ES2366199T3 - SEPARATION OF GLP-1 AND PEPTIDES ASSOCIATED BY ION EXCHANGE CHROMATOGRAPHY. - Google Patents

Abstract

Cation exchange chromatography process for the purification of a GLP-1 peptide from a mixture comprising said GLP-1 peptide and related impurities, comprising the step of: separating said GLP-1 peptide and said related impurities from said mixture by elution in a solution consisting essentially of an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepped gradient or isocratically in a salt component and / or with a linear or stepped pH gradient or at a constant pH value, where the gradient of the pH or the pH value should be in the range where said GLP-1 peptide has a net global or positive local charge different from the positive local or global network of said related impurities to remove said related impurities; where the organic modifier is selected from C1-6 alkanol and C2-6 glycol; and where the related impurities are impurities that have a global or different local net charge and are selected from the group consisting of truncated forms, extended forms, forms with extra amino acids, derivatives, esters, deamidated forms, incorrectly folded forms, forms with undesired glycosylation and forms with sialylation.

Description

Field of the Invention

[0001] The present invention relates to an ion exchange chromatography process for purifying GLP-1 or an analogue or derivative of a mixture containing said GLP-1 and related impurities, and an industrial method that includes such a chromatography process. ion exchange

Background

[0002] For the purification and analysis of proteins and peptides, chromatography is a well known and widely used method. Several different chromatographic principles are applied, among this ion exchange chromatography (IEC). The IEC principle includes two different methods: anion exchange and cation exchange according to the charge of the ligands in the ion exchange resin. A conventional IEC purification process usually consists of one or more: balancing sections, application or loading sections, washing sections, elution sections, and regeneration sections (cf. Remington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton, PA, 1990, or Remington: The Science and Practice of Pharmacy, 19th Edition (1995)).

[0003] The main principle of elution in IEC in industrial purification processes is salt component gradients in an aqueous buffer solution at constant pH, either as linear or linear gradients (cf. S. Bjørn and L. Thim, Activation of Coagulation Factor VII to VIIa, Res. Discl. No. 269, 564-565, 1986). Isocratic elution is possible, but rarely used. Organic solvents or modifiers have occasionally been added to solutions to have the protein or peptide in the desired form or only in solution (cf. KH Jørgensen, Process for Purifying Insulin, US Patent No. 3,907,676, Sept. 23, 1975; and J Brange, O. Hallund and E. Sørensen, Chemical Stability of Insulin 5. Isolation, Characterization and Identification of Insulin Transformation Products, Acta Pharm. Nord. 4 (4), 223-232, 1992).

[0004] Glucagon-like peptide-1 (GLP-1) (cf. Schmidt et al. In Diabetology 28 704-707, 1985) and analogues as well as derivatives thereof can be used in the treatment of diabetes, as described in WO 98/08871. A GLP-1 peptide and related analogs are readily dissolved in aqueous solvents and maintained in a monomized form. The traditional IEC purification of GLP-1 with salt gradients in aqueous solvents can, however, be problematic due to the lack of selectivity between a target fraction of GLP-1 and related impurities.

[0005] WO 87/06941 (The General Hospital Corporation) discloses peptide fragments comprising GLP-1 (7-37) and functional derivatives thereof and their use as an insulinotropic agent.

[0006] WO 90/11296 (The General Hospital Corporation) discloses peptide fragments comprising GLP-1 (7-36) and functional derivatives thereof and has an insulinotropic activity that exceeds the insulinotropic activity of GLP-1 (1-36 ) or GLP-1 (1-37) and its use as insulinotropic agents.

[0007] WO 91/11457 (Buckley et al.) Discloses analogs of the active peptides GLP-1 7-34, 7-35, 7-36, and 7-37.

[0008] WO 98/08871 discloses GLP-1 derivatives in which a lipophilic substituent is attached to at least one amino acid residue. The lipophilic substituents are in particular long chain groups containing for example 12-24 carbon atoms.

[0009] WO 98/08872 discloses GLP-2 derivatives in which a lipophilic substituent is attached to at least one amino acid residue. The lipophilic substituents are in particular long chain groups containing for example 12-24 carbon atoms.

[0010] WO 96/32414 discloses GLP-2 analogs.

[0011] EP 0699686-A2 (Eli Lilly & Co.) discloses certain N-terminal truncated fragments of GLP-1 that are provided as being biologically active.

[0012] EP 0708179-A2 (Eli Lilly & Co.) discloses GLP-1 analogues and derivatives that include an N-terminal imidazole group and optionally a C6-C10 straight chain acyl group attached to the lysine residue in the position 3. 4.

[0013] EP 0207727-A2 refers to a process for recovering glucagon from the pancreas glands.

[0014] J. Chromatography 237, 417-28, 1982 describes the separation of peptides by HPLC in a linked phase of weak anion exchange.

[0015] In US 3907676 a process to reduce the antigenicity of insulin is described where an anion exchange chromatography system is used.

[0016] US 5101013 refers to a process for the isolation of basic proteins such as insulins from a protein mixture where a strongly acidic cationic exchanger is used.

[0017] US 4361 51 0 concerns methods for producing blood coagulation complexes comprising coagulation factors II, VII, IX and X, where the methods include anion exchange and elution wash phases.

Description of the invention

[0018] Unlike the techniques described above IEC for purification of any protein or peptide, which consists of one or more balancing phases, application or loading phases, washing phases, elution phases, and regeneration phases, the The present invention relates to the use of an organic modifier for the purification of a GLP-1 peptide and all related analogs etc. by IEC. By adding an organic modifier especially to the elution section of the IEC purification phase, an increase in selectivity and efficiency is obtained in comparison with the same execution with aqueous buffers, both for anionic and cationic exchange chromatography. The equilibration solution and the sample for application may or may not contain the organic modifier. The use of an organic modifier has the additional advantage that no salt is needed or very low concentrations of salt are needed for elution, especially for cation exchange chromatography compared to an aqueous chromatographic system.

[0019] In a broad aspect the present invention relates to a cation exchange chromatography process for purification of a peptide from a mixture comprising said peptide and related impurities, comprising the phase of:

separating said peptide and said related impurities from said mixture by elution in a solution comprising an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepped gradient or isocratically in salt component and / or with a linear or stepped pH gradient or at a constant pH value, where the pH gradient or pH value should be in the range where said peptide has a positive global or local charge network different from the positive local or global charge network of said related impurities to remove said related impurities.

[0020] In another broad aspect the present invention relates to a cation exchange chromatography process for the purification of a peptide from a mixture comprising said peptide and related impurities, comprising the phase of:

separating said peptide and said related impurities from said mixture by elution in a solution consisting essentially of an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepped gradient or isocratically in salt component and / or with a linear or stepped pH gradient or at a constant pH value, where the pH gradient or pH value should be in the range where said peptide has a positive global or local charge network different from the local charge network or positive overall of said related impurities to remove said related impurities.

[0021] In another broad aspect the present invention relates to an anion exchange chromatography process for purification of a peptide from a mixture comprising said peptide and related impurities, comprising the phase of:

separating said peptide and said related impurities from said mixture by elution in a solution comprising an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepped gradient or isocratically in salt component and / or with a linear or stepped pH gradient or at a constant pH value, where the pH gradient or pH value should be in the range where said peptide has a negative global or local charge network different from the negative local or global charge network of said related impurities to remove said related impurities.

[0022] In another broad aspect the present invention relates to an anion exchange chromatography process for purifying a peptide from a mixture comprising said peptide and related impurities, comprising the phase of:

separating said peptide and said related impurities from said mixture by elution in a solution consisting essentially of an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepped gradient or isocratically in salt component and / or with a linear or stepped pH gradient or at a constant pH value, where the pH gradient or pH value should be in the range where said peptide has a negative global or local charge network, different from the local charge network or overall refusal of said related impurities to remove said related impurities.

[0023] One aspect of the present invention relates to a cation exchange chromatography process for purifying a GLP-1 peptide from a mixture comprising said peptide and related impurities, comprising the phase of:

separating said GLP-1 peptide and said related impurities from said mixture by elution in a solution comprising an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepped gradient or isocratically in salt component and / or with a linear or stepped pH gradient or at a constant pH value, where the pH gradient or pH value should be in the range where said peptide has a positive global or local charge network different from the local charge network or positive overall of said related impurities to remove said related impurities.

[0024] Another aspect of the present invention relates to a cation exchange chromatography process for purifying a GLP-1 peptide from a mixture comprising said peptide and related impurities, comprising the phase of:

separating said GLP-1 peptide and said related impurities from said mixture by elution in a solution consisting essentially of an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepped gradient or isocratically in component of salt and / or with a linear or stepped pH gradient or at a constant pH value, where the pH gradient or pH value should be in the range where said peptide has a positive global or local charge network different from the network of positive local or global charge of said related impurities to eliminate said related impurities.

[0025] Another aspect of the present invention relates to an anion exchange chromatography process for purifying a GLP-1 peptide from a mixture comprising said peptide and related impurities, comprising the phase of:

separating said GLP-1 peptide and said related impurities from said mixture by elution in a solution comprising an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepped gradient or isocratically in salt component and / or with a linear or stepped pH gradient or at a constant pH value, where the pH gradient or pH value should be in the range where said peptide has a negative global or local charge network different from the local charge network or overall refusal of said related impurities to remove said related impurities.

[0026] Another aspect of the present invention relates to an anion exchange chromatography process for purifying a GLP-1 peptide from a mixture comprising said peptide and related impurities, comprising the phase of:

separating said GLP-1 peptide and said related impurities from said mixture by elution in a solution consisting essentially of an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepped gradient or isocratically in component of salt and / or with a linear or stepped pH gradient or at a constant pH value, where the pH gradient or pH value should be in the range where said peptide has a negative global or local net charge different from the charge network local or global refusal of said related impurities to eliminate said related impurities.

[0027] Elution in the above aspects may also be possible by changing the content of organic modifier in the elution section, which is considered an embodiment of the present invention.

[0028] The linear or stepped gradient in the salt component would be of a concentration lower than a higher concentration in both IEC modes.

[0029] In the previous aspects of the present process elution could also be considered a washing phase of related impurities.

[0030] In an embodiment of the present invention the ratio of organic modifier to water, on a weight percent basis, is 1:99 to 99: 1, such as 1:99 to 80:20, 20:80 at 80:20, 30:70 for 70:30, 35:50 to 50:35, or 40:50 to

50:40 Each of these proportions constitutes an alternative embodiment of the present invention.

[0031] In the present invention the organic modifier is selected from C1-6 alkanol and C2-6 glycol, more preferably methanol, ethanol, propanoles and butane and hexyl glycols, most preferably ethanol and 2-propanol. Each of these organic modifiers constitutes an alternative embodiment of the present invention.

[0032] In another embodiment of the present invention the stepped or linear pH gradient for the anion exchange chromatography process ranges from a higher pH to a lower pH.

[0033] In another embodiment of the present invention the stepped or linear pH gradient for the cation exchange chromatography process ranges from a lower pH to a higher pH.

[0034] In another embodiment of the present invention the salt component is selected from any inorganic or organic salt and its derivative mixtures, preferably NaCl, KCl, NH4Cl, CaCl2, sodium acetate, potassium acetate, ammonium acetate, sodium citrate , potassium citrate, ammonium citrate, sodium sulfate, potassium sulfate, ammonium sulfate, calcium acetate or derived mixtures, most preferred sodium acetate, potassium acetate, ammonium acetate, NaCl, NH4Cl, KCI. Each of these salt components constitutes an alternative embodiment of the present invention.

[0035] In another embodiment of the present invention the gradient in the salt component is a stepped gradient in the salt component.

[0036] In another embodiment of the present invention the salt component is present in a stepped concentration selected from the range of 0.1 mmol / kg to 3000 mmol / kg, preferably 1 mmol / kg to 1000 mmol / kg, more preferably 5 mmol / kg to 500, most preferably 20 mmol / kg to 300 mmol / kg. Each of these ranges constitutes an alternative embodiment of the present invention.

[0037] In another embodiment of the present invention the gradient in the salt component is a linear gradient in the salt component.

[0038] In another embodiment of the present invention the salt component is present in a selected linear concentration of 0.1 mmol / kg to 3000 mmol / kg, preferably 1 mmol / kg to 1000 mmol / kg, more preferably 5 mmol / kg to 500, most preferably 20 mmol / kg to 300 mmol / kg. Each of these linear concentrations constitutes an alternative embodiment of the present invention.

[0039] In another embodiment of the present invention no salt component is present.

[0040] In another embodiment of the present invention the buffer is selected from citrate buffers, phosphate buffers, tris buffers, borate buffers, lactate buffers, glycine glycine buffers, arginine buffers, carbonate buffers, buffers. acetate, glutamate buffers, ammonium buffers, glycine buffers, alkylamine buffers, aminoethyl alcohol buffers, ethylenediamine buffers, trietanol amine, imidazole buffers, pyridine buffers and barbiturate buffers and their derivative mixtures, preferably acidic citric, sodium citrate, potassium citrate, sodium phosphate, potassium phosphate, phosphorous acid, glutamic acid, sodium glutamate, potassium glutamate, glycine, sodium carbonate, tris-hydroxymethyl amino methane and boric acid and their derived mixtures. Each of these buffers constitutes an alternative embodiment of the present invention.

[0041] In another embodiment of the present invention the buffer is present in a concentration selected from the range of 0.1 mmol / kg to 500 mmol / kg, preferably 1 mmol / kg to 200 mmol / kg, more preferably 5 mmol / kg at 100 mmol / kg, most preferably 10 mmol / kg at 50 mmol / kg. Each of these ranges constitutes an alternative embodiment of the present invention.

[0042] In another embodiment of the present invention no buffer is present.

[0043] In another embodiment of the present invention the GLP-1 peptide to be purified is selected from GLP-1 (7-37), GLP-1 (7-36) amide as well as analogs and derivatives thereof , in particular but not limited to human glucagon-like peptide-1,

Arg26-GLP-1 (7-37); Arg34-GLP-1 (7-37); Lys36-GLP-1 (7-37);

Arg26,34Lys36-GLP-1 (7-37); Arg26,34Lys38-GLP-1 (7-38); Arg26,34Lys39-GLP-1 (7-39); Arg26,34Lys40-GLP-1 (7-40); Arg26Lys36-GLP-1 (7-37); Arg34Lys36-GLP-1 (7-37); Arg26Lys39-GLP-1 (7-39); Arg34Lys40-GLP-1 (7-40); Arg26,34Lys36,39-GLP-1 (7-39); Arg26,34Lys36,40-GLP-1 (7-40); Gly8Arg26-GLP-1 (7-37); Gly8Arg34-GLP-1 (7-37); Gly8Lys36-GLP-1 (7-37); Gly8Arg26,34Lys36-GLP-1 (7-37); Gly8Arg26,34Lys39-GLP-1 (7-39); Gly8Arg26,34Lys40-GLP-1 (7-40); Gly8Arg26Lys36-GLP-1 (7-37); Gly8Arg34Lys36-GLP-1 (7-37); Gly8Arg26Lys39-GLP-1 (7-39); Gly8Arg34Lys40-GLP-1 (7-40); Gly8Arg26,34Lys36,39-GLP-1 (7-39); Gly8Arg26,34Lys36,40-GLP-1 (7-40); Arg26,34Lys38GLP-1 (7-38); Arg26,34Lys39GLP-1 (7-39); Arg26,34Lys40GLP-1 (7-40); Arg26,34Lys41GLP-1 (7-41); Arg26,34Lys42GLP-1 (7-42); Arg26,34Lys43GLP-1 (7-43); Arg28,34Lys44GLP-1 (7-44); Arg26,34Lys45GLP-1 (7-45); Arg26,34Lys38GLP-1 (1-38); Arg26,34Lys39GLP-1 (1-39); Arg26,34Lys40GLP-1 (1-40); Arg26,34Lys41GLP-1 (1-41); Arg26,34Lys42GLP-1 (1-42); Arg26,34Lys43GLP-1 (1-43); Arg26,34Lys44GLP-1 (1-44); Arg26,34Lys45GLP-1 (1-45); Arg26,34Lys38GLP-1 (2-38); Arg26,34Lys39GLP-1 (2-39); Arg26,34Lys40GLP-1 (2-40); Arg26,34Lys41GLP-1 (2-41); Arg26,34Lys42GLP-1 (2-42); Arg26,34Lys43GLP-1 (2-43); Arg26,34Lys44GLP-1 (2-44); Arg26,34Lys45GLP-1 (2-45); Arg26,34Lys38GLP-1 (3-38); Arg26,34Lys39GLP-1 (3-39); Arg26,34Lys40GLP-1 (3-40); Arg26,34Lys41GLP-1 (3-41); Arg26,34Lys42GLP-1 (3-42); Arg26,34Lys43GLP-1 (3-43); Arg26,34Lys44GLP-1 (3-44); Arg26,34Lys45GLP-1 (3-45); Arg26,34Lys38GLP-1 (4-38); Arg26,34Lys39GLP-1 (4-39);

