Process for synthesizing glucagon-like peptide 2 (GLP-2) analogues

Abstract

The present invention relates to a process for obtaining glucagon-like peptide 2 (GLP-2) analogues, such as grepaglutide. In particular, the process described herein uses a multi-step purification method for GLP-2 analogues synthesized by solid phase peptide synthesis (SPPS).

Description

[Technical field]

[0001] The present invention relates to a process for obtaining glucagon-like peptide-2 (GLP-2) analogues. [Background technology]

[0002] Human GLP-2 is a 33 amino acid peptide with the following sequence: Hy-His-Ala-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-OH (SEQ ID NO: 10) (where Hy is hydrogen). It is derived from specific post-translational processing of proglucagon in enteroendocrine L-cells of the intestine and in specific regions of the brainstem. GLP-2 binds to a single G protein-coupled receptor belonging to the class II glucagon-secretin family.

[0003] GLP-2 has been reported to induce significant proliferation of the small intestinal mucosal epithelium through stimulation of stem cell proliferation in the crypts and inhibition of apoptosis in the villi (Drucker et al., 1996, Proc. Natl. Acad. Sci. USA 93:7911-7916). GLP-2 also has the effect of promoting colonic growth. In addition, GLP-2 inhibits gastric emptying and gastric acid secretion (Wojdemann et al., 1999, J. Clin. Endocrinol. Metab. 84:2513-2517), enhances intestinal barrier function (Benjamin et al., 2000, Gut 47:112-119), stimulates intestinal hexose transport via upregulation of glucose transporters (Cheeseman, 1997, Am. J. Physiol. R 1965-71), and increases intestinal blood flow (Guan et al., 2003, Gastroenterology, 125:136-147).

[0004] It has been recognized in the art that glucagon-like peptide-2 receptor analogs have therapeutic potential for the treatment of intestinal diseases. However, native hGLP-2, a 33-amino acid gastrointestinal peptide, is not very useful in clinical practice due to its very short half-life in humans, about 7 minutes for full-length GLP-2 [1-33] and about 27 minutes for truncated GLP-2 [3-33]. In large part, the short half-life is due to degradation by the enzyme dipeptidyl peptidase IV (DPP-IV). Thus, the art has attempted to develop GLP-2 receptor agonists with better pharmacokinetic properties, and in particular to improve the half-life of the GLP-2 molecule. As an example, GLP-2 analogs with substitutions have been suggested, such as a GLP-2 analog ([hGly2]GLP-2, teduglutide) containing a Gly substitution at position 2 that extends the half-life from 7 minutes (native GLP-2) to about 2 hours. Acylation of peptide drugs with fatty acid chains has also proven beneficial for extending systemic circulation without destroying biological efficacy, as well as for enhancing enzymatic stability.However, these efforts improve the pharmacokinetics of GLP-2 analogs, which are sometimes described in the art as "long-acting", but it must be kept in mind that this is in comparison with native hGLP-2, whose half-life is on the order of hours rather than minutes.This means that GLP-2 analogs still need to be administered to patients once or multiple times per day.

[0005] WO2006 / 117565 discloses [hGly 2] GLP-2 analogs are described that contain one or more substitutions compared to GLP-2 and have improved biological activity in vivo and / or improved chemical stability, for example as assessed in an in vitro stability assay. In particular, GLP-2 analogs are described that have substitutions at one or more of positions 8, 16, 24 and / or 28 of the wild-type GLP-2 sequence, optionally combined with further substitutions at positions 2 and one or more of positions 3, 5, 7, 10 and 11, and / or with one or more deletions of amino acids 31-33. These substitutions can also be combined with the addition of N- or C-terminal stabilizing peptide sequences.

[0006] Among the molecules disclosed in WO2006 / 117565 is ZP1848 (also called grepaglutide), which has been designed to improve chemical stability and / or biological activity. Dosage regimes for GLP-2 analogs, including ZP1848 and its metabolite (i.e., grepaglutide), are described in WO2018 / 229252. Summary of the Invention

[0007] Broadly, the present invention relates to improved methods for synthesizing and purifying glucagon-like peptide-2 (GLP-2) analogs, such as ZP1848. Accordingly, the present invention provides a method for producing a glucagon-like peptide 2 (GLP-2) analogue synthesized by solid phase peptide synthesis (SPPS), the GLP-2 analogue having the following formula: R 1 -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z 2 -R 2 (SEQ ID NO:11) (In the formula, R 1 is hydrogen, C 1~4alkyl (e.g., methyl), acetyl, formyl, benzoyl, or trifluoroacetyl; X5 is Ser or Thr; X11 is Ala or Ser, R 2 NH 2 or OH, Z 2 is a peptide sequence of 1 to 6 amino acid units of either Lys or none or a pharma- ceutically acceptable salt or derivative thereof.

[0008] In a first aspect, the present invention provides a method for producing a GLP-2 analogue synthesized by solid phase peptide synthesis (SPPS), comprising the steps of: i) loading the crude GLP-2 analog onto a column; ii) adjusting the pH of the column with a first buffer system; Step ii) provides a method comprising passing the isoelectric point of the peptide while it is on the column.

[0009] In the context of this specification, the step of adjusting the pH of the column, as shown in FIG. 1, is referred to as "Pre-RPC1". During this Pre-RPC1, O→N transacylation occurs. It is understood that the transacylation occurs while the peptide is on the column. In other words, step ii) involves passing the isoelectric point of the peptide while it is on the column and performing O→N transacylation.

[0010] More specifically, N→O transacylation occurs during peptide cleavage of GLP-2 analogs in published methods, and such transacylation is recognized in the art, and it is known that adjusting the pH of the peptide in solution to neutral and back to acidic prior to subsequent purification steps reduces these impurities and effectively performs the O→N transacylation.

[0011] Before the present invention, this pH adjustment was performed in solution. However, with the GLP-2 analogue of the present invention, undesirable precipitation is observed. Surprisingly, the present inventors have found that this O→N acyl group transfer can be performed on column, which can avoid the loss of material and the complicated processing caused by precipitation.

[0012] The isoelectric passing of peptides on a column constitutes, to the best of the inventors' knowledge, a new general methodology, which in effect carries out this "conversion" or "purification" step on a column rather than in solution.

[0013] In other words, the present invention provides a method for performing O→N acyl group transactivation on a GLP-2 analog while the GLP-2 analog is on the column (pre-RPC1). The use of phosphate buffer for this type of peptide synthesis is desirable because it is known to efficiently remove unwanted oligomers and C-terminal deamidation products. However, GLP-2 analogs of the claimed structure are known to precipitate in phosphate buffer, making the use of phosphate buffer problematic for O→N acyl transfer. It is therefore even more surprising that we can avoid material loss by using phosphate buffer. This performance of on-column acyl transfer using a pH adjustment step provides the additional advantage of eliminating a process step (separate pH adjustment in solution).

[0014] Preferably, the first buffer system is a phosphate buffer system, in other words the first buffer system is based on phosphate buffer / phosphoric acid. The isoelectric point pH (I) of a peptide can be calculated using methods known in the art or can be experimentally measured. It is the pH at which the overall charge of the molecule is zero (neutral charge) or is electrically neutral on statistical average.

[0015] In the present invention, passing the isoelectric point is understood to mean raising the pH of the column from an acidic pH, i.e. passing the isoelectric point of the peptide while it is on the column can include adjusting the acidic pH of the column to a neutral pH (pH 7), preferably a slightly basic (alkaline) pH, for example ≧7.2, for example about 7.5.

[0016] Thus, in some embodiments, the process for purifying a GLP-2 analogue synthesized by solid phase peptide synthesis (SPPS) comprises: i) loading the crude GLP-2 analog onto a column; ii) adjusting the pH of the column with a first buffer system; Step ii) involves the sequential use of buffers to increase the pH of the column above 7.5 and then to decrease the pH of the column to an acidic pH. [Pre-RPC1] An acidic pH is less than pH 7, such as less than pH 5, for example less than pH 3.

[0017] Preferably the pH of the column is reduced to < 2.5, ie below pH 2.5. Advantageously, step ii) involves on-column O→N acyl group transacylations to hydrolyze acetylated and trifluoroacetylated impurities, which allows for the avoidance of undesired precipitations while still achieving the desired chemical transformation.

[0018] In the examples described herein, the column is a C18 column, but the invention is not so limited. Suitable columns will be apparent to those of skill in the art. In some embodiments, the column is a C18 column. In some embodiments, the column is a C8 column.

[0019] Advantageously, this on-column O→N transacylation can be accomplished on the same column as the subsequent purification step. The present invention further provides a method for purifying a GLP-2 analogue synthesized by SPPS via a four-step chromatographic purification process (referred to herein as RPC1-4). As described above, the on-column O→N acyl group transfer (Pre-RPC1) can be performed on the same column as the first "purification" step (RPC1). Alternatively, the four-step chromatographic purification process can be performed on a GLP-2 analogue synthesized by SPPS that has already been subjected to a pH adjustment process in solution to perform the O→N acyl group transfer.

