Method for formulating a composition containing a glucagon-like peptide-2 (GLP-2) analog.

Abstract

A method for making stable liquid pharmaceutical formulations containing glucagon-like peptide 2 (GLP-2) analogues, and in particular a method for making formulations containing ZP1848 (grepaglutide) or ZP1846 (ersiglutide) based on a one-pot formulation.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a stable liquid pharmaceutical formulation containing a glucagon-like peptide 2 (GLP-2) analog, and more particularly to a method for producing a formulation containing ZP1848 (glepaglutide) and / or ZP1846 (elsiglutide), including its metabolites. [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). Human GLP-2 is produced as a result of specific post-translational processing of proglucagon in enteroendocrine L cells of the intestine and in specific regions of the brainstem. GLP-2 binds to one G protein-coupled receptor belonging to the class II glucagon secretin family.

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

[0004] In this field, glucagon-like peptide-2 receptor analogs are recognized as having therapeutic potential for treating intestinal diseases. However, natural hGLP-2, a gastrointestinal peptide consisting of 33 amino acids, is not useful in clinical settings because its half-life in humans is extremely short: approximately 7 minutes for full-length GLP-2 [1-33] and 27 minutes for cleaved GLP-2 [3-33]. This short half-life is mainly due to degradation by the enzyme dipeptidyl peptidase IV (DPP-IV). Therefore, in this field, attempts have been made to develop GLP-2 receptor agonists with better pharmacokinetic properties, particularly attempts to improve the half-life of the GLP-2 molecule. For example, GLP-2 analogs with substitutions, such as a Gly substitution at the 2-position ([hGly2]GLP-2, teduglutide), have been proposed, and this substitution increases the half-life from 7 minutes (natural GLP-2) to approximately 2 hours. Acylation of peptide drugs in fatty acid chains has also been proven beneficial in extending systemic circulation and increasing enzyme stability without interfering with biological capabilities. However, while these attempts have improved the pharmacokinetics of GLP-2 analogs, and they are sometimes described as "long-acting" in this art, it must be kept in mind that this improvement is in comparison to natural hGLP-2, which has a half-life on the order of hours, not minutes. This, in turn, means that GLP-2 analogs still need to be administered to patients once or multiple times a day. Teduglutide is approved for the treatment of short bowel syndrome under the names Gattex (in the United States) and Revestive (in Europe).

[0005] WO2006 / 117565 (Zealand Pharma A / S) describes GLP-2 analogs containing one or more additional substitutions compared to [hGly2]GLP-2, which have improved in vivo biological activity and / or improved chemical stability as assessed, for example, by in vitro stability assays. Of the molecules disclosed in WO2006 / 117565, ZP1848 (glepaglutide) and ZP1846 (elsiglutide) are disclosed in conjunction with their medical uses for the treatment of gastrointestinal disorders and for improving side effects of chemotherapy and radiotherapy. Dosage schemes for GLP-2 analogs, including ZP1848 and ZP1846 and their metabolites, are described in WO2018 / 229252. Ready-to-use formulations of ZP1848 and ZP1846 are described in WO2020 / 065064 and WO2020 / 065063. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] In this field, challenges remain, particularly in improving manufacturing methods used for the formulation of synthesized GLP-2 analogs, and in addressing problems arising from the unusual properties of these molecules. Providing improved methods for producing GLP-2 analog formulations, especially improved methods for use in delivery devices such as pre-filled syringes, infusion pumps, wearable syringes, or auto-injectors, is also considered a goal in the field of GLP-2 analog formulations. [Means for solving the problem]

[0007] In general, the present invention relates to a method for producing pharmaceutical formulations of GLP-2 analogs, and in particular a method for producing stable liquid pharmaceutical formulations containing ZP1848 (glepaglutide) and ZP1846 (elsiglutide) and / or their metabolites. Accordingly, the present invention relates to the development of a novel method ("Method B" or "One-Pot Formulation Method") that improves upon the technical problems arising from conventional manufacturing methods, "Method A," or "Two-Pot Formulation Method" or "Two-Solution Formulation Method." Please refer to Figure 1. To date, ZP1848 and ZP1846 active pharmaceutical ingredients have been produced using "Method A." However, Method A using ZP1848 or ZP1846 presents several technical challenges, including excessive foaming during dissolution when adding the GLP-2 analog to other components of the formulation, formation of hard clumps of the active pharmaceutical ingredient during dissolution, deviations from visual control, and formation of higher molecular weight peptide oligomers. Clumps of active ingredients (APIs) can become hidden within the foam layer, leading to failures in visual process control crucial for clear solution and API dissolution. Therefore, foaming and clump formation, as well as the need for manual stirring, can be problematic when scaling up the manufacturing process.

[0008] Method A is a traditional formulation method for peptides. During formulation development, using a peptide stock solution dissolved in water is common in the art because it allows for safe and easy handling of the peptide and facilitates the combination of various excipients. Method A works well on a small scale. Furthermore, generally, with peptides, if the pI exceeds a certain level in the formulation, Method A is preferable to avoid local pI and precipitation of the peptide, which can make redissolution difficult or impossible during the formulation process.

[0009] While not bound by any particular explanation, the inventors believe that using Method A contributes to the specific physicochemical properties of ZP1848 and ZP1846, particularly the amphiphilic nature of the peptide structure, and that the problematic, unusual behavior of ZP1848 and ZP1846 leads to the observed foaming and clumping. This property of ZP1848 and ZP1846 results in an unusual tendency to form gels at higher concentrations, for example, at the solid / water interface during dissolution, which can in turn contribute to unwanted clumping in the formulation process. In Method A for ZP1848 and related GLP-2 analogues, since they require the dissolution of the active pharmaceutical ingredient as a 50 mg / mL solution (i.e., about 40% of the final liquid volume of the formulation), these problems combine to make the dissolution of the active pharmaceutical ingredient significantly slow and difficult, especially when the concentration of the GLP-2 analogue in the final formulation is higher (e.g., 20 mg / mL or higher). This dissolution method results in excessive foaming and clumping. The clumps of API are then hidden within the foam layer, leading to a failure of critical visual process control regarding the dissolution of the clear solution and the active pharmaceutical ingredient.