Arg26,34Lys40GLP-1 (4-40); Arg26,34Lys41GLP-1 (4-41); Arg26,34Lys42GLP-1 (4-42); Arg26,34Lys43GLP-1 (4-43); Arg26,34Lys44GLP-1 (4-44); Arg26,34Lys45GLP-1 (4-45); Arg26,34Lys38GLP-1 (5-38); Arg26,34Lys39GLP-1 (5-39); Arg26,34Lys40GLP-1 (5-40); Arg26,34Lys41GLP-1 (5-41); Arg26,34Lys42GLP-1 (5-42); Arg26,34Lys43GLP-1 (5-43); Arg26,34Lys44GLP-1 (5-44); Arg26,34Lys45GLP-1 (5-45); Arg26,34Lys38GLP-1 (6-38); Arg26,34Lys39GLP-1 (6-39); Arg26,34Lys40GLP-1 (6-40); Arg26,34Lys41GLP-1 (6-41); Arg26,34Lys42GLP-1 (6-42); Arg26,34Lys43GLP-1 (6-43); Arg26,34Lys44GLP-1 (6-44); Arg26,34Lys45GLP-1 (6-45); Arg26Lys38GLP-1 (1-38); Arg34Lys38GLP-1 (1-38); Arg26,34Lys36,38GLP-1 (1-38); Arg26Lys38GLP-1 (7-38); Arg34Lys38GLP-1 (7-38); Arg26,34Lys36,38GLP-1 (7-38); Arg26,34Lys38GLP-1 (7-38); Arg26Lys39GLP-1 (1-39); Arg34Lys39GLP-1 (1-39); Arg26,34Lys36,39GLP-1 (1-39); Arg26Lys39GLP-1 (7-39); Arg34Lys39GLP-1 (7-39); Arg26,34Lys36,39GLP-1 (7-39); Arg26-GLP-1 (8-37); Arg34-GLP-1 (8-37); Lys36-GLP-1 (8-37); Arg26,34Lys36-GLP-1 (8-37); Arg26,34Lys38GLP-1 (8-38); Arg26,34Lys39-GLP-1 (8-39); Arg26,34Lys40-GLP-1 (8-40); Arg26Lys36-GLP-1 (8-37); Arg34Lys36-GLP-1 (8-37); Arg26Lys39-GLP-1 (8-39); Arg34Lys40-GLP-1 (8-40); Arg26,34Lys36,39-GLP-1 (8-39); Arg26,34Lys36,40-GLP-1 (8-40); Gly8Arg26-GLP-1 (8-37); Gly8Arg34-GLP-1 (8-37); Gly8Lys36-GLP-1 (8-37); Gly8Arg26,34Lys36-GLP-1 (8-37); Gly8Arg26,34Lys39-GLP-1 (8-39); Gly8Arg26,34Lys40-GLP-1 (8-40); Gly8Arg26Lys36-GLP-1 (8-37); Gly8Arg34Lys36-GLP-1 (8-37); Gly8Arg26Lys39-GLP-1 (8-39); Gly8Arg34Lys40-GLP-1 (8-40); Gly8Arg26,34Lys36,39-GLP-1 (8-39); Gly8Arg26,34Lys36,40-GLP-1 (8-40); Argzsv’Lys38GLP-1 (8-38); Arg26,34Lys39GLP-1 (8-39); Arg26,34Lys40GLP-1 (8-40); Arg26,34Lys41GLP-1 (8-41); Arg26,34Lys42GLP-1 (8-42);

Arg26,34Lys43GLP-1 (8-43); Arg26,34Lys44GLP-1 (8-44); Arg26,34Lys45GLP-1 (8-45); Arg26Lys38GLP-1 (8-38); Arg34Lys38GLP-1 (8-38); Arg26,34Lys36,38GLP-1 (8-38); Arg26,34Lys38GLP-1 (8-38); Arg26Lys39GLP-1 (8-39); Arg34Lys39GLP-1 (8-39); Arg26,34Lys36,39GLP-1 (8-39); Arg26-GLP-1 (8-37), Arg34-GLP-1 (8-37), Lys36-GLP-1 (8-37), Arg26,34Lys36-GLP-1 (8-37), Arg26Lys36-GLP-1 (8-37), Arg34Lys36-GLP-1 (8-37), Gly8Arg26-GLP-1 (8-37),

Gly8Arg34-GLP-1 (8-37), Gly8Lys36-GLP-1 (8-37), Gly8Arg26,34Lys36-GLP-1 (8-37), Gly8Arg26Lys36-GLP-1 (8-37), Gly8Arg34Lys36-GLP-1 (8-37); Arg26Lys38-GLP-1 (8-38), Arg26,34Lys38-GLP-1 (8-38), Arg26,34Lys36,38-GLP-1 (8-38), Gly8Arg26Lys38-GLP-1 (8-38); Gly8Arg26,34Lys36,38-GLP-1 (8-38); Arg34Lys40-GLP-1 (8-40), Arg26,34Lys36,40-GLP-1 (8-40), Gly8Arg34Lys40-GLP-1 (8-40); GIy8Arg26,34Lys36,40-GLP-1 (8-40); Arg26-GLP-1 (8-36); Arg34-GLP-1 (8-36); Arg26,34Lys36-GLP-1 (8-36); Arg26-GLP-1 (8-36) amide; Arg34-GLP-1 (8-36) amide; Arg26,34Lys36-GLP-1 (8-36) amide; Arg26-GLP-1 (8-37); Arg34-GLP-1 (8-37); Arg26,34Lys36-GLP-1 (8-37); Arg26-GLP-1 (8-38); Arg34-GLP-1 (8-38); Arg26,34Lys38GLP-1 (8-38); Arg26-GLP-1 (8-39); Arg34-GLP-1 (8-39); Arg26,34Lys39-GLP-1 (8-39); Gly8Arg26-GLP-1 (8-36); Gly8Arg34-GLP-1 (8-36); Gly8Arg26,34Lys36-GLP-1 (8-36); Gly8Arg26-GLP-1 (8-36) amide; Gly8Arg34-GLP-1 (8-36) amide; Gly8Arg26,34Lys36-GLP-1 (8-36) amide; Gly8Arg26-GLP-1 (8-37); Gly8Arg34-GLP-1 (8-37); Gly8Arg26,34Lys36-GLP-1 (8-37); Gly8Arg26-GLP-1 (8-38); Gly8Arg34-GLP-1 (8-38); Gly8Arg26,34Lys38GLP-1 (8-38); Gly8Arg26-GLP-1 (8-39); Gly8Arg34-GLP-1 (8-39); Gly8Arg26,34Lys39-GLP-1 (8-39); Val8Arg26-GLP-1 (8-36); Val8Arg34-GLP-1 (8-36); Val8Arg26,34Lys36-GLP-1 (8-36);

Val8Arg26-GLP-1 (8-36) amide; Val8Arg34-GLP-1 (8-36) amide; Val8Arg26,34Lys36 GLP-1 (8-36) amide; Val8Arg26-GLP-1 (8-37);

Val8Arg34-GLP-1 (8-37); Val8Arg26,34Lys36-GLP-1 (8-37); Val8Arg26-GLP-1 (8-38); Val8Arg34-GLP-1 (8-38); Val8Arg26,34Lys38GLP-1 (8-38); Val8Arg26-GLP-1 (8-39); Val8Arg34-GLP-1 (8-39); Val8Arg26,34Lys39-GLP-1 (8-39); Ser8Arg26-GLP-1 (8-36); Ser8Arg34-GLP-1 (8-36); Ser8Arg26,34Lys38-GLP-1 (8-36); Ser8Arg26-GLP-1 (8-36) amide; Ser8Arg34-GLP-1 (8-36) amide; Ser8Arg26,34Lys36-GLP-1 (8-36) amide; Ser8Arg26-GLP-1 (8-37); Ser8Arg34-GLP-1 (8-37); Ser8Arg26,34Lys36-GLP-1 (8-37); Ser8Arg26-GLP-1 (8-38); Ser8Arg34-GLP-1 (8-38); Ser8Arg26,34Lys38GLP-1 (8-38); Ser8Arg26-GLP-1 (8-39); Ser8Arg34-GLP-1 (8-39); Ser8Arg26,34Lys39-GLP-1 (8-39); Thr8Arg26-GLP-1 (8-36); Thr8Arg34-GLP-1 (8-36); Thr8Arg26,34Lys36-GLP-1 (8-36); Thr8Arg26-GLP-1 (8-36) amide; Thr8Arg34-GLP-1 (8-36) amide; Thr8Arg26,34Lys36-GLP-1 (8-36) amide; Thr8Arg26-GLP-1 (8-37); Thr8Arg34-GLP-1 (8-37); Thr8Arg26,34Lys36-GLP-1 (8-37); Thr8Arg26-GLP-1 (8-38); Thr8Arg34-GLP-1 (8-38); Thr8Arg26,34Lys38-GLP-1 (8-38); Thr8Arg26-GLP-1 (8-39); Thr8Arg34-GLP-1 (8-39); Thr8Arg26,34Lys39-GLP-1 (8-39); Val8Glu35Arg26,34Lys36-GLP-1 (8-36); Val8Glu35Arg26,34Lys36-GLP-1 (8-36) amide; Val8Glu36Arg26,34Lys37-GLP-1 (8-37); Val8Glu37Arg26,34Lys38-GLP-1 (8-38); Val8Glu38Arg26,34Lys39-GLP-1 (8-39); Val8Glu35Arg26,34Lys38-GLP-1 (8-36); Val8Glu35Arg26,34Lys36-GLP-1 (8-36) amide; Val8Glu36Arg26,34Lys37-GLP-1 (8-37); Val8Glu37Arg26,34Lys38-GLP-1 (8-38); Val8Glu38Arg26,34Lys39-GLP-1 (8-39); Val8Asp35Arg26,34Lys36-GLP-1 (8-36); Val8Asp35Arg26,34Lys36-GLP-1 (8-36) amide;

Val8Asp36Arg26,34Lys37-GLP-1 (8-37); Val8Asp37Arg26,34Lys38-GLP-1 (8-38); Val8Asp38Arg26,34Lys39-GLP-1 (8-39); Val8Asp35Arg26,34Lys36-GLP-1 (8-36);

Val8Asp35Arg26,34Lys36-GLP-1 (8-36) amide; Val8Asp36Arg26,34Lys37-GLP-1 (8-37); Val8Asp37Arg26,34Lys38-GLP-1 (8-38); Val8Asp38Arg26,34Lys39-GLP-1 (8-39); Ser8Glu35Arg26,34Lys36-GLP-1 (8-36); Ser8Glu35Arg26,34Lys36-GLP-1 (8-36) amide; Ser8Glu36Arg26,34Lys37-GLP-1 (8-37); Ser8Glu37Arg26,34Lys38-GLP-1 (8-38); Ser8Glu38Arg26,34Lys39-GLP-1 (8-39); Ser8Glu35Arg26,34Lys36-GLP-1 (8-36); Ser8Glu35Arg26,34Lys36-GLP-1 (8-36) amide; Ser8Glu38Arg26,34Lys37-GLP-1 (8-37); Ser8Glu37Arg26,34Lys38-GLP-1 (8-38); Ser8Glu38Arg26,34Lys39-GLP-1 (8-39); Ser8Asp35Arg26,34Lys36-GLP-1 (8-36); Ser8Asp35Arg26,34Lys36-GLP-1 (8-36) amide; Ser8Asp36Arg26,34Lys37-GLP-1 (8-37); Ser8Asp37Arg26,34Lys38 -GLP-1 (8-38); Ser8Asp38Arg26,34Lys39-GLP-1 (8-39); Ser8Asp35Arg26,34Lys36-GLP-1 (8-36); Ser8Asp35Arg26,34Lys36-GLP-1 (8-36) amide; Ser8Asp36Arg26,34Lys37-GLP-1 (8-37); Ser8Asp37Arg26,34Lys38-GLP-1 (8-38); Ser8Asp38Arg26,34Lys39-GLP-1 (8-39); Thr8Glu35Arg26,34Lys36-GLP-1 (8-36); Thr8Glu35Arg26,34Lys36-GLP-1 (8-36) amide; Thr8Glu36Arg26,34Lys37-GLP-1 (8-37); Thr8Glu37Arg26,34Lys38-GLP-1 (8-38); Thr8Glu38Arg26,34Lys39-GLP-1 (8-39); Thr8Glu35Arg26,34Lys36-GLP-1 (8-36); Thr8Glu35Arg26,34Lys36-GLP-1 (8-36) amide; Thr8Glu36Arg26,34Lys37-GLP-1 (8-37); Thr8Glu37Arg26,34Lys38-GLP-1 (8-38); Thr8Glu38Arg26,34Lys39-GLP-1 (8-39); Thr8Asp35Arg26,34Lys36-GLP-1 (8-36); Thr8Asp35Arg26,34Lys36-GLP-1 (8-36) amide; Thr8Asp36Arg26,34Lys37-GLP-1 (8-37); Thr8Asp37Arg26,34Lys38-GLP-1 (8-38); Thr8Asp38Arg26,34Lys39-GLP-1 (8-39); Thr8Asp35Arg26,34Lys36-GLP-1 (8-36); Thr8Asp35Arg26,34Lys36-GLP-1 (8-36) amide; Thr8Asp36Arg26,34Lys37-GLP-1 (8-37); Thr8Asp37Arg26,34Lys38-GLP-1 (8-38); Thr8Asp38 Arg26,34Lys39-GLP-1 (8-39); Gly8Glu35Arg26,34Lys36-GLP-1 (8-36); Gly8Glu35Arg26,34Lys36-GLP-1 (8-36) amide; Gly8Glu36Arg26,34Lys37-GLP-1 (8-37); Gly8Glu37Arg26,34Lys38-GLP-1 (8-38); Gly8Glu38Arg26,34Lys39-GLP-1 (8-39); Gly8Glu35Arg26,34Lys36-GLP-1 (8-36); Gly8Glu35Arg26,34Lys36-GLP-1 (8-36) amide; Gly8Glu36Arg26,34Lys37-GLP-1 (8-37); Gly8Glu37Arg26,34Lys38-GLP-1 (8-38); Gly8Glu38Arg26,34Lys39-GLP-1 (8-39); Gly8Asp35Arg26,34Lys36-GLP-1 (8-36); Gly8Asp35Arg26,34Lys36-GLP-1 (8-36) amide; Gly8Asp36Arg26,34Lys37-GLP-1 (8-37);

Gly8Asp37Arg26,34Lys38-GLP-1 (8-38); Gly8Asp38Arg26,34Lys39-GLP-1 (8-39); Gly8Asp35Arg26,34Lys36-GLP-1 (8-36); Gly8Asp35Arg26,34Lys36-GLP-1 (8-36) amide; Gly8Asp36Arg26,34Lys37-GLP-1 (8-37); Gly8Asp37Arg26,34Lys38 -GLP-1 (8-38); Gly8Asp38Arg26,34Lys39-GLP-1 (8-39); Arg26,34Lys18-GLP-1 (8-36); Arg26,34Lys18-GLP-1 (8-36) amide; Arg26,34Lys18-GLP-1 (8-37); Arg26,34Lys18-GLP-1 (8-38); Gly8Asp19Arg26,34Lys18-GLP-1 (8-36); Gly8Asp17Arg26,34Lys18-GLP-1 (8-36); Gly8Asp19Arg26,34Lys18-GLP-1 (8-36) amide; Gly8Asp17Arg26,34Lys18-GLP-1 (8-36) amide; Gly8Asp19Arg26,34Lys18-GLP-1 (8-37); Gly8Asp19Arg26,34Lys18-GLP-1 (8-38); Gly8Asp17Arg26,34Lys18-GLP-1 (8-38); Arg26,34Lys23-GLP-1 (8.36) Arg26,34Lys23-GLP-1 (8-36) amide; Arg26,34Lys23GLP-1 (8-37); Arg26,34Lys23GLP-1 (8-38); Gly8Asp24Arg26,34Lys23-GLP-1 (8-36); Gly8Asp22Arg26,34Lys23-GLP-1 (8-36); Gly8Asp24Arg26,34Lys23-GLP-1 (8-36) amide; Gly8Asp22Arg26,34Lys23-GLP-1 (8-36) amide; Gly8Asp24Arg26,34Lys23-GLP-1 (8-37); Gly8Asp24Arg28,34Lys23-GLP-1 (8-38); Gly8Asp22Arg26,34Lys23-GLP-1 (8-38); Arg26,34Lys27-GLP-1 (8-36); Arg26,34Lys27-GLP-1 (8-36) amide; Arg26,34Lys27-GLP-1 (8-37); Arg26,34Lys27 -GLP-1 (8-38); Gly8Asp28Arg26,34Lys27-GLP-1 (8-36); Gly8Asp26Arg26,34Lys27-GLP-1 (8-36); Gly8Asp28Arg26,34Lys27-GLP-1 (8-36) amide; Gly8Asp26Arg26,34Lys27-GLP-1 (8-36) amide; Gly8Asp28Arg26,34Lys27-GLP-1 (8-37); Gly8Asp28Arg26,34Lys27-GLP-1 (8-38); Gly8Asp26Arg26,34Lys27-GLP-1 (8-38); Arg26,34Lys18-GLP-1 (8-36); Arg26,34Lys18-GLP-1 (8-36) amide; Arg26,34Lys18GLP-1 (8-37); Arg26,34Lys18GLP-1 (8-38); Val8Asp19Arg26,34Lys18-GLP-1 (8-36); Val8Asp17Arg26,34Lys18-GLP-1 (8-36); Val8Asp19Arg26,34Lys18-GLP-1 (8-36) amide; Val8Asp17Arg26,34Lys18-GLP-1 (8-36) amide; Val8Asp19Arg26,34Lys18-GLP-1 (8-37); Val8Asp19Arg26,34Lys18-GLP-1 (8-38); Val8Asp17Arg26,34Lys18-GLP-1 (8-38); Arg26,34Lys23-GLP-1 (8-36); Arg26,34Lys23-GLP-1 (8-36) amide; Arg26,34Lys23-GLP-1 (8-37); Arg26,34Lys23-GLP-1 (8-38); Val8Asp24Arg26,34Lys23-GLP-1 (8-36); Val8Asp22Arg26,34Lys23-GLP-1 (8-36); Val8Asp24Arg26,34Lys23-GLP-1 (8-36) amide; Val8Asp22Arg26,34Lys23-GLP-1 (8-36) amide; Val8Asp24Arg26,34Lys23-GLP-1 (8-37); Val8Asp24Arg26,34Lys23-GLP-1 (8-38); Val8Asp22Arg26,34Lys23-GLP-1 (8-38); Arg26,34Lys27-GLP-1 (8-36); Arg26,34Lys27-GLP-1 (8-36) amide; Arg26,34Lys27-GLP-1 (8-37); Arg26,34Lys27-GLP-1 (8-38);

