Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Insulin; related peptides
Reexamination Certificate
1999-04-13
2001-01-30
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Insulin; related peptides
C530S381000, C530S412000, C530S416000, C530S417000
Reexamination Certificate
active
06180757
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the process of chromatographically separating glycosylated proteins or protein precursors from non-glycosylated proteins or protein precursors using a Ca
++
containing eluant.
BACKGROUND AND DESCRIPTION OF THE INVENTION
In the manufacture of proteins or protein precursors the separation of glycosylated proteins from non-glycosylated proteins represents an independent field of research. The industrial implications of the present invention relate to the optimisation of protein purification. It is the purpose of the optimised purification to achieve an end product to be used commercially comprising non-glycosylated proteins, substantially free from glycosylated proteins.
Proteins or protein precursors may originate from yeast expression systems. A correlation between a high expression level and an increase in glycosylated protein precursors has been observed. As a consequence the need for a more efficient process of separating the non-glycosylated proteins from the glycosylated proteins has become even more apparent.
In the yeast expression system a yeast organism produces proteins or protein precursors synthesised intracellularly. The yeast host organism is transformed by an expression vehicle harbouring DNA encoding the desired protein. The process comprises preparing a culture of the transformed yeast host organism, growing the culture and recovering the protein from the culture medium.
Prior art recovery of the desired protein comprised multiple steps of purification using the process of chromatography, such as ion exchange chromatography involving an eluant containing salt. By using the method of reverse phase high performance liquid chromatography (RP-HPLC) the procedure of separating non-glycosylated proteins from glycosylated proteins can be carried out. The eluant used for the eluation of the test samples is preferably an organic solvent containing salt, such as KCl. Although various salts have been applied to the organic solvent in the prior art the effect of a Ca
++
containing eluant has never been disclosed.
In the present context the term eluant is synonymous with the buffer used for eluation. The present invention provides an improved purification of the desired protein by using a Ca
++
containing eluant.
SUMMARY OF THE INVENTION
The present invention provides an improved process of separating glycosylated proteins from non-glycosylated proteins. Accordingly the present invention relates to a process of separating glycosylated proteins from non-glycosylated proteins by subjecting a solution comprising glycosylated and non-glycosylated proteins to RP-HPLC (reverse phase high performance liquid chromatography) using a Ca
++
containing eluant, and obtaining a fraction comprising non-glycosylated proteins, said fraction substantially free from glycosylated proteins.
In the present invention the term protein refers to all proteins or protein precursors and the term glycosylated proteins includes glycosylated proteins or protein precursors. In a preferred embodiment the protein is insulin or an analogue or precursor thereof.
Another object of the present invention is a fraction obtainable by using the process according to the invention comprising non-glycosylated proteins substantially free from glycosylated proteins.
REFERENCES:
patent: 3649456 (1972-03-01), Benneville et al.
patent: 4269605 (1981-05-01), Dean et al.
patent: 5633350 (1997-05-01), Fischer et al.
Green, Esq. Reza
Low Christopher S. F.
Lu Stephan
Novo Nordisk A S
Zelson, Esq. Steve T.
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