[0020] In some embodiments, the present invention can provide a five step chromatographic purification process for purifying a GLP-2 analogue synthesized by SPPS, the method comprising: (1) Pre-RPC1 i) loading a GLP-2 analog onto a column; ii) adjusting the pH of the column with a first buffer system; Step ii) involves the sequential use of a buffer to increase the pH of the column above 7.5 and then to decrease the pH of the column to an acidic pH; (2) RPC1 i) eluting the pool containing the GLP-2 analogue using a first buffer system, then (3) RPC2 i) loading the pool containing the GLP-2 analogue obtained in step (2) onto a column; ii) washing the column with a second buffer system containing trifluoroacetic acid and eluting the pool containing the GLP-2 analogue; and (4) RPC3 i) loading the pool containing the GLP-2 analogue obtained in step (3) onto a column; ii) washing the column with a third buffer system comprising acetic acid / ammonium acetate and eluting the pool containing the GLP-2 analogue; and (5) RPC4 i) loading the pool containing the GLP-2 analogue obtained in step (4) onto a column; ii) washing the column with a fourth buffer system comprising acetic acid / ammonium acetate and eluting the pool containing the GLP-2 analogue.

[0021] It is understood that the GLP-2 analogue-containing pool obtained at any step may be loaded directly onto a column for the next step or may be concentrated by evaporation. In some embodiments, the method further comprises a desalting step in which the GLP-2 analogue obtained in step (5) is loaded onto a column and washed with a buffer system comprising 10 mM AcOH and acetonitrile.

[0022] In a further aspect, the present invention relates to a method for synthesizing a GLP-2 analogue by solid phase peptide synthesis (SPPS), comprising: 2 is a peptide sequence of 1 to 6 amino acid units of Lys, and Z 2 At least one of the Lys units in is protected with a trityl protecting group during synthesis.

[0023] The trityl group is

[0024] [ka]

[0025] It is. Preferably, Z 2 is a peptide sequence of 2 to 6 amino acid units of Lys, and the method comprises the steps of: i) coupling the first P-Lys(Trt)-OH to a solid peptide resin using a linker; ii) removing the P group from the Lys(Trt) amino acid unit; iii) attaching the second P-Lys(Trt)-OH to the Lys(Trt) amino acid unit bound to the solid peptide resin using a linker; iv) removing the P group from the second Lys(Trt) amino acid unit; v) coupling subsequent amino acid units until the GLP-2 analog is synthesized; vi) cleaving the GLP-2 analog from the solid peptide resin; vii) purifying the GLP-2 analogue, Each P is a protecting group, for example Fmoc.

[0026] Use of this Lys(Trt) strategy during SPPS results in the disappearance of all Lys(Boc)→Lys(t-Bu) adduct t-Bu peaks present in the Lys(Boc) crude product, including the most problematic peak present at relative retention time 1.1.

[0027] In some embodiments, Z 2 is a peptide sequence of six amino acid units of Lys, and Lys 39 and Lys 38 is attached as P-Lys(Trt)-OH. 2 The remaining Lys amino acid units are attached as P-Lys(Boc)-OH. That is, the lysine tail during synthesis is [K(Boc)] 4 [K(Trt)] 2 It is.

[0028] Surprisingly, the inventors have found that it is possible to trityl-protect two of the six amino acid units of Lys, e.g., Lys 39 and Lys 38 It has been found that protecting Lys is sufficient to prevent the formation of t-Bu impurities from the crude product. 39 and Lys 38 The rationale for using Lys(Trt) in Fmoc / t-Bu SPPS is that i) the use of Lys(Boc) in Fmoc / t-Bu SPPS results in the formation of Lys-t-Bu adducts (Pawlas et al., Peptides, 2014, 108), and ii) the Lys(Boc) side chain closest to the C-terminus (i.e., Lys in the peptides described herein).39 and Lys 38 ) is responsible for the formation of an additional t-Bu impurity that elutes closest to the main peak, as revealed in unpublished work by the PolyPeptide Group. We have observed that Lys(Boc) residues further away from the C-terminus cause the formation of a +56 Da adduct that elutes further away and is therefore less problematic during downstream processing. Therefore, for these reasons, it is recommended to focus on those Lys positions that are understood to be involved in the formation of the critical Lys-t-Bu adduct, namely Lys 38 and Lys 39 It is reasonable to use Lys(Trt) only for

[0029] Embodiments of the invention will now be described hereinafter by way of example, and not by way of limitation, with reference to the accompanying figures. However, various further aspects and embodiments of the invention will be apparent to those skilled in the art in light of the present disclosure.

[0030] As used herein, "and / or" is considered a specific disclosure of each of the two specified features or components with or without the other feature or component. For example, "A and / or B" is considered a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein.

[0031] Unless the context requires otherwise, the feature descriptions and definitions set forth above are not intended to be limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described. [Brief description of the drawings]

[0032] [Figure 1] FIG. 1 shows an overview of the O→N acyl group transfer (Pre-RPC1) and four-step (RPC1-4) chromatographic purification process for purifying GLP-2 analogues synthesized by SPPS. [Diagram 2] Figure 1 shows three syntheses of ZP1848 by +56 Da EIC MS. Main peak at approximately 28 min. Top panel: Purified ZP1848 from synthesis with [K(Boc)]6. Note remaining +56 Da impurity. Middle panel: Crude product from synthesis with [K(Boc)]6. Bottom panel: Crude product from synthesis with [K(Trt)]6. [Diagram 3] Figure 1 shows two Fmoc-K6-NH2 crude products by +56Da EIC MS. Top panel: Crude product from [K(Boc)]6 resin. Bottom panel: Crude product from [K(Boc)]4[K(Trt)]2 resin. [Figure 4] Graphs showing ZP1848 after cleavage: Top graph: after 1.5 h in cleavage solution (same procedure as in Example 1.11); Bottom graph: after pH treatment with RPC1 (same procedure as in Example 1.12). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] The present invention provides a method for the purification of GLP-2 analogues. As described herein, the purification steps (unless otherwise stated) are carried out on a column. Preferably, the column is a C18 column.

[0034] Chromatographic purification methods are well known in the art. In one aspect, the present invention relates to the surprising insight that purification steps, including chemical modification (i.e., acyl group translocation), can be performed on a column rather than in solution. The present invention further provides a four-step chromatographic purification process for GLP-2 analogs. As described herein, the four-step chromatographic purification process produces GLP-2 with low levels of impurities in high yields.

[0035] Thus, the process of the invention for purifying a GLP-2 analogue preferably comprises a series of column-based processes. These processes include the steps of loading a solution containing the GLP-2 analogue onto a column, followed by washing and elution with a buffer system, in each case obtaining a pool containing the GLP-2 analogue. Each washing process comprises two or more steps. It is understood that in chromatographic purification, the change of eluent (buffer) is usually achieved by applying a gradient.

[0036] In some embodiments, the process for purifying a GLP-2 analog synthesized by solid phase peptide synthesis (SPPS) comprises the following steps: i) loading the GLP-2 analog onto a column; ii) adjusting the pH of the column by washing with a first buffer system; The first buffer system comprises: Buffer A: 0.1% H 3 PO 4 and, Buffer B: MeCN, Buffer C: 45mM H 3 PO 4 , pH 2.2 + 100 mM NaCl, Buffer D: 45mM H 3 PO 4 , pH 7.5-8.0 + 100 mM NaCl, The column was pre-equilibrated with a mixture of 95% buffer C and 5% buffer B. The washing sequence is as follows: Step 1: A mixture of 95% buffer C and 5% buffer B, Step 2: A mixture of 95% buffer D and 5% buffer B until the pH is higher than 7.5; Step 3: A mixture of 95% buffer C and 5% buffer B until the pH is below 2.5.

[0037] In step 1, as is common in column-based purification, the column is homogenized, or equilibrated. Steps 2 and 3 may be referred to herein as "Pre-RPC1." As described herein, pH adjustment in Pre-RPC1 advantageously induces O→N acyl group transfer, which is conventionally performed in solution, to remove impurities resulting from unwanted N→O acyl group transfer during synthesis.

[0038] After step 3, the column may be washed with a solution containing 93% A and 7% B. Preferably, the washing further comprises: Step 4: Increasing the content of Buffer B from 7% to 22% (Buffer A from 93% to 78%), preferably linearly over 12 column volumes; and Step 5: Preferably involves obtaining a pool containing the GLP-2 analogue, which is a mixture of 22% Buffer B and 78% Buffer A until elution is complete.

[0039] Steps 4 and 5 are the elution of the GLP-2 analogue and are referred to herein as the "RPC1" or "first dimension" chromatographic purification of the peptide. This purification is the result of differences in the interactions between the peptide and impurities with the column medium and elution medium.

[0040] In some embodiments, the process for purifying a GLP-2 analog synthesized by solid phase peptide synthesis (SPPS) comprises the following steps: iii) loading the pool containing the GLP-2 analogue obtained in step ii) onto a column; iv) washing the column with a second buffer system; The second buffer system is Buffer A: 0.1% TFA, Buffer B: containing MeCN, The column was pre-equilibrated with a mixture of 90% buffer A and 10% buffer B. The washing sequence is as follows: Step 1: A mixture of 90% buffer A and 10% buffer B, Step 2: Increasing the content of buffer B from 10% to 19% (buffer A from 90% to 81%), preferably linearly over one column volume; and Step 3: Linearly increasing the content of buffer B from 19% to 30% (buffer A from 81% to 70%), preferably over 12 column volumes, then continuing to wash the column to elute the pool containing the GLP-2 analogue.

