Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-08-10
2003-04-01
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S402000, C530S403000, C530S813000, C530S815000, C424S501000, C424S489000, C424S094100, C424S094200, C424S094500, C424S094600, C435S039000, C435S174000, C435S178000, C435S181000, C435S183000, C435S188000, C514S002600, C514S004300
Reexamination Certificate
active
06541606
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention relates to methods for the stabilization, storage and delivery of biologically active macromolecules, such as proteins, peptides and nucleic acids. In particular, this invention relates to protein or nucleic acid crystals, formulations and compositions comprising them. Methods are provided for the crystallization of proteins and nucleic acids and for the preparation of stabilized protein or nucleic acid crystals for use in dry or slurry formulations. The crystals, crystal formulations and compositions of this invention can be reconstituted with a diluent for the parenteral administration of biologically active macromolecular components.
The methods of this invention are useful for preparing crystals of “naked” DNA and RNA sequences that code for therapeutic or immunogenic proteins and can be administered parenterally. The dissolving DNA and RNA molecules, subsequently taken up by the cells and used to express the protein with the proper glycosylation pattern, can be either therapeutic or immunogenic. Alternatively, the present invention is useful for preparing crystals, crystal formulations and compositions of sense and antisense polynucleotides of RNA or DNA.
The present invention is further directed to encapsulating proteins, glycoproteins, enzymes, antibodies, hormones and peptide crystals or crystal formulations into compositions for biological delivery to humans and animals. According to this invention, protein crystals or crystal formulations are encapsulated within a matrix comprising a polymeric carrier to form a composition. The formulations and compositions enhance preservation of the native biologically active tertiary structure of the proteins and create a reservoir which can slowly release active protein where and when it is needed. Such polymeric carriers include biocompatible and biodegradable polymers. The biologically active protein is subsequently released in a controlled manner over a period of time, as determined by the particular encapsulation technique, polymer formulation, crystal geometry, crystal solubility, crystal crosslinking and formulation conditions used. Methods are provided for crystallizing proteins, preparing stabilized formulations using pharmaceutical ingredients or excipients and optionally encapsulating them in a polymeric carrier to produce compositions and using such protein crystal formulations and compositions for biomedical applications, including delivery of therapeutic proteins and vaccines. Additional uses for the protein crystal formulations and compositions of this invention involve protein delivery in human food, agricultural feeds, veterinary compositions, diagnostics, cosmetics and personal care compositions.
BACKGROUND OF THE INVENTION
Proteins are used in a wide range of applications in the fields of pharmaceuticals, veterinary products, cosmetics and other consumer products, foods, feeds, diagnostics, industrial chemistry and decontamination. At times, such uses have been limited by constraints inherent in proteins themselves or imposed by the environment or media in which they are used. Such constraints may result in poor stability of the proteins, variability of performance or high cost.
It is imperative that the higher order three-dimensional architecture or tertiary structure of a protein be preserved until such time that the individual protein molecules are required to perform their unique function. To date, a limiting factor for use of proteins, particularly in therapeutic regimens, remains the sensitivity of protein structure to chemical and physical denaturation encountered during delivery.
Various approaches have been employed to overcome these barriers. However, these approaches often incur either loss of protein activity or the additional expense of protein stabilizing carriers or formulations.
One approach to overcoming barriers to the widespread use of proteins is crosslinked enzyme crystal (“CLEC™”) technology [N. L. St. Clair and M. A. Navia,
J. Am. Chem. Soc.,
114, pp. 4314-16 (1992)]. See also PCT patent application PCT/US91/05415. Crosslinked enzyme crystals retain their activity in environments that are normally incompatible with enzyme function. Such environments include prolonged exposure to proteases, organic solvents, high temperature or extremes of pH. In such environments, crosslinked enzyme crystals remain insoluble, stable and active.
Despite recent progress in protein technology generally, two problems which are discussed below continue to limit the use of biological macromolecules in industry and medicine. The first problem relates to molecular stability and sensitivity of higher order tertiary structures to chemical and physical denaturation during manufacturing and storage. Second, the field of biological delivery of therapeutic proteins requires that vehicles be provided which release native proteins, such as proteins, glycoproteins, enzymes, antibodies, hormones, nucleic acids and peptides at a rate that is consistent with the needs of the particular patient or the disease process.
Macromolecule Stability
Numerous factors differentiate biological macromolecules from conventional chemical entities, such as for example, their size, conformation and amphiphilic nature. Macromolecules are not only susceptible to chemical, but also physical degradation. They are sensitive to a variety of environmental factors, such as temperature, oxidizing agents, pH, freezing, shaking and shear stress [Cholewinski, M., Luckel, B. and Horn, H.,
Acta Helv.,
71, 405 (1996)]. In considering a macromolecule for drug development, stability factors must be considered when choosing a production process.
Maintenance of biological activity during the development and manufacture of pharmaceutical products depends on the inherent stability of the macromolecule, as well as the stabilization techniques employed. A range of protein stabilization techniques exist; including:
a) Addition of chemical “stabilizers” to the aqueous solution or suspension of protein. For example, U.S. Pat. Nos. 4,297,344 discloses stabilization of coagulation factors II and VIII, antithrombin III and plasminogen against heat by adding selected amino acids. U.S. Pat. No. 4,783,441 discloses a method for stabilizing proteins by adding surface-active substances. U.S. Pat. No. 4,812,557 discloses a method for stabilizing interleukin-2 using human serum albumin. The drawback of such methods is that each formulation is specific to the protein of interest and requires significant development efforts.
b) Freeze/thaw methods in which the preparation is mixed with a cryoprotectant and stored at very low temperatures. However, not all proteins will survive a freeze/thaw cycle.
c) Cold storage with cryoprotectant additive, normally glycerol.
d) Storage in the glass form, as described in U.S. Pat. No. 5,098,893. In this case, proteins are dissolved in water-soluble or water-swellable substances which are in amorphous or glassy state.
e) The most widely used method for the stabilization of proteins is freeze-drying or lyophilization [Carpenter, J. F., Pical, M. J., Chang, B. S. and Randolph, T. W.,
Pharm. Res.,
14:(8) 969 (1997)]. Whenever sufficient protein stability cannot be achieved in aqueous solution, lyophilization provides the most viable alternative. One disadvantage of lyophilization is that it requires sophisticated processing, is time consuming and expensive [Carpenter, J. F., Pical, M. J., Chang, B. S. and Randolph, T. W.,
Pharm. Res.,
14:(8) 969 (1997) and literature cited therein]. In addition, if lyophilization is not carried out carefully, most preparations are at least partially denatured by the freezing and dehydration steps of the technique. The result is frequently irreversible aggregation of a portion of protein molecules, rendering a formulation unacceptable for parenteral administration.
The vast majority of protein formulations produced by the above-described techniques require cold storage, sometimes as low as −20° C. Exposure to eleva
Khalaf Nazar K.
Margolin Alexey L.
Rakestraw Scott L.
Shenoy Bhami C.
St. Clair Nancy L.
Altus Biologics Inc.
Fish & Neave
Haley Jr. James F.
Low Christopher S. F.
Mohamed Abdel A.
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