Protein formulations

Abstract

To provide protein formulations.SOLUTION: The present disclosure provides solid protein formulations that are stable over a variety of temperatures for extended time periods. The present disclosure also provides methods of making and using these formulations. In one embodiment, the present invention provides a solid protein formulation comprising a stabilizer, a sugar alcohol, a sugar and a surfactant. In another embodiment, the present invention provides a solid protein formulation comprising darbepoetin alfa, histidine, mannitol, sucrose, and polysorbate-80.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 017,560, filed June 26, 2014, which is incorporated herein by reference.

[0002] The field of the invention relates to compositions and methods relating to protein formulations. [Background technology]

[0003] Therapeutic protein drugs can be unstable and vulnerable to environmental and other external influences, which can lead to changes that reduce their activity. Attempts to formulate therapeutic proteins can often lead to formulations that cause discomfort to patients (e.g., low pH formulations).

[0004] In some cases, therapeutic protein formulations are stabilized by adding additional proteins to the formulation. For example, human serum albumin (HSA) is used as a stabilizer. However, adding additional proteins to pharmaceutical products is not ideal for various reasons, including the risk of adverse immune responses or viral contamination. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there is a need for protein formulations that provide long-term stability at a variety of temperatures and minimize the pain associated with injection. [Means for solving the problem]

[0006] Summary of the Invention In one embodiment, the present invention provides a solid protein formulation comprising a stabilizer, a sugar alcohol, a sugar, and a surfactant. In another embodiment, the present invention provides a solid protein formulation comprising darbepoetin alfa, histidine, mannitol, sucrose, and polysorbate-80. In another embodiment, the present invention provides a solid protein formulation comprising sodium glutamate, mannitol, sucrose, and polysorbate 20. In another embodiment, the present invention provides a solid protein formulation comprising histidine, mannitol, sucrose, and polysorbate 20. In a further embodiment, the present invention provides a method for preparing a solid protein formulation, the method comprising: a) diluting the protein with a lyophilization buffer; and b) lyophilizing the diluted protein. In another embodiment, the present invention provides a kit comprising a solid protein formulation and a reconstitution buffer. [Brief explanation of the drawings]

[0007] [Figure 1] Graph showing comparative results of denaturation of Aranesp using detection of antibody 9G8A without (A) and with (B) polysorbate-80 at 37° C. [Figure 2] 1 is a graph showing the comparative results of denaturation of Aranesp using detection of antibody 9G8A with polysorbate-80 at 45° C. [Figure 3] 1 is a graph showing a comparison of the % main peak by SEC of Aranesp® without polysorbate-80 in a lyophilized formulation compared to a liquid formulation at 37° C. and 45° C. [Figure 4] 1 shows SEC-HPLC overlays of polysorbate-80-free samples reconstituted with 4% sorbitol at 24 months at 45° C. (A) shows a magnified sample overlay, and (B) shows a non-magnified sample overlay. [Figure 5] 1 is a graph showing the comparative results of % main peak by SEC of Aranesp® without polysorbate-80 in a lyophilized formulation at 45° C. [Figure 6] 1 is a graph showing the comparative results of % main peak by SEC of Aranesp® containing polysorbate-80 in a lyophilized formulation at 45° C. [Figure 7] 1 is a graph showing a comparison of the pH of Aranesp® with and without polysorbate-80 in lyophilized and liquid formulations at 45° C. [Figure 8] 1 is a graph showing comparative results of % Aranesp oxidation after storage at 4° C. for up to 24 months. [Figure 9] Graph showing the comparative results of % oxidation of Aranesp in liquid and frozen formulations after storage at 29° C. for up to 24 months, where P62N500 and P62N500T are liquid formulations and the other samples are lyophilized formulations. [Figure 10] 1 is a graph showing the comparative results of % oxidation of Aranesp in frozen formulations only after storage at 29° C. [Figure 11] Graph showing the comparative results of % oxidation of Aranesp in liquid and frozen formulations after storage at 37° C. for up to 24 months, where P62N500 and P62N500T are liquid formulations and the rest are lyophilized formulations. [Figure 12] 1 is a graph showing the comparative results of % oxidation of Aranesp in frozen formulations only after storage at 37° C. [Figure 13] Graph showing the comparative results of % oxidation of Aranesp in liquid and frozen formulations after storage at 45° C. for up to 24 months, where P62N500 and P62N500T are liquid formulations and the remaining samples are lyophilized formulations. [Figure 14] 1 is a graph showing the comparative results of % oxidation of Aranesp in frozen formulations only after storage at 37° C. for up to 24 months. [Figure 15]1 is a graph showing a comparison of degradation and truncation species by non-reducing reverse-phase HPLC among Aranesp liquid formulations and Aranesp (500 μg / mL) without polysorbate-80 in three formulations lyophilized and reconstituted in 4% sorbitol after storage at 45° C. for 24 months. [Figure 16] 1 is a graph showing a comparison of Aranesp liquid formulations, lyophilized and reconstituted in 4% sorbitol, and polysorbate-80-free Aranesp in three forms for degradation and truncation species by non-reducing reverse-phase HPLC after storage at 37° C. for 24 months. [Figure 17] 1 is a graph showing SEC-HPLC % main peak results for samples containing no polysorbate 80 at 4° C. [Figure 18] FIG. 1 is a graph showing SEC-HPLC % main peak results for samples containing polysorbate 80 at 4° C. [Figure 19] 1 is a graph showing SEC-HPLC % main peak results for samples containing no polysorbate 80 at 29° C. [Figure 20] Figure 20A is a graph showing SEC-HPLC main peak % results for samples containing polysorbate 80 at 29°C. Figure 20B is a graph showing SEC-HPLC main peak % results for samples not containing polysorbate 80 at 29°C. [Figure 21] 1 is a graph showing SEC-HPLC % main peak results for samples containing polysorbate 80 at 37° C. [Figure 22] 1 is a graph showing a comparison of Aranesp® 9G8A results between CZ and glass vials at different temperatures. CZ containers showed higher denaturation at higher temperatures (25° C., 37° C., 45° C.). [Figure 23] 1 is a graph showing a comparison of % HMW by SEC of Aranesp® in two different containers with three different reconstitution diluents at different temperatures. [Figure 24]1 is a graph showing a comparison of the % main peak by SEC of Aranesp® in two different containers with three different reconstitution diluents at different temperatures. [Figure 25] 1 is a graph showing the results of HIAC particle counts per mL of protein sample results at different temperatures. [Figure 26] Figure 1 shows visual observations of lyophilized samples. At 12 months, samples showed a small amount of lyophilized cake in the CZ vial at 4°C. The vial with the blue cap contains the protein sample. The vial with the green cap contains the placebo sample. [Figure 27] 1 is a graph showing a comparison of Aranesp® concentrations (μg / mL) at different temperatures. [Figure 28] 1 is a graph showing the comparative results of % oxidation of Aranesp after storage in two different containers (CZ and glass vials) at four different temperatures (4° C., 29° C., 37° C., and 45° C.). [Figure 29] 1 is a graph showing the comparison of truncated species of Aranesp by non-reducing reverse phase HPLC after storage in two different containers at four different temperatures over a 12 month period. [Figure 30] 1 is a graph showing a comparison of the pH of Aranesp in two different containers (CZ and glass vials) at four different temperatures (4° C., 29° C., 37° C., and 45° C.). [Figure 31] 1 is a graph showing a comparison of the osmolality of Aranesp in two different containers (CZ and glass vials) at four different temperatures (4° C., 29° C., 37° C., and 45° C.). [Figure 32] 1 is a graph showing the results of % low molecular weight analysis by SEC of 100 μg / mL and 500 μg / mL samples of two different lyophilized formulations at 4° C. and 37° C. for up to 12 weeks of sample analysis. [Figure 33]1 is a graph showing the results of % high molecular weight analysis by SEC of samples up to 12 weeks at 4° C. and 37° C. for 100 μg / mL and 500 μg / mL samples of two different lyophilized formulations. [Figure 34] 1 is a graph showing the % main peak results of sample analysis by SEC up to 12 weeks at 4° C. and 37° C. for 100 μg / mL and 500 μg / mL samples of two different lyophilized formulations. [Figure 35] 1 is a graph showing the results of % heavy chain analysis by CE-SDS of samples at 4° C. and 37° C. for up to 12 weeks for 100 μg / mL and 500 μg / mL samples of two different lyophilized formulations. [Figure 36] 1 is a graph showing the results of % light chain analysis by CE-SDS for 100 μg / mL and 500 μg / mL samples of two different lyophilized formulations at 4° C. and 37° C. for up to 12 weeks of samples. [Figure 37] 1 is a graph showing the results of % non-glycosylated heavy chain analysis by CE-SDS for 100 μg / mL and 500 μg / mL samples of two different lyophilized formulations at 4° C. and 37° C. for up to 12 weeks of samples. [Figure 38] FIG. 1 shows the results of CE-SDS electropherograms of EPO mAb for 500 μg / mL samples of two different lyophilized formulations, H=histidine and G=glutamate, after storage at 4° C. and 37° C. for 7 weeks. [Figure 39] 1 shows the results of DSC scans of pre-frozen samples in GMST and HMST buffers. Black = 0.1 mg / mL anti-EPO mAb 8C10-GMST, red = 0.1 mg / mL anti-EPO mAb 8C10-HMST, green = 0.5 mg / mL anti-EPO mAb 8C10-GMST, and blue = 0.5 mg / mL anti-EPO mAb 8C10-HMST. The signal of the 0.1 mg / mL sample was normalized to 0.5 mg / mL (the same normalization was used for all of the 0.1 mg / mL samples). [Figure 40]1 is a graph showing the results of DSC scans of 0.1 mg / mL anti-EPO mAb 8C10-GMST samples under different conditions: black = before freezing, red = frozen (T=0), green = frozen (T=7 weeks at 4° C.), blue = frozen (T=7 weeks at 37° C.), cyan = frozen (T=12 weeks at 4° C.), and dark yellow = frozen (T=12 weeks at 37° C.). [Figure 41] 1 is a graph showing the results of DSC scans of 0.1 mg / mL anti-EPO mAb 8C10-HMST samples under different conditions: black = before freezing, red = frozen (T=0), green = frozen (T=7 weeks at 4° C.), blue = frozen (T=7 weeks at 37° C.). [Figure 42] 1 is a graph showing the results of DSC scans of 0.5 mg / mL anti-EPO mAb 8C10-GMST samples under different conditions: black = before freezing, red = frozen (T=0), green = frozen (T=7 weeks at 4° C.), blue = frozen (T=7 weeks at 37° C.), cyan = frozen (T=12 weeks at 4° C.), and dark yellow = frozen (T=12 weeks at 37° C.). [Figure 43] 1 is a graph showing the results of DSC scans of 0.5 mg / mL anti-EPO mAb 8C10-HMST samples under different conditions: black = before freezing, red = frozen (T=0), green = frozen (T=7 weeks at 4° C.), blue = frozen (T=7 weeks at 37° C.). [Figure 44] 1 is a graph showing the results of a size distribution analysis of an anti-EPO mAb sample at T=0 (magnified 10x). [Figure 45] Graphs showing the results of AUC analysis of anti-EPO mAb samples. Glutamate buffer is the top panel, and histidine buffer is the bottom panel; 4°C is shown in blue, and 37°C is shown in red. Pre-frozen samples are at T=0 in all graphs. DETAILED DESCRIPTION OF THE INVENTION

