from the first peptide
`fragment, optionally by adding a secondary amine selected
`from the group consisting of piperidine, piperazine, morpho-
`line and dicyclohexylamine;
`(f) coupling the second peptide fragment to the support-con-
`jugated first peptide fragment by adding the purified second
`peptide fragment to the N-terminally deprotected, support-
`conjugated first peptide fragment;
`(g) cleaving the support-conjugated first peptide fragment
`coupled to the second peptide fragment from the support; and
`(h) purifying the cleaved first peptide fragment coupled to the
`second peptide fragment, optionally by reversed-phase high-
`pressure liquid chromatography.
`[0033] According to one embodiment, the support is a func-
`tionalized polymer, optionally selected from the group con-
`sisting of polystyrene, polydimethylacrylarnide and polyeth-
`yleneglycol.
`[0034] Usually, the C-terminal amino acid of the first pep-
`tide fragment and/or the second peptide fragment is attached
`to the functionalized polymer by means ofa linker, optionally
`4-hydroxymethylphenoxyacetic acid (HMPA).
`[0035]
`In some embodiments, the first peptide fragment
`and/or the second peptide fragment are cleaved from the
`support by means of an acid, optionally selected from the
`group consisting of trifluoroacetic acid (TFA),
`trifluo-
`romethanesulfonic acid (TFMSA), hydrogen bromide (HBr),
`hydrogen chloride (HCl) and hydrogen fluoride (HF), or by
`means of a base, optionally a hydroxide.
`[0036]
`In another aspect, the present invention relates to a
`method of preparing a pharmaceutical composition contain-
`ing a peptide comprising the amino acid sequence His-Gly-
`Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-
`Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-
`Thr-Lys-Ile-Thr-Asp (SEQ ID NO: 1 ), the method comprising
`the steps of:
`(a) preparing the peptide according to the method of the
`present invention as described above; and
`
`(b) preparing a pharmaceutical composition containing the
`peptide prepared in step (a).
`[0037]
`In one embodiment, the pharmaceutical composi-
`tion further comprises a buffer, optionally a phosphate buffer
`in an amount sufficient to adjust the pH of the composition to
`a physiologically tolerable level, e.g. at a pH between from
`about 6 to about 9 or between from about 6.5 to about 8 or
`between from about 7 to about 7.5.
`
`In another embodiment, the pharmaceutical compo-
`[0038]
`sition further comprises L-histidine.
`[0039]
`Further, the pharmaceutical composition may com-
`prise a bulking agent which is optionally selected from the
`group consisting of mannitol and sucrose.
`[0040] According to a specific embodiment, the pharma-
`ceutical composition is provided as an injectable dosage
`form.
`
`invention
`the present
`In a further embodiment,
`[0041]
`relates to a peptide comprising the amino acid sequence His-
`Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-
`Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-
`Gln-Thr-Lys-Ile-Thr-Asp (SEQ ID NO:l), the peptide being
`obtainable by the method of the present
`invention as
`described above.
`
`FIGURE LEGENDS
`
`FIG. 1 depicts an HPLC of crude teduglutide mol-
`[0042]
`ecule assembled using the standard Fmoc solid phase proce-
`dure (linear assembly). Two major peaks were observed,
`namely the crude teduglutide peptide (peak 1) and the [beta] -
`Asp by-product (peak 2). This HPLC shows that the purity of
`the crude peptide assembled was 52% by HPLC and con-
`tained 24% of the [beta]-Asp analogue.
`[0043]
`FIG. 2 depicts an HPLC of crude teduglutide mol-
`ecule assembled using the standard Fmoc-solid phase proce-
`dure with extended piperidine treatment for the last 4 amino
`acids. Two major peaks were observed, namely the crude
`teduglutide peptide (peak 1) and the [beta]-Asp by-product
`(peak 2). This HPLC shows that the purity of the crude pep-
`tide assembled was only 39% by HPLC and contained 45% of
`the [beta]-Asp analogue.
`[0044]
`FIG. 3 depicts an HPLC of crude teduglutide mol-
`ecule assembled using the method according to the present
`invention (fragment-based assembly). A major peak and a
`smaller peak were observed, namely the crude teduglutide
`peptide (peak 1) and the [beta]-Asp by-product (peak 2). This
`HPLC shows that the purity of the crude peptide assembled
`was 59% by HPLC and contained only 17% of the [beta]-Asp
`analogue.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0045] The present invention is based on the finding that
`potential aspartimide by-product formation during the syn-
`thesis of teduglutide can be reduced or even avoided by pre-
`paring the peptide via a fragment based assembly. The
`method according to the present invention is capable of pro-
`viding teduglutide in high yield and/or a high purity. Further-
`more, the method according to the present invention can be
`used to prepare teduglutide at industrial process scale with a
`high yield and/or a high purity. It has further been found that
`a crude purity of at least 80% can be achieved by preparing
`teduglutide according to the method according to the present
`invention.