[0010] Therefore, Method A requires a relatively high peptide concentration in the API solution. At high peptide concentrations (e.g., at least 20 mg / mL or preferably at least 50 mg / mL or higher), ZP1848 and ZP1846 can be affected by vigorous stirring by mechanical means, such as those typically used in peptide formulation processes. For this reason, Method A has historically employed manual stirring of the peptide active pharmaceutical ingredient during the dissolution process. Manual stirring is unsuitable for scaling up to industrial scale and is a subjective method that cannot be verified by process validation.

[0011] In Method A, the excipients and active ingredients are dissolved in separate solutions and tanks in parallel processes and subsequently mixed with two solutions of the excipients and peptide active ingredient, respectively, prior to an optional step of adjusting the volume and / or pH of the formulation (i.e., mixed solution) to obtain a final formulation ready for packaging and clinical use.

[0012] The options for avoiding the technical problems of Method A described above are further limited by the mannitol concentration. The excipient solution contains mannitol at a concentration close to the upper limit of solubility, and therefore, it is not possible to solve the problems of Method A by reducing the volume of the excipient solution and consequently increasing the volume available in the API solution.

[0013] In summary, this means that the current method A has technical problems, and scaling up from the current 10-liter batch size cannot be done using method A.

[0014] In the present invention, the “one-pot” formulation method or “Method B” means that only one tank or container is used, in which water, excipients, and the active pharmaceutical ingredient are added sequentially in any particular order, and the method includes an optional step of adjusting the volume and / or pH of the formulation to obtain a final formulation ready for packaging and clinical use. This can be contrasted with the “two-pot formulation” method, as described above, where the parallel dissolution of the API and excipients in two solutions is a limitation. In Method B, only one pot / tank may be used based on the ratio of water, excipients, and active pharmaceutical ingredient, thereby the method is simpler, easier to handle, more robust to deviations, scale-up feasible, and results in a final formulation with improved stability.

[0015] In Method B described in the following examples, the excipients (mannitol, histidine) are dissolved in, for example, 80% of the final volume; the GLP-2 analog drug substance is added as a dry powder to, for example, approximately 25 mg / ml; the volume is adjusted to 90%; the pH is adjusted as needed; and the volume is adjusted to 100% to obtain the final glepaglutide (20 mg / mL) formulation.

[0016] Therefore, in the first embodiment, the present invention relates to a method for producing a stable liquid pharmaceutical formulation comprising a glucagon-like peptide 2 (GLP-2) analog or a pharmaceutically acceptable salt or derivative thereof, wherein the GLP-2 analog is of 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 (Sequence ID 11) (In the formula, R 1 is hydrogen, C 1~4 Alkyl (e.g., methyl), acetyl, formyl, benzoyl, or trifluoroacetyl, X5 is either Ser or Thr. X11 is either Ala or Ser. R 2 is NH2 or OH, Z 2 (This is a peptide sequence consisting of 1 to 6 amino acid units of Lys, or is not present.) Represented by, The liquid pharmaceutical formulation contains a GLP-2 analog at a concentration of approximately 2 mg / mL to approximately 30 mg / mL, and an excipient comprising (i) a histidine buffer at a concentration of approximately 5 mM to approximately 50 mM, (ii) mannitol as a nonionic tonicity regulator at a concentration of approximately 90 mM to approximately 360 mM, and (iii) an appropriate amount of arginine to provide a formulation having a pH of approximately 6.6 to approximately 7.4. The method is (a) A step of mixing the excipient with a first volume of water for injection to obtain an excipient solution that is 70-90% of the volume of the final liquid formulation, (b) Adding the GLP-2 analog drug substance to the excipient solution to obtain a solution of the excipient and the GLP-2 analog at a volume of 70-90% of the final liquid formulation, (c) Adjusting the pH and volume of the liquid composition as necessary to obtain a liquid formulation with 100% of the volume of the final liquid pharmaceutical formulation. and The present invention provides a final aqueous liquid pharmaceutical formulation containing a glucagon-like peptide 2 (GLP-2) analog. Provide a method.

[0017] In some embodiments, the method does not require stirring when the GLP-2 solution composition is added to the excipient solution, which helps to minimize the harmful effects of excessive foaming. Generally, in step (b), the GLP-2 analog API is added as a lyophilized composition, typically in 1 to 5 parts, preferably in 3 parts. Typically, the GLP-2 analog API is in powder form. Alternatively or additionally, the adjustment of the volume of the liquid composition in step (c) includes a first addition of sterile water for injection to adjust the pH, and a second addition of sterile water for injection to increase the volume of the liquid composition to near the final volume, and optionally, the adjustment of the pH of the liquid composition is carried out by adding arginine and / or acetic acid.

[0018] Advantageously, in this manufacturing method, the excipient solution constitutes approximately 80% of the volume of the final liquid formulation, and the first and second additions each contribute approximately 10% of the volume of the final liquid formulation. In the alternative case, the excipient solution constitutes approximately 75% of the volume of the final liquid formulation, and the first and second additions each contribute approximately 15% and 10% of the volume of the final liquid formulation, respectively.

[0019] Depending on the circumstances, the manufacturing method may further include the steps of sterile filtering the final liquid pharmaceutical formulation (e.g., to remove microorganisms), and / or dividing the final liquid pharmaceutical formulation into doses, and / or performing quality control testing on the final liquid pharmaceutical formulation, and comparing the results of the quality control testing with references, such as regulatory approvals or references that are the same as or consistent with the literature.

[0020] The manufacturing method of the present invention also enables the scale-up of pilot-scale production of GLP-2 analog liquid formulations to be used beforehand. This allows for batch sizes exceeding 10 liters of the final liquid pharmaceutical formulation to be produced, for example, in the liquid volume range of 10 to 50 liters of the final liquid pharmaceutical formulation, allowing for batches of 15 liters, 20 liters, 30 liters, 40 liters, or even 50 liters of the final liquid pharmaceutical formulation, optionally allowing for batch sizes of 10 to 20 liters and optionally 20 to 50 liters of the final liquid pharmaceutical formulation. In some cases, the method is carried out in a 10-liter or 20-liter tank.

