Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
2001-01-22
2004-11-09
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S407000, C530S411000, C530S418000, C435S068100, C514S002600, C514S012200
Reexamination Certificate
active
06815533
ABSTRACT:
This invention relates to the bulk manufacturing process of activated protein C.
Protein C is a serine protease and naturally occurring anticoagulant that plays a role in the regulation of hemostasis by inactivating Factors Va and VIIIa in the coagulation cascade. Human protein C circulates as a 2-chain zymogen which is activated in vivo by thrombin and thrombomodulin on phospholipid surfaces resulting in activated protein C (aPC). The protein C enzyme system represents a major physiological mechanism of anticoagulation.
Recombinant human protein C is generally produced by mammalian cell culture and purified by conventional chromatography techniques. When these techniques are performed at a laboratory scale, the resulting solution of aPC is readily handled and processed by standard techniques such as freeze drying. For production on a commercial scale, however, the aPC production and purification procedure generates a large volume of solution which is immediately shipped to formulation and filling operations in sterile bags. This method of handling large volumes of aPC is not desirable because the solution is neither stable nor easily dispensed. Furthermore, the necessity to handle large solutions of aPC links the schedule, scale, and site of commercial purification operations to the scale, schedule, and site of the commercial formulation fill/finish operations.
A stable preformulation form of aPC is preferred. Freeze-drying usually is a viable option for rendering a protein in a bulk biological solution in the solid form as an amorphous powder. However, amorphous proteins can be unstable for extended periods. The large volumes of solution generated in the commercial manufacturing process renders freeze drying as an impractical technique due to the expense and the extended time needed to process the solution. Additionally, the freeze-dried protein can be fluffy, dusty, and difficult to handle as an amorphous solid powder (Akers and Schmidt,
BioPharm
10(4):29-31, 1997).
Frozen solutions of aPC are not suitable for a commercial setting because containers freeze and thaw unevenly, generating concentration gradients and autoproteolysis. Freezing of large volumes of solution requires a significant amount of time and the resulting “blocks” of frozen solution are difficult to handle. Moreover, containers of frozen aPC are not readily dispensed for filling or sampling.
Therefore, commercial production of aPC generates a large volume of a biologically active solution that is difficult to process. A stable preformulation form is preferred to facilitate handling and to maintain the product integrity of the solution during the manufacturing process. Traditional “block” freezing or commercial freeze drying procedures are not practical and do not solve this problem. Thus, there remains the need to identify a stable, preformulation form of the aPC solution generated in the manufacturing process that is suitable for storage, handling, and recovery.
The present invention provides cryogranules of activated protein C. The invention further provides a process of preparing cryogranules which comprises providing an aqueous solution of activated protein C, dividing the aqueous solution of aPC into droplets, and freezing said droplets in liquid nitrogen forming cryogranules.
The invention further provides a process of thawing the cryogranules of activated protein C, adding a pharmaceutically acceptable excipient, dispensing the solution into unit dosage receptacles, and lyophilizing the solution.
For purposes of the present invention, as disclosed and claimed herein, the following terms are defined below.
aPC or activated protein C whether recombinant or plasma derived—aPC includes and is preferably human protein C although aPC may also include other species or derivatives having protein C proteolytic, amidolytic, esterolytic, and biological (anticoagulant or pro-fibrinolytic) activities. Examples of protein C derivatives are described by Gerlitz, et al., U.S. Pat. No. 5,453,373, and Foster, et al., U.S. Pat. No. 5,516,650, the entire teachings of which are hereby included by reference.
r-hPC—recombinant human protein C zymogen, produced in prokaryotic cells, eukaryotic cells or transgenic animals.
r-aPC—recombinant human activated protein C produced by activating r-hPC in vitro or by direct secretion of the activated form of protein C from procaryotic cells, eukaryotic cells, or transgenic animals [Cottingham, WO97/20043] including, for example, secretion from human kidney 293 cells as a zymogen then purified and activated by techniques well known to the skilled artisan demonstrated in Yan, U.S. Pat. No. 4,981,952, the entire teachings of which are herein incorporated by reference.
Zymogen—refers to secreted, inactive forms, whether one chain or two chains of protein C.
Cryogranules—frozen, discrete granules from solutions or slurries of activated protein C formed after contact with a cryogenic material such as liquid nitrogen.
Pharmaceutical formulation—a formulation or solution that is appropriate to be given as a therapeutic agent.
Aqueous solution—a liquid solvent that contains water. Aqueous solutions may be comprised solely of water, or may be comprised of water plus one or more miscible solvents, and may contain dissolved solutes such as sugars or other excipients. The more commonly-used miscible solvents are the short-chain organic alcohols, such as, methanol, ethanol, propanol, short-chain ketones, such as acetone, and polyalcohols, such as glycerol.
Pharmaceutically acceptable bulking agent—agents such as but not limited to sucrose, trehalose and raffinose which provide a pharmaceutically elegant formulation which has a uniform appearance and is readily solubilized when resuspended with the appropriate solute. A pharmaceutically elegant formulation is desired for parenteral administration.
The present invention provides a process for producing cryogranules of activated protein C. Cryogranules preserve the biological activity of aPC and are suitable for storage, handling, and recovery of aPC during the manufacturing process.
Preserving the biological activity of recombinantly produced proteins during the bulk manufacturing process and bulk distribution to product manufacturing facilities is a critical issue in the biotechnology process. Unlike most bulk manufacturing processes which prepare a solid bulk distribution form, proteins produced by recombinant technology, generate large volumes of solution during the cell culture and purification process. Generally, this bulk solution must be transported to formulation and filling operations. Bulk biological solutions are often frozen because of their instability in solution at refrigerated or higher temperatures. Nonetheless, freezing is slow and nonuniform and, in some cases, causes considerable degradation which may adversely affect product quality. Freeze-thaw procedures in recombinant protein manufacturing can denature and/or inactivate many enzymes. Redissolution of frozen proteins does not always readily occur. Therefore, traditional freezing of large volumes of solutions containing recombinant proteins is not only unpredictable and generally results in a decrease in yield and a loss of activity of the protein but also results in a preformulation form that is problematic for formulation and filling operations. (Pikal,
BioPharm
3(8):18-27, 1990; Pikal,
Biopharm
3(9):26-30, 1990).
Proteins often present unique stability problems when traditional methods of freezing or freeze-drying are utilized. Aggregation leading to insoluble protein is a common observation which can adversely affect enzymatic activity. Furthermore, serine proteases such as aPC can autodegrade, leading to decreased functionality. Previous studies describing the production of aPC have not addressed the unique problems associated with commercial production, storage, and handling of the bulk protein substance (for example, U.S. Pat. Nos. 5,270,040, 5,550,036, 5,681,932 and Japanese patent application JP7165605). Therefore, the applicants are the first to prov
Baker Jeffrey Clayton
Jones Nancy Delores
Barrett Brian P.
Eli Lilly and Company
Hostettler Danica
Low Christopher S. F.
Mohamed Abdel A.
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