Val8Asp28Arg26,34Lys27-GLP-1 (8-36); Val8Asp26Arg26,34Lys27-GLP-1 (8-36); Val8Asp28Arg26,34Lys27-GLP-1 (8-36) amide; Val8Asp26Arg26,34Lys27-GLP-1 (8-36) amide; Val8Asp28Arg26,34Lys27-GLP-1 (8-37); Val8Asp28Arg26,34Lys26,34-GLP-1 (8-38); Val8Asp26Arg26,34Lys27-GLP-1 (8-38); Arg26,34Lys18-GLP-1 (8-36); Arg26,34Lys18-GLP-1 (8-36) amide; Arg26,34Lys18GLP-1 (8-37); Arg26,34Lys18GLP-1 (8-38); Ser8Asp19Arg26,34Lys18-GLP-1 (8-36); Ser8Asp17Arg26,34Lys18-GLP-1 (8-36); Ser8Asp19Arg26,34Lys18-GLP-1 (8-36) amide; Se8Asp17Arg26,34Lys18-GLP-1 (8-36) amide; Ser8Asp19Arg26,34Lys18-GLP-1 (8-37); Ser8Asp19Arg26,34Lys18-GLP-1 (8-38); Ser8Asp17Arg26,34Lys18-GLP-1 (8-38); Arg26,34Lys23-GLP-1 (8-36); Arg26,34Lys23-GLP-1 (8-36) amide; Arg26,34Lys23-GLP-1 (8-37); Arg26,34Lys23-GLP-1 (8-38); Se8Asp24Arg26,34Lys23-GLP-1 (8-36); Ser8Asp22Arg26,34Lys23-GLP-1 (8-36); Ser8Asp24Arg26,34Lys23-GLP-1 (8-36) amide; Ser8Asp22Arg26,34Lys23-GLP-1 (8-36) amide; Ser8Asp24Arg26,34Lys23-GLP-1 (8-37); Se8Asp24Arg26,34Lys23-GLP-1 (8-38); Se8Asp22Arg26,34Lys23-GLP-1 (8-38); Arg26,34Lys27-GLP-1 (8-36); Arg26,34Lys27-GLP-1 (8-36) amide; Arg26,34Lys27-GLP-1 (8-37); Arg26,34Lys27-GLP-1 (8-38); Se8Asp28Arg26,34Lys27-GLP-1 (8-36); Ser8Asp26Arg26,34Lys27-GLP-1 (8-36); Ser8Asp28Arg26,34Lys27-GLP-1 (8-36) amide; Ser8Asp26Arg26,34Lys27-GLP-1 (8-36) amide; Ser8Asp28Arg26,34Lys27GLP-1 (8-37); Ser8Asp28Arg26,34Lys27GLP-1 (8-38); Ser8Asp26Arg26,34Lys27GLP-1 (8-38); Arg26,34Lys18-GLP-1 (8-36); Arg26,34Lys18-GLP-1 (8-36) amide; Arg26,34Lys18GLP-1 (8-37); Arg26,34Lys18GLP-1 (8-38); Thr8Asp19Arg26,34Lys18-GLP-1 (8-36); Thr8Asp17Arg26,34Lys18-GLP-1 (8-36); Thr8Asp19Arg26,34Lys18-GLP-1 (8-36) amide; Thr8Asp17Arg26,34Lys18-GLP-1 (8-36) amide; Thr8Asp19Arg26,34Lys18-GLP-1 (8-37); Thr8Asp19Arg26,34Lys18-GLP-1 (8-38); Thr8Asp17Arg26,34Lys18-GLP-1 (8-38); Arg26,30Lys23-GLP-1 (8-36); Arg26,34Lys23-GLP-1 (8-36) amide; Arg26,34Lys23GLP-1 (8-37); Arg26,34Lys23GLP-1 (8-38);

Thr8Asp24Arg26,34Lys23-GLP-1 (8-36); Thr8Asp22Arg26,34Lys23-GLP-1 (8-36); Thr8Asp24Arg26,34Lys23-GLP-1 (8-36) amide; Thr8Asp22Arg26,34Lys23-GLP-1 (8-36) amide; Thr8Asp24Arg26,34Lys23GLP-1 (8-37); Thr8Asp24Arg26,34Lys23GLP-1 (8-38); Thr8Asp22Arg26,34Lys23GLP-1 (8-38); Arg26,34Lys26-GLP-1 (8-36); Arg26,34Lys27-GLP-1 (8-36) amide; Arg26,34Lys27-GLP-1 (8-37);

Arg26,34Lys27GLP-1 (8-38); Thr8Asp28Arg26,34Lys27-GLP-1 (8-36); Thr8Asp26Arg26,34Lys27-GLP-1 (8-36); Thr8Asp28Arg26,34Lys27-GLP-1 (8-36) amide; Thr8Asp26Arg26,34Lys27-GLP-1 (8-36) amide; Thr8Asp28Arg26,34Lys27-GLP-1 (8-37); Thr8Asp28Arg26,34Lys27-GLP-1 (8-38); Thr8Asp26Arg26,34Lys27-GLP-1 (8-38); Arg26Lys36-GLP-1 (8-36); Arg34Lys36-GLP-1 (8-36); Arg26Lys36-GLP-1 (8-37); Arg34Lys36-GLP-1 (8-37); Arg26Lys37-GLP-1 (8-37); Arg34Lys37-GLP-1 (8-37); Arg26Lys39-GLP-1 (8-39); Arg34Lys39-GLP-1 (8-39); Arg26,34Lys36,39-GLP-1 (8-39); Arg26Lys18-GLP-1 (8-36); Arg34Lys18-GLP-1 (8-36); Arg26Lys18-GLP-1 (8-37); Arg34Lys18-GLP-1 (8-37); Arg26Lys18-GLP-1 (8-38); Arg34Lys18-GLP-1 (8-38); Arg26Lys18-GLP-1 (8-39); Arg34Lys18-GLP-1 (8-39); Arg26Lys23-GLP-1 (8-36); Arg34Lys23-GLP-1 (8-36); Arg26Lys23GLP-1 (8-37); Arg34Lys23GLP-1 (8-37); Arg26Lys23GLP-1 (8-38); Arg34Lys23GLP-1 (8-38); Arg26Lys23GLP-1 (8-39); Arg34Lys23GLP-1 (8-39); Arg26Lys27GLP-1 (8-36); Arg34Lys27-GLP-1 (8-36); Arg26Lys27GLP-1 (8-37); Arg34Lys27GLP-1 (8-37); Arg26Lys27GLP-1 (8-38); Arg34Lys27GLP-1 (8-38); Arg26Lys27GLP-1 (8-39); Arg34Lys27GLP-1 (8-39); Arg26,34Lys18,36-GLP-1 (8-36); Arg26,34Lys18-GLP-1 (8-37); Arg26,34Lys18,37-GLP-1 (8-37); Arg26,34Lys18,38-GLP-1 (8-38); Arg26,34Lys18,39-GLP-1 (8-39); Arg26,34Lys23,36-GLP-1 (8-36); Arg26,34Lys23GLP-1 (8-37); Arg26,34Lys23,37-GLP-1 (8-37);

Arg26,34Lys23,38-GLP-1 (8-38); Arg28,34Lys23,39-GLP-1 (8-39); Arg26,34Lys27,36-GLP-1 (8-36); Arg26,34Lys27-GLP-1 (8-37); Arg26,34Lys27,37-GLP-1 (8-37); Arg26,34Lys27,38-GLP-1 (8-38); Arg26,34Lys27,39-GLP-1 (8-39); Gly8GLP-1 (8-36); Gly8GLP-1 (8-37); Gly8GLP-1 (8-38); Gly8GLP-1 (8-39); Gly8Arg26Lys36-GLP-1 (8-36); Gly8Arg34Lys36-GLP-1 (8-36); Gly8Arg26Lys36-GLP-1 (8-37); Gly8Arg34Lys36-GLP-1 (8-37); Gly8Arg26Lys37-GLP-1 (8-37); Gly8Arg34Lys37-GLP-1 (8-37); Gly8Arg26Lys39-GLP-1 (8-39); Gly8Arg34Lys39-GLP-1 (8-39); Gly8Arg26,34Lys36,39-GLP-1 (8-39); Gly8Arg26Lys18-GLP-1 (8-36); Gly8Arg34Lys18-GLP-1 (8-36); Gly8Arg26Lys18-GLP-1 (8-37); Gly8Arg34Lys18-GLP-1 (8-37); Gly8Arg26Lys18-GLP-1 (8-38); Gly8Arg34Lys18-GLP-1 (8-38); Gly8Arg26Lys18-GLP-1 (8-39); Gly8Arg34Lys18-GLP-1 (8-39); Gly8Arg26Lys23-GLP-1 (8-36); Gly8Arg34Lys23-GLP-1 (8-36); Gly8Arg26Lys23-GLP-1 (8-37); Gly8Arg34Lys23-GLP-1 (8-37); Gly8Arg26Lys23-GLP-1 (8-38); Gly8Arg34Lys23-GLP-1 (8-38); Gly8Arg26Lys23-GLP-1 (8-39); Gly8Arg34Lys23-GLP-1 (8-39); Gly8Arg26Lys27-GLP-1 (8-36);

Gly8Arg34Lys27-GLP-1 (8-36); Gly8Arg26Lys27-GLP-1 (8-37); Gly8Arg34Lys27GLP-1 (8-37); Gly8Arg26Lys27GLP-1 (8-38); Gly8Arg34Lys27GLP-1 (8-38); Gly8Arg26Lys27GLP-1 (8-39); Gly8Arg34Lys27GLP-1 (8-39); Gly8Arg26,34Lys18,36-GLP-1 (8-36); Gly8Arg26,34Lys18GLP-1 (8-37); Gly8Arg18.37Lys18.37GLP-1 (8-37); Gly8Arg26,34Lys18,38GLP-1 (8-38); Gly8Arg26,34Lys18,39GLP-1 (8-39); Gly8Arg26,34Lys23,36-GLP-1 (8-36); Gly8Arg26,34Lys23GLP-1 (8-37); Gly8Arg26,34Lys23,37GLP-1 (8-37); Gly8Arg26,34Lys23,38GLP-1 (8-38); Gly8Arg26,34Lys23,39GLP-1 (8-39); Gly8Arg26,34Lys27,36-GLP-1 (8-36); Gly8Arg26,34Lys27GLP-1 (8-37); Gly8Arg26,34Lys27,37GLP-1 (8-37); Gly8Arg26,34Lys27,38GLP-1 (8-38); Gly8Arg26,34Lys27,39GLP-1 (8-39); Val8GLP-1 (8-36); Val8GLP-1 (8-37); Val8GLP-1 (8-38); Val8GLP-1 (8-39) Val8Arg26Lys36-GLP-1 (8-36); Val8Arg34Lys36-GLP-1 (7-36); Val8Arg26Lys36-GLP-1 (8-37); Val8Arg34Lys36-GLP-1 (8-37); Val8Arg26Lys37-GLP-1 (8-37); Val8Arg34Lys37-GLP-1 (8-37); Val8Arg26Lys39-GLP-1 (8-39); Val8Arg34Lys39-GLP-1 (8-39); Val8Arg26,34Lys36,39GLP-1 (8-39); Val8Arg26Lys18-GLP-1 (8-36); Val8Arg34Lys18-GLP-1 (8-36); Val8Arg26Lys18-GLP-1 (8-37); Val8Arg34Lys18-GLP-1 (8-37); Val8Arg26Lys18-GLP-1 (8-38); Val8Arg34Lys18-GLP-1 (8-38); Val8Arg26Lys18-GLP-1 (8-39); Val8Arg34Lys18GLP-1 (8-39); Val8Arg26Lys23-GLP-1 (8-36); Val8Arg34Lys23-GLP-1 (8-36); Val8Arg26Lys23GLP-1 (8-37); Val8Arg34Lys23GLP-1 (8-37); Val8Arg26Lys23GLP-1 (8-38); Val8Arg34Lys23GLP-1 (8-38); Val8Arg26Lys23GLP-1 (8-39); Val8Arg34Lys23GLP-1 (8-39); Val8Arg26Lys27-GLP-1 (8-36); Val8Arg34Lys27-GLP-1 (8-36); Val8Arg26Lys27GLP-1 (8-37); Val8Arg34Lys27GLP-1 (8-37); Val8Arg26Lys26GLP-1 (8-38); Val8Arg34Lys27GLP-1 (8-38); Val8Arg26Lys27GLP-1 (8-39); Val8Arg34Lys27GLP-1 (8-39); Val8Arg26,34Lys18,36-GLP-1 (8-36); Val8Arg26,34Lys18GLP-1 (8-37); Val8Arg26,34Lys18,37GLP-1 (8-37); Val8Arg26,34Lys18,38GLP-1 (8-38); Val8Arg26,34Lys18,39GLP-1 (8-39); Val8Arg26,34Lys23,26-GLP-1 (8-36); Val8Arg26,34Lys23GLP-1 (8-37); Val8Arg26,34Lys23,37GLP-1 (8-37); Val8Arg26,34Lys23,38GLP-1 (8-38); Val8Arg26,34Lys23,39GLP-1 (8-39); Val8Arg26,34Lys27,36-GLP-1 (8-36); Val8Arg26,34Lys27GLP-1 (8-37); Val8Arg26,34Lys27,37GLP-1 (8-37); Val8Arg26,34Lys27,38GLP-1 (8-38); Val8Arg26,34Lys27,39GLP-1 (8-39); Val8GLP-1 (7-37); Thr8GLP-1 (7-37);

Met8GLP-1 (7-37); Gly8GLP-1 (7-37); Val8GLP-1 (7-36) amide; Thr8GLP-1 (7-36) amide; Met8GLP-1 (7-36) amide; Gly8GLP-1 (7-36) amide; Lys26 (N-tetradecanoyl) -GLP-1 (7-37); Lys34 (N-tetradecanoyl) -GLP-1 (7-37); Lys26,34-bis (N-tetradecanoyl) -GLP-1 (7-37); Gly8Lys26 (N-tetradecanoyl) -GLP-1 (7-37); Gly8Lys34 (N-tetradecanoyl) -GLP-1 (7-37); Gly8Lys26,34-bis (N-tetradecanoyl) -GLP-1 (7-37); Arg26Lys34 (N-tetradecanoyl) -GLP-1 (7-37); Lys26 (N-tetradecanoyl) -G LP-1 (7-38); Lys34 (N-tetradecanoyl) -GLP-1 (7-38); Lys26,34-bis (N-tetradecanoyl) -GLP-1 (7-38); Gly8Lys26 (N-tetradecanoyl) -GLP-1 (7-38); Gly8Lys34 (N-tetradecanoyl) -GLP-1 (7-38); Gly8Lys26,34-bis (N-tetradecanoyl) -GLP-1 (7-38); Arg26Lys34 (N-tetradecanoyl) -GLP-1 (7-38); Lys26 (N-tetradecanoyl) -GLP-1 (7-39); Lys34 (N-tetradecanoyl) -GLP-1 (7-39); Lys26,34-bis (N-tetradecanoyl) -GLP-1 (7-39); Gly8Lys26 (N-tetradecanoyl) -GLP-1 (7-39); Gly8Lys34 (N-tetradecanoyl) -GLP-1 (7-39); Gly8Lys26,34-bis (N-tetradecanoyl) -GLP-1 (7-39); Arg26Lys34 (N-tetradecanoyl) -GLP-1 (7-39); Lys26 (N-tetradecanoyl) -GLP-1 (7-40); Lys34 (N-tetradecanoyl) -GLP-1 (7-40); Lys26,34-bis (N-tetradecanoyl) -GLP-1 (7-40); Gly8Lys26 (N-tetradecanoyl) -GLP-1 (7-40); Gly8Lys34 (N-tetradecanoyl) -GLP-1 (7-40); Gly8Lys26,34-bis (N-tetradecanoyl) -GLP-1 (7-40); Arg26Lys34 (N-tetradecanoyl) -GLP-1 (7-40); Lys26 (N-tetradecanoyl) -GLP-1 (7-36); Lys34 (N-tetradecanoyl) -GLP-1 (7-36); Lys26,34-bis (N-tetradecanoyl) -GLP-1 (7-36); Gly8Lys26 (N-tetradecanoyl) -GLP-1 (7-36); Gly8Lys34 (N-tetradecanoyl) -GLP-1 (7-36); Gly8Lys26,34-bis (N-tetradecanoyl) -GLP-1 (7-36); Arg26Lys34 (N-tetradecanoyl) -GLP-1 (7-36); Lys26 (N-tetradecanoyl) -G LP-1 (7-35); Lys34 (N-tetradecanoyl) -GLP-1 (7-35); Lys26,34-bis (N-tetradecanoyl) -GLP-1 (7-35); Gly8Lys26 (N-tetradecanoyl) -GLP-1 (7-35);

Gly8Lys34 (Nε-tetradecanoyl) -GLP-1 (7-35); Gly8Lys26,34-bis (N-tetradecanoyl) -GLP-1 (7-35); Arg26Lys34 (N-tetradecanoyl) -GLP-1 (7-35); Lys26 (N-tetradecanoyl) -GLP-1 (7-36) amide; Lys34 (N-tetradecanoyl) -GLP-1 (7-36) amide; Lys26,34-bis (N-tetradecanoyl) -GLP-1 (7-36) amide; Gly8Lys26 (N-tetradecanoyl) -GLP-1 (7-36) amide; Gly8Lys34 (N-tetradecanoyl) -GLP-1 (7-36) amide; Gly8Lys26,34-bis (N-tetradecanoyl) -GLP-1 (7-36) amide; Arg26Lys34 (N-tetradecanoyl) -GLP-1 (7-36) amide; Gly8Arg26Lys34 (N-tetradecanoyl) -GLP-1 (7-37); Lys26 (N-tetradecanoyl) Arg34-GLP-1 (7-37); Gly8Lys26 (N-tetradecanoyl) Arg34-GLP-1 (7-37); Arg26,34Lys36 (N-tetradecanoyl) -GLP-1 (7-37); Gly8Arg26,34Lys36 (N-tetradecanoyl) -GLP-1 (7-37); Gly8Arg26Lys34 (N-tetradecanoyl) -GLP-1 (7-38); Lys26 (N-tetradecanoyl) Arg34-GLP-1 (7-38); Gly8Lys26 (N-tetradecanoyl) Arg34-GLP-1 (7-38); Arg26,34Lys36 (N-tetradecanoyl) -GLP-1 (7-38); Arg26,34Lys36 (N-tetradecanoyl) -GLP-1 (7-38); Gly8Arg26,34Lys36 (N-tetradecanoyl) -GLP-1 (7-38); Gly8Arg26Lys34 (N-tetradecanoyl) -GLP-1 (7-39); Lys26 (N-tetradecanoyl) Arg34-GLP-1 (7-39); Gly8Lys26 (N-tetradecanoyl) Arg34-GLP-1 (7-39); Arg26,34Lys36 (N-tetradecanoyl) -GLP-1 (7-39); Gly8Arg26,34Lys36 (N-tetradecanoyl) -GLP-1 (7-39); Gly8Arg26Lys34 (N-tetradecanoyl) -GLP-1 (7-40); Lys26 (N-tetradecanoyl) Arg34-GLP-1 (7-40);

Gly8Lys26 (N-tetradecanoyl) Arg34-GLP-1 (7-40); Arg26,34Lys36 (N-tetradecanoyl) -GLP-1 (7-40); Gly8Arg26,34Lys36 (N-tetradecanoyl) -GLP-1 (7-40);