[0008] Detailed Description of the Invention The present invention relates to protein formulations. The present invention further provides compositions, kits, and methods related to protein formulations.

[0009] definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular terms. Generally, the nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, gene and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout the specification, unless otherwise indicated. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), and Ausubel et al., Current Protocols in See Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The terminology used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparations, formulations, and delivery, and treatment of patients.

[0010] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0011] A "glycoprotein" is a protein having oligosaccharides covalently attached to polypeptide side chains.

[0012] An "immunoglobulin" is a tetrameric molecule. In naturally occurring immunoglobulins, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), incorporated herein by reference in its entirety for all purposes. The variable regions of each light / heavy chain pair form the antibody binding site, such that an intact immunoglobulin has two binding sites.

[0013] "Antibody" refers to an intact immunoglobulin or, unless otherwise specified, an antigen-binding portion thereof that is comparable to an intact antibody for specific binding. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include, inter alia, Fab, Fab', F(ab'), Fv, domain antibodies (dAbs), fragments containing the complementarity-determining regions (CDRs), single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide.

[0014] Fragments or analogs of antibodies can be readily prepared by those of skill in the art using techniques well known in the art, and such fragments or analogs are contemplated for use with the formulations of the present invention.

[0015] formulation The present invention relates to protein formulations, and in particular to stable solid protein formulations. While certain aspects of the present invention are directed to protein formulations that can be stored and used under various environmental conditions (e.g., a wide temperature range), the present invention provides formulations that maintain protein stability under a variety of these environmental conditions. In addition to the specific formulation components described herein, the formulations of the present invention may further include one or more other pharmaceutically acceptable carriers, additives, or stabilizers, such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), provided that they do not adversely affect the desired characteristics of the formulation.

[0016] In certain embodiments, the formulation contains an amino acid as a stabilizer. Non-limiting examples of amino acid stabilizers contemplated for use in the present invention are histidine, tryptophan, methionine, leucine, phenylalanine, serine, glutamic acid, arginine, or lysine. In a specific embodiment, the amino acid stabilizer is histidine. The concentration of the amino acid stabilizer in the formulation of the present invention can range from 0.1 mM to 100 mM before solidification, depending on the desired properties. In one embodiment, the formulation does not contain an additional stabilizing protein (e.g., albumin).

[0017] In one embodiment, the histidine concentration is 0.1 mM to 100 mM. In another embodiment, the histidine concentration is 0.1 mM to 50 mM. In another embodiment, the histidine concentration is 0.1 mM to 40 mM. In another embodiment, the histidine concentration is 0.1 mM to 30 mM. In another embodiment, the histidine concentration is 0.1 mM to 20 mM. In another embodiment, the histidine concentration is 0.1 mM to 15 mM. In another embodiment, the histidine concentration is 0.1 mM to 10 mM. In another embodiment, the histidine concentration is 0.1 mM to 5 mM. In another embodiment, the histidine concentration is 0.1 mM to 1 mM. In another embodiment, the histidine concentration is 1 mM to 100 mM. In another embodiment, the histidine concentration is 1 mM to 50 mM. In another embodiment, the histidine concentration is 1 mM to 40 mM. In another embodiment, the histidine concentration is 1 mM to 30 mM. In another embodiment, the histidine concentration is 1 mM to 20 mM. In another embodiment, the histidine concentration is 1 mM to 15 mM. In another embodiment, the histidine concentration is 1 mM to 10 mM. In another embodiment, the histidine concentration is 5 mM to 50 mM. In another embodiment, the histidine concentration is 5 mM to 25 mM. In another embodiment, the histidine concentration is 5 mM to 15 mM. In another embodiment, the histidine concentration is 0.1 mM. In another embodiment, the histidine concentration is 0.5 mM. In another embodiment, the histidine concentration is 1 mM. In another embodiment, the concentration of histidine is 2 mM. In another embodiment, the concentration of histidine is 3 mM. In another embodiment, the concentration of histidine is 4 mM. In another embodiment, the concentration of histidine is 5 mM. In another embodiment, the concentration of histidine is 6 mM. In another embodiment, the concentration of histidine is 7 mM. In another embodiment, the concentration of histidine is 8 mM. In another embodiment, the concentration of histidine is 9 mM. In another embodiment, the concentration of histidine is 10 mM. In another embodiment, the concentration of histidine is 11 mM. In another embodiment, the concentration of histidine is 12 mM.In another embodiment, the concentration of histidine is 13 mM. In another embodiment, the concentration of histidine is 14 mM. In another embodiment, the concentration of histidine is 15 mM.

[0018] In one embodiment, the histidine concentration is about 0.1 mM to about 100 mM. In another embodiment, the histidine concentration is about 0.1 mM to about 50 mM. In another embodiment, the histidine concentration is about 0.1 mM to about 40 mM. In another embodiment, the histidine concentration is about 0.1 mM to about 30 mM. In another embodiment, the histidine concentration is about 0.1 mM to about 20 mM. In another embodiment, the histidine concentration is about 0.1 mM to about 15 mM. In another embodiment, the histidine concentration is about 0.1 mM to about 10 mM. In another embodiment, the histidine concentration is about 0.1 mM to about 5 mM. In another embodiment, the histidine concentration is about 0.1 mM to about 1 mM. In another embodiment, the histidine concentration is about 1 mM to about 100 mM. In another embodiment, the histidine concentration is about 1 mM to about 50 mM. In another embodiment, the histidine concentration is about 1 mM to about 40 mM. In another embodiment, the histidine concentration is about 1 mM to about 30 mM. In another embodiment, the histidine concentration is about 1 mM to about 20 mM. In another embodiment, the histidine concentration is about 1 mM to about 15 mM. In another embodiment, the histidine concentration is about 1 mM to about 10 mM. In another embodiment, the histidine concentration is about 5 mM to about 50 mM. In another embodiment, the histidine concentration is about 5 mM to about 25 mM. In another embodiment, the histidine concentration is about 5 mM to about 15 mM. In another embodiment, the concentration of histidine is about 0.1 mM. In another embodiment, the concentration of histidine is about 0.5 mM. In another embodiment, the concentration of histidine is about 1 mM. In another embodiment, the concentration of histidine is about 2 mM. In another embodiment, the concentration of histidine is about 3 mM. In another embodiment, the concentration of histidine is about 4 mM. In another embodiment, the concentration of histidine is about 5 mM. In another embodiment, the concentration of histidine is about 6 mM. In another embodiment, the concentration of histidine is about 7 mM. In another embodiment, the concentration of histidine is about 8 mM. In another embodiment, the concentration of histidine is about 9 mM. In another embodiment, the concentration of histidine is about 10 mM. In another embodiment, the concentration of histidine is about 11 mM.In another embodiment, the concentration of histidine is about 12 mM. In another embodiment, the concentration of histidine is about 13 mM. In another embodiment, the concentration of histidine is about 14 mM. In another embodiment, the concentration of histidine is about 15 mM.