[0041] Steps iii) and iv) are sometimes referred to herein as "RPC2" or "2-dimension". In some embodiments, the process for purifying a GLP-2 analog synthesized by solid phase peptide synthesis (SPPS) comprises the following steps: v) loading the pool containing the GLP-2 analogue obtained in step iv) onto a column; iv) washing the column with a third buffer system; The column was pre-equilibrated with a mixture of 85% buffer A and 15% buffer B. The third buffer system is Buffer A: 100mM NH 4 OAc+0.5% AcOH, Buffer B: containing MeCN, The washing sequence is as follows: Step 1: A mixture of 85% buffer A and 15% buffer B, Step 2: Increasing the content of buffer B from 15% to 29% (buffer A from 85% to 71%), preferably linearly over one column volume; Step 3: Increasing the content of Buffer B from 29% to 37% (Buffer A from 71% to 63%), preferably linearly over 10 column volumes; and Step 4: Elute the pool containing the GLP-2 analogue with a mixture of 30% Buffer A and 70% Buffer B.

[0042] Steps v) and vi) are sometimes referred to herein as "RPC3" or "three-dimensional." In some embodiments, the process for purifying a GLP-2 analog synthesized by solid phase peptide synthesis (SPPS) comprises the following steps: vii) loading the pool containing the GLP-2 analogue obtained in step vi) onto a column; viii) washing the column with a fourth buffer system; The fourth buffer system is Buffer A: 5mM NH 4 OAc+0.1% AcOH, Buffer B: MeCN, Buffer C: 100mM NH 4 OAc+0.5% AcOH, The column was pre-equilibrated with a mixture of 90% buffer C and 10% buffer B. The washing sequence is as follows: Step 1: A mixture of 90% Buffer C and 10% Buffer B, preferably in one column volume; Step 2: A mixture of 90% Buffer A and 10% Buffer B, preferably in one column volume; Step 3: Increasing the content of buffer B from 10% to 13% (buffer A from 90% to 87%), preferably linearly over one column volume; and Step 4: Linearly increasing the content of Buffer B from 13% to 25% (Buffer A from 87% to 75%), preferably over 12 column volumes, to elute the pool containing the GLP-2 analogue.

[0043] Steps vii) and vii) are sometimes referred to herein as "RPC4" or the "fourth dimension." In some embodiments, the process for purifying a GLP-2 analog synthesized by solid phase peptide synthesis (SPPS) comprises the following steps: ix) loading the pool containing the GLP-2 analogue obtained in step viii) onto a column; x) washing the column with a desalting buffer system; The desalting buffer system is Buffer A: 10 mM AcOH, Buffer B: containing MeCN, The column was pre-equilibrated with a mixture of 95% A and 5% B. The washing sequence is as follows: Step 1: A mixture of 95% A and 5% B, and Step 2: Increasing the content of buffer B from 5% to 50% (buffer A from 95% to 50%), preferably linearly over one column volume.

[0044] Steps ix) and x) are sometimes referred to as "desalting" or "SPE". The process for purifying a GLP-2 analogue synthesized by solid phase peptide synthesis (SPPS) may further comprise a lyophilization step.

[0045] Preferably, the column is a C18 column, although the invention is not so limited. Any suitable column can be used. In a further aspect, the present invention relates to a method for synthesizing a GLP-2 analogue by solid phase peptide synthesis (SPPS), comprising: 2 is a peptide sequence of 1 to 6 amino acid units of Lys, and Z 2 At least one of the Lys units in is protected with a trityl protecting group during synthesis.

[0046] Z 2 In some embodiments, where R is a peptide sequence of 1 to 6 amino acid units of Lys, a method for synthesizing a GLP-2 analog by solid phase peptide synthesis (SPPS) comprises the steps of: i) coupling Fmoc-Lys(Trt)-OH to a solid peptide resin using a linker; ii) removing the Fmoc groups from the Lys(Trt) amino acid units; iii) coupling subsequent amino acid units until the GLP-2 analog is synthesized; iv) cleaving the GLP-2 analogue from the solid peptide resin; and v) purifying the GLP-2 analogue.

[0047] Z 2 In some embodiments, where R is a peptide sequence of 2 to 6 amino acid units of Lys, the method of synthesizing a GLP-2 analog comprises the steps of: i) coupling a first Fmoc-Lys(Trt)-OH to a solid peptide resin using a linker; ii) removing the Fmoc groups from the Lys(Trt) amino acid units; iii) attaching a second Fmoc-Lys(Trt)-OH to the Lys(Trt) amino acid unit bound to the solid peptide resin using a linker; iv) removing the Fmoc group from the second Lys(Trt) amino acid unit; v) coupling subsequent amino acid units until the GLP-2 analog is synthesized; vi) cleaving the GLP-2 analog from the solid peptide resin; vii) purifying the GLP-2 analogue.

[0048] Therefore, Z 2 If is a peptide sequence of six amino acid units of Lys, then the motif is [K(Boc)] 4 [K(Trt)] 2 In another embodiment, the motif is [K(Boc)] 4 [K(Trt)] 2 , [K(Boc)] 3 [K(Trt)] 3 , [K(Boc)] 2 [K(Trt)] 4 , [K(Boc)] 1 [K(Trt)] 5 , or [K(Trt)] 6 That is, the subsequent steps of the Lys amino acid unit attached in (v) may be protected with Trt or Boc.

[0049] In some embodiments, the motif is [K(Boc)] 4 [K(Trt)] 2 or [K(Trt)] 6 In some embodiments, the motif is [K(Boc)] 4 [K(Trt)] 2 It is. definition Unless otherwise stated, the following definitions are set forth for specific terms used in the above description.

[0050] Throughout the specification and claims, the conventional one-letter and three-letter codes for natural amino acids are used. All amino acid residues in the peptides of the invention are preferably of the L-configuration. GLP-2 analogue The glucagon-like peptide 2 (GLP-2) analogs of the present invention have the following formula: R 1 -Z 1 -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z 2 -R 2 (In the formula, R 1 is hydrogen, C 1~4 alkyl (e.g., methyl), acetyl, formyl, benzoyl, or trifluoroacetyl; X5 is Ser or Thr; X11 is Ala or Ser, R 2 NH 2 or OH, Z 1 and Z 2 are independently absent or a peptide sequence of 1 to 6 amino acid units of Lys), or a pharma- ceutically acceptable salt or derivative thereof.

[0051] Z 1 and Z 2 is independently present and / or absent or a peptide sequence of 1 to 6 amino acid units of Lys, i.e. 1, 2, 3, 4, 5 or 6 Lys residues. The Lys residues may have either the D- or L-configuration, but preferably have the L-configuration. Particularly preferred sequences Z are sequences of 4, 5 or 6 consecutive lysine residues, in particular 6 consecutive lysine residues. Exemplary sequences Z are given in WO01 / 04156.

[0052] In some embodiments, R 1 is hydrogen. In some embodiments, X5 is Thr. In some embodiments, X11 is Ala. In some embodiments, R 2 NH 2 It is.

[0053] In some embodiments, Z 1 does not exist. In some embodiments, Z 2 is a peptide sequence of 1 to 6 amino acid units of Lys. 2 is a peptide sequence of 2 to 6 amino acid units of Lys. 2 is a peptide sequence of 3 to 6 amino acid units of Lys. 2 is a peptide sequence of 4 to 6 amino acid units of Lys. 2 is a peptide sequence of 5 to 6 amino acid units of Lys. 2 is a peptide sequence of six amino acid units of Lys. 2 is a peptide sequence of 1-2 amino acid units of Lys.

[0054] In some embodiments, the glucagon-like peptide 2 (GLP-2) analogs of the present invention have the following formula: R 1 -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z 2 -R 2 (In the formula, R 1 is hydrogen, C 1~4 alkyl (e.g., methyl), acetyl, formyl, benzoyl, or trifluoroacetyl; X5 is Ser or Thr; X11 is Ala or Ser, R 2 NH 2 or OH, Z 2 is a peptide sequence of 1 to 6 amino acid units of either Lys or none or a pharma- ceutically acceptable salt or derivative thereof.

[0055] In some embodiments, Z 2 is a peptide sequence of 1 to 6 amino acid units of Lys. In some embodiments of the invention, in the above formula, X5 is Thr and / or X11 is Ala. Examples of these glucagon-like peptide 2 (GLP-2) analogs are: ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH 2 (SEQ ID NO:1) ZP2949 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKK-OH (SEQ ID NO:2); ZP2711 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKK-OH (SEQ ID NO: 3); ZP2469 H-HGEGTFSSELATILDALAARDFIAWLIATKITDK-OH (SEQ ID NO: 4); ZP1857 H-HGEGTFSSELATILDALAARDFIAWLIATKITD-NH 2 (SEQ ID NO:5); or ZP2530 H-HGEGTFSSELATILDALAARDFIAWLIATKITD-OH (SEQ ID NO: 6) Includes.

[0056] In some embodiments of the invention, the glucagon-like peptide 2 (GLP-2) analogue is ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH 2 (Sequence Number 1).

[0057] That is, the GLP-2 analog has the following formula: H-His 1 -Gly 2 -Glu 3 -Gly 4 -Thr 5 -Phe 6 -Ser 7 -Ser 8 -Glu 9 -Leu 10 -Ala 11 -Thr 12 -Ile 13 -Leu 14 -Asp 15 -Ala 16 -Leu 17 -Ala 18 -Ala 19 -Arg 20 -Asp 21 -Phe 22 -Ile 23 -Ala 24 -Trp 25 -Leu 26 -Ile 27 -Ala 28 -Thr 29 -Lys 30 -Ile 31 -Thr 32-Asp 33 -Lys 34 -Lys 35 -Lys 36 -Lys 37 -Lys 38 -Lys 39 -NH 2 It is expressed as:

[0058] In some embodiments of the invention, in the above formula, X5 is Ser and / or X11 is Ser. Examples of these glucagon-like peptide 2 (GLP-2) analogs include: ZP1846 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH 2 (SEQ ID NO:7); ZP1855 H-HGEGSFSSELSTILDALAARDFIAWLIATKITD-NH 2 (SEQ ID NO:8); or ZP2242 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDK-OH (SEQ ID NO: 9) Includes.