Lys26 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-37); Lys34 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-37); Lys26,34-bis (N- (ω-carboxynonadecanoyl)) - GLP-1 (7-37); Gly8Lys26 (N- (ω-carboxinonadecanoyl)) -GLP-1 (7-37); Gly8Lys34 (N- (ω-carboxinonadecanoyl)) -GLP-1 (7-37); Gly8Lys26,34-bis (N- (ω-carboxynonadecanoyl)) - GLP-1 (7-37); Lys26 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-38); Lys34 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-38); Lys26,34-bis (N- (ω-carboxynonadecanoyl)) - GLP-1 (7-38); Gly8Lys26 (N- (ω-carboxynonadecanoyl)) -GLP-1 (7-38); Gly8Lys34 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-38); Gly8Lys26,34-bis (N- (ω-carboxynonadecanoyl)) - GLP-1 (7-38); Lys26 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-39); Lys34 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-39); Lys26,34-bis (N- (ω-carboxynonadecanoyl)) - GLP-1 (7-39); Gly8Lys26 (N- (ω-carboxinonadecanoyl)) - G LP-1 (7-39); Gly8Lys34 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-39); Gly8Lys26,34-bis (N- (ω-carboxynonadecanoyl)) - GLP-1 (7-39); Lys26 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-40); Lys34 (N- (ω-carboxinonadecanoyl)) -GLP-1 (7-40); Lys26,34-bis (N- (ω-carboxynonadecanoyl)) - GLP-1 (7-40); Gly8Lys26 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-40); Gly8Lys34 (N- (ω-carboxynonadecanoyl)) -GLP-1 (7-40); Gly8Lys26,34-bis (N- (ω-carboxynonadecanoyl)) - GLP-1 (7-40); Lys26 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-36); Lys34 (N- (ω-carboxinonadecanoyl)) -GLP-1 (7-36); Lys26,34-bis (N- (ω-carboxynonadecanoyl)) - GLP-1 (7-36); Gly8Lys26 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-36); Gly8Lys34 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-36); Gly8Lys26,34-bis (N- (ω-carboxynonadecanoyl)) - GLP-1 (7-36); Lys26 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-36) amide; Lys34 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-36) amide; Lys26,34-bis (N- (ω-carboxynonadecanoyl)) - GLP-1 (7-36) amide; Gly8Lys26 (N- (ω-carboxynonadecanoyl)) - GLP-1 (7-36) amide; Gly8Lys34 (N- (ω-carboxynonadecanoyl)) - GLP-1 (7-36) amide;

Gly8Lys26,34-bis (N- (ω-carboxynonadecanoyl)) - GLP-1 (7-36) amide; Lys26 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-35); Lys34 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-35); Lys26,34-bis (N- (ω-carboxynonadecanoyl)) - GLP-1 (7-35); Gly8Lys26 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-35); Gly8Lys34 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-35); Gly8Lys26,34-bis (N- (ω-carboxynonadecanoyl)) - GLP-1 (7-35); Arg26Lys34 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-37); Gly8Arg26Lys34 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-37); Lys26 (N- (ω-carboxinonadecanoyl)) Arg34-GLP-1 (7-37); Gly8Lys26 (N- (ω-carboxinonadecanoyl)) Arg34-GLP-1 (7-37); Arg26,34Lys36 (N- (ω-carboxynonadecanoyl)) - GLP-1 (7-37); Gly8Arg21.34Lys36 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-37); Arg26Lys34 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-38); Gly8Arg26Lys34 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-38); Lys26 (N- (ω-carboxinonadecanoyl)) Arg34-GLP-1 (7-38); Gly8Lys26 (N- (ω-carboxinonadecanoyl)) Arg34-GLP-1 (7-38); Arg26,34Lys36 (N- (ω-carboxynonadecanoyl)) - GLP-1 (7-38); Arg26,34Lys38 (N- (ω-carboxynonadecanoyl)) - GLP-1 (7-38); Gly8Arg26,34Lys36 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-38); Arg26Lys34 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-39); Gly8Arg26Lys34 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-39); Lys26 (N- (ω-carboxinonadecanoyl)) Arg34-GLP-1 (7-39); Gly8Lys26 (N- (ω-carboxinonadecanoyl)) Arg34-GLP-1 (7-39); Arg26,34Lys36 (N- (ω-carboxynonadecanoyl)) - GLP-1 (7-39); Gly8Arg26,34Lys16 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-39);

Arg34Lys26 (N- (-glutamyl (N-hexadecanoyl))) - GLP-1 (7-37) -OH, Lys26,34-bis (N- (-glutamyl (N-hexadecanoil))) -GLP-1 (7-37) -OH, Lys26,34-bis (N- (-glutamyl (N-tetradecanoyl))) - GLP-1 (7-37) -OH, Arg26,34Lys38 (N - (-glutamyl (N-tetradecanoyl))) - GLP-1 (7-38) -OH, Arg26,34Lys38 (N- (-glutamyl (N-hexadecanoyl))) - G LP-1 (7-38) -OH,

Arg26Lys34 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-40); Gly8Arg26Lys34 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-40);

Lys26 (N- (ω-carboxinonadecanoyl)) Arg34-GLP-1 (7-40); Gly8Lys26 (N- (ω-carboxinonadecanoyl)) Arg34-GLP-1 (7-40);

Arg26,34Lys31 (N- (ω-carboxinonadecanoyl)) - GLP-1 (7-40); Gly8Arg26,34Lys36 (N- (- carboxinonadecanoyl)) - GLP-1 (7-40 Lys34 (N- (7-deoxicoloil)) - GLP-1 (7-37); Lys26,34-bis (N - (7-deoxicoloil)) - GLP-1 (7-37); Gly8Lys26 (N- (7-deoxicoloil)) - GLP-1 (7-37); Gly8Lys34 (N- (7-deoxicoloil)) - GLP-1 (7-37); Gly8Lys26,34-bis (N- (7-deoxicoloil)) - GLP-1 (7-37); Arg26Lys34 (N- (7-deoxicoloil)) - GLP-1 ( 7-37); Lys26 (N- (7-deoxicoloil)) - GLP-1 (7-38); Lys34 (N- (7-deoxicoloil)) - GLP-1 (7-38); Lys26,34 -bis (N- (7-deoxicoloil)) - GLP-1 (7-38); Gly8Lys26 (N- (7-deoxicoloil)) - GLP-1 (7-38); Gly8Lys34 (N - (7 -deoxicoloyl)) - GLP-1 (7-38); Gly8Lys26,34-bis (N- (7-deoxicoloil)) - GLP-1 (7-38); Arg26Lys34 (N- (7-deoxicoloil)) -GLP-1 (7-38); Lys26 (N- (7-deoxicoloil)) - GLP-1 (7-39); Lys34 (N- (7-deoxicoloil)) - GLP-1 (7-39 ); Lys26,34-bis (N- (7-deoxicoloil)) - GLP-1 (7-39); Gly8Lys26 (N- (7-deoxicoloil)) - GLP-1 (7-39); Gly8Lys34 ( N- (7-deoxicoloil)) - GLP-1 (7-39); Gly8Lys26,34-bis (N- (7-deoxicoloil)) - GLP-1 (7-39); Arg26Lys34 (N- ( 7-deoxicoloyl)) - GLP-1 (7-39); Lys26 (N- (7-deoxicoloi l)) - GLP-1 (7-40); Lys34 (N- (7-deoxicoloyl)) - GLP-1 (7-40); Lys26,34-bis (N- (7-deoxicoloyl)) - GLP-1 (7-40); Gly8Lys26 (N- (7-deoxicoloyl)) - GLP-1 (7-40); Gly8Lys34 (N- (7-deoxicoloyl)) - GLP-1 (7-40); Gly8Lys26,34-bis (N- (7-deoxicoloyl)) - GLP-1 (7-40); Arg26Lys34 (N- (7-deoxicoloyl)) - GLP-1 (7-40); Lys26 (N- (7-deoxicoloyl)) - GLP-1 (7-36); Lys34 (N- (7-deoxicoloyl)) - GLP-1 (7-36); Lys26,34-bis (N- (7-deoxicoloyl)) - GLP-1 (7-36); Gly8Lys26 (N- (7-deoxicoloyl)) - GLP-1 (7-36); Gly8Lys34 (N- (7-deoxicoloyl)) - GLP-1 (7-36); Gly8Lys26,34-bis (N- (7-deoxicoloyl)) - GLP-1 (7-36); Arg26Lys34 (N- (7-deoxicoloyl)) - GLP-1 (7-36); Lys26 (N- (7-deoxicoloyl)) - GLP-1 (7-35); Lys34 (N- (7-deoxicoloyl)) - GLP-1 (7-35); Lys26,34-bis (N- (7-deoxicoloyl)) - GLP-1 (7-35); Gly8Lys26 (N- (7-deoxicoloyl)) - GLP-1 (7-35); Gly8Lys34 (N- (7-deoxicoloyl)) - GLP-1 (7-35); Gly8Lys26,34-bis (N- (7-deoxicoloyl)) - GLP-1 (7-35); Arg26Lys34 (N- (7-deoxicoloyl)) - GLP-1 (7-35); Lys26 (N- (7-deoxicoloyl)) - GLP-1 (7-36) amide; Lys34 (N- (7-deoxicoloyl)) - GLP-1 (7-36) amide; Lys26,34-bis (N- (7-deoxicoloyl)) - GLP-1 (7-36) amide; Gly8Lys26 (N- (7-deoxicoloyl)) - GLP-1 (7-36) amide; Gly8Lys34 (N- (7-deoxicoloyl)) - GLP-1 (7-36) amide; Gly8Lys26,34-bis (N- (7-deoxicoloyl)) - GLP-1 (7-36) amide; Arg26Lys34 (N- (7-deoxicoloyl)) - GLP-1 (7-36) amide; Gly8Arg26Lys34 (N- (7-deoxicoloyl)) - GLP-1 (7-37); Lys26 (N- (7-deoxicoloyl)) Arg34-GLP-1 (7-37); Gly8Lys26 (N- (7-deoxicoloil)) Arg34-GLP-1 (7-37); Arg26,34Lys36 (N- (7-deoxicoloyl)) - GLP-1 (7-37); Gly8Arg26,34Lys36 (N- (7-deoxicoloyl)) - GLP-1 (7-37); Lys26 (N- (colyl)) - GLP-1 (7-37); Lys34 (N- (colyl)) - GLP-1 (7-37); Lys26,34-bis (N- (coloyl)) - GLP-1 (7-37); Gly8Lys26 (N- (colyl)) - GLP-1 (7-37); Gly8Lys34 (N- (coloyl)) - GLP-1 (7-37); Gly8Lys26,34-bis (N- (colyl)) - GLP-1 (7-37); Arg26Lys34 (N- (coloyl)) - GLP-1 (7-37); Gly8Arg26Lys34 (N- (7-deoxicoloyl)) - GLP-1 (7-38);

Lys26 (N- (7-deoxicoloyl)) Arg34-GLP-1 (7-38); Gly8Lys26 (N- (7-deoxicoloyl)) Arg34-GLP-1 (7-38); Arg26,34Lys36 (N- (7-deoxicoloyl)) - GLP-1 (7-38); Arg26,34Lys38 (N- (7-deoxicoloyl)) - GLP-1 (7-38); Gly8Arg26,34Lys36 (N- (7-deoxicoloyl)) - GLP-1 (7-38); Lys26 (N- (colyl)) - GLP-1 (7-38); Lys34 (N- (coloyl)) - GLP-1 (7-38); Lys26,34-bis (N- (coloyl)) - GLP-1 (7-38); Gly8Lys26 (N- (coloyl)) - GLP-1 (7-38); Gly8Lys34 (N- (coloyl)) - GLP-1 (7-38); Gly8Lys26,34-bis (N- (coloyl)) - GLP-1 (7-38); Arg26Lys34 (N- (coloyl)) - GLP-1 (7-38); Gly8Arg26Lys34 (N- (7-deoxicoloyl)) - GLP-1 (7-39); Lys26 (N- (7-deoxicoloyl)) Arg34-GLP-1 (7-39); Gly8Lys26 (N- (7-deoxicoloil)) Arg34-GLP-1 (7-39); Arg26,34Lys36 (N- (7-deoxicoloyl)) - GLP-1 (7-39); Gly8Arg26,34Lys36 (N- (7-deoxicoloyl)) - GLP-1 (7-39); Lys26 (N- (colyl)) - GLP-1 (7-39); Lys34 (N- (colyl)) - GLP-1 (7-39); Lys26,34-bis (N- (coloyl)) - GLP-1 (7-39); Gly8Lys26 (N- (colyl)) - GLP-1 (7-39); Gly8Lys34 (N- (colyl)) - GLP-1 (7-39); Gly8Lys26,34-bis (N- (colyl)) - GLP-1 (7-39); Arg26Lys34 (N- (coloyl)) - GLP-1 (7-39); Gly8Arg26Lys34 (N- (7-deoxicoloyl)) - GLP-1 (7-40); Lys26 (N- (7-deoxicoloyl)) Arg34-GLP-1 (7-40); Gly8Lys26 (N- (7-deoxicoloil)) Arg34-GLP-1 (7-40); Arg26,34Lys36 (N- (7-deoxicoloyl)) - GLP-1 (7-40); Gly8Arg21,34Lys36 (N- (7-deoxicoloyl)) - GLP-1 (7-40); Lys26 (N- (colyl)) - GLP-1 (7-40); Lys34 (N- (colyl)) - GLP-1 (7-40); Lys26,34-bis (N- (coloyl)) - GLP-1 (7-40); Gly8Lys26 (N- (colyl)) - GLP-1 (7-40); Gly8Lys34 (N- (coloyl)) - GLP-1 (7-40); Gly8Lys26,34-bis (N- (colyl)) - GLP-1 (7-40); Arg26Lys34 (N- (colyl)) - GLP-1 (7-40); Lys26 (N- (colyl)) - GLP-1 (7-36); Lys34 (N- (colyl)) - GLP-1 (7-36); Lys26,34-bis (N- (coloyl)) - GLP-1 (7-36); Gly8Lys26 (N- (coloyl)) - GLP-1 (7-36); Gly8Lys34 (N- (coloyl)) - GLP-1 (7-36); Gly8Lys26,34-bis (N- (colyl)) - GLP-1 (7-36); Arg26Lys34 (N- (coloyl)) - GLP-1 (7-36); Lys26 (N- (colyl)) - GLP-1 (7-35); Lys34 (N- (colyl)) - GLP-1 (7-35); Lys26,34-bis (N- (coloyl)) - GLP-1 (7-35); Gly8Lys26 (N- (colyl)) - GLP-1 (7-35); Gly8Lys34 (N- (coloyl)) - GLP-1 (7-35); Gly8Lys26,34-bis (N- (coloyl)) - GLP-1 (7-35); Arg26Lys34 (N- (colyl)) - GLP-1 (7-35); Lys26 (N- (coloyl)) - GLP-1 (7-36) amide; Lys34 (N- (coloyl)) - GLP-1 (7-36) amide; Lys26,34-bis (N- (coloyl)) - GLP-1 (7-36) amide; Gly8Lys26 (N- (coloyl)) - GLP-1 (7-36) amide; Gly8Lys34 (N- (coloyl)) - GLP-1 (7-36) amide; Gly8Lys26,34-bis (N- (coloyl)) - GLP-1 (7-36) amide; Arg26Lys34 (N- (coloyl)) - GLP-1 (7-36) amide; Gly8Arg26Lys34 (N- (coloil)) - GLP-1 (7-37); Lys26 (N- (colyl)) Arg34-GLP-1 (7-37); Gly8Lys26 (N- (colyl)) Arg34-GLP-1 (7-37); Arg26,34Lys36 (N- (colyl)) - GLP-1 (7-37); Gly8Arg26,34Lys36 (N- (colyl)) - GLP-1 (7-37); Lys26 (N- (lithocolyl)) - GLP-1 (7-37); Lys34 (N- (lithocolyl)) - GLP-1 (7-37); Lys26,34-bis (N- (lithocolyl)) - GLP-1 (7-37); Gly8Lys26 (N- (lithocol)) - GLP-1 (7-37); Gly8Lys34 (N- (lithocol)) - GLP-1 (7-37); Gly8Lys26,34-bis (N- (lithocolyl)) - GLP-1 (7-37); Arg26Lys34 (N- (lithocol)) - GLP-1 (7-37); Gly8Arg26Lys34 (N- (colyl)) - GLP-1 (7-38); Lys26 (N- (colyl)) Arg34-GLP-1 (7-38); Gly8Lys26 (N- (colyl)) Arg34-GLP-1 (7-38); Arg26,34Lys36 (N (colyl)) - GLP-1 (7-38);

Arg26,34Lys38 (N- (colyl)) - GLP-1 (7-38); Gly8Arg26,34Lys36 (N- (colyl)) - GLP-1 (7-38); Lys26 (N- (lithocolyl)) - GLP-1 (7-38); Lys34 (N- (lithocolyl)) - GLP-1 (7-38); Lys26,34-bis (N- (lithocolyl)) - GLP-1 (7-38); Gly8Lys26 (N- (lithocol)) - GLP-1 (7-38); Gly8Lys34 (N- (lithocol)) - GLP-1 (7-38); Gly8Lys26,34-bis (N- (lithocolyl)) - GLP-1 (7-38); Arg26Lys34 (N- (lithocol)) - GLP-1 (7-38); Gly8Arg26Lys34 (N- (coloil)) - GLP-1 (7-39); Lys26 (N- (colyl)) Arg34-GLP-1 (7-39); Gly8Lys26 (N- (colyl)) Arg34-GLP-1 (7-39); Arg26,34Lys36 (N- (colyl)) - GLP-1 (7-39); Gly8Arg26,34Lys36 (N- (colyl)) - GLP-1 (7-39); Lys26 (N- (lithocolyl)) - GLP-1 (7-39); Lys34 (N- (lithocolyl)) - GLP-1 (7-39); Lys26,34-bis (N- (lithocolyl)) - GLP-1 (7-39); Gly8Lys26 (N- (lithocol)) - GLP-1 (7-39); Gly8Lys34 (N- (lithocol)) - GLP-1 (7-39); Gly8Lys26,34-bis (N- (lithocolyl)) - GLP-1 (7-39); Arg26Lys34 (N- (lithocol)) - GLP-1 (7-39); Gly8Arg26Lys34 (N- (coloil)) - GLP-1 (7-40); Lys26 (N- (colyl)) Arg34-GLP-1 (7-40); Gly8Lys26 (N- (colyl)) Arg34-GLP-1 (7-40); Arg26,34Lys36 (N- (colyl)) - GLP-1 (7-40); Gly8Arg26,34Lys36 (N- (colyl)) - GLP-1 (7-40); Lys26 (N- (lithocolyl)) - GLP-1 (7-40); Lys34 (N- (lithocolyl)) - GLP-1 (7-40); Lys26,34-bis (N- (lithocolyl)) - GLP-1 (7-40); Gly8Lys26 (N- (lithocol)) - GLP-1 (7-40); Gly8Lys34 (N- (lithocol)) - GLP-1 (7-40); Gly8Lys26,34-bis (N- (lithocolyl)) - GLP-1 (7-40); Arg26Lys34 (N- (lithocol)) - GLP-1 (7-37);