[0019] In a further embodiment, the formulation may include a non-amino acid buffer. In a specific embodiment, the non-amino acid buffer is sodium succinate. The concentration of the non-amino acid buffer in the formulation of the present invention may range from 0.1 mM to 100 mM before solidification, depending on the desired properties.

[0020] In one embodiment, the concentration of sodium succinate is 0.1 mM to 100 mM. In another embodiment, the concentration of sodium succinate is 0.1 mM to 50 mM. In another embodiment, the concentration of sodium succinate is 0.1 mM to 40 mM. In another embodiment, the concentration of sodium succinate is 0.1 mM to 30 mM. In another embodiment, the concentration of sodium succinate is 0.1 mM to 20 mM. In another embodiment, the concentration of sodium succinate is 0.1 mM to 15 mM. In another embodiment, the concentration of sodium succinate is 0.1 mM to 10 mM. In another embodiment, the concentration of sodium succinate is 0.1 mM to 5 mM. In another embodiment, the concentration of sodium succinate is 0.1 mM to 1 mM. In another embodiment, the concentration of sodium succinate is 1 mM to 100 mM. In another embodiment, the concentration of sodium succinate is 1 mM to 50 mM. In another embodiment, the concentration of sodium succinate is 1 mM to 40 mM. In another embodiment, the concentration of sodium succinate is 1 mM to 30 mM. In another embodiment, the concentration of sodium succinate is 1 mM to 20 mM. In another embodiment, the concentration of sodium succinate is 1 mM to 15 mM. In another embodiment, the concentration of sodium succinate is 1 mM to 10 mM. In another embodiment, the concentration of sodium succinate is 5 mM to 50 mM. In another embodiment, the concentration of sodium succinate is 5 mM to 25 mM. In another embodiment, the concentration of sodium succinate is 5 mM to 15 mM. In another embodiment, the concentration of sodium succinate is 0.1 mM. In another embodiment, the concentration of sodium succinate is 0.5 mM. In another embodiment, the concentration of sodium succinate is 1 mM. In another embodiment, the concentration of sodium succinate is 2 mM. In another embodiment, the concentration of sodium succinate is 3 mM. In another embodiment, the concentration of sodium succinate is 4 mM. In another embodiment, the concentration of sodium succinate is 5 mM. In another embodiment, the concentration of sodium succinate is 6 mM. In another embodiment, the concentration of sodium succinate is 7 mM. In another embodiment, the concentration of sodium succinate is 8 mM.In another embodiment, the concentration of sodium succinate is 9 mM. In another embodiment, the concentration of sodium succinate is 10 mM. In another embodiment, the concentration of sodium succinate is 11 mM. In another embodiment, the concentration of sodium succinate is 12 mM. In another embodiment, the concentration of sodium succinate is 13 mM. In another embodiment, the concentration of sodium succinate is 14 mM. In another embodiment, the concentration of sodium succinate is 15 mM.

[0021] In one embodiment, the concentration of sodium succinate is about 0.1 mM to about 100 mM. In another embodiment, the concentration of sodium succinate is about 0.1 mM to about 50 mM. In another embodiment, the concentration of sodium succinate is about 0.1 mM to about 40 mM. In another embodiment, the concentration of sodium succinate is about 0.1 mM to about 30 mM. In another embodiment, the concentration of sodium succinate is about 0.1 mM to about 20 mM. In another embodiment, the concentration of sodium succinate is about 0.1 mM to about 15 mM. In another embodiment, the concentration of sodium succinate is about 0.1 mM to about 10 mM. In another embodiment, the concentration of sodium succinate is about 0.1 mM to about 5 mM. In another embodiment, the concentration of sodium succinate is about 0.1 mM to about 1 mM. In another embodiment, the concentration of sodium succinate is about 1 mM to about 100 mM. In another embodiment, the concentration of sodium succinate is about 1 mM to about 50 mM. In another embodiment, the concentration of sodium succinate is about 1 mM to about 40 mM. In another embodiment, the concentration of sodium succinate is about 1 mM to about 30 mM. In another embodiment, the concentration of sodium succinate is about 1 mM to about 20 mM. In another embodiment, the concentration of sodium succinate is about 1 mM to about 15 mM. In another embodiment, the concentration of sodium succinate is about 1 mM to about 10 mM. In another embodiment, the concentration of sodium succinate is about 5 mM to about 50 mM. In another embodiment, the concentration of sodium succinate is about 5 mM to about 25 mM. In another embodiment, the concentration of sodium succinate is about 5 mM to about 15 mM. In another embodiment, the concentration of sodium succinate is about 0.1 mM. In another embodiment, the concentration of sodium succinate is about 0.5 mM. In another embodiment, the concentration of sodium succinate is about 1 mM. In another embodiment, the concentration of sodium succinate is about 2 mM. In another embodiment, the concentration of sodium succinate is about 3 mM. In another embodiment, the concentration of sodium succinate is about 4 mM. In another embodiment, the concentration of sodium succinate is about 5 mM. In another embodiment, the concentration of sodium succinate is about 6 mM.In another embodiment, the concentration of sodium succinate is about 7 mM. In another embodiment, the concentration of sodium succinate is about 8 mM. In another embodiment, the concentration of sodium succinate is about 9 mM. In another embodiment, the concentration of sodium succinate is about 10 mM. In another embodiment, the concentration of sodium succinate is about 11 mM. In another embodiment, the concentration of sodium succinate is about 12 mM. In another embodiment, the concentration of sodium succinate is about 13 mM. In another embodiment, the concentration of sodium succinate is about 14 mM. In another embodiment, the concentration of sodium succinate is about 15 mM.

[0022] In a further embodiment, the buffering agent is monosodium glutamate, the concentration of which may range from 0.1 mM to 100 mM before solidification, depending on the desired properties.

[0023] In one embodiment, the concentration of monosodium glutamate is 0.1 mM to 100 mM. In another embodiment, the concentration of monosodium glutamate is 0.1 mM to 50 mM. In another embodiment, the concentration of monosodium glutamate is 0.1 mM to 40 mM. In another embodiment, The concentration of monosodium glutamate is 0.1 mM to 30 mM. In another embodiment, the concentration of monosodium glutamate is 0.1 mM to 20 mM. In another embodiment, the concentration of monosodium glutamate is 0.1 mM to 15 mM. In another embodiment, the concentration of monosodium glutamate is 0.1 mM to 10 mM. In another embodiment, the concentration of monosodium glutamate is 0.1 mM to 5 mM. In another embodiment, the concentration of monosodium glutamate is 0.1 mM to 1 mM. In another embodiment, the concentration of monosodium glutamate is 1 mM to 100 mM. In another embodiment, the concentration of monosodium glutamate is 1 mM to 50 mM. In another embodiment, the concentration of monosodium glutamate is 1 mM to 40 mM. In another embodiment, the concentration of monosodium glutamate is 1 mM to 30 mM. In another embodiment, the concentration of monosodium glutamate is 1 mM to 20 mM. In another embodiment, the concentration of monosodium glutamate is 1 mM to 15 mM. In another embodiment, the concentration of monosodium glutamate is 1 mM to 10 mM. In another embodiment, the concentration of monosodium glutamate is 5 mM to 50 mM. In another embodiment, the concentration of monosodium glutamate is 5 mM to 25 mM. In another embodiment, the concentration of monosodium glutamate is 5 mM to 15 mM. In another embodiment, the concentration of monosodium glutamate is 0.1 mM. In another embodiment, the concentration of monosodium glutamate is 0.5 mM. In another embodiment, the concentration of monosodium glutamate is 1 mM. In another embodiment, the concentration of monosodium glutamate is 2 mM. In another embodiment, the concentration of monosodium glutamate is 3 mM. In another embodiment, the concentration of monosodium glutamate is 4 mM. In another embodiment, the concentration of monosodium glutamate is 5 mM. In another embodiment, the concentration of monosodium glutamate is 6 mM. In another embodiment, the concentration of monosodium glutamate is 7 mM. In another embodiment, the concentration of monosodium glutamate is 8 mM. In another embodiment, the concentration of monosodium glutamate is 9 mM. In another embodiment, the concentration of monosodium glutamate is 10 mM.In another embodiment, the concentration of monosodium glutamate is 11 mM. In another embodiment, the concentration of monosodium glutamate is 12 mM. In another embodiment, the concentration of monosodium glutamate is 13 mM. In another embodiment, the concentration of monosodium glutamate is 14 mM. In another embodiment, the concentration of monosodium glutamate is 15 mM.