`
`Page 7
`
`Page 7
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`

`
`US 2013/0310314 A1
`
`Nov. 21,2013
`
`Purity and yield are important aspects of any route
`[0046]
`of peptide synthesis. Purity is represented by the degree of
`presence ofpharrnacologically active related impurities (such
`as the aspartimide by-product). In peptide synthesis repeated
`purifications at each step lead to a lower yield of the final
`peptide. The present
`invention provides a method that
`achieves a higher purity together with enhanced yield of the
`target peptide teduglutide through solid phase methodology
`when compared with known solid phase synthetic methods.
`[0047] The methods of the present invention will now be
`described with respect to particular embodiments and with
`reference to certain drawings but the invention is not limited
`thereto.
`
`[0048] Where the term “comprising” is used in the present
`description and claims, it does not exclude other elements or
`steps. For the purposes of the present invention, the term
`“consisting of” is considered to be an optional embodiment of
`the term “comprising of”. If hereinafter a group is defined to
`comprise at least a certain number of embodiments, this is
`also to be understood to disclose a group which optionally
`consists only of these embodiments.
`[0049] Where an indefinite or definite article is used when
`referring to a singular noun e. g. “a” or “an”, “the”, this
`includes a plural of that noun unless specifically stated.
`[0050] The term “about” in the context ofthe present inven-
`tion denotes an interval of accuracy that the person skilled in
`the art will understand to still ensure the technical effect ofthe
`
`feature in question. The term typically indicates deviation
`from the indicated numerical value of 110%, and in some
`embodiments 15%.
`
`Furthermore, the terms first, second, third and the
`[0051]
`like in the description and in the claims, are used for distin-
`guishing between similar elements and not necessarily for
`describing a sequential or chronological order. It is to be
`understood that the terms so used are interchangeable under
`appropriate circumstances and that the embodiments of the
`invention described herein are capable of operation in other
`sequences than described or illustrated herein.
`[0052]
`Further definitions of term will be given in the fol-
`lowing in the context of which the terms are used.
`[0053]
`In one embodiment, the present invention relates to
`a method of preparing a peptide comprising the amino acid
`sequence His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-
`Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-
`Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp (SEQ ID NO:l), the
`method comprising:
`(a) providing a first peptide fragment comprising the amino
`acid sequence X-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-
`Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-
`Ile-Gln-Thr-Lys-Ile-Thr-Asp (SEQ ID NO:2), wherein X is a
`first protection group and the C-terminal residue of the first
`peptide fragment is conjugated to a support;
`(b) providing a second peptide fragment comprising the
`amino acid sequence Y-His-Gly-Asp-Gly (SEQ ID NO:3),
`whereinY is a second protection group;
`(c) removing the first protection group from the first peptide
`fragment; and
`(d) coupling the second peptide fragment to the N-terminally
`deprotected, support-conjugated first peptide fragment.
`[0054] Hence, the present invention is directed to a method
`of preparing teduglutide which is a single-chain and non-
`glycosylated 33-amino acid peptide having the following
`sequence:
`
`Hisl — Glyz — Asp3 — Gly4 —Ser5 — Pheé — Ser7 —
`
`(SEQ ID NO5 1)
`
`Asps — Glug — Metlo — Asn“ — Thrlz — H213 — Lew” —
`
`Aspls — Asnlé — Lew” — Alum — Ala” — Argzo —
`
`Aspzl — Phezz — H223 — Asn24 — Trpzs — 1.61426 —
`
`Ile27 — Glnzs — Thrzg — Lys30 — H231 — Thr32 — Asp“
`
`[0055] This analog of GLP-2 (glucagon-like peptide 2) dif-
`fers from native GLP-2 by a change in one amino acid, i.e.
`alanine is replaced by glycine in position 2.
`[0056] The terms “polypeptide”, “peptide”, “oligopeptide”
`and “protein” are used interchangeably herein to refer to a
`polymer or oligomer of consecutive amino acid residues. The
`amino acids in such a polymer are joined together by the
`peptide bonds between the carboxyl and amino groups of
`adjacent amino acid residues. As used herein,
`the term
`“amino acid” refers not only to amino acid molecules or
`amino acid residues per se, but also to a list of abbreviations,
`letters, characters or words representing amino acid residues,
`e.g. amino acid residues being part of a peptide. Amino acids
`may be referred to herein by either their commonly known
`three letter symbols or by the one-letter symbols recom-
`mended by the IUPAC-IUB Biochemical Nomenclature
`Commission.
`
`[0057] Unless noted otherwise, the N-terminus of the pep-
`tide (e.g. at position 1) may be —H or a peptide bond (e.g., it
`is linked to an N-terminal blocking/protection group or to
`another amino acid or peptide fragment). Unless noted oth-
`erwise, the C-terminus ofthe peptide (e. g. at position 33) may
`be —OH or a peptide bond (e.g., it is linked to a C-terminal
`blocking/protection group or to another amino acid or peptide
`fragment).