[0021] Depending on the circumstances, mechanical stirring, particularly gentle stirring, may be used in step (b) to dissolve the GLP-2 analog, for example, by using a stirring blade. Depending on the circumstances, one or more steps in the method may be carried out under nitrogen. The formation of oligomers and / or impurities in the glepaglutide formulation is reduced in batches prepared with nitrogen gas compared to batches prepared with or without oxygen. For example, this may be achieved by purging the formulation with nitrogen or by carrying out the method under a nitrogen gas overlay.

[0022] Since the formulations produced using the method of the present invention are for administration to patients (particularly human patients), the method may include a step of performing visual control after step (a) to determine whether the excipient has dissolved (completely), and / or a step of performing visual control after step (b) to determine whether the GLP-2 analog and the excipient have dissolved substantially completely, and / or a step of performing visual control after step (c) to determine whether a solution of the GLP-2 analog and the excipient has been prepared by the method.

[0023] In some cases, step (b) includes washing the container or transport bag or container containing the lyophilized GLP-2 analog to remove residual GLP-2 analog. Alternatively or additionally, by adding the GLP-2 analog in step (b), for example, to ensure visual control of the clear solution at the end of the method, foaming of the composition containing the GLP-2 analog and excipient and / or formation of residual lumps of the GLP-2 analog are reduced, and / or by adding the GLP-2 analog in step (b), the level of covalently bound high molecular weight species in the final liquid pharmaceutical formulation is reduced.

[0024] In common with the aim of providing a formulation with storage stability of the GLP-2 analog, the method of the present invention results in a formulation that is stable for at least 18 months when stored at 2 - 8°C. By way of example, the formulation may contain a GLP-2 analog or a pharmaceutically acceptable salt or derivative thereof at a concentration of about 20 mg / mL, a histidine buffer at a concentration of about 15 mM, mannitol at a concentration of about 230 mM, and an appropriate amount of arginine to obtain a pH of about 7.0. Generally, the histidine buffer is L-histidine and the mannitol is D-mannitol.

[0025] The method of the present invention may also be accompanied by downstream steps, for example, filling the formulation into a prefilled syringe, injection pen, or syringe device. In a further aspect, the present invention is a method for reducing the formation of covalently bound oligomeric products of a glucagon-like peptide 2 (GLP-2) analog or a pharmaceutically acceptable salt or derivative thereof in a stable liquid pharmaceutical formulation, wherein the GLP-2 analog has 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 1~4 Alkyl (e.g., methyl), acetyl, formyl, benzoyl, or trifluoroacetyl, X5 is either Ser or Thr. X11 is either Ala or Ser. R 2 is NH2 or OH, Z 2 (This is a peptide sequence consisting of 1 to 6 amino acid units of Lys, or is not present.) Represented by, The liquid pharmaceutical preparation contains a GLP-2 analog at a concentration of approximately 2 mg / mL to approximately 30 mg / mL, and an excipient comprising (i) a histidine buffer at a concentration of approximately 5 mM to approximately 50 mM, (ii) mannitol as a nonionic tonicity regulator at a concentration of approximately 90 mM to approximately 360 mM, and (iii) an appropriate amount of arginine to obtain a preparation having a pH of approximately 6.6 to approximately 7.4. The method is (a) A step of mixing the excipient with a first volume of water for injection to obtain an excipient solution that is 70-90% of the volume of the final liquid formulation, (b) Adding the GLP-2 analog drug substance to the excipient solution to obtain a solution of the excipient and the GLP-2 analog at a volume of 70-90% of the final liquid formulation, (c) Adjusting the pH and volume of the liquid composition as necessary to obtain a liquid formulation with 100% of the volume of the final liquid pharmaceutical formulation. and The present invention provides a final aqueous liquid pharmaceutical formulation containing a glucagon-like peptide 2 (GLP-2) analog. Provide a method.

[0026] In a further embodiment, the present invention relates to the use of a formulation that reduces the formation of covalent oligomer products of a glucagon-like peptide 2 (GLP-2) analog or a pharmaceutically acceptable salt or derivative thereof, wherein the GLP-2 analog is of 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 (Sequence ID 11) (In the formula, R 1 is hydrogen, C 1~4 Alkyl (e.g., methyl), acetyl, formyl, benzoyl, or trifluoroacetyl, X5 is either Ser or Thr. X11 is either Ala or Ser. R 2 is NH2 or OH, Z 2 (This is a peptide sequence consisting of 1 to 6 amino acid units of Lys, or is not present.) Represented by, The liquid pharmaceutical preparation contains a GLP-2 analog at a concentration of approximately 2 mg / mL to approximately 30 mg / mL, and an excipient comprising (i) a histidine buffer at a concentration of approximately 5 mM to approximately 50 mM, (ii) mannitol as a nonionic tonicity regulator at a concentration of approximately 90 mM to approximately 360 mM, and (iii) an appropriate amount of arginine to obtain a preparation having a pH of approximately 6.6 to approximately 7.4. Use (a) A step of mixing the excipient with a first volume of water for injection to obtain an excipient solution that is 70-90% of the volume of the final liquid formulation, (b) Adding the GLP-2 analog drug substance to the excipient solution to obtain a solution of the excipient and the GLP-2 analog at a volume of 70-90% of the final liquid formulation, (c) Adjusting the pH and volume of the liquid composition as necessary to obtain a liquid formulation with 100% of the volume of the final liquid pharmaceutical formulation. and The method provides a final aqueous liquid pharmaceutical formulation containing a glucagon-like peptide 2 (GLP-2) analog. Provides usage.

[0027] In some embodiments, the formulation contains 5% or less of a GLP-2 analog in the form of a covalent oligomeric product. Alternatively or additionally, the total acetate concentration derived from the GLP-2 analog in the formulation is 11% acetate or less per 1 mg of GLP-2 analog. Alternatively or additionally, the formation of covalent oligomers of the GLP-2 analog depends inversely to the concentration of the GLP-2 analog in the formulation.

[0028] Hereinafter, embodiments of the present invention are described as non-limiting examples in reference to the accompanying drawings. However, various further aspects and embodiments of the present invention will become apparent to those skilled in the art in consideration of this disclosure.

[0029] While the present invention has been described in conjunction with the embodiments described above, numerous equivalent modifications and variations will be apparent to those skilled in the art. Therefore, the embodiments of the present invention described herein are considered illustrative and not limiting. Various modifications to the embodiments described can be made without departing from the spirit and scope of the invention. All documents referenced herein are incorporated by reference as a whole for all purposes.