[0024] In one embodiment, the concentration of monosodium glutamate is about 0.1 mM to about 100 mM. In another embodiment, the concentration of monosodium glutamate is about 0.1 mM to about 50 mM. In another embodiment, the concentration of monosodium glutamate is about 0.1 mM to about 40 mM. In another embodiment, the concentration of monosodium glutamate is about 0.1 mM to about 30 mM. In another embodiment, the concentration of monosodium glutamate is about 0.1 mM to about 20 mM. In another embodiment, the concentration of monosodium glutamate is about 0.1 mM to about 15 mM. In another embodiment, the concentration of monosodium glutamate is about 0.1 mM to about 10 mM. In another embodiment, the concentration of monosodium glutamate is about 0.1 mM to about 5 mM. In another embodiment, the concentration of monosodium glutamate is about 0.1 mM to about 1 mM. In another embodiment, the concentration of monosodium glutamate is about 1 mM to about 100 mM. In another embodiment, the concentration of monosodium glutamate is about 1 mM to about 50 mM. In another embodiment, the concentration of monosodium glutamate is about 1 mM to about 40 mM. In another embodiment, the concentration of monosodium glutamate is about 1 mM to about 30 mM. In another embodiment, the concentration of monosodium glutamate is about 1 mM to about 20 mM. In another embodiment, the concentration of monosodium glutamate is about 1 mM to about 15 mM. In another embodiment, the concentration of monosodium glutamate is about 1 mM to about 10 mM. In another embodiment, the concentration of monosodium glutamate is about 5 mM to about 50 mM. In another embodiment, the concentration of monosodium glutamate is about 5 mM to about 25 mM. In another embodiment, the concentration of monosodium glutamate is about 5 mM to about 15 mM. In another embodiment, the concentration of monosodium glutamate is about 0.1 mM. In another embodiment, the concentration of monosodium glutamate is about 0.5 mM. In another embodiment, the concentration of monosodium glutamate is about 1 mM. In another embodiment, the concentration of monosodium glutamate is about 2 mM. In another embodiment, the concentration of monosodium glutamate is about 3 mM. In another embodiment, the concentration of monosodium glutamate is about 4 mM. In another embodiment, the concentration of monosodium glutamate is about 5 mM.In another embodiment, the concentration of monosodium glutamate is about 6 mM. In another embodiment, the concentration of monosodium glutamate is about 7 mM. In another embodiment, the concentration of monosodium glutamate is about 8 mM. In another embodiment, the concentration of monosodium glutamate is about 9 mM. In another embodiment, the concentration of monosodium glutamate is about 10 mM. In another embodiment, the concentration of monosodium glutamate is about 11 mM. In another embodiment, the concentration of monosodium glutamate is about 12 mM. In another embodiment, the concentration of monosodium glutamate is about 13 mM. In another embodiment, the concentration of monosodium glutamate is about 14 mM. In another embodiment, the concentration of monosodium glutamate is about 15 mM.

[0025] In further embodiments, the formulation may include an inorganic or organic salt. In certain embodiments, these salts function as buffers in the protein formulation. Non-limiting examples of inorganic salts include sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, and sodium bicarbonate. Non-limiting examples of organic salts include sodium citrate, potassium citrate, and sodium acetate. In a specific embodiment, the inorganic salt is sodium phosphate. The concentration of inorganic or organic salt in the formulation of the present invention may range from 0.1 mM to 100 mM before solidification, depending on the desired properties.

[0026] In one embodiment, the concentration of sodium phosphate is 0.1 mM to 100 mM. In another embodiment, the concentration of sodium phosphate is 0.1 mM to 50 mM. In another embodiment, the concentration of sodium phosphate is 0.1 mM to 40 mM. In another embodiment, the concentration of sodium phosphate is 0.1 mM to 30 mM. In another embodiment, the concentration of sodium phosphate is 0.1 mM to 20 mM. In another embodiment, the concentration of sodium phosphate is 0.1 mM to 15 mM. In another embodiment, the concentration of sodium phosphate is 0.1 mM to 10 mM. In another embodiment, the concentration of sodium phosphate is 0.1 mM to 5 mM. In another embodiment, the concentration of sodium phosphate is 0.1 mM to 1 mM. In another embodiment, the concentration of sodium phosphate is 1 mM to 100 mM. In another embodiment, the concentration of sodium phosphate is 1 mM to 50 mM. In another embodiment, the concentration of sodium phosphate is 1 mM to 40 mM. In another embodiment, the concentration of sodium phosphate is 1 mM to 30 mM. In another embodiment, the concentration of sodium phosphate is 1 mM to 20 mM. In another embodiment, the concentration of sodium phosphate is 1 mM to 15 mM. In another embodiment, the concentration of sodium phosphate is 1 mM to 10 mM. In another embodiment, the concentration of sodium phosphate is 5 mM to 50 mM. In another embodiment, the concentration of sodium phosphate is 5 mM to 25 mM. In another embodiment, the concentration of sodium phosphate is 5 mM to 15 mM. In another embodiment, the concentration of sodium phosphate is 0.1 mM. In another embodiment, the concentration of sodium phosphate is 0.5 mM. In another embodiment, the concentration of sodium phosphate is 1 mM. In another embodiment, the concentration of sodium phosphate is 2 mM. In another embodiment, the concentration of sodium phosphate is 3 mM. In another embodiment, the concentration of sodium phosphate is 4 mM. In another embodiment, the concentration of sodium phosphate is 5 mM. In another embodiment, the concentration of sodium phosphate is 6 mM. In another embodiment, the concentration of sodium phosphate is 7 mM. In another embodiment, the concentration of sodium phosphate is 8 mM. In another embodiment, the concentration of sodium phosphate is 9 mM.In another embodiment, the concentration of sodium phosphate is 10 mM. In another embodiment, the concentration of sodium phosphate is 11 mM. In another embodiment, the concentration of sodium phosphate is 12 mM. In another embodiment, the concentration of sodium phosphate is 13 mM. In another embodiment, the concentration of sodium phosphate is 14 mM. In another embodiment, the concentration of sodium phosphate is 15 mM.

[0027] In one embodiment, the sodium phosphate concentration is about 0.1 mM to about 100 mM. In another embodiment, the sodium phosphate concentration is about 0.1 mM to about 50 mM. In another embodiment, the sodium phosphate concentration is about 0.1 mM to about 40 mM. In another embodiment, the sodium phosphate concentration is about 0.1 mM to about 30 mM. In another embodiment, the sodium phosphate concentration is about 0.1 mM to about 20 mM. In another embodiment, the sodium phosphate concentration is about 0.1 mM to about 15 mM. In another embodiment, the sodium phosphate concentration is about 0.1 mM to about 10 mM. In another embodiment, the sodium phosphate concentration is about 0.1 mM to about 5 mM. In another embodiment, the sodium phosphate concentration is about 0.1 mM to about 1 mM. In another embodiment, the sodium phosphate concentration is about 1 mM to about 100 mM. In another embodiment, the sodium phosphate concentration is about 1 mM to about 50 mM. In another embodiment, the sodium phosphate concentration is about 1 mM to about 40 mM. In another embodiment, the sodium phosphate concentration is about 1 mM to about 30 mM. In another embodiment, the sodium phosphate concentration is about 1 mM to about 20 mM. In another embodiment, the sodium phosphate concentration is about 1 mM to about 15 mM. In another embodiment, the sodium phosphate concentration is about 1 mM to about 10 mM. In another embodiment, the sodium phosphate concentration is about 5 mM to about 50 mM. In another embodiment, the sodium phosphate concentration is about 5 mM to about 25 mM. In another embodiment, the sodium phosphate concentration is about 5 mM to about 15 mM. In another embodiment, the sodium phosphate concentration is about 0.1 mM. In another embodiment, the sodium phosphate concentration is about 0.5 mM. In another embodiment, the sodium phosphate concentration is about 1 mM. In another embodiment, the concentration of sodium phosphate is about 2 mM. In another embodiment, the concentration of sodium phosphate is about 3 mM. In another embodiment, the concentration of sodium phosphate is about 4 mM. In another embodiment, the concentration of sodium phosphate is about 5 mM. In another embodiment, the concentration of sodium phosphate is about 6 mM. In another embodiment, the concentration of sodium phosphate is about 7 mM.In another embodiment, the concentration of sodium phosphate is about 8 mM. In another embodiment, the concentration of sodium phosphate is about 9 mM. In another embodiment, the concentration of sodium phosphate is about 10 mM. In another embodiment, the concentration of sodium phosphate is about 11 mM. In another embodiment, the concentration of sodium phosphate is about 12 mM. In another embodiment, the concentration of sodium phosphate is about 13 mM. In another embodiment, the concentration of sodium phosphate is about 14 mM. In another embodiment, the concentration of sodium phosphate is about 15 mM.

[0028] In certain embodiments, the protein formulation comprises a sugar alcohol. Non-limiting examples of sugar alcohols contemplated for use in the present invention include mannitol, xylitol, sorbitol, maltitol, lactitol, glycerol, erythritol, or arabitol. In a specific embodiment, the sugar alcohol is mannitol. The concentration of the sugar alcohol in the formulation of the present invention can range from 0.1% to 10% mM before solidification, depending on the desired properties.

[0029] In one embodiment, the mannitol concentration is about 0.1% to about 10%. In another embodiment, the mannitol concentration is about 1% to 10%. In another embodiment, the mannitol concentration is about 1% to about 5%. In another embodiment, the mannitol concentration is about 1% to about 3%. In another embodiment, the mannitol concentration is about 3% to about 5%. In another embodiment, the mannitol concentration is about 5% to about 10%. In another embodiment, the mannitol concentration is about 0.1%. In another embodiment, the mannitol concentration is about 0.3%. In another embodiment, the mannitol concentration is about 0.5%. In another embodiment, the mannitol concentration is about 1%. In another embodiment, the mannitol concentration is about 2%. In another embodiment, the mannitol concentration is about 3%. In another embodiment, the mannitol concentration is about 4%. In another embodiment, the mannitol concentration is about 5%. In another embodiment, the concentration of mannitol is about 6%. In another embodiment, the concentration of mannitol is about 7%. In another embodiment, the concentration of mannitol is about 8%. In another embodiment, the concentration of mannitol is about 9%. In another embodiment, the concentration of mannitol is about 10%.