[0059] In one embodiment of the invention, the glucagon-like peptide 2 (GLP-2) analog is ZP1846 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH 2 (Sequence number 7).

[0060] That is, the GLP-2 analog has the following formula: H-His 1 -Gly 2 -Glu 3 -Gly 4 -Ser 5 -Phe 6 -Ser 7 -Ser 8 -Glu 9 -Leu 10 -Ser 11 -Thr 12 -Ile 13 -Leu 14 -Asp15 -Ala 16 -Leu 17 -Ala 18 -Ala 19 -Arg 20 -Asp 21 -Phe 22 -Ile 23 -Ala 24 -Trp 25 -Leu 26 -Ile 27 -Ala 28 -Thr 29 -Lys 30 -Ile 31 -Thr 32 -Asp 33 -Lys 34 -Lys 35 -Lys 36 -Lys 37 -Lys 38 -Lys 39 -NH 2 It is expressed as:

[0061] In some embodiments, the GLP-analogue is: ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH 2 (SEQ ID NO:1) ZP2949 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKK-OH (SEQ ID NO:2); ZP2711 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKK-OH (SEQ ID NO: 3); ZP2469 H-HGEGTFSSELATILDALAARDFIAWLIATKITDK-OH (SEQ ID NO: 4); ZP1846 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH 2 (SEQ ID NO:7); ZP1855 H-HGEGSFSSELSTILDALAARDFIAWLIATKITD-NH 2 (SEQ ID NO:8); or ZP2242 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDK-OH (SEQ ID NO: 9) is selected from.

[0062] The present invention relates to methods for producing and purifying GLP2 analogues. These peptides are intended to be used as drug substances. The present invention includes the GLP2 analogues obtained by the methods of the present invention.

[0063] It should be understood that the peptide (drug substance) of the present invention may be provided in the form of a salt or other derivative. Thus, it is understood that after purification and any further steps, the peptide can finally be obtained as a salt or other derivative. Salts include pharmaceutically acceptable salts such as acid addition salts and base salts. Examples of acid addition salts include hydrochloride, citrate, chloride salts and acetate salts. Preferably, the salt is an acetate salt. In general, it is preferred that the salt is not a chloride salt. Examples of base salts include those in which the cation is an alkali metal such as sodium and potassium, an alkaline earth metal such as calcium, and an ammonium ion. + N(R 3 ) 3 (R 4 ) (where R 3 and R 4 each independently represents an optionally substituted C 1~6 -alkyl, optionally substituted C 2~6 -alkenyl, optionally substituted aryl, or optionally substituted heteroaryl). Other examples of pharma- ceutically acceptable salts are described in "Remington's Pharmaceutical Sciences", 17th Edition, Alfonso R. Gennaro (ed.), Mark Publishing Company, Easton, PA, USA, 1985 and more recent editions, and in the Encyclopaedia of Pharmaceutical Technology.

[0064] Preferably, the salt is an acetate salt, which can be obtained after steps RPC1 to RPC4 and subsequent desalting, as described herein. In a preferred embodiment, the acetate of the GLP-2 analog of the present invention is selected from the group consisting of ZP1848-acetate, ZP2949-acetate, ZP2711-acetate, ZP2469-acetate, ZP1857-acetate, ZP2530-acetate, ZP1846-acetate, ZP1855-acetate and ZP2242-acetate. In the context of this specification, the term "ZP1848-acetate" refers to the ZP1848 molecule in the form of acetate. The acetate of the GLP-2 analog can be represented by the formula (GLP-2 analog), x (CH3COOH), where x is between 1.0 and 8.0, i.e., x is 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 or 8.0. In any composition of acetate salt of GLP-2 analogue, there may be molecules with different numbers of acetate molecules, and therefore x is not necessarily an integer. In some cases, x is 4.0 to 8.0, or x is 6.0 to 8.0, or x is 4.0 to 6.5. In some cases, x is 4.0 to 6.0, or x is 2.0 to 7.0, or x is 3.0 to 6.0, or x is 4.0 to 6.0, or x is 4.0 to 8.0. Further discussion of acetate salt of GLP-2 analogue as defined in the present invention can be found in WO2020 / 265064, the disclosure of which is incorporated herein by reference.

[0065] In a preferred embodiment, the GLP-2 analogue is finally ZP1848-acetate or H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH 2 Acetate (SEQ ID NO: 1) or (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH 2 ), x(CH3COOH) (wherein x is 1.0 to 8.0).

[0066] A solid composition comprising an acetate salt of a glucagon-like peptide 2 (GLP-2) analog can be obtained, for example, by lyophilization. The solid composition is useful for formulation with excipients used to make liquid formulations. For example, a compound of the formula: (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2),x(CH3COOH) (wherein x is 1.0 to 8.0) It is possible to obtain a solid composition comprising an acetate salt of a glucagon-like peptide 2 (GLP-2) analog having the formula:

[0067] Assuming an upper limit of 8.0 acetate molecules per GLP-2 analogue, the acetate content corresponds to less than 11% acetate and can be formulated to have a viscosity between 0.8-2.0 mPa / s measured at 25°C.

[0068] The range of the number of acetate molecules associated with each molecule of the GLP-2 analog defines the molecular weight range of this component of the formulation. For example, in the case of acetate of ZP1848, the range of the number of acetate molecules associated with each molecule of the GLP-2 analog defines the molecular weight range of ZP1848-acetate. As an example, one acetate equivalent for each molecule of ZP1848 results in a molecular weight of 4316+60=4376 Da. Thus, the molecular weights of ZP1848 with increasing acetate equivalents are as follows: 1 acetate equivalent=4376 Da; 2 acetate equivalents=4436 Da; 3 acetate equivalents=4496 Da; 4 acetate equivalents=4556 Da; 5 acetate equivalents=4616 Da; 6 acetate equivalents=4676 Da; 7 acetate equivalents=4736 Da and 8 acetate equivalents=4796 Da. As a result, the molecular weight ranges are defined as follows: 1-8 eq. acetate = 4376 Da to 4796 Da, 4-8 eq. acetate = 4556 Da to 4796 Da, and 6-8 eq. acetate = 4676 Da to 4796 Da. Further discussion of the acetate salts of the GLP-2 analogues defined in the present invention can be found in WO2020 / 265064, the disclosure of which is incorporated herein by reference.

[0069] Other derivatives of the GLP-2 analogues of the present invention include Mn 2+ and Zn 2+Coordination complexes with metal ions such as , esters such as in vivo hydrolyzable esters, free acids or free bases, hydrates, or lipids. Esters can be formed between hydroxyl or carboxylic acid groups present in the compounds and appropriate carboxylic acid or alcohol reaction partners using techniques well known in the art. Medical conditions The GLP-2 analogue formulations of the present invention are useful as pharmaceuticals, for example, as described on page 26 of WO2020 / 065064 ("Medical Conditions"), the contents of which are incorporated by reference in their entirety. EXAMPLES

[0070] material and method Standard equipment and materials for manufacturing pharmaceutical preparations of therapeutic peptides and meeting Good Manufacturing Practice (GMP) standards known in the art are used throughout.

[0071] Example 1 General Peptide Synthesis 1.1 First-stage download via Ramage linker addition and substoichiometric download DEGAM-resin (6.18 kg, 4.20 moles, 1.0 equiv., 0.68 mmol / g) was added to the reactor. DMF (25 L, 50-58 °C) was then added. After stirring for 20 more minutes, the resin was drained and additional DMF (25 L, 50-58 °C) was added to the reactor and the mixture was stirred. For deprotonation of the resin, piperidine (2.5 L) was added and the mixture was stirred for 15 minutes. Additional DMF was added and the reactor was drained. The resin was washed once batchwise with DMF. It was then washed continuously with DMF until the washings gave a negative chloranil test (indicating the absence of piperidine in the washings). Finally, the resin was washed once batchwise with DMF.

[0072] Fmoc-Ramage-OH linker and Oxyma (0.9 equiv. each, 3.78 mol) were dissolved in DMF (8 L) at 50-58 °C. This solution was added to the reactor containing the deprotonated resin. Additional DMF (17 L) was added. DIC (2.25 equiv.) was then added to the reactor in four portions at 5 min intervals, with the temperature maintained at 50-58 °C, for a total addition time of 15 min. The first three portions contained 13% each of the total DIC added, and the last portion contained 61%. After the first addition of DIC, the mixture was stirred for a total of 45 min. Extra DMF was then added and the reactor was drained. Finally, the resin was washed batchwise twice with DMF.

[0073] AcOH and Oxyma (2 equiv. each, 8.40 mol) were dissolved in 8 L of DMF at 50-58 °C and added to the reactor. Additional DMF (17 L) was added, followed by DIC (5.0 equiv.) in four portions at 3 min intervals for a total of 9 min under stirring, with other conditions as previously described for the linker coupling. After the last addition of DIC, the reaction mixture was stirred for 6 min. Extra DMF was added and the reactor was drained. Finally, the resin was washed once batchwise with DMF. 1.2 Fmoc-Lys 39 Second-stage download via addition of (Trt)-OH and competitive co-capping DMF (25 L, 50-58 °C) was added to the resin obtained in the previous step with stirring. Piperidine (2.5 L) was then added to the reactor and the mixture was stirred for 20 min. The reactor was drained. The same amount of DMF was added and the treatment with piperidine was repeated. An extra amount of DMF was then added and the reactor was drained. The resin was washed once batchwise with DMF. It was then washed continuously with DMF until a negative chloranil test was obtained. The resin was then washed once batchwise with DMF.