[0030] As used herein, “and / or” is used as a specific disclosure of each of two specified features or elements, including or excluding the other. For example, “A and / or B” is used as a specific disclosure of (i)A, (ii)B, and (iii)A and B, as if each were described separately herein.

[0031] Unless otherwise indicated, the above descriptions and definitions of features are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described herein. [Brief explanation of the drawing]

[0032] [Figure 1]This is a work diagram for the novel "one-pot" formulation method B, compared to the traditional "two-pot" method A. [Figure 2] This is an image of foaming that occurs during mixing in the two-pot method A. [Modes for carrying out the invention]

[0033] Unless otherwise specified, the following definitions refer to the specific terms used in the above description. Throughout the description and claims, conventional one-letter and three-letter codes are used for natural amino acids. All amino acid residues in the peptide of the present invention are preferably L-configured amino acid residues, but D-configured amino acids may also be present. GLP-2 analogue A glucagon-like peptide 2 (GLP-2) analog or a pharmaceutically acceptable salt or derivative thereof that can be formulated using the method of the present invention is given by 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 either Ser or Thr. X11 is either Ala or Ser. R 2 is NH2 or OH, Z 1 and Z 2 (These are peptide sequences of 1 to 6 amino acid units of Lys that do not exist independently.) It is represented by [this].

[0034] Z 1 and Z 2 This is either 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 a D-configuration or an L-configuration, but preferably an L-configuration. Particularly preferred sequence Z is a sequence of 4, 5, or 6 consecutive lysine residues, and especially a sequence of 6 consecutive lysine residues. An exemplary sequence Z is shown in WO01 / 04156.

[0035] In some embodiments, R 1 is hydrogen. In some embodiments, X5 is Thr. In some embodiments, X11 is Ala. In some embodiments, R 2 It is NH2.

[0036] In some aspects, Z 1 It does not exist. In some aspects, Z 2 This is a peptide sequence of 1 to 6 amino acid units of Lys. In some embodiments, Z 2 This is a peptide sequence of 2-6 amino acid units of Lys. In some embodiments, Z 2 This is a peptide sequence of 3-6 amino acid units of Lys. In some embodiments, Z 2 This is a peptide sequence of 4-6 amino acid units of Lys. In some embodiments, Z 2 This is a peptide sequence of 5-6 amino acid units of Lys. In some embodiments, Z 2 This is a 6-amino acid peptide sequence of Lys. In some embodiments, Z 2 This is a peptide sequence of 1-2 amino acids from Lys.

[0037] In some embodiments, a GLP-2 analog or a pharmaceutically acceptable salt or derivative thereof is defined by 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 (Sequence ID 11) (In the formula, R 1 is hydrogen, C 1~4 Alkyl (e.g., methyl), acetyl, formyl, benzoyl, or trifluoroacetyl, X5 is either Ser or Thr. X11 is either Ala or Ser. R 2 is NH2 or OH, Z 2 (This is a peptide sequence consisting of 1 to 6 amino acid units of Lys, or is not present.) It is represented by [this].

[0038] In some aspects, Z 2 This is a peptide sequence of 1 to 6 amino acids from Lys. In some embodiments of the present invention, X5 is Thr and / or X11 is Ala in the above formula. Examples of these glucagon-like peptide 2 (GLP-2) analogs include the following: ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 (Sequence ID 1) ZP2949 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKK-OH (Sequence No. 2); ZP2711 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKK-OH (Sequence No. 3); ZP2469 H-HGEGTFSSELATILDALAARDFIAWLIATKITDK-OH (Sequence No. 4); ZP1857 H-HGEGTFSSELATILDALAARDFIAWLIATKITD-NH2 (Sequence ID 5); or ZP2530 H-HGEGTFSSELATILDALAARDFIAWLIATKITD-OH (Sequence ID 6) In some embodiments of the present invention, a glucagon-like peptide 2 (GLP-2) analog is ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 (Sequence ID 1) That is the case.

[0039] In other words, the GLP-2 analog is given by 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 -NH2 is represented by

[0040] It will be understood that the "H-" at the N-terminus indicates a free N-terminal amine (NH2-) group. The "NH2-" at the C-terminus indicates the C-terminal amide group. The terms ZP1848 and glepaglutide may be used interchangeably.

[0041] In some embodiments of the present invention, in the above formula, X5 is Ser and / or X11 is Ser. Examples of these glucagon-like peptide 2 (GLP-2) analogs include the following. ZP1846 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO: 7), ZP1855 H-HGEGSFSSELSTILDALAARDFIAWLIATKITD-NH2 (SEQ ID NO: 8), or ZP2242 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDK-OH (SEQ ID NO: 9) In an embodiment of the present invention, the glucagon-like peptide 2 (GLP-2) analog is ZP1846 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO: 7).

[0042] 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 [[ID=4l]]-Leu 10 -Ser 11 -Thr 12 -Ile 13 -Leu 14 -Asp 15 -Ala 16 -Leu 17 -Ala 18 -Ala19 -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 -NH2 It is represented by [this].

[0043] The N-terminal "H-" indicates a free N-terminal amine (NH2-) group. The C-terminal "NH2-" indicates a C-terminal amide group. The terms ZP1846 and ercigurtide can be used interchangeably.

[0044] This invention relates to a method for producing and purifying GLP2 analogs. These peptides are intended for use as active pharmaceutical ingredients. This invention includes GLP analogs obtained by the method of this invention.

[0045] It should be understood that the peptide (API) of the present invention may be provided in the form of a salt or other derivative. Therefore, it will be understood that after further steps of purification and optional selection, the peptide may ultimately be obtained as a salt or other derivative. Salts include pharmaceutically acceptable salts such as acid addition salts and basic salts. Examples of acid addition salts include hydrochloride salts, citrate salts, chloride salts, and acetate salts. Preferably, the salt is an acetate salt. Examples of basic salts include cations of alkali metals such as sodium and potassium, alkaline earth metals such as calcium, and ammonium ions. + N(R 3)3(R 4 )(wherein, R 3 and R 4 C is independently and, depending on the case, substituted. 1~6 -alkyl, and in some cases substituted C 2~6 Examples of salts selected from alkenyls (which may be substituted with aryls, or which may be substituted with heteroaryls) are also included. Other examples of pharmaceutically acceptable salts are listed in Remington's Pharmaceutical Sciences, 17th edition, edited by Alfonso R. Gennaro, Mark Publishing Company, Easton, PA, USA, 1985 and subsequent editions, as well as in the Encyclopaedia of Pharmaceutical Technology.

