Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
2000-03-10
2002-09-17
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S412000, C530S350000, C530S300000, C530S416000, C530S417000, C435S007100, C210S088000
Reexamination Certificate
active
06451987
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an ion exchange chromatography process for purifying a peptide from a mixture containing said peptide and related impurities, and to an industrial method including such ion exchange chromatography process.
2. 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). Also, the change in pH may occasionally be employed to elute the target protein (cf. J. Lamy, J. Lamy, J. Weill, Arch. Biochem. Biophys. 193, 140-149, 1979).
BRIEF SUMMARY 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 present invention relates to a novel elution technique which is the combination of elution in a solution comprising an organic modifier with the subsequent elution in an aqueous solution at the same or a different pH optionally followed by a regeneration step. The equilibration solution and the sample for application may or may not contain the organic modifier. The elution of the peptide occurs at non-denaturing conditions (in a solution free of organic modifier). Moreover, the elution of the peptide is performed in a single peak.
Accordingly, 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 steps of:
a) eluting said related impurities of said mixture in a solution comprising an organic modifier, water, optionally a salt component and optionally a buffer, at a linear or step gradient or isocratically in salt component, and at pH-values optionally maintained with a buffer so that said peptide has a positive local or overall net charge and said related impurities have a local or overall positive net charge which is lower than the positive net charge of said peptide so as to remove said related impurities,
b) subsequently, eluting said peptide by a step or linear change to an aqueous solvent optionally with a salt component, at the same or higher pH-values optionally maintained with a buffer.
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 steps of:
a) eluting said related impurities of said mixture in a solution consisting essentially of an organic modifier, water, optionally a salt component and optionally a buffer, at a linear or step gradient or isocratically in salt component, and at pH-values optionally maintained with a buffer so that said peptide has a positive local or overall net charge and said related impurities have a local or overall positive net charge which is lower than the positive net charge of said peptide so as to remove said related impurities,
b) subsequently, eluting said peptide by a step or linear change to an aqueous solvent optionally with a salt component, at the same or higher pH-values optionally maintained with a buffer.
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 steps of:
a) eluting said related impurities of said mixture in a solution comprising an organic modifier, water, optionally a salt component and optionally a buffer, at a linear or step gradient or isocratically in salt component, and at pH-values optionally maintained with a buffer so that said peptide has a negative local or overall net charge and said related impurities have a local or overall negative net charge which is lower than the negative net charge of said peptide so as to remove said related impurities,
b) subsequently, eluting said peptide by a step or linear change to an aqueous solvent optionally with a salt component, at the same or lower pH-values optionally maintained with a buffer.
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 steps of:
a) eluting said related impurities of said mixture in a solution consisting essentially of an organic modifier, water, optionally a salt component and optionally a buffer, at a linear or step gradient or isocratically in salt component, and at pH-values optionally maintained with a buffer so that said peptide has a negative local or overall net charge and said related impurities have a local or overall negative net charge which is lower than the negative net charge of said peptide so as to remove said related impurities,
b) subsequently, eluting said peptide by a step or linear change to an aqueous solvent optionally with a salt component, at the same or lower pH-values optionally maintained with a buffer.
In the above aspects of the present process the elution in step a) could also be considered a washing step of related impurities.
The elution of the peptide in step b) occurs at non-denaturing conditions (in a solution free of organic modifier). Thus, the peptide is eluted to an aqueous solution with a solution comprising water and optionally a salt component, an acid or base, and/or a buffer, but without the presence of an organic modifier.
In one embodiment of the present invention the ratio of organic modifier to water, on a weight percent basis, is from 1:99 to 99:1, such as from 1:99 to 80:20, 20:80 to 80:20, 30:70 to 70:30, 35:50 to 50:35, or 40:50 to 50:40. Each of these ranges constitutes an alternative embodiment of the present invention.
In further embodiments of the present invention the organic modifier is selected from C
1-6
-alkanol, C
1-6
-alkenol or C
1-6
-alkynol, urea, guanidine, or C
1-6
-alkanoic acid, such as acetic acid, C
2-6
-glycol, C
3-7
-polyalcohol including sugars, preferably C
1-6
-alkanol and C
2-6
-glycol, more preferably methanol, ethanol, propanols and butanols and hexyl glycols, most preferably ethanol and 2-propanol. Each of these organic modifiers constitutes an alternative embodiment of the present invention.
In a further embodiment of the present invention the salt component in step a) is selected from any organic or inorganic salt and mixtures thereof, preferably NaCl, KCl, NH
4
Cl, CaCl
2
, sodium acetate, potassium acetate, ammonium acetate, sodium citrate, potassium citrate, ammonium citrate, sodium sulphate, potassium sulphate, ammonium sulphate, calcium acetate or mixtures thereof, most preferr
Bork, Esq. Richard W.
Green, Esq. Reza
Low Christopher S. F.
Novo Nordisk A S
Robinson Hope A.
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