[0030] In one embodiment, the mannitol concentration is 0.1% to 10%. In one embodiment, the mannitol concentration is 1% to 10%. In another embodiment, the mannitol concentration is 1% to 5%. In another embodiment, the mannitol concentration is 1% to 3%. In another embodiment, the mannitol concentration is 3% to 5%. In another embodiment, the mannitol concentration is 5% to 10%. In another embodiment, the mannitol concentration is 0.1%. In another embodiment, the mannitol concentration is 0.3%. In another embodiment, the mannitol concentration is 0.5%. In another embodiment, the mannitol concentration is 1%. In another embodiment, the mannitol concentration is 2%. In another embodiment, the mannitol concentration is 3%. In another embodiment, the mannitol concentration is 4%. In another embodiment, the mannitol concentration is 5%. In another embodiment, the mannitol concentration is 6%. In another embodiment, the concentration of mannitol is 7%. In another embodiment, the concentration of mannitol is 8%. In another embodiment, the concentration of mannitol is 9%. In another embodiment, the concentration of mannitol is 10%.

[0031] In certain embodiments, the formulation comprises a sugar. Non-limiting examples of sugars include sucrose, maltose, lactose, glucose, fructose, galactose, mannose, arabinose, xylose, ribose, rhamnose, trehalose, sorbose, melezitose, raffinose, thioglucose, thiomannose, thiofructose, octa-O-acetyl-thiotrehalose, thiosucrose, or thiomaltose. In a specific embodiment, the sugar is sucrose. The sugar concentration in the formulation of the present invention may range from 0.05% to 10% mM before solidification, depending on the desired properties.

[0032] In one embodiment, the sucrose concentration is about 0.05% to about 10%. In another embodiment, the sucrose concentration is about 0.5% to about 5%. In another embodiment, the sucrose concentration is about 1% to about 5%. In another embodiment, the sucrose concentration is about 1% to about 10%. In another embodiment, the sucrose concentration is about 1% to about 5%. In another embodiment, the sucrose concentration is about 1% to about 3%. In another embodiment, the sucrose concentration is about 3% to about 5%. In another embodiment, the sucrose concentration is about 5% to about 10%. In another embodiment, the sucrose concentration is about 0.05%. In another embodiment, the sucrose concentration is about 0.1%. In another embodiment, the sucrose concentration is about 0.3%. In another embodiment, the sucrose concentration is about 0.5%. In another embodiment, the sucrose concentration is about 0.7%. In another embodiment, the sucrose concentration is about 1%. In another embodiment, the sucrose concentration is about 2%. In another embodiment, the sucrose concentration is about 3%. In another embodiment, the sucrose concentration is about 4%. In another embodiment, the sucrose concentration is about 5%. In another embodiment, the sucrose concentration is about 6%. In another embodiment, the sucrose concentration is about 7%. In another embodiment, the sucrose concentration is about 8%. In another embodiment, the sucrose concentration is about 9%. In another embodiment, the sucrose concentration is about 10%.

[0033] In one embodiment, the sucrose concentration is 0.05% to 10%. In another embodiment, the sucrose concentration is 0.5% to 5%. In another embodiment, the sucrose concentration is 1% to 5%. In another embodiment, the sucrose concentration is 1% to 3%. In another embodiment, the sucrose concentration is 3% to 5%. In another embodiment, the sucrose concentration is 5% to 10%. In another embodiment, the sucrose concentration is 0.1%. In another embodiment, the sucrose concentration is 0.3%. In another embodiment, the sucrose concentration is 0.5%. In another embodiment, the sucrose concentration is 0.7%. In another embodiment, the sucrose concentration is 1%. In another embodiment, the sucrose concentration is 2%. In another embodiment, the sucrose concentration is 3%. In another embodiment, the sucrose concentration is 4%. In another embodiment, the sucrose concentration is 5%. In another embodiment, the sucrose concentration is 6%. In another embodiment, the concentration of sucrose is 7%. In another embodiment, the concentration of sucrose is 8%. In another embodiment, the concentration of sucrose is 9%. In another embodiment, the concentration of sucrose is 10%.

[0034] In certain embodiments, the formulation includes a surfactant, i.e., a surface-active agent. Non-limiting examples of surfactants include polysorbates (e.g., polysorbate-80, polysorbate-20, polysorbate-21, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-81, or polysorbate-85), poloxamers (e.g., poloxamer 188), Triton, sodium dodecyl sulfate (SDS), sodium laurel sulfate, sodium octyl glycoside, lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine, lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine, linoleyl-, myristyl-, or cetyl-betaine, lauroamidopropyl-, cocamidopropyl-, linoleyl-, or stearyl-sarcosine. Examples of surfactants include myristamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl), myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl- or disodium methyl oleyl taurate, polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.). In a specific embodiment, the surfactant is polysorbate-80. The concentration of surfactant in the formulations of the present invention before solidification can range from 0.0001% to 0.1%, depending on the desired properties.

[0035] In one embodiment, the concentration of polysorbate 80 is about 0.0001% to about 0.1%. In another embodiment, the concentration of polysorbate 80 is about 0.001% to about 0.5%. In another embodiment, the concentration of polysorbate 80 is about 0.001% to about 0.1%. In another embodiment, the concentration of polysorbate 80 is about 0.005% to about 0.1%. In another embodiment, the concentration of polysorbate 80 is about 0.005% to about 0.05%. In another embodiment, the concentration of polysorbate 80 is about 0.01% to about 0.1%. In another embodiment, the concentration of polysorbate 80 is about 0.5% to about 0.1%. In another embodiment, the concentration of polysorbate 80 is about 0.009%. In another embodiment, the concentration of polysorbate 80 is about 0.008%. In another embodiment, the concentration of polysorbate 80 is about 0.007%. In another embodiment, the concentration of polysorbate 80 is about 0.006%. In another embodiment, the concentration of polysorbate 80 is about 0.005%. In another embodiment, the concentration of polysorbate 80 is about 0.004%. In another embodiment, the concentration of polysorbate 80 is about 0.003%. In another embodiment, the concentration of polysorbate 80 is about 0.002%. In another embodiment, the concentration of polysorbate 80 is about 0.001%. In another embodiment, the concentration of polysorbate 80 is about 0.01%. In another embodiment, the concentration of polysorbate 80 is about 0.02%. In another embodiment, the concentration of polysorbate 80 is about 0.03%. In another embodiment, the concentration of polysorbate 80 is about 0.04%. In another embodiment, the concentration of polysorbate 80 is about 0.05%. In another embodiment, the concentration of polysorbate 80 is about 0.06%. In another embodiment, the concentration of polysorbate 80 is about 0.07%. In another embodiment, the concentration of polysorbate 80 is about 0.08%. In another embodiment, the concentration of polysorbate 80 is about 0.09%. In another embodiment, the concentration of polysorbate 80 is about 0.1%. In another embodiment, the concentration of polysorbate 80 is about 0.5%.

[0036] In one embodiment, the concentration of polysorbate 80 is 0.0001% to 0.01%. In another embodiment, the concentration of polysorbate 80 is 0.001% to 0.5%. In another embodiment, the concentration of polysorbate 80 is 0.001% to 0.1%. In another embodiment, the concentration of polysorbate 80 is 0.005% to 0.1%. In another embodiment, the concentration of polysorbate 80 is 0.005% to 0.05%. In another embodiment, the concentration of polysorbate 80 is 0.01% to 0.1%. In another embodiment, the concentration of polysorbate 80 is 0.5% to 0.1%. In another embodiment, the concentration of polysorbate 80 is 0.009%. In another embodiment, the concentration of polysorbate 80 is 0.008%. In another embodiment, the concentration of polysorbate 80 is 0.007%. In another embodiment, the concentration of polysorbate 80 is 0.006%. In another embodiment, the concentration of polysorbate 80 is 0.005%. In another embodiment, the concentration of polysorbate 80 is 0.004%. In another embodiment, the concentration of polysorbate 80 is 0.003%. In another embodiment, the concentration of polysorbate 80 is 0.002%. In another embodiment, the concentration of polysorbate 80 is 0.001%. In another embodiment, the concentration of polysorbate 80 is 0.01%. In another embodiment, the concentration of polysorbate 80 is 0.02%. In another embodiment, the concentration of polysorbate 80 is 0.03%. In another embodiment, the concentration of polysorbate 80 is 0.04%. In another embodiment, the concentration of polysorbate 80 is 0.05%. In another embodiment, the concentration of polysorbate 80 is 0.06%. In another embodiment, the concentration of polysorbate 80 is 0.07%. In another embodiment, the concentration of polysorbate 80 is 0.08%. In another embodiment, the concentration of polysorbate 80 is 0.09%. In another embodiment, the concentration of polysorbate 80 is 0.1%. In another embodiment, the concentration of polysorbate 80 is 0.5%.