[0046] In preferred embodiments, the acetate salts of the GLP-2 analogs of the present invention are 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 this context, the term "ZP1848-acetate" refers to the ZP1848 molecule in acetate form. The acetate salts of the GLP-2 analogs can be represented by the following formula: (GLP-2 analog), x(CH3COOH) (wherein x is 1.0 to 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 GLP-2 analog acetates, x is not necessarily an integer, as molecules with different numbers of acetate molecules may exist. In some cases, x is from 4.0 to 8.0, x is from 6.0 to 8.0, or x is from 4.0 to 6.5. In other cases, x is from 4.0 to 6.0, x is from 2.0 to 6.0, x is from 2.0 to 7.0, x is from 3.0 to 6.0, x is from 4.0 to 6.0, or x is from 4.0 to 8.0. Further consideration of GLP-2 analog acetates as defined herein can be found in WO2020 / 265064, the disclosure of which is incorporated herein by reference.

[0047] In a preferred embodiment, the GLP-2 analog is ZP1848 acetate or H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 acetate (SEQ ID NO: 1) or (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2),x(CH3COOH) (wherein x is 1.0 to 8.0). This is the final result.

[0048] A solid composition containing an acetate of a glucagon-like peptide-2 (GLP-2) analog may be obtained, for example, by freeze-drying. This solid composition is useful for formulation using excipients used to produce liquid formulations. For example, the following formula: (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2),x(CH3COOH)(where x is between 1.0 and 8.0) A solid composition containing the acetate of a glucagon-like peptide 2 (GLP-2) analog having the properties of [the compound] may be obtained.

[0049] The upper limit of 8.0 acetate molecules per GLP-2 analog corresponds to an acetate content of less than 11%, and when this is formulated, it is possible to have a viscosity between 0.8 and 2.0 mPa / sec as measured at 25°C.

[0050] The range of the number of acetate molecules bound to each molecule of the GLP-2 analog determines the molecular weight range of this component in the formulation. For example, in the case of ZP1848 acetate, the range of the number of acetate molecules bound to each molecule of the GLP-2 analog determines the molecular weight range of ZP1848-acetate. For example, the molecular weight of each molecule of ZP1848 and 1 acetate equivalent is 4316 + 60 = 4376 Da. Therefore, when increasing the acetate equivalent of ZP1848, the molecular weights are 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. This then defines the molecular weight ranges as follows: 1-8 acetate equivalents = 4376 Da-4796 Da; 4-8 acetate equivalents = 4556 Da-4796 Da; and 6-8 acetate equivalents = 4676 Da-4796 Da. Further consideration of acetates of GLP-2 analogs as defined in this invention can be found in WO2020 / 265064, the disclosure of which is incorporated herein by reference.

[0051] Other derivatives of the GLP-2 analog of the present invention include Mn 2+ and Zn 2+These include coordination compounds having metal ions, esters such as hydrolyzable esters in vivo, free acids or bases, hydrates, or lipids. Esters can be formed between a hydroxyl or carboxylic acid group present in the compound and a suitable carboxylic acid or alcohol reaction partner using techniques known in the art. GLP-2 analog formulations The GLP-2 analog formulations that can be prepared using the method of the present invention are ready-to-use formulations disclosed in WO2020 / 065064 using Method A. As used herein, the term “ready-to-use” refers to a formulation that does not require composition or dilution with a predetermined amount of diluent, such as water for injection or other suitable diluent, before use via a specified route of administration.

[0052] These liquid formulations of GLP-2 analogs contain a buffer, a nonionic tonic regulator, and an appropriate amount of arginine to obtain the pH of the final formulation. Following standard pharmaceutical practices, the formulations are sterile and / or free of reducing agents. In some cases, the liquid formulations are aqueous.

[0053] A preferred stable liquid formulation is disclosed in WO2002 / 065064, which comprises a GLP-2 analog at a concentration of about 2 mg / mL to about 30 mg / mL, and an excipient comprising (i) a histidine buffer present at a concentration of about 5 mM to about 50 mM, (ii) mannitol as a nonionic tonicity regulator present at a concentration of about 90 mM to about 360 mM, and (iii) an appropriate amount of arginine to obtain a formulation having a pH of about 6.6 to about 7.4.

[0054] As used herein, the term “buffer” means a pharmaceutically acceptable excipient that stabilizes the pH of a pharmaceutical formulation. In the formulations prepared according to the present invention, the buffer is a histidine buffer, for example, L-histidine. Generally, the histidine buffer is present at a concentration of about 5 mM to about 50 mM, more preferably about 5 mM to about 25 mM, and most preferably about 15 mM.

[0055] As used herein, the term “tonic modifier” means a pharmaceutically acceptable isotonic agent used to modulate the tonicity of a formulation. The formulations prepared according to the present invention are preferably isotonic, i.e., they have an osmotic pressure substantially the same as that of human serum. The tonic modifier used in the formulations prepared according to the present invention is mannitol, e.g., D-mannitol. The concentration of the tonic modifier will depend on the concentrations of other formulation components, especially when the formulation is intended to be isotonic. Typically, nonionic tonic modifiers will be used at concentrations of about 90 mM to about 360 mM, more preferably about 150 mM to about 250 mM, and most preferably about 230 mM.

[0056] Generally, the components and amounts of the liquid formulations prepared according to the present invention are selected to obtain formulations having a pH of about 6.6 to about 7.4, more preferably about 6.8 to about 7.2, and most preferably about 7.0. The pH may be adjusted by adding an appropriate amount (qs) of arginine to bring it within the desired pH range. It is preferable not to use hydrochloric acid or sodium hydroxide for pH adjustment.

[0057] In a preferred embodiment, the liquid formulation prepared by the method of the present invention contains approximately 20 mg / mL of ZP1848 acetate or H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 acetate (SEQ ID NO: 1) or ZP1846 acetate or H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 acetate (SEQ ID NO: 7), approximately 15 mM histidine buffer, approximately 230 mM mannitol, and an appropriate amount of arginine to obtain a pH of approximately 7.0.