[0074] Fmoc-Lys 39(Trt)-OH and Oxyma (1.5 equiv. each, 6.30 mol) were dissolved in 8 L of DMF at 50-58 °C and added to the reactor containing the deprotected resin. Additional DMF (17 L) and AcOH (2.70 equiv., 11.34 mol) were added to the reactor. DIC (3.75 equiv.) was then added in four portions at 5 min intervals with stirring while maintaining the temperature at 50-58 °C. The first three portions contained 13% each of the total DIC, and the last portion contained 61%. After the last portion of DIC was added, the mixture was stirred for 30 min (total stirring time 45 min). Coupling was confirmed by Kaiser test (a negative test indicates no free amino groups on the resin) and additional DMF was added to the reactor. The reactor was then drained and the resin was washed once batchwise with DMF and drained. 1.3 Fmoc-Lys 38 (Trt)-OH, Fmoc-Lys 37 (Boc)-OH, Fmoc-Lys 36 (Boc)-OH, Fmoc-Lys 35 (Boc)-OH, Fmoc-Lys 34 Addition of (Boc)-OH DMF (25 L, 53° C.) was added to the resin obtained in the previous step with stirring. Piperidine (625 ml) was then added to the reactor and the mixture was stirred for 10 minutes. After that, piperidine (1875 ml) was added and the mixture was stirred for 10 minutes. The reactor was then drained. The steps involving deprotection with piperidine were repeated twice. After the last deprotection step, the mixture was diluted with DMF. The reactor was then drained and the resin was washed once batchwise with DMF. The resin was continuously washed with DMF until a negative chloranil test was obtained. The resin was then washed once batchwise with DMF.

[0075] The Fmoc-protected amino acid and Oxyma (2.0 equiv., 4.20 mol each) were dissolved in DMF (8 L) at 50-58 °C and added to the reactor containing the deprotected resin. Additional DMF (17 L) was added, followed by DIC (5.0 equiv.) in portions as described for the addition of the Ramage linker (Example 1.1). After the addition of the first portion of DIC, coupling was allowed to proceed with stirring for a total of 35-40 min at 50-58 °C. Coupling was confirmed by Kaiser test. The resin was then subjected to capping by adding AcOH (2 equiv., 5 mol) to the reaction and stirring for 2 min. Extra DMF was added and the reactor was drained. Finally, the resin was washed once batchwise with DMF. 1.4 Fmoc-Asp 33 Addition of (OtBu)-OH DMF (25 L, 53° C.) was added to the resin obtained in the previous step with stirring. Then piperidine (625 ml) was added to the reactor and the mixture was stirred for 5 minutes. After that piperidine (1875 ml) was added to the reactor and the mixture was stirred for 5 minutes. The reactor was then drained. The two-step deprotection was repeated, but with 10 minutes of stirring after each piperidine addition. After the last deprotection step with piperidine, the mixture was diluted with DMF. The reactor was then drained and the resin was washed once batchwise with DMF. The resin was continuously washed with DMF until a negative chloranil test was obtained. Then, one batchwise washing of the resin with DMF was performed.

[0076] The Fmoc-protected amino acid and Oxyma (2.0 equiv., 4.20 mol each) were dissolved in DMF (8 L) at 50-58 °C and added to the reactor containing the deprotected resin. Additional DMF (17 L) was added. DIC (5.0 equiv.) was then added portionwise to the reactor as described in Example 1.1. After the addition of the first portion of DIC, coupling was carried out with stirring at 50-58 °C for 35-40 min. Coupling was confirmed by Kaiser test and the resin was subjected to capping with AcOH (2 equiv.) for 2 min and washed with DMF as described in Example 1.3. 1.5 Fmoc-Thr 32 (tBu)-OH, Fmoc-Thr29 (tBu)-OH, Fmoc-Ala 28 -OH, Fmoc-Leu 26 -OH, Fmoc-Ile 23 -OH, Fmoc-Arg 20 Addition of (Pbf)-OH Oxyma (711 g) was dissolved in DMF (8 L) at 50-58 °C and added to the reactor containing the resin, followed by DMF (17 L). Fmoc deprotection was performed by adding piperidine (625 ml) to the reactor containing Oxyma / DMF and resin and stirring the mixture for 5 minutes. Piperidine (1875 ml) was then added to the reactor and the mixture was stirred for 5 minutes. The reactor was drained. The two-step deprotection was repeated. After the final deprotection step with piperidine, the mixture was diluted with DMF. The reactor was then drained and the resin was washed once in a batchwise fashion. The resin was washed continuously with DMF until a negative chloranil test was obtained. One batchwise wash of the resin with DMF was then performed.

[0077] The Fmoc-protected amino acid and Oxyma (2.0 equiv. each, 4.20 mol) were dissolved in DMF (8 L) at 50-58 °C and added to the reactor containing the deprotected resin. Additional DMF (17 L) was added and DIC (5.0 equiv.) was added portionwise to the reactor as described in Example 1.1. After the addition of the first portion of DIC, the mixture was allowed to react with stirring at 50-58 °C for 30 min. Additional DMF was added and then the reactor was drained. One batchwise wash of the resin with DMF was performed.

[0078] The coupling step was repeated as above, except that the reaction was allowed to proceed for 35-40 min after the addition of the first portion of DIC. 20 (Pbf)-OH was coupled a third time using 1 equivalent each of amino acid, Oxyma and DIC. Coupling was confirmed by Kaiser test and the resin was subjected to capping with AcOH (2 equivalents) for 2 minutes as described in Example 1.3. Extra DMF was added and the reactor was drained. Finally, the resin was washed once batchwise with DMF. 1.6 Fmoc-Lys30 Addition of (Boc)-OH The peptide resin was treated with Oxyma and piperidine as described in Example 1.4, except that the two-step deprotection was carried out with stirring for 10 minutes after the first addition of piperidine and 19 minutes after the second addition of piperidine.

[0079] A two-step deprotection with piperidine was performed by repeated addition of Oxyma and stirring the resin for 10 minutes after each addition of piperidine. Additional DMF was added and the reactor was then drained. One batch wash of the resin with DMF was performed. The resin was continuously washed with DMF until a negative chloranil test was obtained. One batch wash of the resin with DMF was then performed.

[0080] Fmoc-protected amino acid and Oxyma (2.0 equiv., 4.2 mol each) were dissolved in DMF (8 L) at 50-58 °C and added to the reactor containing the deprotected resin. Additional DMF (17 L) was added. DIC (5.0 equiv.) was then added portionwise to the reactor as described in Example 1.1. After the addition of the first portion of DIC, coupling was carried out with stirring at 50-58 °C for 35-40 min. Coupling was then confirmed by Kaiser test and the resin was subjected to capping with AcOH (2 equiv.) for 2 min. Extra DMF was added and the reactor was drained. Finally, the resin was washed once batchwise with DMF. 1.7 Fmoc-Ser 8 (tBu)-OH and Fmoc-Thr 5 Addition of (tBu)-OH Oxyma was added to the reactor as described in Example 1.5. Fmoc deprotection was performed by adding piperidine (625 ml) to the reactor containing Oxyma / DMF and resin and stirring the mixture for 10 minutes. Piperidine (1875 ml) was then added to the reactor and the mixture was stirred for 10 minutes. The reactor was drained. The two-step deprotection was repeated twice. After deprotection, the mixture was diluted with DMF. The reactor was then drained and the resin was washed once batchwise with DMF. The resin was continuously washed with DMF until a negative chloranil test was obtained. The resin was then washed once batchwise with DMF.

[0081] Fmoc-protected amino acid and Oxyma (2.0 equiv., 4.20 mol each) were dissolved in DMF (8 L) at 50-58 °C and added to the reactor containing the deprotected resin. Additional DMF (17 L) was added and DIC (5.0 equiv.) was added portionwise to the reactor as described in Example 1.1. After the addition of the first portion of DIC, coupling was carried out with stirring at 50-58 °C for 35-40 min. Coupling was confirmed by Kaiser test and the resin was subjected to capping with AcOH (2 equiv.) for 2 min. Extra DMF was added and the reactor was drained. Finally, the resin was washed once batchwise with DMF. 1.8 Addition of Fmoc-Gly4-OH Oxyma was added to the reactor and deprotection of the Fmoc group by treatment with piperidine was carried out as described in Example 1.7, including application of the chloranil test.

[0082] The Fmoc-protected amino acid and Oxyma (2.0 equiv., 4.20 mol each) were dissolved in DMF (8 L) at 50-58°C and added to the reactor containing the deprotected resin. Additional DMF (17 L) was added and DIC (5.0 equiv.) was added portionwise to the reactor as described in Example 1.1. Coupling was carried out with stirring for 30 min after the addition of the first portion of DIC. Additional DMF was added and then the reactor was drained. One batchwise wash of the resin with DMF was performed.