[0037] In one embodiment, the concentration of polysorbate 20 is about 0.0001% to about 0.1%. In another embodiment, the concentration of polysorbate 20 is about 0.001% to about 0.5%. In another embodiment, the concentration of polysorbate 20 is about 0.001% to about 0.1%. In another embodiment, the concentration of polysorbate 20 is about 0.005% to about 0.1%. In another embodiment, the concentration of polysorbate 20 is about 0.005% to about 0.05%. In another embodiment, the concentration of polysorbate 20 is about 0.01% to about 0.1%. In another embodiment, the concentration of polysorbate 20 is about 0.5% to about 0.1%. In another embodiment, the concentration of polysorbate 20 is about 0.009%. In another embodiment, the concentration of polysorbate 20 is about 0.008%. In another embodiment, the concentration of polysorbate 20 is about 0.007%. In another embodiment, the concentration of polysorbate 20 is about 0.006%. In another embodiment, the concentration of polysorbate 20 is about 0.005%. In another embodiment, the concentration of polysorbate 20 is about 0.004%. In another embodiment, the concentration of polysorbate 20 is about 0.003%. In another embodiment, the concentration of polysorbate 20 is about 0.002%. In another embodiment, the concentration of polysorbate 20 is about 0.001%. In another embodiment, the concentration of polysorbate 20 is about 0.01%. In another embodiment, the concentration of polysorbate 20 is about 0.02%. In another embodiment, the concentration of polysorbate 20 is about 0.03%. In another embodiment, the concentration of polysorbate 20 is about 0.04%. In another embodiment, the concentration of polysorbate 20 is about 0.05%. In another embodiment, the concentration of polysorbate 20 is about 0.06%. In another embodiment, the concentration of polysorbate 20 is about 0.07%. In another embodiment, the concentration of polysorbate 20 is about 0.08%. In another embodiment, the concentration of polysorbate 20 is about 0.09%. In another embodiment, the concentration of polysorbate 20 is about 0.1%. In another embodiment, the concentration of polysorbate 20 is about 0.5%.

[0038] In one embodiment, the concentration of polysorbate 20 is 0.0001% to 0.01%. In another embodiment, the concentration of polysorbate 20 is 0.001% to 0.5%. In another embodiment, the concentration of polysorbate 20 is 0.001% to 0.1%. In another embodiment, the concentration of polysorbate 20 is 0.005% to 0.1%. In another embodiment, the concentration of polysorbate 20 is 0.005% to 0.05%. In another embodiment, the concentration of polysorbate 20 is 0.01% to 0.1%. In another embodiment, the concentration of polysorbate 20 is 0.5% to 0.1%. In another embodiment, the concentration of polysorbate 20 is 0.009%. In another embodiment, the concentration of polysorbate 20 is 0.008%. In another embodiment, the concentration of polysorbate 20 is 0.007%. In another embodiment, the concentration of polysorbate 20 is 0.006%. In another embodiment, the concentration of polysorbate 20 is 0.005%. In another embodiment, the concentration of polysorbate 20 is 0.004%. In another embodiment, the concentration of polysorbate 20 is 0.003%. In another embodiment, the concentration of polysorbate 20 is 0.002%. In another embodiment, the concentration of polysorbate 20 is 0.001%. In another embodiment, the concentration of polysorbate 20 is 0.01%. In another embodiment, the concentration of polysorbate 20 is 0.02%. In another embodiment, the concentration of polysorbate 20 is 0.03%. In another embodiment, the concentration of polysorbate 20 is 0.04%. In another embodiment, the concentration of polysorbate 20 is 0.05%. In another embodiment, the concentration of polysorbate 20 is 0.06%. In another embodiment, the concentration of polysorbate 20 is 0.07%. In another embodiment, the concentration of polysorbate 20 is 0.08%. In another embodiment, the concentration of polysorbate 20 is 0.09%. In another embodiment, the concentration of polysorbate 20 is 0.1%. In another embodiment, the concentration of polysorbate 20 is 0.5%.

[0039] In one embodiment, the pH is about 5.0 to about 8.0 before solidification. In another embodiment, the pH is about 4.0 to about 10.0. In another embodiment, the pH is about 5.0 to about 7.0. In another embodiment, the pH is about 5.0 to about 9.0. In another embodiment, the pH is about 6.0 to about 9.0. In another embodiment, the pH is about 5.0. In another embodiment, the pH is about 5.5. In another embodiment, the pH is about 6.0. In another embodiment, the pH is about 6.5. In another embodiment, the pH is about 7.0. In another embodiment, the pH is about 7.5. In another embodiment, the pH is about 8.0. In another embodiment, the pH is about 8.5. In another embodiment, the pH is about 9.0. In another embodiment, the pH is about 9.5. In another embodiment, the pH is about 10.0.

[0040] In one embodiment, the pH is 6.0-8.0 before solidification. In another embodiment, the pH is 4.0-10.0. In another embodiment, the pH is 5.0-7.0. In another embodiment, the pH is 5.0-9.0. In another embodiment, the pH is 6.0-9.0. In another embodiment, the pH is 5.0. In another embodiment, the pH is 5.1. In another embodiment, the pH is 5.2. In another embodiment, the pH is 5.3. In another embodiment, the pH is 5.4. In another embodiment, the pH is 5.5. In another embodiment, the pH is 5.6. In another embodiment, the pH is 5.7. In another embodiment, the pH is 5.8. In another embodiment, the pH is 5.9. In another embodiment, the pH is 6.0. In another embodiment, the pH is 6.1. In another embodiment, the pH is 6.2. In another embodiment, the pH is 6.3. In another embodiment, the pH is 6.4. In another embodiment, the pH is 6.5. In another embodiment, the pH is 6.6. In another embodiment, the pH is 6.7. In another embodiment, the pH is 6.8. In another embodiment, the pH is 6.9. In another embodiment, the pH is 7.0. In another embodiment, the pH is 7.5. In another embodiment, the pH is 8.0. In another embodiment, the pH is 8.5. In another embodiment, the pH is 9.0. In another embodiment, the pH is 9.5. In another embodiment, the pH is 10.0.

[0041] In one embodiment, the protein concentration is about 0.01 mg / ml to about 10 mg / ml. In one embodiment, the protein concentration is about 0.01 mg / ml to about 1 mg / ml. In one embodiment, the protein concentration is about 0.1 mg / ml to about 100 mg / ml before solidification. In another embodiment, the protein concentration is about 1 mg / ml to about 100 mg / ml. In another embodiment, the protein concentration is about 1 mg / ml to about 50 mg / ml. In another embodiment, the protein concentration is about 1 mg / ml to about 25 mg / ml. In another embodiment, the protein concentration is about 1 mg / ml to about 10 mg / ml. In another embodiment, the protein concentration is about 5 mg / ml to about 50 mg / ml. In another embodiment, the protein concentration is about 5 mg / ml to about 25 mg / ml. In another embodiment, the protein concentration is about 10 mg / ml to about 100 mg / ml. In another embodiment, the protein concentration is about 10 mg / ml to about 50 mg / ml. In another embodiment, the protein concentration is about 10 mg / ml to about 25 mg / ml. In another embodiment, the protein concentration is about 0.01 mg / ml. In another embodiment, the protein concentration is about 0.05 mg / ml. In another embodiment, the protein concentration is about 0.1 mg / ml. In another embodiment, the protein concentration is about 0.2 mg / ml. In another embodiment, the protein concentration is about 0.3 mg / ml. In another embodiment, the protein concentration is about 0.4 mg / ml. In another embodiment, the protein concentration is about 0.5 mg / ml. In another embodiment, the protein concentration is about 1 mg / ml. In another embodiment, the protein concentration is about 2 mg / ml. In another embodiment, the protein concentration is about 3 mg / ml. In another embodiment, the protein concentration is about 4 mg / ml. In another embodiment, the protein concentration is about 5 mg / ml. In another embodiment, the protein concentration is about 6 mg / ml. In another embodiment, the protein concentration is about 7 mg / ml. In another embodiment, the protein concentration is about 8 mg / ml. In another embodiment, the protein concentration is about 9 mg / ml.In another embodiment, the protein concentration is about 10 mg / ml. In another embodiment, the protein concentration is about 20 mg / ml. In another embodiment, the protein concentration is about 30 mg / ml. In another embodiment, the protein concentration is about 40 mg / ml. In another embodiment, the protein concentration is about 50 mg / ml. In another embodiment, the protein concentration is about 60 mg / ml. In another embodiment, the protein concentration is about 70 mg / ml. In another embodiment, the protein concentration is about 80 mg / ml. In another embodiment, the protein concentration is about 90 mg / ml. In another embodiment, the protein concentration is about 100 mg / ml.