[0058] In a further embodiment, the liquid formulation prepared by the method of the present invention has a concentration of approximately 20 mg / mL, as follows: (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2),x(CH3COOH) or H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH2,x(CH3COOH) (where x is between 1.0 and 8.0) The solution contains an acetate of a glucagon-like peptide-2 (GLP-2) analog, a histidine buffer at a concentration of approximately 15 mM, and mannitol at a concentration of approximately 230 mM, with a pH of approximately 7.0.

[0059] In some cases, the liquid formulations prepared according to the present invention further contain a preservative. In some cases, the preservative is selected from the group consisting of benzalkonium chloride, chlorobutanol, methylparaben, and potassium sorbate. Generally, the preservative is present at a concentration of about 0.1% to about 1% of the final formulation volume.

[0060] Generally, glucagon-like peptide 2 (GLP-2) analog formulations are administered to patients parenterally, preferably by injection, most typically by subcutaneous, intramuscular, intravenous, or intraperitoneal injection. Subcutaneous administration is preferred. The injection may be administered by a physician, nurse, or other healthcare professional, or by the patient themselves. As described herein, in some embodiments, formulations prepared by the method of the present invention have a viscosity that facilitates filling of the formulation into pre-filled syringes, injection pens, or other syringe devices. This may have the advantage that the dose of the formulation to be administered to the patient is predetermined, for example, without the need to measure from a vial used multiple times. Delivery of pharmaceutical products In some embodiments, the present invention relates to a ready-to-use formulation of a GLP-2 analog, which is intended for parenteral administration and is suitable for use in, for example, vials, pre-filled syringes, infusion pumps, wearable syringes, disposable auto-injectors, or dose-adjustable auto-injectors. Medical condition The GLP-2 analog formulations of the present invention are useful as pharmaceuticals for preventing or treating individuals suffering from gastrointestinal disorders, including those affecting the upper gastrointestinal tract, including the esophagus, by administering an effective amount of the GLP-2 analog or a salt thereof as described herein. Gastrointestinal disorders include ulcers of any etiology (e.g., peptic ulcers, drug-induced ulcers, infections or other pathogen-related ulcers), digestive disorders, malabsorption syndromes, short bowel syndrome, blind duct syndrome, inflammatory bowel disease, celiac (e.g., resulting from gluten-sensitive enteropathy or celiac disease), tropical sprue, hypogammaglobulinemia sprue, enteritis, focal enteritis (Crohn's disease), ulcerative colitis, small bowel injury, and chemotherapy-induced diarrhea / mucositis (CID). [Examples]

[0061] The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention. GLP-2 analogs administered according to the dosage regimen described herein may be prepared by methods such as solid-phase peptide synthesis as described in WO2006 / 117565, the contents of which are clearly incorporated by reference as a whole. Analysis method For the analysis of covalently bonded oligomers, a Dionex Ultimate3000 HPLC system was used with a linear gradient and a flow rate of 0.5 ml / min. The mobile phase was 0.1% TFA in 45% acetonitrile and 55% milli-Q water. A wavelength of 215 nm was used for detection. The injection volume was 4 μg of peptide. The column used was a TSKgel SuperSW2000 (TSK Bioscience) with a particle size of 4 μm and dimensions of 300 × 4.6 mm. The total run time was 25 minutes.

[0062] For the evaluation of the chemical stability of peptide monomers (impurities), a C18 column with an acidic mobile phase and an acetonitrile gradient was used. Formulations of the ZP1848 active pharmaceutical ingredient have traditionally been manufactured using a conventional two-solution formulation method, referred to herein as "Method A." Method A, in the case of glepaglutide, resulted in technical problems associated with excessive foaming, formation of hard clumps of the active ingredient during dissolution, deviations from visual control, and the formation of higher molecular weight peptide oligomers. Foaming and clumping, as well as the need for manual stirring, can be particularly problematic in scale-up. In this embodiment, the inventors compare the conventional method (Method A) with a novel method (Method B) in relation to solving the aforementioned technical problems of the conventional method.

[0063] Example 1: Methods A and B (10L batch capacity) material and method Method A: In a tank, 23.27 g of L-histidine, 419 g of mannitol, and 8.71 g of L-arginine were successively added to 5.1 kg of water for injection (WFI) to obtain the excipient solution (Solution 1, Figure 1). The mixture was stirred until a clear and homogeneous solution was obtained. Next, 200 g of glepaglutide (corrected for purity and content) was added to another beaker containing 3.0 kg of WFI to obtain the API solution (ZP1848, Figure 1). The peptide transport bag was rinsed with an additional 1.0 L of WFI, and this rinse water was added to the API solution. The mixture was gently stirred manually with a spoon until the lumps disappeared and a clear and homogeneous API solution was obtained. The obtained API solution was added to the excipient solution to obtain the excipient / API solution (Solution 1 + ZP1848, Figure 1), and the pH was adjusted to 7.0 using 1 M acetic acid or 250 mM L-arginine. WFI was added until the volume reached 10 L. The mixture was gently stirred until the excipient / API solution was clear and homogeneous. Method B: In a tank, 7.2 kg of WFI was successively added to 23.27 g of L-histidine, 419 g of mannitol, and 8.71 g of L-arginine to obtain the excipient solution (Solution 1, Figure 1). The mixture was stirred until a clear and homogeneous solution was obtained. 200 g of glepaglutide (corrected for purity and content) was added directly to this excipient solution to obtain the excipient / API solution (Solution 1 + ZP1848, Figure 1). The peptide transport bag was rinsed with an additional 100 ml of WFI. The sides of the tank were rinsed with an additional 1 L of WFI. The rinse water was added to the excipient / API solution. The mixture was stirred until a clear and homogeneous solution was obtained. The pH was adjusted to 7.0 using 1 M acetic acid or 250 mM L-arginine. WFI was added until 10 L was reached. The mixture was stirred until a clear and homogeneous solution was obtained. Results and Discussion Table 1 shows the results of the visual evaluation of the two method embodiments.