[0083] The above coupling step was repeated for 35-40 min after the addition of the first portion of DIC. Then, coupling was confirmed by Kaiser test and the resin was subjected to capping with AcOH (2 equiv.) for 2 min. Extra DMF was added and the reactor was drained. Finally, the resin was washed once batchwise with DMF. 1.9 Boc-His 1 (Trt)-Gly 2 Addition of -OH Oxyma (711 g) was dissolved in DMF (8 L) at 50-58 °C and added to the reactor containing the resin. Additional DMF (17 L) was added. Fmoc deprotection was performed by adding piperidine (625 ml) to the reactor containing Oxyma / DMF and resin and stirring the mixture for 5 minutes. Piperidine (1875 ml) was then added to the reactor and the mixture was stirred for 5 minutes. The reactor was drained.

[0084] The addition of Oxyma was repeated, stirring the resin for 10 minutes after each addition of piperidine, to perform a two-step deprotection with piperidine. Additional DMF was added and the reactor was then drained. One batch wash of the resin with DMF was performed. The resin was continuously washed with DMF until a negative chloranil test was obtained. One batch wash of the resin with DMF was then performed.

[0085] Boc-His 1 (Trt)-Gly 2 -OH and Oxyma (1.5 equiv. each, 3.15 mol) were dissolved in DMF (8 L) at 50-58 °C and added to the reactor containing the deprotected resin. Additional DMF (17 L) was added. DIC (3.75 equiv.) was then added portionwise to the reactor as described in Example 1.1. After the addition of the first portion of DIC, coupling was carried out with stirring for 60 min at 50-58 °C. Coupling was then confirmed by Kaiser test. Extra DMF was added to the reactor and the reactor was drained. Finally, the resin was washed once batchwise with DMF. 1.10 Remaining amino acids: Fmoc-Glu 3 (OtBu)-OH, Fmoc-Phe 6-OH, Fmoc-Ser 7 (tBu)-OH, Fmoc-Glu 9 (OtBu)-OH, Fmoc-Leu 10 -OH, Fmoc-Ala 11 -OH, Fmoc-Thr 12 (tBu)-OH, Fmoc-Ile 13 -OH, Fmoc-Leu 14 -OH, Fmoc-Asp 15 (OtBu)-OH, Fmoc-Ala 16 -OH, Fmoc-Leu 17 -OH, Fmoc-Ala 18 -OH, Fmoc-Ala 19 -OH, Fmoc-Asp 21 (OtBu)-OH, Fmoc-Phe 22 -OH, Fmoc-Ala 24 -OH, Fmoc-Trp 25 (Boc)-OH, Fmoc-Ile 27 -OH, Fmoc-Ile 31 Addition of -OH Oxyma treatment and deprotection with piperidine was performed as described in Example 1.9. After deprotection, additional DMF was added. The reactor was then drained. One batch wash of the resin with DMF was performed. The resin was continuously washed with DMF until a negative chloranil test was obtained. One batch wash of the resin with DMF was then performed.

[0086] Fmoc amino acid and Oxyma (2.0 equiv., 4.20 mol each) were dissolved in DMF (8 L) at 50-58 °C and added to the reactor containing the deprotected resin. Additional DMF (17 L) was added to the reactor and DIC (5.0 equiv.) was added as described in Example 1.1. After the addition of the first portion of DIC, coupling was carried out with stirring at 50-58 °C for 35-40 min. Coupling was then confirmed by Kaiser test and the resin was subjected to capping with AcOH (2 equiv.) for 2 min. Extra DMF was added and the reactor was drained. Finally, the resin was washed once batchwise with DMF. 1.11 Peptide deprotection and cleavage from the resin After the synthesis was completed, the protected peptide resin was washed three times batchwise with DMF, two times with DMF at 50-58 °C, and once with DMF at room temperature. The peptide resin was then washed five times with isopropanol. The peptide resin was finally dried at 25-35 °C and stored at 2-8 °C.

[0087] TFA (68.1 kg), DTT (1.75 L), TIS (1.25 L) and water (1.25 L) were added to the reactor. Peptide resin (10 kg equivalent to 0.8 moles) prepared as described in Examples 1.1-1.10 was added to the solution. The resulting mixture was left under stirring at 25°C for 135 min. The temperature was then lowered to below 0°C and cold (below 0°C) MTBE (150 L) was slowly added to the reactor. During the addition of MTBE, the temperature was maintained below 10°C while the cleaved peptide precipitated from the resin. After complete precipitation, the mixture was left under stirring at below 10°C for 45±15 min. The mixture was then filtered and washed twice with MTBE and once with a solution containing a mixture of MTBE (3 volumes) and acetonitrile (1 volume). After filtration, the resulting filter cake was dried under vacuum for 1-3 h. 1.12 PreRPC1 (O→N acyl group transfer) Traditionally, O→N acyl group transfer is carried out in solution using the following procedure: The dried resin is washed with AcOH / MeCN / H 2 O+1%NH 4 A solution containing OAc (w / w) (10% / 50% / 40%) was added. The mixture was left under stirring overnight. The mixture was then filtered and the filter cake was washed with the same solution. The combined filtrates containing the crude peptide from the two cleavage rounds (up to 3600 g, equivalent to approximately 0.8 moles of peptide) were stored at 5° C.

[0088] According to the method of the present invention, O→N acyl group transfer is carried out on-column, as described herein, particularly in Example 3. 1.13 Purified RPC1 (one-dimensional) Buffers used in RPC1: ·Buffer A: 0.1%H 3 PO 4 ·Buffer B: MeCN ·Buffer C: 45mM H 3 PO 4 , pH 2.2 + 100mM NaCl ·Buffer D: 45mM H 3 PO 4 , pH 7.7-8.0 + 100mM NaCl The solution from the deprotection and cleavage steps was diluted 5 times in volume with 0.2 M aqueous ammonium acetate. The solution was filtered and applied up to 15 g / L column volume onto a column packed with C18 silica gel (column dimensions 45x(50-45cm)) pre-equilibrated with a solution containing 95% C and 5% B. After application of the peptide solution, the column was washed with the equilibration solution and then with a solution containing 95% D and 5% B until the pH of the eluate was above 7.5. Prior to elution, the column was washed with a solution containing 95% C and 5% B until the pH was below 2.5. (Pre-RPC1) The column was then washed with a solution containing 93% A and 7% B. The adsorbed peptides were eluted by applying a gradient that changed the mobile phase from 7% B (93% A) to 22% B (78% A) in 12 column volumes. The gradient was kept at 22% B (78% A). Isocratic elution was then performed at 22% B until the UV of the eluted peak reached 40-45%. The elution was monitored at 280 nm and the collected fractions were analyzed by RP-HPLC. This process was repeated to purify the remaining pool from the deprotection and cleavage steps, and fractions showing peptide purity of 85% or more were pooled. Fractions with purity of 55% or more and 85% or less were rechromatographed by applying up to 15 g / L column volume to the C18 column after diluting the combined fractions to 2 volumes with water. Fractions from the primary run collected before the main peak were eluted by changing the mobile phase from 10% B (90% A) to 14% B (86% A) in one column volume, then a gradient was applied changing from 14% B (86% A) to 23% B (77% A) in nine column volumes, followed by isocratic elution at 23% B (77% A) until the UV of the eluted peak reached 95%.

[0089] Fractions from the primary run collected after the main peak were eluted by changing the mobile phase from 10% B (90% A) to 14% B (86% A) in one column volume, then a gradient was applied changing from 14% B (86% A) to 22% B (78% A) in nine column volumes, followed by isocratic elution at 22% B (78% A) until the UV of the eluted peak reached 90%. The fractions after rechromatography with a purity of 85% or higher were mixed with the main pool to obtain the final pool of RPC1. Thus, a process was described that included three primary runs as well as one front rerun and one back rerun to purify 3.6 kg of crude peptide, obtaining the final RPC1 pool. 1.14 Purified RPC2 (2D) Buffers used in RPC2: ·Buffer A: 0.1% TFA ·Buffer B: MeCN The final pool from RPC1 was diluted to 2 volumes with water and applied up to 13 g / L column volume to an RPC column (column dimensions: 45x(50-45cm)) packed with C18 silica gel, pre-equilibrated with a solution containing 90% A and 10% B. After application of the peptides, the column was washed with 5 column volumes of equilibration solution. The adsorbed peptides were eluted with a gradient in which the mobile phase was changed from 10% B (90% A) to 19% B (81% A) during one column volume, and then from 19% B (81% A) to 30% B (70% A) during 12 column volumes. An isocratic elution was then performed until the UV of the eluted peak reached 40-45% of the maximum value. The gradient was then changed to 60% B (40% A) and held at this value until all peptides were eluted. The elution was monitored at 280 nm and the collected fractions were analyzed by RP-HPLC. This process was repeated to purify the remaining pool from RPC1, with a purity of 92 or higher and the impurity des-Ile 27 -Ala 28 Fractions showing less than 0.5% purity were pooled. After dilution with water, the purity was greater than 50% but less than 92% and / or the impurity des-Ile 27 -Ala 28Fractions collected before the main peak with a purity of >0.5% but <1.5% as well as fractions collected after the main peak with a purity of >50% but <92% were rechromatographed by application of up to 20 g / L column volume on a C18 column and eluted by application of a mobile phase gradient changing from 10% B (90% A) to 20% B (80% A) in one column volume, then from 20% B (80% A) to 30% B (70% A) in 9 column volumes, followed by an isocratic elution with 30% B (70% A) until the UV of the eluted peak reached 45%.

