Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Stablizing an enzyme by forming a mixture – an adduct or a...
Reexamination Certificate
1997-12-22
2001-03-20
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Stablizing an enzyme by forming a mixture, an adduct or a...
C424S094500, C435S193000
Reexamination Certificate
active
06204036
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to stable transglutaminase preparations, in particular stable preparations of factor XIII, processes for producing stable transglutaminases, and their therapeutic uses.
BACKGROUND OF THE INVENTION
Transglutaminases are Ca
2+
dependent enzymes that catalyze the formation of isopeptide bonds in proteins between the side chain &ggr;-carboxamide group of glutamine and the side chain &egr;-amino group of lysine. Transglutaminases are found both extracellularly and intracellularly. Examples of transglutaminases include Factor XIII, epidermal transglutaminases, type II transglutaminases (tissue type transglutaminase, liver transglutaminases), and type I transglutaminases (keratinocyte transglutaminase).
Factor XIII (F XIII, fibrin-stabilizing factor), is a transglutaminase found as a proenzyme in plasma, platelets and monocytes/macrophages. It is important, inter alia, for ensuring efficient blood coagulation and would healing. F XIII, isolated from placenta or plasma or in the form of fresh frozen plasma, has ben employed for many years for treatment of factor XIII deficiency. It recently has become possible to prepare factor XIII using recombinant DNA technology. As used herein, rF XIII refers to recombinant factor XIII.
Commercially available purified or partially purified transglutaminase or F XIII preparations contain added stabilizers, such as human serum albumin (HSA). The use of protein stabilizers is problematic, since it decreases protein specific activity, increases the protein load when administered to patients, and may interfere with assessment of purity. It may also make the protein preparation immunogenic. These disadvantages of protein stabilizers make them particularly disadvantageous for stabilizing highly purified proteins, such as recombinant proteins. Additionally, use of protein stabilizers causes potential contamination with viral antigens when albumin, for example, is added.
The composition and activity of protein preparations used in therapy must be stable over relatively long periods of time. It is only rarely possible to achieve this stability in solution and, therefore, such products are frequently marketed in the dry state. Mild freeze-drying (lyophilization) is the method of choice for drying such products. However, even when this method is used, stable preparations fulfilling the requirements for integrity and durability are difficult to achieve.
Freeze-drying of unstabilized transglutaminase solutions leads, for example, to a marked drop in activity and to considerable turbidity. Formulations based on albumin and containing relatively high concentrations of salts have, therefore, been previously described for use with purified F XIII preparations. See, for example, DE-C-2063 070 and JP 53/59018. These formulations, however, suffer from the disadvantage described above of containing foreign protein, with all the problems attached thereto.
The freeze-drying of rF XIII in the presence of glycine or arginine and non-reducing sugars has been described. See WO 90/03147. Neither the stability, solubility, nor clarity of the reconstituted lyophilisate was described, however. The products obtained were stored at −20° C., indicating that the lyophilized material probably had inadequate stability at 4° C.
It is apparent, therefore, that a stable lyophilized formulation for transglutaminases, in particular for F XIII, is greatly to be desired. It is desirable such a formulation can be administered locally (e.g. topically) or parenterally, is stable at 2-8° C. (or higher) and does not require the addition of protein stabilizers, for example, HSA. Furthermore, the lyophilisate should be readily soluble and, following dissolution, should yield a stable, non-turbid solution.
SUMMARY OF THE INVENTION
The present invention comprises, inter alia, a stable lyophilized transglutaminase formulation, comprising at least one additive selected from the group consisting of: D- and L- amino acids and salts, derivatives, homologs, dimers, and oligomers thereof; sugars or sugar alcohols; surface-active agents; and reducing agents, with the proviso that the additive is neither glycine nor arginine, and wherein the formulation is readily soluble without any turbidity.
The instant invention further comprehends a formulation wherein the transglutaminase is selected from the group consisting of Factor XIII, and biologically active fragments, derivatives, and muteins thereof.
The instant invention further comprehends a formulation, wherein the Factor XIII is recombinant Factor XIII or is isolated from plasma, placenta, thrombocyte, or macrophages/monocytes. The instant invention also comprehends a formulation, wherein the amino acid is selected from the group consisting of His, Glu, Met, Thr, Lys, Ala, Ile, or Cys, and the salts, derivatives, homologs, dimers and oligomers thereof. The formulation additionally includes a formulation wherein the sugar or sugar alcohol is selected from the group consisting of sucrose, maltose, trehalose, lactose, sorbitol, mannitol, and the derivatives, and homologs thereof and a formulation, which further includes an amino acid selected from the group consisting of His, Glu, Ile and Ala.
The formulation of the present invention also includes a formulation wherein the surface-active agent is selected from the group consisting of Tween 80, Tween 20, PEG, cetyl alcohol, PVP, PVA, lanolin alcohol, and sorbitan monooleate, and wherein the reducing agent is selected from the group consisting of cystein, N-acetyl-cysteine, thioglycerol, sodium sulfide, and glutathione, and wherein the reducing agent is present in combination with a chelating agent.
The formulation of the present invention also includes a formulation comprising an amino acid, a sugar or sugar alcohol, and a surface active-substance, and a further formulation wherein the sugar is sucrose and the amino acid is His, as well as a further formulation comprising an additive selected from the group consisting of Tween 20, Tween 80, and PEG, as well as a further formulation, comprising sucrose, His, PEG, and Ile.
The instant invention also includes a formulation comprising a surface active agent, wherein the agent is PEG, and a further formulation comprising a sugar and a reducing agent, and an additional formulation wherein the sugar is sucrose, and wherein the reducing agent is selected from the group consisting of cystein, N-acetyl cysteine, and thioglycerol, as well as an additional formulation further comprising an amino acid and a chelating agent.
The instant invention also comprehends a formulation, wherein the concentration of the transglutaminase is in the range from about 0.003 to about 50 mg/ml, and wherein the concentration of the amino acid, salt, derivative, and homolog thereof is in the range from about 0.01% to about 10% (w/v), and additionally, wherein the concentration of the amino acid, salt, derivative, and homolog thereof is in the range from about 0.1% to about 3% (w/v).
The present invention further includes a formulation, wherein the concentration of the sugar and sugar alcohol is between about 0.1% and about 20% (w/v), and additionally wherein the concentration of the sugar and sugar alcohol is between about 0.2% and about 10% (w/v).
The instant invention also includes a formulation, wherein the concentration of the surface-active agent is between about 0.00001% and about 5% (w/v), and wherein the concentration of the surface-active agent is between about 0.0002% and about 0.1% (w/v).
The present invention also comprehends a formulation, wherein the concentration of the reducing agent is between about 0.001% and about 2% (w/v), as well as a formulation, wherein the concentration of the reducing agent is between about 0.005% and about 0.5% (w/v).
The instant invention further includes a formulation, wherein the pH of the formulation is in a range from about 6 to about 9, or wherein the pH of the formulation is in a range from about 7 and about 8.
The formulation of the present invention also comprehends using a bor
Karges Hermann
Metzner Hubert
Aventis Behring GmbH
Foley & Lardner
Lankford , Jr. Leon B.
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