Lys26 (N- (lithocolyl)) - GLP-1 (7-36); Lys34 (N- (lithocolyl)) - GLP-1 (7-36); Lys26,34-bis (N- (lithocolyl)) - GLP-1 (7-36); Gly8Lys26 (N- (lithocol)) - GLP-1 (7-36); Gly8Lys34 (N- (lithocol)) - GLP-1 (7-36); Gly8Lys26,34-bis (N- (lithocolyl)) - GLP-1 (7-36); Arg26 Lys34 (N- (lithocol)) - GLP-1 (7-36); Lys26 (N- (lithocolyl)) - GLP-1 (7-35); Lys34 (N- (lithocolyl)) GLP-1 (7-35); Lys26,34-bis (N- (lithocolyl)) - GLP-1 (7-35); Gly8Lys26 (N- (lithocol)) - GLP-1 (7-35); Gly8Lys34 (N- (lithocol)) - GLP-1 (7-35); Gly8Lys26,34-bis (N- (lithocolyl)) -GLP-1 (7-35); Arg26Lys34 (N- (lithocol)) - GLP-1 (7-35); Lys26 (N- (lithocolyl)) - GLP-1 (7-36) amide; Lys34 (N- (lithocolyl)) - GLP-1 (7-36) amide; Lys26,34-bis (N- (lithocolyl)) - GLP-1 (7-36) amide; Gly8Lys26 (N- (lithocolyl)) - GLP-1 (7-36) amide; Gly8Lys34 (N- (lithocolyl)) - GLP-1 (7-36) amide; Gly8Lys26,34-bis (N- (lithocolyl)) - GLP-1 (7-36) amide; Arg26Lys34 (N- (lithocolyl)) - GLP-1 (7-36) amide; Gly8Arg26Lys34 (N- (lithocolyl)) - GLP-1 (7-37); Lys26 (N- (lithocolyl)) Arg34-GLP-1 (7-37); Gly8Lys26 (N- (lithocol)) Arg34-GLP-1 (7-37); Arg26,34Lys36 (N- (lithocolyl)) - GLP-1 (7-37); Arg26,34Lys38 (N- (lithocolyl)) - GLP-1 (7-37); Gly8Arg26,34Lys36 (N- (lithocolyl)) - GLP-1 (7-37); Gly8Arg26Lys34 (N- (lithocolyl)) - GLP-1 (7-38); Lys26 (N- (lithocolyl)) Arg34-GLP-1 (7-38); Gly8Lys26 (N- (lithocol)) Arg34-GLP-1 (7-38); Arg26,34Lys36 (N- (lithocolyl)) - GLP-1 (7-38); Arg26,34Lys38 (N- (lithocolyl)) - GLP-1 (7-38); Gly8Arg26,34Lys36 (N- (lithocolyl)) - GLP-1 (7-38); Gly8Arg26Lys34 (N- (lithocolyl)) - GLP-1 (7-39); Lys26 (N- (lithocolyl)) Arg34-GLP-1 (7-39); Gly8Lys26 (N- (lithocol)) Arg34-GLP-1 (7-39); Arg26,34Lys36 (N- (lithocolyl)) - GLP-1 (7-39); Gly8Arg26,34Lys36 (N- (lithocolyl)) - GLP-1 (7-39); Gly8Arg26Lys34 (N- (lithocolyl)) - GLP-1 (7-40); Lys26 (N- (lithocolyl)) Arg34-GLP-1 (7-40); Gly8Lys26 (N- (lithocol)) Arg34-GLP-1 (7-40); Arg26,34Lys36 (N - (lithocol)) - GLP-1 (7-40) and Gly8Arg26,34Lys36 (N- (lithocolyl)) - GLP-1 (7-40).

Each of these GLP-1 peptides constitutes an alternative embodiment of the present invention.

[0044] In the present invention the related impurities of GLP-1 to be removed are selected in truncated forms, all types of extended forms (extra amino acids, various derivatives including esters etc.), deamidated forms, incorrectly folded forms, forms with unwanted glycosylation including sialylation. As an example illustrating the present invention, histidine has a predominantly positive net charge below pH ~ 6.5, so for the cation exchange the gradient of pH elution with a solvent comprising an organic solvent or modifier could be initiated below pH 6.5 to eliminate a missing truncated form of histidine and terminate the gradient above pH 6.5 thus subsequently eluting the target GLP-1 fraction. As an alternative, elution separating the truncated form of the target GLP-1 fraction using an organic modifier could be performed below pH 6.5 simply by a salt component (gradient or isocratically) under elution conditions. As another alternative, the elution separating the truncated form of the target GLP-1 fraction using organic modifier could be performed below pH 6.5 by a gradient in the organic modifier of a lower content than a higher one. As a second example, the C-terminal amino acid carboxyl group has a predominantly negative net charge above pH ~ 3.1,

thus for the anion exchange the gradient of pH elution with a solvent comprising an organic modifier could be started above pH 3.1 to eliminate an extended form to an amide and terminate the gradient below pH 3.1 thus subsequently eluting the fraction of GLP- 1 goal Alternatively, elution separating the amide form from the target GLP-1 fraction using an organic modifier could be performed above pH 3.1 simply by a salt component (gradient or isocratically) under elution conditions. As a third example, aspartic acid has a predominantly negative net charge above pH4.4, so for anion exchange the elution gradient of pH with a solvent comprising an organic modifier could be initiated above pH 4.4 to eliminate a truncated form missing aspartic acid and finish the gradient below pH 4.4 thus subsequently eluting the target GLP-1 fraction. Alternatively, elution separating the truncated form of the target GLP-1 fraction using organic modifier could be performed above pH 4.4 simply by a salt component (gradient or isocratically) under elution conditions. As a fourth example, glutamic acid has a predominantly negative net charge above pH4.4, so for anionic exchange of the pH elution gradient with a solvent comprising an organic modifier could be initiated above pH 4.4 to elute the fraction of GLP-1 goal and finish the gradient below pH

4.4 thus subsequently eliminating an extended form comprising an extra glutamic acid residue. Alternatively, elution separating the extended form of the target GLP-1 fraction using organic modifier could be performed above pH 4.4 simply by a salt component (gradient or isocratically) under elution conditions. As a fifth example, the amino group of the N-terminal amino acid has a predominantly net positive charge below pH ~ 8.0, so for cation exchange the gradient of pH elution with a solvent comprising an organic modifier could be initiated below pH 8.0 to eliminate an extended form with an unwanted acyl group and finish the gradient above pH 8.0 thus subsequently eluting the target GLP-1 fraction. Alternatively, elution separating the acylated form from the target GLP-1 fraction using organic modifier could be performed below pH 8.0 simply by a salt component (gradient or isocratically) under elution conditions. As a sixth example, the amino group of the N-terminal amino acid has a predominantly positive net charge below pH ~ 8.0, so for the cation exchange the pH elution gradient with a solvent comprising an organic modifier could be initiated below pH 8.0 to elute the target GLP-1 fraction that extends with a desired acyl group and terminate the gradient above pH 8.0 thus subsequently eliminating the unwanted unstretched form. Alternatively, elution separating the meta acylated GLP-1 fraction from the non-acylated form using organic modifier could be performed below pH 8.0 simply by a salt component (gradient or isocratically) under elution conditions. As a seventh example, tyrosine has a predominantly negative net charge above pH 10.0, so for anion exchange the gradient of pH elution with a solvent comprising an organic modifier could be initiated above pH 10.0 to eliminate a truncated form at which is missing a tyrosine residue and finish the gradient below pH 10.0 thus subsequently eluting the target GLP-1 fraction. Alternatively, elution separating the truncated form of the target GLP-1 fraction using organic modifier could be performed above pH 10.0 simply by a salt component (gradient or isocratically) under elution conditions. As an eighth example, lysine has a predominantly positive net charge below pH ~ 10.0, so for cation exchange the pH elution gradient with a solvent comprising an organic modifier could be initiated below pH 10.0 to elute a fraction of GLP-1 target acylated in the side chain of the lysine residue and finish the gradient above pH 10.0 thus subsequently eliminating an unwanted un acylated form. Alternatively, the elution separating the meta acylated GLP-1 fraction from the non-acylated form using organic modifier could be performed below pH 10.0 simply by a salt component (gradient or isocratically) under elution conditions. As a ninth example, arginine has a predominantly positive net charge below pH ~ 12.0, so for anion exchange the pH elution gradient with a solvent comprising an organic modifier could be initiated below pH 12.0 to elute the fraction of GLP-1 goal and finish the gradient above pH 12.0 thus subsequently eliminating an unwanted form comprising an extra arginine residue. Alternatively, elution separating the target GLP-1 fraction from the form comprising an extra arginine residue using organic modifier could be performed below pH 12.0 simply by a salt component (gradient or isocratically) under elution conditions. (The pKA values used in these examples are from: L. Stryer. Biochemistry, 3rd edition, W.H. Freeman and Company, New York, Table 2-1 page 21).

[0045] In another embodiment of the present invention the impurities to be removed are not related to GLP-1.

[0046] Specific examples of the GLP-1 peptide of the above-mentioned method are separation of Arg34GLP-1 (7-37) and Arg34GLP-1 (9-37) by cation exchange chromatography, GLP-1 (7-37) and GLP -1 (7-36) amide by anion exchange chromatography, GLP-1 (15-37) and GLP-1 (16-37) by anion exchange chromatography, Arg34GLP-1 (7-37) and Arg34Lys26 (N -Glu) GLP-1 (7-37) by anion exchange chromatography, Arg34Lys26 (N- (-Glu- (Ntetradecanoil))) GLP-1 (7-37) and Arg34Lys26 (N- ( -Glu- (N-tetradecanoyl))) Gly37 (N- (-Glu- (N-tetradecanoyl))) GLP-1 (7-37) by cation exchange chromatography, Gly37 (N- ( -Glu- (N-tetradecanoyl))) GLP-1 (7-37) and GLP-1 (7-37) by cation exchange chromatography, GLP-1 (19-37) and GLP-1 (20-37 ) by anion exchange chromatography, Arg34Lys26 (N- (-Glu- (N-tetradecanoyl))) GLP-1 (7-37) and Arg34GLP-1 (7-37) by cation exchange chromatography, and GLP -1 (7-37) and Arg34GLP-1 (7-37) by cation exchange chromatography using salt and / or pH gradients.

[0047] GLP-1 peptides or peptides can be produced by a method comprising culturing or fermenting a host cell with a DNA sequence encoding the GLP-1 peptide or peptide and capable of expressing said peptide in a nutrient medium suitable under conditions that allow the expression of the peptide, after which the resulting peptide or GLP-1 peptide is recovered from the culture or fermentation broth. From now on, the crop will be used to cover both the crop and the fermentation and the like.

[0048] The medium used to grow the cells may be any conventional means suitable for the growth of host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or can be prepared according to published recipes (eg in catalogs of the American Type Culture Collection). The GLP-1 peptide or peptide produced by the cells can then be recovered from the culture medium by conventional procedures including, optionally cell lysis, separating the host cells from the medium by centrifugation or filtration, precipitation of the protein components from the supernatant or filtered by a salt, for example ammonium sulfate, purification by conventional purification techniques, such as chromatographic techniques, if necessary, purification by ion exchange chromatography according to the present invention, and subsequently, undergoing analytical tests, for example PAGE, IEF, if necessary, subjecting to further purification, if necessary, and isolation of the pure peptide or GLP-1 peptide.

[0049] During the recovery of the resulting peptide or GLP-1 peptide from the culture medium, but before purification by ion exchange chromatography according to the present invention, the mixture comprising the GLP-1 peptide or peptide and related impurities may optionally be chemically modified by conventional techniques, for example by alkylation, acylation, ester formation or amide formation or the like.

[0050] The DNA sequence encoding the parent peptide or GLP-1 peptide may suitably be of genomic or cDNA origin, for example obtained by preparing a genomic or cDNA library and selecting for coding DNA sequences for all or part. of the GLP-1 peptide or peptide by hybridization using synthetic oligonucleotide probes according to standard techniques (see, for example, Sambrook, J, Fritsch, EF and Maniatis, T, molecular cloning: a laboratory, manual dock port laboratory pressure cold, New York, 1989). The DNA sequence encoding the GLP-1 peptide or peptide can also be prepared synthetically by established standard methods, for example the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, or the described method by Matthes et al., EMBO Journal 3 (1984), 801-805. The DNA sequence can also be prepared by polymerase chain reaction using specific primers, for example as described in US 4,683,202 or Saiki et al., Science 239 (1988), 487-491.

[0051] The DNA sequence can be inserted into any vector that can conveniently be subjected to recombinant DNA procedures, and the choice of the vector will often depend on the host cell into which it is to be introduced. Thus, the vector can be an autonomous replication vector, that is a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, for example a plasmid. Alternatively, the vector may be one that, when introduced into a host cell, is integrated into the genome of the host cell and replicated with the chromosome (s) into which it has been integrated.

[0052] The vector is preferably an expression vector in which the DNA sequence encoding the GLP-1 peptide or peptide is operably linked to additional segments required for DNA transcription, such as a promoter. The promoter can be any DNA sequence that shows transcriptional activity in the host cell of choice and can be derived from genes encoding heterologous proteins or homologous to the host cell. Examples of promoters suitable for directing the transcription of the DNA encoding the GLP-1 peptide or peptide of the invention in a variety of host cells are well known in the art, cf. for example, Sambrook et al., supra.

[0053] The DNA sequence encoding the GLP-1 peptide or peptide may also, if necessary, be operatively connected to a suitable terminator, polyadenylation signals, transcriptional enhancer sequences, and translational enhancer sequences. The recombinant vector of the invention may further comprise a DNA sequence allowing the vector to replicate in the host cell in question.

[0054] The vector can also comprise a selectable marker, for example a gene whose product complements a defect in the host cell or one that confers resistance to a drug, for example ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin or methotrexate.

[0055] To direct a GLP-1 peptide or peptide into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) can be provided in the recombinant vector. The secretory signal sequence binds to the DNA sequence encoding the GLP-1 peptide in the correct reading frame. Secretory signal sequences are commonly located at 5 'to the DNA sequence encoding the GLP-1 peptide or peptide. The secretory signal sequence may be that normally associated with the GLP-1 peptide or peptide or it may be from a gene encoding another secreted protein.

[0056] The methods used to link the DNA sequences encoding the GLP-1 peptide or peptide, the promoter and optionally the terminator and / or secretory signal sequence, respectively, and to insert them into suitable vectors with the information necessary for replication, they are known to those skilled in the art (cf., for example, Sambrook et al., supra).

[0057] The host cell into which the DNA sequence is introduced or the recombinant vector can be any cell that is capable of producing the present GLP-1 peptide or peptide and includes bacteria, viruses, for example baculovirus, yeast, fungi, cells of insect and upper eukaryotic cells. Examples of suitable host cells well known and used in the art are, without limitation, E. coli, Saccharomyces cerevisiae, or mammalian BHK or CHO cell lines.

[0058] Some of the GLP-1 peptides or peptides may be produced according to conventional organic peptide synthetic chemistry. The resulting synthetic mixture can then be chemically modified, for example by alkylation, acylation, ester formation or amide formation or the like, and purified, or purified as is and then chemically modified as mentioned above.

Factor VIIa Preparation

[0059] Human purified Factor VIIa suitable for use in the present invention is preferably made by recombinant DNA technology, for example as described by Hagen et al., Proc.Natl.Acad.Sci. USA 83: 2412-2416, 1986 or as described in European Patent No. 200,421 (ZymoGenetics). Factor VIIa produced by recombinant technology may be authentic factor VIIa or a factor VIIa more or less modified provided that such factor VIIa has substantially the same biological activity for blood coagulation as authentic factor VIIa. Such modified factor VIIa can be produced by modifying the nucleic acid sequence encoding factor VII either by altering the amino acid codons or by removing some of the amino acid codons in the nucleic acid encoding the natural FVII by known means, for example by site specific mutagenesis.

[0060] Factor VII can also be produced by the methods described by Broze and Majerus, J.Biol.Chem. 255 (4): 1242-1247, 1980 and Hedner and Kisiel, J.Clin.Invest. 71: 1836-1841, 1983. These methods produce factor VII without detectable amounts of other blood coagulation factors. An even more purified factor VII preparation can be obtained by including an additional gel filtration as the final purification phase. Factor VII is then converted into FVIIa activated by known means, for example by different plasma proteins, such as factor XIIa, IX a or Xa. Alternatively, as described by Bjoern et al. (Research Disclosure, 269 September 1986, pp. 564565), factor VII can be activated by passing it through an ion exchange chromatography column, such as Mono Q (r) (Pharmacia fine Chemicals) or the like.

Modified or inactivated FVIIa (FVIIai) can be produced by the following methods:

[0061] International application No. WO 92/15686 refers to modified Factor VIIa, polynucleic acid and mammalian cell lines for the production of modified Factor VIIa, and compositions comprising Factor VIIa modified to inhibit blood coagulation.

[0062] The modified Factor VII can be encoded by a polynucleotide molecule comprising two regions encoding operably linked sequences encoding, respectively, a prepro peptide and a Gla domain of a plasma vitamin K-dependent protein, and a Factor VII protein without gla VII domain, where after the expression of said polynucleotide it encodes a modified Factor VII molecule that does not significantly activate plasma factors X or IX, and is capable of binding the tissue factor.

[0063] The catalytic activity of factor VIIa can be inhibited by chemical derivatization of the catalytic center, or triad. Derivatization can be performed by reacting Factor VII with an irreversible inhibitor such as an organophosphorus compound, a sulfonyl fluoride, a peptide halomethyl ketone or azapeptide, or by acylation, for example. Preferred peptide halomethyl ketones include PPACK (D-Phe-Pro-Arg chloromethyl ketone; (see US Patent No. 4,318,904, incorporated herein by reference), D-Phe-Phe-Arg and Phe-Phe-Arg chloromethyl ketone (FFR-cmk); and DEGRck (dansyl-Glu-Gly-Arg chloromethyl ketone).