`[0058] Unless noted otherwise, all amino acid position
`numbers are the position numbers according to the base
`sequence of teduglutide as represented by SEQ ID NO:l.
`[0059]
`In the context of the present invention, the term
`“peptide fragment” refers to a specific fragment of tedug-
`lutide, in particular a first peptide fragment comprising the
`amino acid sequence X-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-
`Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-
`Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp (SEQ ID NO:2), wherein
`X is a first protection group, and a second peptide fragment
`comprising the amino acid sequence Y-His-Gly-Asp-Gly
`(SEQ ID NO:3), whereinY is a second protection group.
`[0060]
`In one embodiment of the present invention, tedug-
`lutide is synthesized by coupling the carboxyl group or C-ter-
`minus of one amino acid or peptide fragment to the amino
`group or N-terminus of another amino acid or peptide frag-
`ment. Chemical peptide synthesis usually starts at the C-ter-
`minal end of the peptide and ends at the N-terminus. This is
`the opposite direction to the direction ofprotein biosynthesis,
`which starts at the N-terminal end. Due to amino acid
`
`excesses typically used to ensure complete coupling during
`each synthesis step, polymerization of amino acids is com-
`mon in reactions where each amino acid is not protected. In
`order to prevent this polymerization, protection groups are
`typically used in the method according to the present inven-
`tion.
`
`Suitable protective groups are well-known to a per-
`[0061]
`son skilled in the art (e.g., see “Fmoc-Solid Phase Peptide
`
`Page 8
`
`Page 8
`
`

`
`US 2013/0310314 A1
`
`Nov. 21,2013
`
`Synthesis-A practical approach”, W. C. Chan, P. D. White,
`Oxford University Press Inc. New York, 2000).
`[0062] Removing the protection groups from the peptide
`fragments can e.g. be accomplished by adding a suitable
`deprotection reagent which depends on the protection group
`being used. Typical standard protection groups for [alpha]-
`amino functions of the coupled amino acids are Boc, which
`can be removed by treatment with a strong acid, or Fmoc,
`which canbe removed with a base. The abbreviations “Fmoc”
`
`and “Boc” as used herein mean 9H-fluoren-9-ylmethoxycar-
`bonyl and t-butyloxycarbonyl, respectively.
`[0063] The t-Boc (“tert-butyloxycarbonyl” or more simply
`“Boc”) group is commonly used for protecting the terminal
`amine of the peptide, typically requiring the use of more acid
`stable groups for side chain protection in orthogonal strate-
`gies. Boc groups can be added to amino acids with Boc
`anhydride and a suitable base.
`is cur-
`[0064]
`Fmoc (9H-fluoren-9-ylmethoxycarbonyl)
`rently a widely used protective group. Fmoc is cleaved under
`very mild basic conditions (e. g. piperidine). This allows mild
`acid labile protection groups that are stable under basic con-
`ditions, such as Boc and t-butyl groups, to be used on the
`side-chains of amino acid residues of the target peptide.
`[0065] According to the method of the present invention,
`the first peptide fragment comprises a first protection group
`and the second peptide fragment comprises a second protec-
`tion group. In some embodiments, the first protection group is
`different from the second protection group. Alternatively, the
`first and second protection groups can be identical.
`[0066]
`Furthermore, also the side chain functionality in the
`peptide pre-sequence is typically protected during the cou-
`pling steps. More than half of the amino acids commonly
`encountered in peptides have side chains that contain reactive
`groups. In peptide synthesis, in particular in solid phase syn-
`thesis, it is usual for all these potentially reactive groups to be
`masked because of the rather harsh conditions employed and
`the need to achieve the highest level of efficiency in all chemi-
`cal reactions. For routine synthesis, protection groups that are
`removed with trifluoroacetic
`acid (TFA)
`are usually
`employed as this allows the peptide to be globally deprotected
`at the same time as it is cleaved or released from the support.
`Furthermore, a wide range of groups is also available which
`can be selectively removed during synthesis (e.g., on the solid
`phase), thus enabling the selective modification of side chains
`of individual residues within the peptide chain.
`[0067]
`For instance, the Boc group can be used for amino
`functions (e.g., Lys and His), tert-butyl esters can be used for
`acidic groups (e.g., Asp and Glu) and tert-butyl ethers can be
`used for hydroxyl groups (e.g., Tyr, Thr and Ser). Further
`suitable protection groups for side chain protection are
`readily available and well-known to a person skilled in the art
`(e.g., see Table 4 on pages 20-25 in “Fmoc-Solid Phase Pep-
`tide Synthesis-A practical approach”, W. C. Chan, P. D.
`White, Oxford University Press Inc. New York, 2000; being
`incorporated herein by reference).
`[0068]
`In one embodiment,