Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2001-05-07
2003-02-11
Carlson, Karen Cochrane (Department: 1652)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S002600, C514S012200, C514S013800, C514S014800, C530S303000, C530S308000, C530S324000, C530S350000, C530S365000, C530S366000, C435S069100
Reexamination Certificate
active
06518241
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method of shock heat treatment of polypeptides, to a stable polypeptide in a fluid medium, to a pharmaceutical composition comprising a stable polypeptide, to use of shock heat treatment for stabilizing a polypeptide, and to an industrial or large scale method of manufacturing a polypeptide comprising applying shock heat treatment during the manufacturing of said polypeptide.
BACKGROUND
Proteins are known to associate in solution. This association is termed aggregation, dimerization, polymerization, precipitation, emulsification, fibrillation, destabilization, inclusion body formation, and more, depending on the protein or the case story.
Changes in the association capabilities can be initiated by breaking of the weak linkages or bonds (e.g., hydrogen bonds), that exist within a protein molecule, and which is responsible for the highly ordered structure of the protein in its stable state. Such destabilized proteins have a looser, more random structure. Denaturation can be brought about in various ways, such as by heating, by vigorous shaking, by treatment with acid, detergents, or other additives. A common consequence of destabilization is loss of biological activity (e.g., loss of the catalytic activity/ability of an enzyme, loss of pharmacological effect of a drug).
The original structure of some proteins can be regenerated upon removal of the destabilizing factor and restoration of conditions favoring a stable state. However, in many cases regeneration is difficult. New techniques to improve such processes without loss of biological function are important.
Protein association is initiated and/or accompanied by structural changes of the protein, i.e. changes in the physical conformation. To be in control of such structural changes is of high importance, because the polypeptide hereby is stabilized.
DESCRIPTION OF THE INVENTION
It has been shown that a short periode of heating (herein designated shock heat treatment) of a protein sample can improve the protein stability without substantial loss of biological activity.
The invention can be used as a unit operation during preparation, purification, and/or formulation of polypeptides. Compared to other methods, it is advantageous that the method of the invention can be applied to change polypeptide conformation in a very fast and non-invasive manner.
In one aspect the invention relates to a method of stabilizing a polypeptide comprising shock heat treatment of said polypeptide.
In another aspect the invention relates to a method for decreasing the association of a polypeptide comprising shock heat treatment of said polypeptide.
In a further aspect the invention relates to a method for arresting the association of a polypeptide comprising shock heat treatment of said polypeptide.
In a further aspect the invention relates to a method for postponement of the association of a polypeptide comprising shock heat treatment of said polypeptide.
In a further aspect the invention relates to a method for prevention of the association of a polypeptide comprising shock heat treatment of said polypeptide.
In a further aspect the invention relates to a method of producing a storage stable polypeptide comprising shock heat treatment of said polypeptide.
In a further aspect the invention relates to a stable polypeptide in a fluid medium, obtainable by a method comprising shock heat treatment of said polypeptide.
In a further aspect the invention relates to a pharmaceutical composition comprising a stable polypeptide in a fluid medium, obtainable by a method comprising shock heat treatment of said polypeptide.
In a further aspect the invention relates to use of shock heat treatment for stabilizing a polypeptide.
In another aspect the invention relates to use of shock heat treatment for decreasing the association of a polypeptide.
In a further aspect the invention relates to use of shock heat treatment for arresting the association of a polypeptide.
In a further aspect the invention relates to use of shock heat treatment for postponement of the association of a polypeptide.
In a further aspect the invention relates to use of shock heat treatment for prevention of the association of a polypeptide.
In a further aspect the invention relates to use of shock heat treatment for producing a storage stable polypeptide.
Industrial or large scale methods of manufacturing polypeptides are intended to include the preparation of the polypeptides, such as by culturing or fermentation, or organic peptide synthesis, recovery of the polypeptides from the culture or from the reaction mixture, the purification of the polypeptides, optionally further modification of the polypeptides and/or formulation of the polypeptide for storage or for making a composition, e.g. a pharmaceutical drug or an industriel enzyme. During the industrial or large scale method of manufacturing a polypeptide, there are several possibilities of applying shock heat treatment, such as the use of a unit operation element, eg. a heating mantle, or internal heating element, e.g. a piezoelectrical element, during recovery and/or purification and/or modification of the polypeptide, or the use of a water bath during formulation of the polypeptide.
In a further aspect the invention relates to an industrial or large scale method of manufacturing a polypeptide comprising applying shock heat treatment during the manufacturing of said polypeptide.
In one embodiment of the invention the polypeptide is in a fluid medium. In another embodiment of the invention the fluid medium is a gas. In a further embodiment of the invention the fluid medium is a liquid. In a further embodiment of the invention the fluid medium is a solution. In a further embodiment of the invention the fluid medium is a suspension. In a further embodiment of the invention the fluid medium may be an organic medium. In a further embodiment of the invention the fluid medium may be an aqueous medium. Preferred examples of the fluid medium are an aqueous solution, aqueous suspension, organic gas, organic solution, or organic suspension, or any mixture thereof. Examples of organic solutions comprising the polypeptide are a C
1-6
-alkanol, such as methanol, ethanol, 2-propanol, tert-butanol and n-butanol; acetonitril; C
3-12
-ketones, e.g. acetone; diols, triols and polyols, e.g. glycerol; phenol; cresols, e.g. m-cresol. Examples of mixtures of an organic and aqueous medium are solutions or suspensions comprising water and a C
1-6
-alkanol. Each of these fluid mediums constitutes an individual embodiment of the present invention.
In a further embodiment of the invention the shock heat treatment is done without changing the chemical structure of the polypeptide. In a further embodiment of the invention the shock heat treatment is done without changing the primary structure of the polypeptide. In a further embodiment of the invention the shock heat treatment is done without substantial loss, preferably without loss, of the biological activity of the polypeptide. In a further embodiment of the invention the shock heat treatment is done without substantial loss, preferably without loss, of the biological activity of the polypeptide, but the chemical structure, such as the primary structure, is changed. For instance, shock heat treatment may lead to deamidation of a protein, but the biological activity is maintained. Precursors and prodrugs of the polypeptide which may or may not have a biological activity are intended to be comprised within the scope of the invention even in such cases where the precursor or prodrug does not have a biological activity.
In a further embodiment of the invention the shock heat treatment is done by applying a heating source, such as a heating mantle, an internal heating element, e.g. a piezoelectrical element, or boiling water.
In a further embodiment of the invention the shock heat treatment is applied for such time which increases the stability of the polypeptide formulation significantly.
In a further embodiment of the invention the shock heat treatment is ap
Carlson Karen Cochrane
Green Reza
Kam Chih-Min
Novo Nordisk A S
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