[0064] [Table 1]

[0065] The novel method solves or improves upon the technical problems associated with glepaglutide formulations in conventional manufacturing method A, such as foaming, failure of visual control, and clumping. The gentle manual stirring process in method A also limits the scaling up of batch volumes from 10 L to larger batch volumes.

[0066] Tables 2 and 3 show the stability results for two batches produced using Method A and two batches produced using Method B.

[0067] [Table 2]

[0068] During stability testing, oligomer formation in glepaglutide formulations was reduced with the novel formulation method B compared to the conventional method A (Table 2).

[0069] [Table 3]

[0070] The stability of the glepaglutide formulation with respect to peptide impurities is exactly the same under the novel formulation method B compared to the conventional method A (Table 3). The difference between the two-pot method (Method A) and the one-pot method (Method B) is that Method A requires gentle manual stirring with a spoon of the API solution and the excipient / API solution to obtain a clear and homogeneous excipient / API solution, whereas Method B does not. In Method B, only one pot / tank can be used based on the ratio of excipient, WFI, and active pharmaceutical ingredient, which makes this method simpler, easier to handle, more robust to deviations, scale-up feasible, and results in a final formulation with improved stability.

[0071] Example 2: Method B (20L batch capacity) material and method Method A: Method A is described above in Example 1. Method B: In a tank, 46.54 g of L-histidine, 0.838 kg of mannitol, and 17.43 g of L-arginine were successively added to 14.4 kg of WFI to obtain the excipient solution (Solution 1, Figure 1). Each excipient beaker was washed with up to 100 ml of WFI. The mixture was stirred until a clear and homogeneous solution was obtained. 400 g of glepaglutide (corrected for purity and content) was added directly to the excipient solution in at least 3 parts, preferably 6 parts, to obtain the excipient / API solution (Solution 1 + ZP1848, Figure 1). The peptide transport bag was then rinsed with an additional 300 ml of WFI. The sides of the tank were then rinsed with an additional 2 L of WFI. The rinse water was added to the excipient / API solution. The mixture was stirred until a clear and homogeneous solution was obtained. The pH was adjusted to 7.0 using 1M acetic acid and / or 250 mM L-arginine. WFI was added until the total weight reached 20.4 kg. The mixture was stirred until a clear, homogeneous solution was obtained.

[0072] [Table 4]

[0073] Even in 20L batches, the novel Method B solved or improved upon the technical problems associated with the previous manufacturing method A, such as foaming, failure of visual control, and clumping of glepaglutide formulations. The gentle manual stirring process in Method A also limits the scaling up of batch volumes from 20L to larger batch volumes.

[0074] The stability results for two batches produced by Method A and Method B are shown in Tables 5 and 6. Table 5 includes data for 10L batch volumes for Methods A and B, compared with scale-up data for a 20L batch volume for Method B.

[0075] [Table 5]

[0076] A direct comparison of oligomer content between Method B(10L) and Method B(20L) was not possible. This is because, in the case of Method B(20L), the total method time (combining mixing and filling time) at ambient temperature is much longer than in the case of Method B(10L), resulting in a relatively slightly higher oligomer content in the 20L batch (Table 5). Considering the longer method time, the amount of oligomer is within an acceptable range.

[0077] [Table 6]

[0078] The stability of the glepaglutide formulation with respect to peptide impurities is exactly the same under the novel formulation method B compared to the conventional method A (Table 6). Example 3: Nitrogen as a process gas material and method In six separate containers suitable for a 15 ml batch volume, 34.95 mg of L-histidine, 628.5 mg of mannitol, and 13.07 mg of L-arginine were successively added to 10.5 ml of MQ water to obtain the excipient solution. The mixture was stirred until a clear and homogeneous solution was obtained. In some batches, this solution was purged with either nitrogen or oxygen gas for 5 minutes (see Table 7). 300 mg of glepaglutide (corrected for purity and content) was added to each container according to Method B. The mixture was gently stirred until the lumps disappeared and a clear and homogeneous solution was obtained. In some batches, this solution was overlaid with nitrogen or oxygen gas (Table 7). The pH was adjusted to 7.0 using 1 M acetic acid or 250 mM L-arginine. MnCl2 and FeCl3 were added to batches 4-6 to a concentration of 50+50 ppm in the final batch volume (see Table 7). MQ water was added to all formulations until 15 ml was reached. Some batches were stirred under a layer of nitrogen or oxygen gas until a clear, homogeneous solution was obtained (Table 7). The batches were filled into vials, and some batches were layered with nitrogen or oxygen gas as shown in Table 7.

[0079] [Table 7]

[0080] Results and Discussion The stability results for batches containing the compositions listed in Table 7 are shown in Tables 8 and 9.

[0081] [Table 8]

[0082] Oligomer formation in glepaglutide formulations is reduced in batches containing nitrogen gas compared to batches containing oxygen or gas-free. Oligomer formation in glepaglutide formulations is increased in batches containing oxygen gas and / or metal salt additives.

[0083] [Table 9]

[0084] The stability of glepaglutide formulations with respect to peptide impurities is exactly the same in batches containing nitrogen gas or oxygen gas, and in batches without nitrogen gas (Batches 1-3, Table 9). This stability is reduced in batches containing metal salt additives (Batches 4-6, Table 9), and this reduction is exacerbated by the combination with oxygen gas, but limited in the combination with nitrogen gas.

[0085] Glepaglutide formulations are susceptible to oxidation in relation to oligomer formation. For example, the influence of oxidizing substances such as iron or manganese from leachates or excipient impurities can be significantly reduced by eliminating the presence of oxygen by using nitrogen as the process gas. This results in more robust stability for glepaglutide formulations.

Claims

1. A method for producing a stable aqueous liquid pharmaceutical preparation containing a glucagon-like peptide 2 (GLP-2) analog, wherein the GLP-2 analog is defined by the following formula: ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH ２ (Sequence No. 1), or ZP1846 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH ２ Represented by (Sequence ID 7), or a pharmaceutically acceptable salt thereof, The aqueous liquid pharmaceutical preparation contains an excipient comprising (i) a GLP-2 analog at a concentration of approximately 2 mg / mL to approximately 30 mg / mL, (ii) a histidine buffer at a concentration of approximately 5 mM to approximately 50 mM, (ii) mannitol as a nonionic tonicity regulator at a concentration of approximately 90 mM to approximately 360 mM, and (iii) an appropriate amount of arginine to obtain a preparation having a pH of approximately 6.6 to approximately 7.

