Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1993-12-22
2001-09-11
McGarry, Sean (Department: 1635)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S021800, C530S350000, C530S399000
Reexamination Certificate
active
06288030
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel stem cell factor compositions and methods. More specifically, the present invention relates to novel lyophilized formulations of stem cell factor and methods for production.
BACKGROUND
Stem cell factor (“SCF”) is an early acting hematopoietic factor. See PCT WO 91/05795, entitled “Stem Cell Factor,” herein incorporated by reference. SCF pharmaceutical compositions are known in the art, PCT WO 91/05795, supra, pages 20-21, however, an SCF pharmaceutical composition with increased shelf-life would be beneficial to both producers and consumers of the product.
In solution, SCF stability is limited by a number of degradation reactions, including cleavage, deamidation, and modification on RP-HPLC. Freeze drying (lyophilization) is considered useful and effective for preservation of many biologically active materials, including proteins. However, lyophilization induces its own stresses, including extreme concentration of the protein during the freezing process and removal of water, which may result in instability of the product. Hence, lyophilization may result in increased rates of crosslinking (covalent oligomer formation) and noncovalent aggregation, in addition to deamidation and oxidation, both of which can occur in the lyophilized state as well as the liquid state. Thus protective agents are often required to enhance stability of the drug by a number of mechanisms, including raising the glass transition of the formulation (that is, the temperature at which the composition changes from a fluid, rubbery and reactive state to a rigid, and, therefore less reactive state); acting as cryo- or lyoprotectants, (that is protective agents during the freezing and/or drying processes); and/or by replacing bound water molecules that are necessary for the conformational stability of the protein.
Amino acids have been noted in some cases to act as stabilizers for freeze-dried protein products. Sodium glutamate and lysine-HC1 have been reported to have cryoprotective effects on the freeze denaturation of a protein, lactate dehydrogenase (Seguro, et al., Cryobiology 27; 70-79 (1990)). Hora, et al. report the use of the L-arginine, L-carnitine chloride, or L-betaine as buffers for rhIL-2 (in Developments in Biological Standardization, Vol. 74, Karger, Basel, 1992). The formulations buffered with betaine showed dimer formation in accelerated testing, while arginine/carnitine-buffered formulations showed poor mechanical stability, leading to aggregation of the protein during production. Thus, the use of amino acids does not predictably enhance stability of lyophilized protein products.
Additionally, incorporation of small amounts of salt may, in some cases, destabilize lyophilized products, including proteins. The salts may be introduced during the pH adjustment of the formulation by addition of strong acids or bases. At the very low concentrations present in such a case, the salt may be trapped in the amorphous phase with the protein and may decrease the glass transition of the formulation or otherwise exert a detrimental effect on the stability of the protein drug. Elimination of such salt may therefore be desirable. Other classes of molecules, including mono- and di-saccharides, and polymers such as PVP have also been reported as stabilizers of lyophilized proteins, but, again, their utility is not predictable for any given protein product.
SUMMARY OF THE INVENTION
The present invention relates to novel formulations of freeze dried SCF which include amino acid buffers. Surprisingly, incorporation of the amino acids histidine and/or glutamic acid as buffers increases SCF stability, as compared to formulations in which inorganic or other types of organic compounds are used to adjust pH. Preferred forms of the present formulations are those in which pH is adjusted by combining histidine and glutamic acid. Adjusting pH by this method avoids the need to titrate with strong acid or base, which creates salts. The present formulations may optionally include additional stabilizers such as sucrose. Such formulations may also optionally include a bulking agent, and/or an osmolarity regulating agent, such as mannitol.
The protective effect of the present amino acid buffer is observed, but the precise mode of action is unknown. One possible explanation relates to the glass transition temperature for histidine. Essentially, the glass transition temperature is that temperature at which the composition (here, containing SCF) changes from being a relatively inflexible, unreactive (and therefore fairly stable) state to a relatively flexible, more reactive (and therefore less stable) state. Because the glass transition temperature of histidine is relatively high compared to non-amino acid buffer components, histidine may contribute to the overall stability of the freeze-dried formulation.
Another mechanism by which amino acids may protect SCF is by co-concentrating with the protein during freezing, thus serving to dilute and protect the protein. It is also possible that the amphiphilic amino acids may specifically interact with the protein to protect it against denaturaturation during the freezing and/or freeze drying processes.
In another aspect of the present invention, sucrose is seen to have a protective effect on lyophilized SCF formulations. Again, the precise mode of action is unknown, but one possible explanation relates to the glass transition temperature, similar to the explanation set forth above for histidine. The glass transition temperature overall depends on the glass transition temperature of the formulation components, as well as their relative proportions, and sucrose also has a relatively high glass transition temperature. Because sucrose co-concentrates with SCF in the frozen state, it may serve to dilute the SCF, and therefore protect against aggregation.
An alternative explanation relates to water replacement. Proteins such as SCF contain bound water molecules, the replacement of which by polyols may increase stability. Sucrose may have superior water replacement properties over other polyols.
The present formulations, using the amino acid histidine and/or the amino acid glutamic acid, and optionally sucrose, and optionally a bulking agent such as mannitol, and further optionally an osmolarity regulating agent, which also may be mannitol, satisfy a need for an SCF formulation with increased stability. The present invention also relates to methods for preparation of such formulations.
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Hora, et al. Developments in Biological Standardization, vol. 74, Karger, Basel, (1992).
Laemmli, Nature 227: 680-685 (1970).
Seguro, Cryobiology 27: 70-79 (1990).
Amgen Inc.
Knight Matthew W.
Lacourciere Karen A.
Levy Ron K.
McGarry Sean
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