[0042] In one embodiment, the protein concentration is 0.01 mg / ml to 10 mg / ml. In one embodiment, the protein concentration is 0.01 mg / ml to 1 mg / ml. In one embodiment, the protein concentration is 0.1 mg / ml to 100 mg / ml before solidification. In another embodiment, the protein concentration is 1 mg / ml to 100 mg / ml. In another embodiment, the protein concentration is 1 mg / ml to 50 mg / ml. In another embodiment, the protein concentration is 1 mg / ml to 25 mg / ml. In another embodiment, the protein concentration is 1 mg / ml to 10 mg / ml. In another embodiment, the protein concentration is 5 mg / ml to 50 mg / ml. In another embodiment, the protein concentration is 5 mg / ml to 25 mg / ml. In another embodiment, the protein concentration is 10 mg / ml to 100 mg / ml. In another embodiment, the protein concentration is 10 mg / ml to 50 mg / ml. In another embodiment, the protein concentration is 10 mg / ml to 25 mg / ml. In another embodiment, the protein concentration is 0.01 mg / ml. In another embodiment, the protein concentration is 0.05 mg / ml. In another embodiment, the protein concentration is 0.1 mg / ml. In another embodiment, the protein concentration is 0.2 mg / ml. In another embodiment, the protein concentration is 0.3 mg / ml. In another embodiment, the protein concentration is 0.4 mg / ml. In another embodiment, the protein concentration is 0.5 mg / ml. In another embodiment, the protein concentration is 1 mg / ml. In another embodiment, the protein concentration is 2 mg / ml. In another embodiment, the protein concentration is 3 mg / ml. In another embodiment, the protein concentration is 4 mg / ml. In another embodiment, the protein concentration is 5 mg / ml. In another embodiment, the protein concentration is 6 mg / ml. In another embodiment, the protein concentration is 7 mg / ml. In another embodiment, the protein concentration is 8 mg / ml, 9 mg / ml, or 10 mg / ml.In another embodiment, the protein concentration is 20 mg / ml. In another embodiment, the protein concentration is 30 mg / ml. In another embodiment, the protein concentration is 40 mg / ml. In another embodiment, the protein concentration is 50 mg / ml. In another embodiment, the protein concentration is 60 mg / ml. In another embodiment, the protein concentration is 70 mg / ml. In another embodiment, the protein concentration is 80 mg / ml. In another embodiment, the protein concentration is 90 mg / ml. In another embodiment, the protein concentration is 100 mg / ml.

[0043] Solid protein formulations include, but are not limited to, protein formulations that start in a liquid state and subsequently remove the liquid (i.e., progress from a hydrated protein solution to a dehydrated protein solution). Non-limiting examples of methods to achieve this include lyophilization (also known as freeze-drying or cryodesication) or spray drying. In lyophilization, a substance is rapidly frozen and dehydrated under vacuum conditions, which causes the liquid to sublimate from the solid phase to the gas phase. Spray drying is a method of producing dry powder from a liquid or slurry by rapid drying with hot air. The processes of lyophilization and spray drying are well known in the art and can be easily optimized to produce the desired results. See also "Lyophilization of Biopharmaceuticals," Biotechnology: Pharmaceutical Aspects, Henry R. Costantino (Editor), Michael J. Pikal (Editor); "Lyophilization: Introduction and Basic Principles," Thomas A. Jennings, CRC Press, 1999; and "Freeze-Drying / Lyophilization of Pharmaceutical & Biological Products," Second Edition: Revised And Expanded (Drugs and the Pharmaceutical Sciences), Louis Rey (Editor), Joan C. May (Editor), CRC Press 2004. In one embodiment, the solid protein formulation is freeze-dried. In another embodiment, the solid protein formulation is spray-dried. Throughout this disclosure, reference may be made to "prior to being solidified," which means the protein formulation in a liquid state before the liquid is removed as described above.

[0044] One aspect of the present invention is to provide protein formulations (both solid and liquid) that are stable and maintain their stability over extended periods of time across a variety of different temperatures, a property that is useful for a variety of different reasons, including but not limited to, ease of storage in a wide variety of clinical settings.

[0045] In one embodiment, the solid protein formulation is stable at 4°C to 45°C for at least 1 month. In one embodiment, the solid protein formulation is stable at 4°C to 45°C for at least 2 months. In one embodiment, the solid protein formulation is stable at 4°C to 45°C for at least 3 months. In one embodiment, the solid protein formulation is stable at 4°C to 45°C for at least 4 months. In one embodiment, the solid protein formulation is stable at 4°C to 45°C for at least 5 months. In one embodiment, the solid protein formulation is stable at 4°C to 45°C for at least 6 months. In one embodiment, the solid protein formulation is stable at 4°C to 45°C for at least 12 months. In one embodiment, the solid protein formulation is stable at 4°C to 45°C for at least 18 months. In one embodiment, the solid protein formulation is stable at 4°C to 45°C for at least 24 months. In one embodiment, the solid protein formulation is stable at 4°C to 45°C for at least 36 months. In one embodiment, the solid protein formulation is stable for at least 48 months at 4° C. to 45° C. In one embodiment, the solid protein formulation is stable for at least 60 months at 4° C. to 45° C.

[0046] In one embodiment, the solid protein formulation is stable at about 4°C to about 45°C for at least about 1 month. In one embodiment, the solid protein formulation is stable at about 4°C to about 45°C for at least about 2 months. In one embodiment, the solid protein formulation is stable at about 4°C to about 45°C for at least about 3 months. In one embodiment, the solid protein formulation is stable at about 4°C to about 45°C for at least about 4 months. In one embodiment, the solid protein formulation is stable at about 4°C to about 45°C for at least about 5 months. In one embodiment, the solid protein formulation is stable at about 4°C to about 45°C for at least about 6 months. In one embodiment, the solid protein formulation is stable at about 4°C to about 45°C for at least about 12 months. In one embodiment, the solid protein formulation is stable at about 4°C to about 45°C for at least about 18 months. In one embodiment, the solid protein formulation is stable at about 4°C to about 45°C for at least about 24 months. In one embodiment, the solid protein formulation is stable for at least about 36 months at about 4° C. to about 45° C. In one embodiment, the solid protein formulation is stable for at least about 48 months at about 4° C. to about 45° C. In one embodiment, the solid protein formulation is stable for at least about 60 months at about 4° C. to about 45° C.

[0047] In one embodiment, the solid protein formulation is stable at about 4°C, about 29°C, about 37°C, and / or about 45°C for at least about one month. In a further embodiment, the solid protein formulation is stable at about 4°C, about 29°C, about 37°C, and / or about 45°C for at least about two months. In a further embodiment, the solid protein formulation is stable at about 4°C, about 29°C, about 37°C, and / or about 45°C for at least about three months. In a further embodiment, the solid protein formulation is stable at about 4°C, about 29°C, about 37°C, and / or about 45°C for at least about four months. In a further embodiment, the solid protein formulation is stable at about 4°C, about 29°C, about 37°C, and / or about 45°C for at least about five months. In a further embodiment, the solid protein formulation is stable at about 4°C, about 29°C, about 37°C, and / or about 45°C for at least about six months. In a further embodiment, the solid protein formulation is stable at about 4°C, about 29°C, about 37°C, and / or about 45°C for at least about 12 months. In a further embodiment, the solid protein formulation is stable at about 4°C, about 29°C, about 37°C, and / or about 45°C for at least about 18 months. In a further embodiment, the solid protein formulation is stable at about 4°C, about 29°C, about 37°C, and / or about 45°C for at least about 24 months. In a further embodiment, the solid protein formulation is stable at about 4°C, about 29°C, about 37°C, and / or about 45°C for at least about 36 months. In a further embodiment, the solid protein formulation is stable at about 4°C, about 29°C, about 37°C, and / or about 45°C for at least about 48 months. In a further embodiment, the solid protein formulation is stable at about 4°C, about 29°C, about 37°C, and / or about 45°C for at least about 60 months.

[0048] In yet a further embodiment, the solid protein formulation is reconstituted with a liquid. In one embodiment, the solid protein formulation is reconstituted with water. In another embodiment, the solid protein formulation is reconstituted with a reconstitution solution comprising sorbitol and sodium chloride. In certain embodiments, the sorbitol concentration is 4%, the sodium chloride concentration is 0.7%, and the pH is 7. In a further embodiment, the solid protein formulation is reconstituted with a reconstitution solution further comprising benzyl alcohol. In a specific embodiment, the benzyl alcohol concentration is 1%. In a further embodiment, the liquid used to reconstitute the solid protein formulation is sterile.

[0049] In a specific embodiment, the present invention provides a solid protein formulation that, prior to solidification, consists of, consists essentially of, or comprises Aranesp (0.5-5 mg / ml), 10 mM histidine, 2% mannitol, 0.5% sucrose, 0.005% polysorbate-80 at pH 7.

[0050] In one specific embodiment, the present invention provides a solid protein formulation that, prior to solidification, consists essentially of, or comprises Aranesp (2 mg / ml), 10 mM histidine, 2% mannitol, 0.5% sucrose, 0.005% polysorbate-80 at pH 7.

[0051] In a specific embodiment, the present invention provides a solid protein formulation that, prior to solidification, consists of, consists essentially of, or comprises Aranesp (0.5-5 mg / ml), 10 mM histidine, 2% mannitol, 0.5% sucrose, 0.005% polysorbate-80 at pH 7.

[0052] In a specific embodiment, the present invention provides a solid protein formulation that, prior to solidification, consists of, consists essentially of, or comprises Aranesp (0.5-5 mg / ml), 10 mM sodium phosphate, 4.5% mannitol, 0.5% sucrose, 0.005% polysorbate-80 at pH 6.

[0053] In a specific embodiment, the present invention provides a solid protein formulation that, prior to solidification, consists of, consists essentially of, or comprises Aranesp (0.5-5 mg / ml), 10 mM sodium phosphate, 2% mannitol, 0.5% sucrose, 0.005% polysorbate-80 at pH 7.