[0064] The catalytic activity of Factor VIIa can also be inhibited by substitution, insertion or deletion of amino acids. In preferred amino acid embodiments, substitutions have been made in the amino acid sequence of the catalytic triad of Factor VII, defined herein as the regions, which contain the amino acids, that contribute to the catalytic site of Factor VIIa. Substitutions, insertions or deletions in the catalytic triad are generally at or adjacent to the amino acids that form the catalytic site. In human and bovine Factor VII proteins, the amino acids, which form a catalytic "triad", are Ser344, Asp242, and His193 (the numbering of the subscript indicating the position in the sequence). Factor VII catalytic sites of other mammalian species can be determined using currently available techniques including, among others, protein isolation and amino acid sequence analysis. Catalytic sites can also be determined by aligning a sequence with the sequence of other serine proteases, particularly chymotrypsin, whose active site has been previously determined (Sigler et al., J. Mol. Biol., 35: 143-164 (1968), incorporated here by reference), and from there determining from said alignment the analogous residues of the active site.

[0065] In preferred embodiments of human and bovine Factor VII, the Ser344 active site residue is modified, substituted with Gly, Met, Thr, or more preferably, Ala. Such substitution could be made separately or in combination with substitution (s) to other sites in the catalytic triad, which includes His193 and AsP242.

[0066] The amino acids, which form the catalytic site in Factor VII, such as Ser344, Asp242, and His193 in human and bovine Factor VII, can either be eliminated or substituted. In the present invention, it is preferred to change only a single amino acid, thus minimizing the probability of increasing the antigenicity of the molecule or inhibiting its ability to bind tissue factor, however two or more amino acid changes (substitutions, additions or deletions) may be made and combinations of substitution (s), addition (s) and deletion (s) can also be made. In a preferred embodiment for human and bovine Factor VII, Ser344 is preferably substituted with Ala, but Gly, Met, Thr or other amino acids can be substituted. It is preferred to replace Asp with Glu and replace His with Lys or Arg. In general, substitutions are chosen to mismatch as little as possible the tertiary structure of the protein. The Dayhoff et al. (in Atlas of Protein Structure 1978, Nat'l Biomed. Res. Found., Washington, D.C.), incorporated herein by reference, can be used as a guide in the selection of other amino acid substitutions. Residue alterations can be introduced as described above at the catalytic site of the appropriate Factor VII sequence of human, bovine or other species and evaluate the resulting protein for a desired level of catalytic activity inhibition and giving as the anticoagulant activity. as described in this case. For the modified Factor VII the catalytic activity will be substantially inhibited, generally less than about 5% of the catalytic activity of the wild type factor VII of the corresponding species, more preferably less than about 1%.

[0067] Modified Factor VII can be produced through the use of recombinant DNA techniques.

[0068] Alterations of the amino acid sequence can be performed by a variety of techniques. The modification of the DNA sequence can be by site specific mutagenesis. Techniques for site specific mutagenesis are well known in the art and are described by, for example, Zoller and Smith (DNA 3: 479-488, 1984). Thus, using the nucleotide and amino acid sequences of Factor VII, the alteration (s) of choice can be introduced.

[0069] Modified FVIIa can also be produced by chemical methods.

FFR-FVIIa (ie D-Phe-Phe-Arg-FVlla)

Example FFR chloromethyl ketone

[0070] Blocking of the active site of FVIIa with FFR chloromethyl ketone.

[0071] Blocking of the active site serine and histidine with chloromethyl ketone is a known method for irreversible inactivation of serine proteases. To optimize the blocking of a given protease it is important to choose a chloromethyl ketone derivative, which reacts specifically with the active site and with a rapid association constant. Such derivatives can be developed by binding to the chloromethyl ketone group of an oligopeptide, which interacts, with the substrate binding site of the particular serine protease of interest.

[0072] Glutamyl-Glycyl-Arginine chloromethyl ketone (EGR-ck or its dansyl derivative, DEGR-ck) (S. Higashi, H. Nishimura, S. Fujii, K. Takada, S. Iwanaga, (1992) J. Biol. Chem. 267, 17990) or Prolyl-Phenyl-Arginine chloromethyl ketone (PFR-ck) (JH Lawson, S. Butenas, K. Mann, (1992) J. Biol. Chem. 267, 4834; J. Contrino, GA Hair, MA Schmeizl, FR Rickles, DL Kreutzer (1994) Am. J. Pathol. 145, 1315) have been applied as inhibitors of the active site of FVIIa. Compared with these chloromethyl ketones, the application of FFRck represents an index increase of 10-70 times.

[0073] The specificity of the reaction with the FFR-chloromethyl ketone derivative of FVIIa was controlled by HPLC and peptide mapping showing that FVlla has reacted with FFR-chloromethyl ketone in a 1: 1 ratio so that> 98% could be recovered as the intended product derivatized to histidine 193.

Inactivation of FVlla by several chloromethyl ketones:

[0074] 3 µM of FVlla were incubated with 12 µM of chloromethyl ketone derivative in 50 mM TrisHCl, 100 mM NaCl, 5 mM CaCl2, 0.01% Tween-80, pH 7.4. Samples were removed at various time intervals as indicated and diluted 20 times for activity measurements in 50 mM TrisHCl, 100 mM NaCl, 5 mM CaCl2, 0.01% Tween-80, pH 7.4 containing 1 mM IIe-pro-Arg- pNA The residual FVIIa activity was measured by the increase in absorbency at 405 nm.

[0075] Normally, the mixture comprising the GLP-1 peptide or peptide and related impurities to be purified by ion exchange chromatography according to the present invention will also contain amino acids, small peptides, large peptides, unrelated proteins, reagents, cell detritus , HCP, endotoxins, and / or viruses depending on whether recombinant DNA techniques and / or chemical modification techniques have been used or if synthetic organic peptide chemistry techniques have been used.

[0076] Thus, any method, such as an industrial method, for producing a pure peptide or GLP-1 peptide, which includes an IEC process according to the present invention is also an aspect of the present application.

[0077] Accordingly, the present invention relates in another aspect to an industrial method for the production of a pure peptide or GLP-1 peptide, the method including a cation exchange chromatography process for purifying a peptide from a mixture comprising said GLP-1 peptide or peptide and related impurities, which include the steps of:

separating said GLP-1 peptide or peptide and said related impurities from said mixture by elution in a solution comprising an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepped gradient or isocratically in component of salt and / or with a linear or stepped pH gradient or at a constant pH value, where the pH gradient or pH value should be in the range where said GLP-1 peptide or peptide has a different positive global or local net charge of the positive net local or global charge of said related impurities to eliminate said related impurities.

[0078] The present invention relates in another aspect to a method for isolating a GLP-1 peptide, including the method of purifying a GLP-1 peptide from a mixture comprising said peptide and related impurities by means of a chromatography process. of cation exchange, the process of cation exchange chromatography comprising the phase of:

separating said GLP-1 peptide and said related impurities from said mixture by elution in a solution comprising an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepped gradient or isocratically in salt component and / or with a linear or stepped gradient

or at a constant pH value, where the pH gradient or pH value should be in the range where said peptide has a positive global or local net charge different from the positive local or global net charge of said related impurities to remove said related impurities ; and subsequently, if necessary, undergo analytical testing and / or further purification, and isolate said peptide in a conventional manner.

[0079] The present invention relates in another aspect to a method for isolating a GLP-1 peptide, including the method of purifying a GLP-1 peptide from a mixture comprising said peptide and unrelated or related impurities by means of a cation exchange chromatography process, the cation exchange chromatography process comprising the phase of:

separating said GLP-1 peptide and said related impurities from said mixture by elution in a solution consisting essentially of an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepped gradient or isocratically in component of salt and / or with a linear or stepped pH gradient or at a constant pH value, where the pH gradient or pH value should be in the range where said peptide has a positive global or local net charge different from the local net charge or positive overall of said related impurities to remove said related impurities; and subsequently, if necessary, undergo analytical testing and / or further purification, and isolate said peptide in a conventional manner.

[0080] The present invention relates in another aspect to an industrial method for the production of a pure peptide or GLP-1 peptide, the method including an anion exchange chromatography process for purifying a peptide from a mixture comprising said peptide or GLP-1 peptide and related impurities, including the steps of:

separating said GLP-1 peptide or peptide and said related impurities from said mixture by elution in a solution comprising an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepped gradient or isocratically in component of salt and / or with a linear or stepped pH gradient or at a constant pH value, where the pH gradient or pH value should be in the range where said GLP-1 peptide or peptide has a different net global or local negative charge of the net local or global negative charge of said related impurities to eliminate said related impurities

[0081] The present invention relates in a further aspect to a method for isolating a GLP-1 peptide, the method including purification of a GLP-1 peptide from a mixture comprising said peptide and related impurities by means of a process of anion exchange chromatography, the process of anion exchange chromatography comprising the phase of:

separating said GLP-1 peptide and said related impurities from said mixture by elution in a solution comprising an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepped gradient or isocratically in salt component and / or with a linear or stepped pH gradient or at a constant pH value, where the pH gradient or pH value should be in the range where said peptide has a negative global or local net charge different from the local or global net charge refusal of said related impurities to eliminate said related impurities; and subsequently, if necessary, undergo analytical testing and / or further purification, and isolate said peptide in a conventional manner.

[0082] The present invention relates in a further aspect to a method for isolating a GLP-1 peptide, the method including purification of a GLP-1 peptide from a mixture comprising said peptide and unrelated or related impurities by means of a anion exchange chromatography process, the anion exchange chromatography process comprising the phase of:

separating said GLP-1 peptide and said related impurities from said mixture by elution in a solution consisting essentially of an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepped gradient or isocratically in component of salt and / or with a linear or stepped pH gradient or at a constant pH value, where the pH gradient or pH value should be in the range where said peptide has a negative global or local net charge different from the local net charge or overall refusal of said related impurities to remove said related impurities; and subsequently, if necessary, undergo analytical testing and / or further purification, and isolate said peptide in a conventional manner.

[0083] Any possible combination of two or more of the embodiments described herein is within the scope of the present invention.

[0084] The term "an organic modifier", as used herein, is intended to include an organic solvent or water-soluble organic compound or derived mixtures, this modifier induces a favorable and changed selectivity between unwanted impurity or impurities ( s) related (s) and the GLP-1 peptide and the ion exchanger. Whether or not a selected modifier induces said selectivity will normally depend on the related impurity or impurities (s), and can be evaluated by trial-and-error. The organic modifier includes but is not limited to C1-6 alkanol, C1-6 alkenol or C1-6 alkynyl, urea, guanidine · HCl, or C1-6 alkanoic acid, such as acetic acid, C2-6 glycol, C3-7 polyalcohol including sugars, or derived mixtures.

[0085] The term, "C1-6-alkanol" "C1-6 -alquinol" or "C1-6 -alkenol", as used in this case alone or in combination is intended to include those C1-6 alkane groups, C1-6 alkene or C1-6 alquina of the length designated in either a cyclic or linear or branched configuration to which a hydroxyl (OH) is linked (cf. Morrison & Boyd, Organic Chemistry, 4th ed). Examples of linear alcohols are methanol, ethanol, n-propanol, allyl alcohol, N-butanol, N-pentanol and N-hexanol. Examples of branched alcohols are 2-propanol and tert-butyl alcohol. Examples of cyclic alcohols are propyl alcohol cycle and 2-cyclohexen-l-ol.

[0086] The term "C1-6-alkanoic acid", as used herein, is intended to include a group of the formula R'COOH where R 'is H or C1-5 alkyl, such as, acetic acid, propionic, butyric, methylbutyric, or valeric (cf. Morrison & Boyd, Organic Chemistry, 4th ed).

[0087] The term "C1-5-alkyl", as used herein, is intended to include a branched or straight alkyl group having one to five carbon atoms. Typical C1-5 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, Isobutyl, secbutyl, tert-butyl, pentyl, isopentyl, and the like (cf. Morrison & Boyd, Organic Chemistry , 4th ed).

[0088] The term "C2-6 -glycol", as used herein, is intended to include a C2-6 alkane containing two hydroxyl groups in different carbon atoms that may or may not be adjacent. A typical C2-6 glycol includes, but is not limited to 1,2-ethanediol, 1,2-propanediol, or 2-methyl-2,4-pentanediol (cf. Morrison & Boyd, Organic Chemistry, 4th ed).

[0089] The term "C2-6-alkane", as used herein, is intended to include a branched or straight alkane group having two to six carbon atoms. Typical C2-6-alkane groups include, but are not limited to ethane, propane, isopropane, butane, isobutane, pentane, hexane and the like (cf. Morrison & Boyd, Organic Chemistry, 4th ed).

[0090] The term "sugars that include C3-7-polyalcohol", as used herein, is intended to include a group of the formula HOCH2 (CHOH) nCH2OH where n is an integer of 1-5, and monosaccharides such as mannose, glucose (cf. Morrison & Boyd, Organic Chemistry, 4th ed).

[0091] The term "GLP-1 peptide", as used herein, is intended to designate GLP-1 (7-37), GLP-1 (7-36) amide as well as analogues and derivatives thereof. , which are capable of being produced by conventional recombinant DNA techniques as well as conventional synthetic methods. Such GLP-1 peptides include but not limited to native glucagon-like peptide 1, for example such peptide fragments comprising GLP-1 (7-37) and functional derivatives thereof as described in WO 87/06941; such peptide fragments comprising GLP-1 (7-36) and functional derivatives thereof as described in WO 90/11296; such analogs of the active GLP-1 peptides 734, 7-35, 7-36, and 7-37 as described in WO 91/11457; such GLP-1 derivatives in which a lipophilic substituent binds to at least one amino acid residue as described in WO 98/08871; such N-terminal truncated fragments of GLP-1 as described in EP 0699686-A2; and such GLP-1 analogs and derivatives that include an N-terminal imidazole group as described in EP 0708179-A2.

[0092] The term "GLP-2 peptide", as used herein, is intended to designate GLP-2 (1-35), GLP-2 (1-34), GLP-2 (1-33) at like analogs and derivatives thereof, which are capable of being produced by conventional recombinant DNA techniques as well as conventional synthetic methods. Such GLP-2 peptides include but not limited to native glucagon-like peptide 2, derivatives of GLP-2 in which a lipophilic substituent is fixed to at least one amino acid residue as described in WO 98/08872, peptide 2 of human glucagon type (hGLP-2), GLP-2 (1-30); GLP-2 (1-31); GLP-2 (1-32); GLP-2 (1-33); GLP-2 (1-34), GLP-2 (1-35), Lys20GLP-2 (1-33), Lys20Arg30GLP-2 (1-33), Arg30Lys34GLP-2 (1-34), Arg30Lys35GLP-2 (1 -35), Arg30,35Lys20GLP-2 (1-35), Arg35GLP-2 (1-35), Lys20 (Ntetradecanoyl) GLP-2 (1-33); Lys20,30-bis (N-tetradecanoyl) GLP-2 (1-33); Lys20 (N-tetradecanoyl) Arg30GLP-2 (1-33); Arg30Lys35 (N-tetradecanoyl) GLP-2 (1-35); Arg30,35Lys20 (N-tetradecanoyl) GLP-2 (1-35); Arg35Lys30 (Ntetradecanoyl) GLP-2 (1-35); Arg30Lys34 (N-tetradecanoyl) GLP-2 (1-34); Lys20 (N- (-carboxinonadecanoyl)) GLP-2 (1-33); Lys20,30-bis (N- (-carboxynonadecanoyl)) GLP-2 (1-33); Lys20 (N- (-carboxinonadecanoyl)) Arg30GLP-2 (1-33); Arg30Lys35 (N- (-carboxynonadecanoyl)) GLP-2 (1-35); Lys30 (N- (-glutamyl (N-tetradecanoi)) hGLP-2, Lys30 (N- (glutamil (N-hexadecanoil))) hGLP-2, Arg30,35Lys20 (N- (- carboxinonadecanoyl)) GLP-2 (1-35); Arg35Lys30 (N- (carboxinonadecanoyl)) GLP-2 (1-35); and Arg30Lys34 (N- (-carboxinonadecanoyl)) GLP-2 (1-34 ).

[0093] The term "analogues" as used herein is intended to designate a peptide where one or more amino acid residues of the parent peptide has been replaced by another amino acid residue and / or where one or more amino acid residues of the parent peptide has been removed and / or where one or more amino acid residues have been added to the parent peptide. Such addition can take place either at the N-terminal end or at the C-terminal end of the parent peptide or both.

[0094] The term "derivatives" as used herein is intended to designate a peptide in which one or more of the amino acid residues of the parent peptide has been chemically modified, for example by alkylation, acylation, ester formation or amide formation or the like.

[0095] The term "salt component" as used herein is intended to include any inorganic or organic salt, including but not limited to NaCl, KCI, NH4 Cl, CaCl2, sodium acetate, potassium acetate, ammonium acetate , sodium citrate, potassium citrate, ammonium citrate, sodium sulfate, potassium sulfate, ammonium sulfate, calcium acetate or derivative mixtures (cf. Remington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton, PA , 1990, or Remington: The Science and Practice of Pharmacy, 19th Edition (1995), or Amersham-Pharmacia Biotech manuals).

[0096] The term "a buffer" as used herein is intended to include any buffer that includes but is not limited to: citrate buffers, phosphate buffers, tris buffers, borate buffers, lactate buffers, buffers of glycyl glycine, arginine buffers, carbonate buffers, acetate buffers, glutamate buffers, ammonium buffers, glycine buffers, alkylamino buffers, aminoethyl alcohol buffers, ethylenediamine buffers, trietanol amine buffers, imidazole buffers Pyridine and barbiturate buffers and their derivative mixtures (cf. Remington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton, PA, 1990, or Remington: The Science and Practice of Pharmacy, 19th Edition (1995), or manuals from Amersham-Pharmacia Biotech).

[0097] The choice of starting pH, buffer and ionic strength is made according to well known techniques such as conventional test tube methods, cf. for example manuals from Amersham-Pharmacia Biotech. The chromatographic ion exchange resin is chosen depending on the specific GLP-1 peptide to be purified and the conditions employed, such as pH, buffer, ionic strength etc., which is known to those skilled in the art (i.e., typically, pH below the isoelectric point (pl) of the GLP-1 peptide for cation exchange resins and pH above pl of the GLP-1 peptide for anion exchange resins, a buffer force sufficient to maintain the desired pH, and an ionic strength sufficiently low possibly induced by salt concentration), and includes but is not limited to Sepharose resins, Sephadex resins, Streamline resins, and Amersham-Pharmacia Biotech Source resins, HyperD resins, Trisacryl resins, and Spherosil resins BioSepra, TSKgel resins and TyoHaas Toyopearl resins, Merck Fractogel EMD resins, Perseptive Biosystems pore resins, BioRAD Macro-Prep resins, Express-ion resins of Whatman etc.