[0090] The purity obtained after rechromatography was 92% or more, and the impurity des-Ile 27 -Ala 28 Fractions with less than 0.5% were combined with the main pool to obtain the final pool of RPC2. 1.15 Purified RPC3 (three-dimensional) Buffers used in RPC3: ·Buffer A: 100mM NH 4 OAc+0.5% AcOH ·Buffer B: MeCN The final pool from RPC2 was diluted to 2x volume with water and applied up to 13 g / L column volume to an RPC column (column dimensions: 45x(50-45cm)) packed with C18 silica gel, pre-equilibrated with a solution containing 85% A and 15% B. After application of the peptide, the column was washed with equilibration solution. The adsorbed peptide was eluted with a gradient in which the mobile phase was changed from 15% B (85% A) to 29% B (71% A) in one column volume, then from 29% B (71% A) to 37% B (63% A) in 10 column volumes. An isocratic elution was then performed until the UV signal reached 30% of the maximum value. The gradient was then changed to 70% B (30% A) and held at this value until the UV signal reached baseline. The elution was monitored at 280 nm, and the pH of the collected fractions was adjusted to 5.8-6.0 with aqueous ammonia. The collected fractions were analyzed by RP-HPLC, and those with peptide purity ≥96.5% and without any impurities >0.5% before the main peak were pooled to obtain the final main pool of RPC3. This process was repeated to purify the remaining pools from RPC2. 1.16 Purified RPC4 (four-dimensional) Buffers used in RPC4: ·Buffer A: 5mM NH 4 OAc+0.1% AcOH ·Buffer B: MeCN ·Buffer C: 100mM NH 4 OAc+0.5% AcOH The final pool from RPC3 was diluted to 2 volumes with water and ammonium acetate was added to the resulting solution to obtain a final concentration in the pool of approximately 100 mM ammonium acetate. A maximum column volume of 15 g / L was applied to an RPC column packed with Amberchrom XT20 (column dimensions: 45x(40-35cm)) pre-equilibrated with 90% C and 10% B. After peptide application, the column was washed with one column volume of equilibration solution and one column volume of 90% A and 10% B. The adsorbed peptides were eluted with a gradient in which the mobile phase was changed from 10% B (90% A) to 13% B (87% A) in one column volume, then from 13% B (87% A) to 25% B (75% A) in 12 column volumes. When the UV signal reached 40% of the maximum, the gradient was changed to 60% B (40% A) and held at this value until all peptides were eluted. Elution was monitored at 280 nm and the pH of the collected fractions was adjusted to 5.8-6.0 with aqueous ammonia. The process was repeated to purify the remaining pool from RPC3. The collected fractions were analyzed by RP-HPLC and fractions with peptide purity ≥98.0% and no impurities >0.5% were pooled. After dilution with water, fractions with purity ≥80.0% and <98.0% and / or no impurities Des-Ser were pooled. 7 / 8 / Fractions collected before the main peak showing more than 0.5% aspartimide and purity more than 80.0% but less than 98.0% and / or impurity Des-Ser 7 / 8Fractions collected after the main peak showing >0.5% aspartimide were rechromatographed on an Amberchrom XT20 column applying up to 15 g / L column volume. The product was eluted by applying a gradient changing the mobile phase from 10% B (90% A) to 13% B (87% A) in one column volume, then from 13% B (87% A) to 25% B (75% A) in 9 column volumes, followed by an isocratic elution with 25% B (75% A) until the UV of the eluted peak reached 30%. The fractions were adjusted to pH 5.8-6.0 with aqueous ammonia and analyzed by RP-HPLC. The pool obtained after rechromatography with a purity of >98.0% and without any impurities >0.5% was mixed with the main pool to obtain the final pool of RPC4. 1.17 Desalination (SPE) Buffer used for desalting: ·Buffer A: 10mM AcOH ·Buffer B: MeCN The final pool from RPC4 was diluted to 2 volumes by adding water and then ammonium acetate was added to obtain a final concentration in the pool of approximately 100 mM. The pool was applied to a column packed with Amberchrom XT20 (column dimensions: 45x40cm), pre-equilibrated with 95% A and 5% B. After application of the peptides, the column was washed with equilibration solution. The adsorbed peptides were eluted with a gradient changing the mobile phase from 5% B (95% A) to 50% B in one column volume. The gradient was held at this value until all peptides were eluted. The elution was monitored at 280 nm and collected fractions were analyzed by RP-HPLC. 1.18 Isolation of purified peptides The product from the desalting step was subjected to evaporation under reduced pressure at 40° C. This process evaporated the MeCN and reduced the peptide solution to approximately 30% of its original volume, which was then diluted with water to obtain a final peptide concentration of about 25 g / L. The concentrated peptide was filtered through a 0.45 / 0.22 μm filter and then isolated by lyophilization to obtain approximately 1.3 kg of purified peptide (overall purification yield of 35% or more) with a purity of 97.75% or more by RP-HPLC and no impurities exceeding 0.5%.

[0091] Example 2 Optimization of crude peptide by using Fmoc-Lys(Trt)-OH During the development of ZP1848 synthesis, Lys(Boc) 6 Note that the presence of results in several tert-butylated by-products (+56 Da), as expected. Notably, several of these eluted close to the main peak and could therefore also be found in the purified solution (Figure 2, top panel). Without wishing to be bound by any theory, we believe that this +56 impurity is related to cleavage of the tert-butyl group and therefore the +56 moiety is located somewhere in the lysine tail, most likely at 37 or 38.

[0092] [Lys(Trt)] 6 When the synthesis of ZP1848 was performed using , tert-butylated by-products were highly removed (Figure 2, bottom panel), but some butylated by-products could nevertheless be observed as a result of migration from protecting groups elsewhere in the synthesis.

[0093] It is not necessary to use Lys(Trt) for each lysine residue in the tail, but it can be used. Fmoc-Lys(Trt)-OH is a more expensive building block than Fmoc-Lys(Boc)-OH, especially on a molar basis, so a cost-effective process may apply this building block to only a portion of the amino acid couplings.

[0094] Furthermore, the present inventors have found that Lys 39 (Boc) and Lys 38 The tert-butylated by-product Lys resulting from the use of (Boc) residues 39 -t-Bu and Lys 38 We observed that -t-Bu was the most difficult impurity to remove from the crude peptide to yield the desired peptide. Changing the protecting groups of these two residues from Boc to Trt afforded Lys. 39 -t-Bu and Lys 38 Therefore, we performed the first two couplings (i.e., Lys 39 and Lys 38 residue) and [K(Boc)] 4 [K(Trt)] 2 It was determined that obtaining this motif would facilitate removal of the tert-butylated by-product that elutes closest to the main peak (Figure 3, peak 9.487), thus providing a purer crude product. This would result in a cleaner purification process, leading to higher yields and a purer final product.

[0095] Example 3 O to N acyl transfer on the column of Pre-RPC1 Upon cleavage of ZP1848 (Example 1.11), N→O acyl group migration occurs. The crude precipitated product was purified by elution with AcOH / MeCN / 1% aqueous NH 4 When dissolved in a 1:5:4 mixture of OAc and left overnight for decarboxylation of Trp(Boc), acetylated (i.e., truncation of the peptide chain) and trifluoroacetylated impurities (i.e., trifluoroacetyl esters of the peptide chain) are also found.

[0096] Adjustment of the pH to neutral and then back to acidic conditions prior to the purification step is known to reduce these impurities and allow O→N acyl group migration, see e.g. (a) Bergmann M, Brand E, Weimann FZ Physiol Chem. 1923;131:1-17; (b) Phillips AP, Baltzly R, J Am Chem Soc. 1947;69:200-204.

[0097] O→N transacylation is usually carried out in solution before loading the peptide product onto the column. To effect the transacylation, the pH is usually lowered using a phosphate buffer / phosphoric acid. However, it was found that ZP1848 precipitates at neutral pH when phosphate buffer / phosphoric acid is used in the peptide product solution (for further details regarding the incompatibility of phosphate buffers, see Example 4 of WO2020 / 065064, the disclosure of which is incorporated herein by reference). Furthermore, due to the high acetic acid content in the peptide product solution prior to purification, it was found that raising the pH to neutral pH without precipitation would require very large amounts of NaOH and was not practical or feasible.

[0098] Furthermore, ZP1848 precipitates at neutral pH in solutions containing NaCl, phosphate, NaOH and high ionic strength. The purpose of this example is to compare the results of performing the O→N acyl group transacylations in solution and on column.

[0099] In Table 4.1 below, various formulations of ZP1848 were prepared at different pH and / or concentrations in 100 mM NaCl and 45 mM phosphate (buffers are described in Example 1.12) to reflect in solution the same conditions present on the column under RPC1.

[0100] [Table 1]

[0101] The data in Table 4.1 clearly show that ZP1848 precipitates when the same conditions used in solution as those used on column during RPC1 are used. Precipitation occurs within minutes of sample preparation. Furthermore, ZP1848 was shown to precipitate even at low concentrations (0.2 and 0.5 mg / mL) at pH 7.5.

[0102] Surprisingly, it was found that it was possible to carry out this pH treatment on the C18 column used for purification, prior to the first purification step. In particular, it was surprising that a phosphate buffer could be used without loss of material. In fact, Pre-RPC1 and RPC1 are carried out on the same column, thus removing a treatment step from the purification sequence.