4. The method is (a) A step of mixing the excipient with a first volume of water for injection to obtain an excipient solution at 70-90% of the volume of the final aqueous liquid pharmaceutical preparation, (b) Adding the GLP-2 analog active pharmaceutical ingredient to the excipient solution to obtain a liquid composition which is a solution of the excipient and the GLP-2 analog at a volume of 70-90% of the final aqueous liquid pharmaceutical formulation, (c) Adjusting the pH and volume of the liquid composition as necessary to obtain the final aqueous liquid pharmaceutical formulation with 100% of the final aqueous liquid pharmaceutical formulation volume. and This invention provides a stable aqueous liquid pharmaceutical formulation that is a final aqueous liquid pharmaceutical formulation containing a glucagon-like peptide 2 (GLP-2) analog. The aforementioned method.

2. The method according to claim 1, wherein stirring is not required when the GLP-2 analog active ingredient is added to the excipient solution.

3. The method according to claim 1 or 2, wherein a GLP-2 analog active pharmaceutical ingredient is added as a lyophilized composition.

4. The method according to claim 1 or 2, wherein the adjustment of the volume of the liquid composition in step (c) comprises the addition of a first water for injection to adjust the pH and a second water for injection to increase the volume of the liquid composition to near the final volume, and optionally, the adjustment of the pH of the liquid composition is carried out by adding arginine and / or acetic acid.

5. The method according to claim 1 or 2, wherein the excipient solution constitutes about 80% of the volume of the final aqueous liquid pharmaceutical formulation, and each of the first and second additions constitutes about 10% of the volume of the final aqueous liquid pharmaceutical formulation.

6. The method according to claim 1 or 2, wherein the excipient solution constitutes about 75% of the volume of the final aqueous liquid pharmaceutical formulation, and the first and second additions each contribute about 15% and about 10% of the volume of the final aqueous liquid pharmaceutical formulation.

7. The method according to claim 1 or 2, wherein the active pharmaceutical ingredient is added in 1 to 5 parts, preferably in 3 parts.

8. The method according to claim 1 or 2, further comprising the step of sterilizing and filtering the final aqueous liquid pharmaceutical formulation.

9. The method according to claim 1 or 2, further comprising the step of dividing the final aqueous liquid pharmaceutical formulation into doses.

10. The method according to claim 1 or 2, further comprising the steps of performing a quality control test on a final aqueous liquid pharmaceutical formulation and comparing the results of the quality control test with a reference.

11. The method according to claim 1 or 2, wherein a batch size of 10 to 50 liters of the final aqueous liquid pharmaceutical formulation is used, and optionally a batch size of 10 to 20 liters of the final aqueous liquid pharmaceutical formulation is used, and optionally a batch size of 20 to 50 liters of the final aqueous liquid pharmaceutical formulation is used.

12. The method according to claim 1 or 2, performed in a 10-liter or 20-liter tank.

13. The method according to claim 1, (i) Mechanical stirring is used in step (b) to dissolve the GLP-2 analog drug substance, for example, using a stirring blade; and / or (ii) The method, wherein the method is carried out under nitrogen.

14. A method according to claim 1 or 2, The step includes, and / or, performing a visual check after step (a) to determine whether the excipient has dissolved. The step includes, and / or, performing a visual check after step (b) to determine whether the GLP-2 analog and excipients have completely dissolved. The method includes a step of performing a visual check after step (c) to determine whether a solution of the GLP-2 analog and excipient was prepared by the method, The aforementioned method.

15. The method according to claim 1 or 2, wherein step (b) includes washing a container or transport bag / container containing the freeze-dried GLP-2 analog to remove any remaining GLP-2 analog.

16. A method according to claim 1 or 2, (i) The addition of the GLP-2 analog in step (b) reduces foaming of the composition containing the GLP-2 analog and excipients, and / or the formation of residual clumps of the GLP-2 analog, and / or (ii) The addition of the GLP-2 analog in step (b) reduces the level of covalently bonded high molecular weight species in the final aqueous liquid pharmaceutical formulation. The aforementioned method.

17. The GLP-2 analog is in the form of an acetate, and the formula is as follows: H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH ２ Acetate (SEQ ID NO: 1) The method according to claim 1 or 2, as represented by...

18. The acetate of a glucagon-like peptide 2 (GLP-2) analog is given by the following formula: (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH ２ ), x (CH ３ COOH) (where x is between 1.0 and 8.0), or (H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH ２ ), x (CH ３ COOH) (where x is between 1.0 and 8.0) The method according to claim 17, having the following characteristics.

19. The method according to claim 18, wherein x is from 2.0 to 6.0 in the formula of the acetate of the glucagon-like peptide-2 analog.

20. The method according to either claim 1 or 2, wherein the formulation is stable for at least 18 months when stored at 2 to 8°C.

21. The method according to claim 1 or 2, wherein the histidine buffer is present at a concentration of about 5 mM to about 25 mM.

22. The method according to claim 21, wherein a histidine buffer is present at a concentration of approximately 15 mM.

23. The method according to claim 1 or 2, wherein mannitol is present as a tonicity modifier at a concentration of about 150 mM to about 250 mM.

24. The method according to claim 1 or 2, wherein the formulation has a pH of about 6.8 to about 7.

2.

25. The method according to claim 24, wherein the formulation has a pH of approximately 7.

0.

26. The method according to claim 1 or 2, wherein the formulation comprises a GLP-2 analog or a pharmaceutically acceptable salt thereof at a concentration of about 20 mg / mL, a histidine buffer at a concentration of about 15 mM, mannitol at a concentration of about 230 mM, and an appropriate amount of arginine to obtain a pH of about 7.

0.

27. The histidine buffer is L-histidine, and / or Mannitol is D-mannitol. The method according to claim 1 or 2.

28. The method according to claim 1 or 2, further comprising the step of filling a pre-filled syringe, injection pen, or syringe device with the formulation.

29. A method according to claim 1 or 2, wherein the method reduces the formation of covalent oligomer products of the GLP-2 analog in a stable aqueous liquid pharmaceutical formulation.

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