[0098] The term "a solution consisting essentially of an organic modifier, water, optionally a salt component and optionally a buffer" as used in this case, is intended to mean a solution containing one or more organic modifiers, water , one or more salt components or no salt component and one or more buffers or no buffer, and optionally one or more other conventional components that the person skilled in the art would consider addition, according to conventional ion exchange chromatography processes.

[0099] The term "related impurities" as used herein is intended to mean one or more impurities with a global or local net charge other than the GLP-1 peptide, for example truncated forms, all types of extended forms ( extra amino acids, various derivatives including esters etc.), deamidated forms, incorrectly folded forms, forms with undesired glycosylation including sialylation, and others. It follows that "unrelated impurities" as used in this case, is intended to cover impurities that are different from related impurities.

[0100] The term "a constant pH value", as used herein is intended to mean that the pH value can be constant, such as in the presence of a buffer or can typically vary within 3 pH units, If no buffer is present.

[0101] The term "with a linear or stepped pH gradient", as used in this case is intended to mean that the pH value changes during the elution from a lower to a higher pH, or from a more pH high to lower one. Such a change in pH is normally generated with a buffer and / or by the addition of an inorganic or organic acid or base, for example HCl, NaOH, H2O acetic acid, NH3, potassium hydroxide, H2SO4, citric acid. A pH gradient for cation exchange would normally be from a lower pH to a higher one, and for anion exchange from a higher pH to a lower one.

[0102] The term "with a linear or stepped gradient or isocratically in salt component", as used in this case is intended to mean that the salt concentration changes during the elution from a lower concentration to a higher one or is constant.

[0103] The present invention also relates to the following aspects:

Aspect 1. A cation exchange chromatography process for purification of a GLP-1 peptide from a mixture comprising said peptide and unrelated or related impurities, comprising the phase of:

separating said GLP-1 peptide and said unrelated or related impurities from said mixture by elution in a solution consisting essentially of an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepwise or isocratically gradient in salt component and / or with a linear or stepped pH gradient or at a constant pH value, where the pH gradient or pH value should be in the range where said peptide has a positive global or local net charge different from the positive local or global net charge of said unrelated or related impurities to eliminate said unrelated or related impurities.

Aspect 2. A cation exchange chromatography process for purification of a GLP-1 peptide from a mixture comprising said peptide and related impurities, comprising the phase of:

separating said GLP-1 peptide and said related impurities from said mixture by elution in a solution consisting essentially of an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepped gradient or isocratically in component of salt and / or with a linear or stepped pH gradient or at a constant pH value, where the pH gradient or pH value should be in the range where said peptide has a positive global or local net charge different from the local net charge or positive overall of said related impurities to remove said related impurities.

Aspect 3. An anion exchange chromatography process for purification of a GLP-1 peptide from a mixture comprising said peptide and unrelated or related impurities, comprising the phase of:

separating said GLP-1 peptide and said unrelated or related impurities from said mixture by elution in a solution consisting essentially of an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepwise or isocratically gradient in salt component and / or with a linear or stepped pH gradient or at a constant pH value, where the pH gradient or pH value should be in the range where said peptide has a negative global or local net charge different from the Negative local or global net charge of such unrelated or related impurities to remove such unrelated or related impurities.

Aspect 4. An anion exchange chromatography process for purifying a GLP-1 peptide from a mixture comprising said peptide and related impurities, comprising the phase of:

separating said GLP-1 peptide and said related impurities from said mixture by elution in a solution consisting essentially of an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepped gradient or isocratically in component of salt and / or with a linear or stepped pH gradient or at a constant pH value, where the pH gradient or pH value should be in the range where said peptide has a negative global or local net charge different from the local net charge or overall refusal of said related impurities to remove said related impurities.

Aspect 5. The process according to any of aspects 1-4 where the ratio of organic modifier to water on a percentage basis by weight is from 1:99 to 99: 1.

Aspect 6. The process according to any of aspects 1-5 wherein said organic modifier is selected from C1-6 alkanol, C1-6 alkenol or C1-6 alkyol, urea, guanidine, or C1-6 alkanoic acid, C2-6 glycol , or C3-7 polyalcohol including sugars.

Aspect 7. The process according to any of aspects 1-6 wherein said salt component is selected from any inorganic or organic salt, preferably NaCl, KCI, NH4 Cl, CaCl2, sodium acetate, potassium acetate, ammonium acetate, sodium citrate, potassium citrate, ammonium citrate, sodium sulfate, potassium sulfate, ammonium sulfate, calcium acetate

or derived mixtures.

Aspect 8. The process according to any of aspects 1-6 where no salt component is present.

Aspect 9. The process according to any of aspects 1-7 where said gradient in salt component is a stepped or linear gradient in the salt component.

Aspect 10. The process according to aspect 9 where said salt component is present in a concentration selected from the range of 0.1 mmol / kg to 3000 mmol / kg.

Aspect 11. The process according to any of aspects 1-10 wherein said buffer is independently selected from citrate buffers, phosphate buffers, tris buffers, borate buffers, lactate buffers, glycyl glycine buffers, arginine buffers, buffers carbonate, acetate buffers, glutamate buffers, ammonium buffers, glycine buffers, alkylamino buffers, aminoethyl alcohol buffers, ethylenediamine buffers, trietanol amine buffers, imidazole buffers, pyridine buffers and barbiturate buffers and mixtures thereof derived.

Aspect 12. The process according to any of aspects 1-11 wherein said buffer is present in a concentration selected from the range of 0.1 mmol / kg to 500 mmol / kg.

Aspect 13. The process according to any of aspects 1-10 where no buffer is present.

Aspect 14. A method for isolating a peptide, the method including purification of a GLP-1 peptide from a mixture comprising said peptide and related impurities by means of a cation exchange chromatography process, the cation exchange chromatography process including the steps from:

separating said GLP-1 peptide and said related impurities from said mixture by elution in a solution consisting essentially of an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepped gradient or isocratically in component of salt and / or with a linear or stepped pH gradient or at a constant pH value, where the pH gradient or pH value should be in the range where said peptide has a positive global or local net charge different from the local net charge or positive overall of said related impurities to remove said related impurities;

and subsequently, if necessary, undergo analytical testing and / or further purification, and isolate said peptide in a conventional manner.

Aspect 15. A method for isolating a peptide, the method including purification of a GLP-1 peptide from a mixture comprising said peptide and related impurities by means of an anion exchange chromatography process, the anion exchange chromatography process including the steps from:

separating said GLP-1 peptide and said related impurities from said mixture by elution in a solution consisting essentially of an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepped gradient or isocratically in component of salt and / or with a linear or stepped pH gradient or at a constant pH value, where the pH gradient or pH value should be in the range where said peptide has a negative global or local net charge different from the local net charge or overall refusal of said related impurities to remove said related impurities; and subsequently, if necessary, undergo analytical testing and / or further purification, and isolate said peptide in a conventional manner.

Aspect 16. The process or method according to any of aspects 1-15 where said peptide to be purified is selected from GLP-1 (7-37), GLP-1 (7-36) amide as well as analogues and derivatives of the same.

Examples

[0104] The present invention is subsequently illustrated by the following examples which, however, should not be construed as limiting the scope of protection. The features described in the preceding description and in the following examples may, both separately and in any combination thereof, be material for the realization of the invention in various forms thereof.

Example 1:

[0105] Arg34GLP-1 (1-37) was expressed in yeast (Sacch. Cerevisiae) by conventional recombinant DNA technology p. ex. as described in WO 98/08871. The fermentation broth of Arg34GLP-1 (7-37) was purified by a capture phase of conventional cation exchange chromatography. The grouping containing Arg34GLP-1 (737) and the truncated form, Arg34GLP-1 (9-37), as an impurity was adjusted to pH 3.1, and 1 ml of the resulting solution was applied to a 6.5 ml column of Ceramic S HyperD F (BioSepra SA) balanced with 32.5 ml 1.54% (w / w) trisodium citrate, 0.6% (w / w) succinic acid, 1.09% (w / w) disodium hydrogen phosphate dihydrate, pH-3.2. The column was washed with

6.5 ml of equilibration solution, and elution was performed with a linear pH gradient of 3.2 to 8.0 (1.54% (w / w) trisodium citrate, 0.6% (w / w) succinic acid, 1.09% (w / w) disodium hydrogen phosphate dihydrate, pH 8.0) followed by 13 ml of isocratic elution at pH 8.0. A chromatogram is shown in Figure 1. No different maximum value or separation between the truncated form and the target GLP-1 fraction was obtained.

Example 2:

[0106] Arg34GLP-1 (7-37), was expressed and captured by cation exchange as described in example 1. The grouping containing Arg34GLP-1 (7-31) and the truncated form, Arg34GLP-1 (9-37 ), as an impurity was adjusted to pH 3.1, and 1 ml of the resulting solution was applied to a 20 ml column of Source 30S (Amersham Pharmacia Biotech) equilibrated with 100 ml of 1.54% (w / w) trisodium citrate, 0.6 % (w / w) succinic acid, 1.09% (w / w) disodium hydrogen phosphate dihydrate, pH-3.2. The column was washed with 20 ml of equilibration solution, and the elution was performed with a linear pH gradient of 3.2 to 8.0 (1.54% (w / w) trisodium citrate, 0.6% (w / w) succinic acid, 1.09% (w / w) disodium hydrogen phosphate dihydrate, pH 8.0) followed by 40 ml of isocratic elution at pH 8.0. No different maximum value or separation between the truncated form and the GLP-1 target fraction were obtained.

Example 3:

[0107] Arg34GLP-1 (7-37) was expressed and captured by cation exchange as described in example 1. The grouping containing Arg34GLP-1 (7-37) and the truncated form, Arg34GLP-1 (9-37) , as an impurity was adjusted to pH 3.1, and 1 ml of the resulting solution was applied to a 20 ml column Source 30S (Amersham Pharmacia Biotech) equilibrated with 100 ml 0.77% (w / w) trisodium citrate, 0.3% (p / p) succinic acid, 0.55% (w / w) disodium hydrogen phosphate dihydrate, pH-3.2. The column was washed with 20 ml of equilibration solution. Elution was performed with a linear pH gradient of 3.2 to 8.0 (0.77% (w / w) of trisodium citrate, 0.3% (w / w) of succinic acid, 0.55% (w / w) of disodium hydrogen phosphate dihydrate) followed by 40 ml of isocratic elution at pH 8.0. Post elution at pH 8.0 was performed with a

5

fifteen

25

35

Four. Five

Linear salt gradient of 0.0 to 1.0 M NaCl (0.77% (w / w) of trisodium citrate, 0.3% (w / w) of succinic acid, 0.55% (w / w) of disodium hydrogen phosphate dihydrate, pH 8.0) followed by 40 ml of isocratic elution at 1.0 M NaCl. No maximum value

or different separation between the truncated form and the fraction of GLP-1 target was obtained.

Example 4:

[0108] Arg34GLP-1 (7-37) was expressed and captured by cation exchange as described in Example 1. 2 volumes of water were added to the cluster containing Arg34GLP-1 (7-37) and the truncated form, Arg34GLP -1 (9-37), as an impurity, and the solution was adjusted to pH 3.5. 25.5 ml of the resulting solution was applied to a 20 ml column Source 30S (Amersham Pharmacia Biotech) equilibrated with 100 ml 0.21% (w / w) trisodium citrate, 0.08% (w / w) succinic acid, 0.15% ( w / w) disodium hydrogen phosphate dihydrate, 45% (w / w) ethanol, pH-3.2. The column was washed with 20 ml equilibration solution, and the elution was performed with a linear pH gradient of 3.2 to 8.0 (0.21% (w / w) of trisodium citrate, 0.08% (w / w) of succinic acid, 0.15 % (w / w) of hydrogen disodium phosphate dihydrate, 45% (w / w) of ethanol, pH 8.0) followed by 40 ml of isocratic elution at pH 8.0. A chromatogram is shown in Figure 2. Different maximum values and separation between the truncated form and the target GLP-1 fraction were obtained by adding ethanol to the chromatographic solutions. Insignificant differences in configuration between this and Example 2 are: different batch, pH, and aqueous dilution of the sample for application, higher loading, and lower buffer concentration.

Example 5:

[0109] Arg34GLP-1 (7-37) was expressed and captured by cation exchange as described in example 1. The grouping containing Arg34GLP-1 (7-37) and the truncated form, Arg34GLP-1 (9-37) , as an impurity was adjusted to pH 3.1, and 5 ml of the resulting solution was applied to a 20 ml column Source 30S (Amersham Pharmacia Biotech) equilibrated with 100 ml 0.85% (w / w) trisodium citrate, 0.33% (p / p) succinic acid, 0.6% (w / w) hydrogen disodium phosphate dihydrate, 45% (w / w) ethanol, pH-3.2. The column was washed with 20 ml equilibration solution, and the elution was performed with a linear pH gradient of -3.2 to -5.0 (0.85% (w / w) trisodium citrate, 0.33% (w / w) succinic acid 0.6% (w / w) of hydrogen disodium phosphate dihydrate, 45% (w / w) of ethanol) followed by 60 ml of isocratic elution at pH 8.0 (0.85% (w / w) of trisodium citrate, 0.33% (p / p) succinic acid, 0.6% (w / w) hydrogen disodium phosphate dihydrate, 45% (w / w) ethanol, pH 8.0). A chromatogram is shown in Figure 3. Different maximum values and separation between the truncated form and the target GLP-1 fraction were obtained by adding ethanol to the chromatographic solutions.

[0110] An RP-HPLC analysis was performed for identification / verification of maximum values collected in a 4.0x250 mm column substituted in C18 120 A silica (YMC) with 5 µm particles. Buffer A consisted of 0.15 M (NH4) 2SO4 in

7.8% (w / w) acetonitrile, pH 2.5, and buffer B contained 63.4% (w / w) acetonitrile. Linear gradients of 37-41% B in 12 min followed by 41-100% B in 15 min was executed at a flow rate of 1 ml / min. The chromatographic temperature was maintained at 60 ° C and UV detection was performed at 214 nm. The analytical results were:

 Arg34GLP1 content (7-37)

Arg34GLP1 content (9-37)

Sample for application Maximum impurity value Main maximum value

36% 2% 50% 13% 45% 1%

[0111] Chromatograms of the sample for application and the eluate are shown in Figures 4 and 5, respectively. The analytical results show a selective separation of the truncated form and a high reduction of the trick form in the main maximum value contained in the target GLP-1 fraction by cation exchange chromatography using organic modifiers.

Example 6:

[0112] Arg34GLP-1 (7-37) was expressed and captured by cation exchange as described in Example 1. The grouping containing Arg34GLP-1 (7-37) and the truncated form, Arg34GLP-1 (9-37) , as an impurity was adjusted to pH 3.1, and 10 ml of the resulting solution was applied to a 20 ml column Source 30S (Amersham Pharmacia Biotech) equilibrated with 100 ml 20 mmol / kg citric acid, 45% (w / w ) of ethanol, pH 3.0. The column was washed with 20 ml of equilibration solution, and the elution was performed with a linear salt gradient of 0 to 250 mmol / kg KCI (20 mmol / kg citric acid, 45% (w / w) ethanol, pH 3.0) followed by 60 ml of isocratic elution at 250 mmol / kg KCl. A chromatogram is shown in Figure 6. Different maximum values and separation between the truncated form and the target GLP-1 fraction were obtained by adding ethanol to the chromatographic solutions.

Example 7:

[0113] Arg34GLP-1 (7-37) was expressed and captured by cation exchange as described in example 1. The grouping containing Arg34GLP-1 (7-37) and the truncated form, Arg34GLP-1 (9-37) , as an impurity was adjusted to pH 3.5, and 10 ml of the resulting solution was applied to a 20 ml column Source 30S (Amersham Pharmacia Biotech) equilibrated with 100 ml 20 mmol / kg citric acid, 45% (w / w ) of ethanol, pH 3.5. The column was washed with 20 ml of equilibration solution, and the elution was performed with a linear salt gradient of 0 to 250 mmol / kg of KCI (20 mmol / kg of citric acid, 45% (w / w) ethanol, pH 3.5) followed by 40 ml of isocratic elution at 250 mmol / kg KCl. Different maximum values and separation between the truncated form and the target GLP-1 fraction similar to example 6 were obtained.

Example 8:

[0114] The fermentation broth of Arg34GLP-1 (7-37) was purified by a conventional cation exchange chromatography capture step followed by a conventional RP-HPLC purification step. 6 volumes of water were added to the cluster containing Arg34GLP-1 (7-37) and the truncated form, Arg34GLP-1 (9-37), as an impurity, and the solution was adjusted to pH 3.5. 170 ml of the resulting solution were applied to a 20 ml column Source 30S (Amersham Pharmacia Biotech) equilibrated with 100 ml 20 mmol / kg citric acid, 37.5 mmol / kg KCl, 45% (w / w) of ethanol, pH 3.5. The column was washed with 20 ml of equilibration solution, and the elution was performed with a linear salt gradient of 37.5 to 162.5 mmol / kg KCl (20 mmol / kg citric acid, 45% (w / w) ethanol , pH 3.5) followed by 20 ml of isocratic elution at 250 mmol / kg KCl (20 mmol / kg citric acid, 45% (w / w) ethanol, pH 3.5). Different maximum values and separation between the truncated form and the GLP-1 target fraction were obtained by adding ethanol to the chromatographic solutions.

Example 9:

[0115] Arg34GLP-1 (7-37) was expressed and captured by anion exchange as described in example 1. 10 ml of the cluster containing Arg34GLP-1 (7-37) and various impurities were applied to a column of 20 ml DEAE HyperD 20 (BioSepra SA) balanced with 100 ml 20 mM hydrogen disodium phosphate dihydrate, pH 7.5. The column was washed with 20 ml equilibration solution, and the elution was performed with a linear salt gradient of 0 to 250 mM NaCl (20 mM hydrogen disodium phosphate dihydrate, pH 7.5) followed by 40 ml of isocratic elution with 1 M NaCl (20 mM disodium hydrogen phosphate dihydrate, pH 7.5). No different maximum value or separation were obtained when the GLP-1 target fraction was eluting throughout the entire maximum value area.

