Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Separation or purification
Reexamination Certificate
2000-03-13
2002-09-03
Carlson, Karen Cochrane (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Separation or purification
C530S416000, C530S417000, C530S324000, C514S012200
Reexamination Certificate
active
06444788
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an ion exchange chromatography process for purifying GLP-1 or an analog or a derivative thereof from a mixture containing said GLP-1 and related impurities, and to an industrial method including such ion exchange chromatography process.
BACKGROUND
For the purification and analysis of proteins and peptides, chromatography is a well-known and widely used method. A number of different chromatographic principles are applied, among these ion exchange chromatography (IEC). The IEC principle includes two different approaches: anion exchange and cation exchange according to the charge of the ligands on the ion exchange resin. A conventional IEC purification process usually consists of one or more: equilibration sections, application or loading sections, wash 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)).
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 step 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 seldom used. Organic solvents or modifiers have occasionally been added to the solutions to keep the protein or peptide on the desired form or just in solution (cf. K. H. Jørgensen, Process for Purifying Insulin, U.S. Pat. No. 3,907,676, Sep. 23, 1975; and J. Brange, O. Hallund and E. Sørensen, Chemical Stability of Insulin 5. Isolation, Characterisation and Identification of Insulin Transformation Products, Acta Pharm. Nord. 4(4), 223-232, 1992).
Glucagon-Like Peptide-1 (GLP-1) (cf. Schmidt et al. in Diabetologia 28 704-707, 1985) and analogues as well as derivatives thereof may be used in the treatment of diabetes, as disclosed in WO 98/08871. A GLP-1 peptide and related analogues are easily dissolved in aqueous solvents and kept in a monomized form. Traditional IEC purification of GLP-1 with salt gradients in aqueous solvents may, however, be problematic due to the lack of selectivity between a GLP-1 target moiety and related impurities.
WO 87/06941 (The General Hospital Corporation) disclose peptide fragments which comprises GLP-1(7-37) and functional derivatives thereof and to its use as an insulinotropic agent.
WO 90/11296 (The General Hospital Corporation) disclose peptide fragments which comprise GLP-1(7-36) and functional derivatives thereof and have an insulinotropic activity which exceeds the insulinotropic activity of GLP-1(1-36) or GLP-1 (1-37) and to their use as insulinotropic agents.
WO 91/11457 (Buckley et al.) discloses analogues of the active GLP-1 peptides 7-34, 7-35, 7-36, and 7-37.
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 e.g. 12∝24 carbon atoms.
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 e.g. 12-24 carbon atoms.
WO 96/32414 discloses GLP-2 analogues.
EP 0699686-A2 (Eli Lilly & Co.) discloses certain N-terminal truncated fragments of GLP-1 that are reported to be biologically active.
EP 0708179-A2 (Eli Lilly & Co.) discloses GLP-1 analogues and derivatives that include an N-terminal imidazole group and optionally an unbranched C
6
-C
10
acyl group in attached to the lysine residue in position 34.
DESCRIPTION OF THE INVENTION
In contrast to the above described IEC techniques for purification of any protein or peptide, consisting of one or more equilibration steps, application or loading steps, wash steps, elution steps, and regeneration steps, the instant invention relates to the use of an organic modifier for the purification of a GLP-1 peptide and all related analogues etc. by IEC. By addition of an organic modifier especially to the elution section of the IEC purification step, an increase in selectivity and efficiency is obtained compared to the same run with aqueous buffers, both for anion and cation 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 or very low concentrations of salt is needed for elution, especially for cation exchange chromatography compared to an aqueous chromatographic system.
In a broad aspect the present invention relates to a cation exchange chromatography process for purifying a peptide from a mixture comprising said peptide and related impurities, comprising the step of:
separating said peptide and said related impurities of said mixture by elution in a solution comprising an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or step gradient or isocratically in salt component and/or with a linear or step pH-gradient or at a constant pH-value, wherein the pH-gradient or pH-value should be in the range where said peptide has a positive local or overall net charge different from the local or overall positive net charge of said related impurities so as to remove said related impurities.
In another broad aspect the present invention relates to a cation exchange chromatography process for purifying a peptide from a mixture comprising said peptide and related impurities, comprising the step of:
separating said peptide and said related impurities of 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 step gradient or isocratically in salt component and/or with a linear or step pH-gradient or at a constant pH-value, wherein the pH-gradient or pH-value should be in the range where said peptide has a positive local or overall net charge different from the local or overall positive net charge of said related impurities so as to remove said related impurities.
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 step of:
separating said peptide and said related impurities of said mixture by elution in a solution comprising an organic modifier, water, optionally a salt component, and optionally a buffer, with a linear or step gradient or isocratically in salt component and/or with a linear or step pH-gradient or at a constant pH-value, wherein the pH-gradient or pH-value should be in the range where said peptide has a negative local or overall net charge different from the local or overall negative net charge of said related impurities so as to remove said related impurities.
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 step of:
separating said peptide and said related impurities of 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 step gradient or isocratically in salt component and/or with a linear or step pH-gradient or at a constant pH-value, wherein the pH-gradient or pH-value should be in the range where said peptide has a negative local or overall net charge different from the local or overall negative net charge of said related impurities so as to remove said related impurities.
In an embodiment of the present invention the peptide to be purified is selected from polypeptides, oligopeptides, proteins, receptors, vira, as well as homologues, analogues and derivatives thereof, preferably glucagon, hGH, insulin, aprotinin, FactorVII, TPA, FactorVIIa (NovoSeven®, availabl
Bork, Esq. Richard
Carlson Karen Cochrane
Green, Esq. Reza
Mitra Rita
Novo Nordisk A S
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