[0103] In the cleavage, the data in Figure 4 show that TFA and acyl impurities, as well as acyl translocation, are reduced after pH treatment on-column. In addition, the yield that may be lost on-column due to the peptide's incompatibility with phosphate buffer at neutral pH (potential precipitation) was also evaluated. In the experiment in Figure 4, 100 mg was used before and after pH treatment, and the amount of ZP1848 was quantified to 89 mg (approximately 10 mg loss can be explained by product handling during cleavage / precipitation). Therefore, the losses on-column are considered negligible, and no yield loss due to pH treatment on-column was observed. In conclusion, performing O→N acyl translocation on-column prevents undesired precipitation while achieving the desired chemical transformation. Furthermore, increasing the pH to neutral increases the yield by approximately 5% due to O→N acyl translocation and hydrolysis of acetylated and trifluoroacetylated impurities. That is, the quantified amount of product eluting from the RPC1 step is approximately 5% higher than the quantified amount in the loading volume.

[0104] Example 4 Purification of crude peptides and removal of unwanted components Using a five-step ("pre" step and four "dimensions") chromatographic purification process (Pre RPC1+RPC1-RPC4) of Fmoc-SPPS as outlined in Example 1, a ZP1848 product with 98.2% purity was obtained. The ZP1848 product was found to contain 0.5% or less impurities when assessed by analytical HPLC with UV or MS detection. Each step of the purification process was developed to remove specific unwanted components such as peptide impurities (i.e. high molecular weight (HMW) truncations, deletions, or other undesired derivatives) or to improve the purity of the final material. These unwanted components may consist of covalently or non-covalently bound impurities and need to be reduced as much as possible because they may alter or inactivate the bioactivity or have unknown side effects. In the ZP1848 product, at least one species of C-terminal deamidation, HMW compounds, and the formation of aspartic acid-related impurities from aspartic acid (e.g., formation of Iso-Asp / Beta-Asp and aspartimides) are particularly undesirable impurities. Removal of specific impurities – C-terminal deamidation products, Lys 39 -OH impurities and aspartic acid related impurities The order of the RPC steps is crucial in that RPC1 must be performed before RPC2 and 3 because it removes impurities that would otherwise obscure essential details found in these two steps.

[0105] Phosphate buffer was used in the first dimension (RPC-1), TFA in the second dimension, and ammonium acetate in the third and fourth dimensions. Acetonitrile (MeCN) was used as the organic modifier in all purification steps. To evaluate the effectiveness of the purification, fractions of the main components after each step were analyzed by HPLC or LC-MS. The results of the obtained purity, the main undesired impurities, and the impurities that were particularly difficult to remove are shown in Table 4.1 below. It can be seen that through successive purification steps, the aspartic acid-related impurities (Iso-Asp / Beta-Asp and aspartimide formation) are reduced, achieving a final product with a purity of more than 98%.

[0106] [Table 2]

[0107] The first RPC is used to isolate the main component and remove certain impurities, namely aspartic acid-related impurities and C-terminal deamidated impurities. Most of the truncated terminal amino acid moieties are also removed in this step. In the crude product before RPC1, the C-terminal deamidated product Lys 39 This purification step is very efficient in removing the -OH impurity, since its level is relatively high at 2.9% and only elutes in the last fractions after running RPC1. When RPC2 was used instead of the C-terminal deamidated product, Lys 39 The -OH impurity also elutes across five fractions containing over 50% ZP1848, indicating that RPC1 and the use of phosphate buffer can reduce this impurity very efficiently, whereas RPC2 fails, compromising overall yield and purity.

[0108] The use of phosphate buffer was particularly suitable for this purpose, whereas TFA and ammonium acetate failed to separate the C-terminal deamidated product from the main product. The second, third and fourth steps improve the purity but are also used to remove aspartic acid-related impurities. Oligomer Removal Oligomers are mainly formed before purification and are observed in the crude solution (cleavage acidic conditions). Table 4.2 shows the content of oligomers throughout the purification of ZP1848. The level of covalently bound oligomer products is 1.8% in the crude solution, but increases slightly to 2.4% after O→N transacylation. Oligomers are mainly removed in RPC1 and partially in RPC2, with levels in the final product being less than 0.1%. The data also show that there is no further generation of oligomers throughout the purification process.

[0109] [Table 3]

[0110] As can be seen from Table 4.2, oligomers are strongly reduced after RPC1 and are present in only trace (i.e. small) amounts after RPC2. The amount of oligomers is not expected to increase during the on-column pH treatment.

Claims

1. A method for producing a GLP-2 analogue, the GLP-2 analogue having the following formula: R １ -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z ２ -R ２ (In the formula, R １ is hydrogen, C １～４ alkyl (e.g., methyl), acetyl, formyl, benzoyl, or trifluoroacetyl; X5 is Ser or Thr, X11 is Ala or Ser, R ２ is NH ２ or OH, Z ２ is absent or is a peptide sequence of 1 to 6 amino acid units of Lys), or a pharmaceutically acceptable salt or derivative thereof, The method involves the following steps: i) loading a crude GLP-2 analogue synthesized by solid phase peptide synthesis (SPPS) onto a column; ii) adjusting the pH of the column with a first buffer system, wherein the first buffer system is a phosphate buffer system; A method wherein step ii) comprises increasing the pH of the column from an acidic pH to a neutral pH of 7 or to a slightly basic pH of ≧7.2 while the GLP2 analogue is on the column.

2. Z ２ The method of claim 1, wherein: is a peptide sequence of 1 to 6 amino acid units of Lys.

3. 3. The method of claim 1 or 2, wherein increasing the acidic pH of the column comprises increasing the pH to a neutral pH of pH 7.

4. 3. The method of claim 1 or 2, wherein increasing the acidic pH of the column comprises increasing the pH to pH > 7.2, such as a pH of about 7.

5.

5. A method as described in claim 1 or 2, wherein step ii) includes the sequential use of a buffer to raise the pH of the column above 7.5 or to 7.5 and then lower the pH of the column to an acidic pH.

6. (1) i) loading a GLP-2 analog onto a column; ii) adjusting the pH of the column with a first buffer system comprising phosphate buffer / phosphoric acid; and iii) eluting the pool containing the GLP-2 analogue; and (2) i) loading the pool containing the GLP-2 analogue obtained in step (1) onto a column; ii) washing the column with a second buffer system containing trifluoroacetic acid to elute the pool containing the GLP-2 analogue; and (3) i) loading the pool containing the GLP-2 analogue obtained in step (2) onto a column; ii) washing the column with a third buffer system comprising acetic acid / ammonium acetate to elute the pool containing the GLP-2 analogue; and (4) i) loading the pool containing the GLP-2 analogue obtained in step (3) onto a column; ii) washing the column with a fourth buffer system comprising acetic acid / ammonium acetate and eluting the pool containing the GLP-2 analogue; 3. The method of claim 1 or 2, comprising:

7. The method further includes a method for synthesizing a GLP-2 analog by solid phase peptide synthesis (SPPS), wherein Z 2 is a peptide sequence of 1 to 6 amino acid units of Lys, and Z ２ 3. The method of claim 1, wherein at least one Lys unit in is protected with a trityl protecting group during synthesis.

8. Z ２ is a peptide sequence of 2 to 6 amino acid units of Lys, and the method is i) coupling the first P-Lys(Trt)-OH to a solid peptide resin using a linker; ii) removing the P group from the Lys(Trt) amino acid unit; iii) attaching the second P-Lys(Trt)-OH to the Lys(Trt) amino acid unit bound to the solid peptide resin using a linker; iv) removing the P group from the second Lys(Trt) amino acid unit; v) coupling subsequent amino acid units until the GLP-2 analogue is synthesized; vi) cleaving the GLP-2 analogue from the solid peptide resin; vii) purifying the GLP-2 analogue; The method of claim 7 , wherein each P is a protecting group.

9. 3. The method of claim 1 or 2, wherein X5 is Thr and / or X11 is Ala.

10. GLP-2 analogues include: ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH ２ (SEQ ID NO: 1) ZP2949 H-HGEGTFSSSELATILDALAARDFIAWLIATKITDKKK-OH (SEQ ID NO: 2); ZP2711 H-HGEGTFSSSELATILDALAARDFIAWLIATKITDKK-OH (SEQ ID NO: 3); ZP2469 H-HGEGTFSSSELATILDALAARDFIAWLIATKITDK-OH (SEQ ID NO: 4); ZP1857 H-HGEGTFSSELATILDALAARDFIAWLIATKITD-NH ２ (SEQ ID NO: 5); and ZP2530 H-HGEGTFSSSELATILDALAARDFIAWLIATKITD-OH (SEQ ID NO: 6) The method of claim 9, wherein the compound is selected from the group consisting of:

11. GLP-2 analogues include: ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH ２ (SEQ ID NO: 1) ZP2949 H-HGEGTFSSSELATILDALAARDFIAWLIATKITDKKK-OH (SEQ ID NO: 2); ZP2711 H-HGEGTFSSSELATILDALAARDFIAWLIATKITDKK-OH (SEQ ID NO: 3); and ZP2469 H-HGEGTFSSSELATILDALAARDFIAWLIATKITDK-OH (SEQ ID NO: 4) The method of claim 9, wherein the compound is selected from the group consisting of:

12. GLP-2 analogues include: ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH ２ (SEQ ID NO: 1).

13. 3. The method of claim 1 or 2, wherein X5 is Ser and / or X11 is Ser.

14. GLP-2 analogues include: ZP1846 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH ２ (SEQ ID NO: 7); ZP1855 H-HGEGSFSSELSTILDALAARDFIAWLIATKITD-NH ２ (SEQ ID NO: 8); and ZP2242 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDK-OH (SEQ ID NO: 9) The method of claim 13, wherein the compound is selected from the group consisting of:

15. GLP-2 analogues include: ZP1846 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH ２ (SEQ ID NO: 7).

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