Example 10:

[0116] Arg34GLP-1 (7-37) was expressed and captured by anion exchange as described in example 1. The grouping containing Arg34GLP-1 (7-37) and various impurities was diluted with three volumes of water, and 40 ml of the resulting solution were applied to a 20 ml column Source 15Q (Amersham Pharmacia Biotech) equilibrated with 100 ml 20 mM Tris-hydroxymethyl amino methane, pH 8.5. The column was washed with 20 ml of equilibration solution, and the elution was performed with a linear salt gradient of 0 to 250 mM NaCl (20 mM Tris-hydroxymethyl amino methane, pH 8.5) followed by 40 ml of isocratic elution with 250 mM NaCl. A chromatogram is shown in Figure 7. No different maximum value or separation were obtained when the GLP-1 target fraction was eluting throughout the entire maximum value area.

Example 11:

[0117] Arg34GLP-1 (7-37) was expressed and captured by anion exchange as described in example 1. Clustering containing Arg34GLP-1 (7-37) and various impurities were diluted with three volumes of water, and 20 ml of the resulting solution were applied to a 20 ml column Source 15Q (Amersham Pharmacia Biotech) equilibrated with 100 ml 20 mmol / kg Tris-hydroxymethyl amino methane, 45% (w / w) ethanol, pH 8.5. The column was washed with 20 ml of equilibration solution, and the elution was performed with a linear salt gradient of 0 to 250 mmol / kg NaCl (20 mmol / kg Trishydroxymethyl amino methane, 45% (w / w) ethanol , pH 8.5) followed by 40 ml of isocratic elution with 250 mmol / kg of NaCl. A chromatogram is shown in Figure 8. Different maximum values and the separation between various impurities and the target GLP-1 fraction were obtained by adding ethanol to the chromatographic solutions. Insignificant differences in configuration between this and example 10 are: different loading and buffer concentration.

Example 12:

[0118] Arg34GLP-1 (7-37) was expressed and captured by anion exchange as described in example 1. The grouping containing Arg34GLP-1 (7-37) and various impurities were diluted with a volume of water and two volumes of ethanol, and 20 ml of the resulting solution were applied to a 20 ml column Source 15Q (Amersham Pharmacia Biotech) equilibrated with 100 ml 20 mmol / kg Tris-hydroxymethyl amino methane, 45% (w / w) ethanol , pH 8.5. The column was washed with 20 ml of equilibration solution, and the elution was performed with a linear salt gradient of 0 to 100 mmol / kg NaCl (20 mmol / kg Tris-hydroxymethyl amino methane, 45% (w / w ) of ethanol, pH 8.5) followed by 40 ml of isocratic elution with 100 mM NaCl. Different maximum values and separation between various impurities and the GLP-1 target fraction were obtained by adding ethanol to the chromatographic solutions.

Example 13:

[0119] Arg34GLP-1 (7-37) was expressed as described in example 1. Arg34GLP-1 (7-37) was isolated from the fermentation broth by a reverse conventional phase chromatography capture stage, and subsequently precipitated to pl (isoelectric point) of Arg34GLP-1 (7-37). 10 g of the precipitate containing Arg34GLP-1 (7-37) and the truncated form, Arg34GLP-1 (9-37), as one of several impurities was suspended in 500 ml of water and dissolved by pH adjustment to 8.3 at a concentration of Arg34GLP-1 (7-37) of approximately 1.6 mg / ml. 5 ml of the resulting solution was adjusted to pH 3.5 and applied to a 20 ml column Source 30S (Amersham Pharmacia Biotech) equilibrated with 60 ml 0.42% w / w citric acid, pH 3.5. The truncated form was not eluted / washed by a linear gradient of 0 to 2 M NaCl (0.42% w / w citric acid, pH 3.5). The target peptide, Arg34GLP-1 (7-37), and the impurity, Arg34GLP-1 (9-37), were eluted at a single maximum value per 40 ml of the 4% w / w NaOH regeneration solvent. A chromatogram is shown in Figure 9. No removal of the truncated impurity was achieved by the low pH washing step with a high conventional saline solution without an organic modifier.

Example 14:

[0120] Arg34GLP-1 (7-37) was isolated from the fermentation broth by reverse conventional phase chromatography and precipitated as described in example 13. 10 g of the precipitate containing Arg34GLP-1 (7-37) and the truncated form , Arg34GLP-1 (9-37), as one of several impurities was suspended in 500 ml water and dissolved by pH adjustment to 8.3 at a concentration of Arg34GLP-1 (7-37) of approximately 1.6 mg / ml. 5 ml of the resulting solution was adjusted to pH

3.5 and applied to a 20 ml column Source 30S (Amersham Pharmacia Biotech) balanced with 60 ml 0.42% w / w citric acid, 34% w / w ethanol, pH 3.5. Elution was performed with a linear salt gradient of 0 to 2.23% w / w KCI (0.42% w / w citric acid, 34% w / w ethanol, pH 3.5). Different maximum values and separation between the truncated form and the GLP-1 target fraction were obtained similar to example 6.

Example 15:

[0121] Arg34GLP-1 (7-37) was isolated from the fermentation broth by reverse conventional phase chromatography and precipitated as described in example 13. 10 g of the precipitate containing Arg34GLP-1 (7-37) and the truncated form , Arg34GLP-1 (9-37), as one of several impurities was suspended in 500 ml water and dissolved by pH adjustment to 8.3 at a concentration of Arg34GLP-1 (7-37) of approximately 1.6 mg / ml. 5 ml of the resulting solution was adjusted to pH

3.5 and applied to a 20 ml column Source 30S (Amersham Pharmacia Biotech) balanced with 60 ml 0.42% w / w citric acid, 29% w / w ethanol, pH 3.5. Elution was performed with a linear salt gradient of 0 to 2.23% w / w KCl (0.42% w / w citric acid, 29% w / w ethanol, pH 3.5). The separation between the truncated form and the GLP-1 target fraction was obtained.

Example 16:

[0122] Arg34GLP-1 (7-37) was isolated from the fermentation broth by reverse conventional phase chromatography and precipitated as described in Example 13. 10 g of the precipitate containing Arg34GLP-1 (7-37) and the truncated form , Arg34GLP-1 (9-37), as one of several impurities was suspended in 500 ml of water and dissolved by adjusting pH to 8.3 at a concentration of Arg34GLP-1 (7-37) of approximately 1.6 mg / ml. 5 ml of the resulting solution was adjusted to pH 3.5 and applied to a 20 ml column Source 30S (Amersham Pharmacia Biotech) equilibrated with 60 ml 0.42% w / w citric acid, 51% w / w ethanol, pH 3.5. Elution was performed with a linear salt gradient of 0 to 2.23% w / w KCI (0.42% w / w citric acid, 51% w / w ethanol, pH 3.5). Different maximum values and the separation between the truncated form and the GLP-1 target fraction were obtained similar to example 6.

Example 17:

[0123] Arg34GLP-1 (7-37) was isolated from the fermentation broth by reverse conventional phase chromatography and precipitated as described in Example 13. 10 g of the precipitate containing Arg34GLP-1 (7-37) and the truncated form , Arg34GLP-1 (9-37), as one of several impurities was suspended in 500 ml water and dissolved by pH adjustment to 8.3 at a concentration of Arg34GLP-1 (7-37) of approximately 1.6 mg / ml. 5 ml of the resulting solution was adjusted to pH

3.5 and applied to a 20 ml column Source 30S (Amersham Pharmacia Biotech) balanced with 60 ml 0.42% w / w citric acid, 71% w / w ethanol, pH 3.5. Elution was performed with a linear salt gradient of 0 to 1.12% w / w KCl (0.42% w / w citric acid, 71% w / w ethanol, pH 3.5). A chromatogram is shown in Figure 10. Different maximum values and the separation between the truncated form and the target fraction of GLP-1 were obtained similar to example 6.

Example 18:

[0124] Arg34GLP-1 (7-37) was isolated from the fermentation broth by reverse conventional phase chromatography and precipitated as described in Example 13. 10 g of the precipitate containing Arg34GLP-1 (7-37) and the truncated form , Arg34GLP-1 (9-37), as one of different impurities was suspended in 500 ml of water and dissolved by pH adjustment to 8.3 at a concentration of Arg34GLP-1 (7-37) of approximately 1.6 mg / ml. 5 ml of the resulting solution was adjusted to pH 3.5 and applied to a 20 ml column Source 30S (Amersham pharmacia Biotech) equilibrated with 60 ml 0.42% w / w citric acid, 40% w / w 2-propanol, pH 3.5 . Elution was performed with a linear salt gradient of 0 to 2.23% w / w KCl (0.42% w / w citric acid, 40% w / w 2-propanol, pH 3.5). A chromatogram is shown in Figure 11. Different maximum values and the separation between the truncated form and the GLP-1 target fraction were obtained.

Example 19:

[0125] Arg34GLP-1 (7-37) was isolated from the fermentation broth by reverse conventional phase chromatography and precipitated as described in Example 13. 10 g of the precipitate containing Arg34GLP-1 (7-37) and the truncated form , Arg34 GLP-1 (9-37), as one of several impurities were suspended in 500 ml of water and dissolved by adjustment to pH at 8.3 at an Arg34GLP-1 concentration (7-37) of approximately 1.6 mg / ml. 5 ml of the resulting solution was adjusted to pH 3.5 and applied to a column of 8 ml Poros 50 HS (PE biosystems) balanced with 24 ml 0.42% w / w citric acid, 51% w / w ethanol, pH 3.5. Elution was performed with a linear salt gradient of 0 to 2.23% w / w KCI (0.42% w / w citric acid, 51% w / w ethanol, pH 3.5). A chromatogram is shown in Figure 12. Different maximum values and the separation between the truncated form and the GLP-1 target fraction were obtained.

Example 20:

[0126] Arg34GLP-1 (7-37) was isolated from the fermentation broth by reverse conventional phase chromatography and precipitated as described in Example 13. 10 g of the precipitate containing Arg34GLP-1 (7-37) and the truncated form , Arg34GLP-1 (9-37), as one of several impurities were suspended in 500 ml of water and dissolved by pH adjustment at 8.3 at an Arg34GLP-1 concentration (7-37) of approximately 1.6 mg / ml. 5 ml of the resulting solution was adjusted to pH

3.5 and applied to an 8 ml column Poros 50 HS (PE biosystems) balanced with 24 ml 0.42% w / w citric acid, 40% w / w 2-propanol, pH 3.5. Elution was performed with a linear salt gradient of 0 to 2.23% w / w KCI (0.42% w / w citric acid, 40% w / w 2-propanol, pH 3.5). Different maximum values and the separation between the truncated form and the GLP-1 target fraction were obtained.

Example 21:

[0127] Arg34GLP-1 (7-37) was isolated from the fermentation broth by reverse conventional phase chromatography and precipitated as described in Example 13. 10 g of the precipitate containing Arg34GLP-1 (7-37) and the truncated form , Arg34GLP-1 (9-37), as one of several impurities was suspended in 500 ml of water and dissolved by adjustment to pH at 8.3 at an Arg34GLP-1 concentration (7-37) of approximately 1.6 mg / ml 5 ml of the resulting solution was adjusted to pH 3.5 and applied to a 20 ml column Source 30S (Amersham pharmacia Biotech) equilibrated with 60 ml 0.42% w / w citric acid, 40% w / w 2 -methyl-2,4-pentanediol, pH 3.5. Elution was performed with a linear salt gradient of 0 to 2.23% w / w KCl (0.42% w / w citric acid, 40% w / w 2-methyl-2,4-pentanediol, pH 3.5 ). A chromatogram is shown in Figure 13. Different maximum values and the separation between the truncated form and the GLP-1 target fraction were obtained.

Example 22:

[0128] Arg34GLP-1 (7-37) was isolated from the fermentation broth by reverse conventional phase chromatography and precipitated as described in example 13. The precipitate was dissolved in water and purified by cation exchange chromatography using a Organic modifier followed by a stage of reverse conventional phase chromatography in ethanol. The purified Arg34GLP-1 (7-37) of the reverse phase eluate was precipitated to the pl of Arg34GLP-1 (7-37). Arg34GLP-1 (7-37) was acylated as described in WO 9808871.

[0129] The resulting solution containing mono-acylated Arg34GLP-1 (7-37) in a concentration of 2 mg / ml, and Arg34GLP1 (7-37) and di-acylated Arg34GLP-1 (7-37) as impurities, was diluted with 3 volumes of water. 5 ml of the diluted solution with pH 6.9 were applied to a 20 ml column Source 30S (Amersham pharmacia Biotech) equilibrated with 60 ml 0.42% w / w citric acid, 64.5% w / w ethanol, pH 3.5. Elution was performed with a linear salt gradient of 0 to 1.30% w / w KCI (0.42% w / w citric acid, 64.5% w / w ethanol, pH 3.5). A chromatogram is shown in Figure 14. The components of GLP-1 eluted as different maximum values and the separation between the three GLP-1 fractions was obtained.

[0130] The RP-HPLC analysis for the identification / verification of maximum values collected was carried out on a column (Fuji-Davison) of 4.0x250 mm in 100 A silica substituted by dimethyl-silyl dimethyl-butyl, with particles of 5 µm. Buffer A consisting of 0.15 M (NH4) 2SO4 in 7.8% (w / w) of acetonitrile, pH 2.5, and buffer B containing 63.4% (w / w) of acetonitrile. The gradient program at a flow rate of 1 ml / min was as follows: linear gradient of 35-57.5% B in 10 min, linear gradient of 57.5-67.5% B in 22 min, linear gradient of 67.5-90 % B in 3 min, isocratic at 90% B in 5 min, linear gradient of 90-35% B in 2 min, and isocratic at 35% B in 5 min. The chromatographic temperature was maintained at 60 ° C and a UV detection was performed at 214 nm. Analytical chromatograms verified the separation of the three GLP-1 fractions.

Example 23:

[0131] Arg34GLP-1 (7-37) was isolated from the fermentation broth by reverse conventional phase chromatography and precipitated as described in Example 13. The precipitate was dissolved in water and purified by cation exchange chromatography using a Organic modifier followed by a stage of reverse conventional phase chromatography in ethanol. The purified Arg34GLP-1 (7-37) of the reverse phase eluate was precipitated to the pl of Arg34GLP-1 (7-37). Arg34GLP-1 (7-37) was acylated as described in WO 9808871.

[0132] The resulting solution containing mono-acylated Arg34GLP-1 (7-37) in a concentration of 2 mg / ml, and Arg34GLP1 (7-37) and di-acylated Arg34GLP-1 (7-37) as impurities, was diluted with 3 volumes of water. 5 ml of the diluted solution with pH 6.9 was applied to a 20 ml column Source 30S (Amersham pharmacia Biotech) equilibrated with 60 ml 0.42% w / w citric acid, 40% w / w 2-propanol, pH 3.5. Elution was performed with a linear salt gradient of 0 to 2.23% w / w KCI (0.42% w / w citric acid, 40% w / w 2-propanol, pH 3.5). The separation between the three fractions of GLP-1 was obtained.

[0133] The RP-HPLC analysis method of Example 22 was used for the identification / verification of maximum values collected.

Claims (10)

1. Cation exchange chromatography process for the purification of a GLP-1 peptide from a mixture comprising said GLP-1 peptide and related impurities, comprising the step of: separating said GLP-1 peptide and said related impurities from said mixture by elution in a solution consisting essentially of an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepped gradient or isocratically in a component of salt and / or with a gradient of the linear or stepped pH or at a constant pH value, where the gradient of the pH or the pH value should be in the range where said GLP-1 peptide has a global net charge or local positive other than the positive local or global network of said related impurities to eliminate said related impurities; where the organic modifier is selected from C1-6 alkanol and C2-6 glycol; and where the related impurities are impurities that have a global or different local net charge and are selected from the group consisting of truncated forms, extended forms, forms with extra amino acids, derivatives, esters, deamidated forms, incorrectly folded forms, forms with undesired glycosylation and forms with sialylation.

2.

Anion exchange chromatography process for the purification of a GLP-1 peptide from a mixture comprising said GLP-1 peptide and related impurities, comprising the step of:

separating said GLP-1 peptide and said related impurities from said mixture by elution in a solution consisting essentially of an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or stepped gradient or isocratically in a component of salt and / or with a linear or stepped pH gradient or at a constant pH value, where the pH gradient or the pH value should be in the range where said GLP-1 peptide has a different local global or negative net charge of the net local or global negative charge of said related impurities to eliminate said related impurities; where the organic modifier is selected from C1-6 alkanol and C2-6 glycol; and where the related impurities are impurities that have a global or different local net charge and are selected from the group consisting of truncated forms, extended forms, forms with extra amino acids, derivatives, esters, deamidated forms, incorrectly folded forms, forms with undesired glycosylation and forms with sialylation.

3.

Process according to one of claims 1 to 2, wherein said GLP-1 peptide to be purified is selected from GLP-1 (737), GLP-1 (7-36) amide, Arg34GLP-1 (7-37); Arg26GLP-1 (7-37); Arg34Lys26 (N- (-Glu- (N-tetradecanoyl))) GLP-1 (7-37); Arg34Lys26 (N- (-Glu- (N-hexadecanoyl))) GLP-1 (7-37); Arg26Lys34 (N- (-Glu- (N-tetradecanoyl))) GLP-1 (7-37); Arg26Lys34 (N- (-Glu- (N-hexadecanoyl))) GLP-1 (7-37); Val8GLP-1 (7-37); Thr8GLP-1 (7-37); Met8GLP-1 (7-37); Gly8GLP-1 (7-37); Val8GLP-1 (7-36) amide; Thr8GLP-1 (7-36) amide; Met8GLP-1 (7-36) amide; and Gly8GLP1 (7-36) amide

Four.

Process according to one of claims 1 to 3, wherein the proportion of the organic modifier with water on a weight percentage basis is from 1:99 to 99: 1.

5.

Process according to claim 4, wherein the proportion of the organic modifier with water on a weight percentage basis is from 30:70 to 70:30.

6.

Process according to claim 4, wherein the proportion of the organic modifier with water on a weight percentage basis is from 35:50 to 50:35.

7.

Process according to claim 4, wherein the proportion of the organic modifier with water on a weight percentage basis is from 40:50 to 50:40.

8.

Process according to one of claims 1 to 7, wherein the organic modifier is selected from C1-6 alkanol and hexylene glycol.

9.

Process according to one of claims 1 to 8, wherein the organic modifier is C1-6 alkanol.

10.

Process according to one of claims 1 to 8, wherein the organic modifier is hexylene glycol.