[0054] In a specific embodiment, the present invention provides a solid protein formulation that, prior to solidification, consists of, consists essentially of, or comprises Aranesp (0.5-5 mg / ml), 10 mM sodium succinate, 2% mannitol, 0.5% sucrose, 0.005% polysorbate-80 at pH 7.

[0055] In a specific embodiment, the present invention provides a solid protein formulation that, prior to solidification, consists of, consists essentially of, or comprises Aranesp (0.5-5 mg / ml), 10 mM sodium succinate, 4.5% mannitol, 0.5% sucrose, 0.005% polysorbate-80 at pH 6.

[0056] In one specific embodiment, the present invention provides a solid protein formulation that, prior to solidification, consists of, consists essentially of, or comprises Aranesp (0.5-5 mg / ml), 20 mM sodium phosphate, 140 mM NaCl, 0.005% polysorbate-80 at pH 6.2.

[0057] In one specific embodiment, the present invention provides a solid protein formulation that, prior to solidification, consists of, consists essentially of, or comprises Aranesp (0.5-5 mg / ml), 10 mM histidine, 2% mannitol, 0.5% sucrose at pH 7.

[0058] In one specific embodiment, the present invention provides a solid protein formulation that, prior to solidification, consists essentially of, or comprises Aranesp (2 mg / ml), 10 mM histidine, 2% mannitol, 0.5% sucrose at pH 7.

[0059] In one specific embodiment, the present invention provides a solid protein formulation that, prior to solidification, consists of, consists essentially of, or comprises Aranesp (0.5-5 mg / ml), 10 mM histidine, 2% mannitol, 0.5% sucrose at pH 7.

[0060] In one specific embodiment, the present invention provides a solid protein formulation that, prior to solidification, consists of, consists essentially of, or comprises Aranesp (0.5-5 mg / ml), 10 mM sodium phosphate, 4.5% mannitol, 0.5% sucrose at pH 6.

[0061] In one specific embodiment, the present invention provides a solid protein formulation that, prior to solidification, consists of, consists essentially of, or comprises Aranesp (0.5-5 mg / ml), 10 mM sodium phosphate, 2% mannitol, 0.5% sucrose at pH 7.

[0062] In one specific embodiment, the present invention provides a solid protein formulation that, prior to solidification, consists of, consists essentially of, or comprises Aranesp (0.5-5 mg / ml), 10 mM sodium succinate, 2% mannitol, 0.5% sucrose at pH 7.

[0063] In one specific embodiment, the present invention provides a solid protein formulation that, prior to solidification, consists of, consists essentially of, or comprises Aranesp (0.5-5 mg / ml), 10 mM sodium succinate, 4.5% mannitol, 0.5% sucrose at pH 6.

[0064] In one specific embodiment, the present invention provides a solid protein formulation that, prior to solidification, consists of, consists essentially of, or comprises Aranesp (0.5-5 mg / ml), 20 mM sodium phosphate, 140 mM NaCl, at pH 6.2.

[0065] In another embodiment, the present invention provides a method for preparing a stable solid protein formulation, comprising: a) diluting the protein with a lyophilization buffer; and b) lyophilizing the diluted protein prior to solidification.

[0066] In certain embodiments, the formulations of the invention comprise a glycoprotein. In one embodiment, the protein is erythropoietin. In another embodiment, the protein is an erythropoietin analog. In a further embodiment, the protein is darbepoetin alfa. In yet a further embodiment, the protein is an antibody. In yet another embodiment, the protein is a monoclonal or polyclonal antibody.

[0067] Erythropoietin is a glycoprotein hormone involved in the maturation of erythroid progenitor cells into red blood cells. It is essential for regulating the level of red blood cells in the circulation. Naturally occurring erythropoietin is produced by the liver during fetal development and by the kidneys in adults, circulates in the blood, and stimulates the production of red blood cells in the bone marrow. Anemia is almost always the result of renal failure due to reduced erythropoietin production from the kidney. Recombinant erythropoietin produced by genetic engineering techniques involving the expression of a protein product from host cells transformed with an erythropoietin-encoding gene has been found to be effective in treating anemia resulting from chronic renal failure.

[0068] The identification, cloning, and expression of the gene encoding erythropoietin are described in U.S. Patent No. 4,703,008. A description of the purification of recombinant erythropoietin from cell culture media supporting the growth of mammalian cells containing a recombinant erythropoietin plasmid is contained, for example, in U.S. Patent No. 4,667,016. Expression and recovery of biologically active recombinant erythropoietin from mammalian cell hosts containing the erythropoietin gene on a recombinant plasmid has resulted in usable quantities of erythropoietin suitable for therapeutic applications. Polynucleotide and polypeptide sequences for several species of erythropoietin are known.

[0069] Currently, several recombinant human erythropoietin pharmaceuticals are commercially available (e.g., Epogen®; epoetin alfa). In addition to epoetin alfa, other epoetins have been developed (e.g., epoetin beta, delta, omega, zeta). In the context of the present invention, when the terms "erythropoietin" or "recombinant human erythropoietin" are used, all forms of erythropoietin are intended to be encompassed.

[0070] Certain formulations containing analogs of human erythropoietin are also encompassed by the present invention. In certain embodiments, the phrase "analog of human erythropoietin" refers to erythropoietin with one or more changes in the amino acid sequence of human erythropoietin, resulting in an increased number of sites for sialic acid attachment. One non-limiting example is darbepoetin alfa (Aranesp®), a hyperglycosylated analog of human erythropoietin. The additional glycosylation sites in these analogs can result in a greater number of carbohydrate chains and a higher sialic acid content than human erythropoietin. Erythropoietin analogs containing amino acid sequences with rearrangements of at least one glycosylation site are also provided. Analogs that include the addition of one or more amino acids to the carboxy terminus of erythropoietin, providing at least one glycosylation site, are also encompassed. Analogs can be generated by site-directed mutagenesis with addition, deletion, or substitution of amino acid residues that increase or alter the sites available for glycosylation. Such analogs may have more carbohydrate chains than human erythropoietin. Additionally, see, for example, U.S. Patent No. 7,217,689.

[0071] In another aspect, the invention provides formulations comprising antibodies, antibody fragments, antibody derivatives, antibody muteins, and antibody variants that specifically bind to human erythropoietin.

[0072] The antibody may comprise any constant region known in the art. The light chain constant region may be, for example, a kappa- or lambda-type light chain constant region, such as a human kappa- or lambda-type light chain constant region. The heavy chain constant region may be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region, such as a human alpha-, delta-, epsilon-, gamma-, or neu-type heavy chain constant region. In one embodiment, the light or heavy chain constant region is a fragment, derivative, mutant, or mutein of a naturally occurring constant region.

[0073] In one embodiment, the antibody further comprises a constant light chain kappa or lambda domain, or a fragment thereof. The sequences of light chain constant regions and the polynucleotides encoding them are well known in the art. In another embodiment, the antibody further comprises a heavy chain constant domain, or a fragment thereof, such as an IgG1 or IgG2 heavy chain constant region, the sequences of which are well known in the art.

[0074] Antibodies include those with the desired isotype (e.g., IgA, IgG1, IgG2, IgG3, IgG4, IgM, IgE, and IgD), as well as Fab or F(ab')2 fragments thereof. Furthermore, if IgG4 is desired, it may be desirable to introduce point mutations into the hinge region to reduce the tendency for intra-H-chain disulfide bonds to form, which can lead to heterogeneity in IgG4 antibodies, as described in Bloom et al., 1997, Protein Science 6:407 (incorporated herein by reference).

[0075] The term "antibody," as broadly described in the definitions section, refers to an intact antibody or an antigen-binding fragment thereof. Antibodies may include complete antibody molecules (including polyclonal, monoclonal, chimeric, humanized, or human versions with full-length heavy and / or light chains) or antigen-binding fragments thereof. Antibody fragments include F(ab')2, Fab, Fab', Fv, Fc, and Fd fragments, which can be incorporated into single-domain antibodies, single-chain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). Antibody polypeptides, such as those disclosed in U.S. Pat. No. 6,703,199, including fibronectin polypeptide monobodies, are also included. Other antibody polypeptides are disclosed in US Patent Application Publication No. 2005 / 0238646, which are single chain polypeptides.

[0076] The present invention provides stable protein formulations. The term stable, in certain non-limiting embodiments, relates to the structure and activity of the protein. In certain embodiments, stable means that the protein maintains the correct amino acid sequence and conformational structure. Non-limiting examples of methods for assaying this include antibody binding assays or gel electrophoresis.

[0077] In certain embodiments, one aspect of stability is percent oxidation measured over storage time. As proteins oxidize, they may lose activity, among other properties. In one embodiment, the protein in a formulation of the invention has a percent oxidation of about 5% to about 10%. In another embodiment, the protein in a formulation of the invention has a percent oxidation of less than about 10%. In another embodiment, the protein in a formulation of the invention has a percent oxidation of less than about 5%. In one embodiment, the protein in a formulation of the invention has a percent oxidation of 5% to 10%. In another embodiment, the protein in a formulation of the invention has a percent oxidation of less than 10%. In another embodiment, the protein in a formulation of the invention has a percent oxidation of less than 5%.

[0078] In yet a further embodiment, stable means that the protein present in the formulation has and maintains a biological effect. Non-limiting examples include bioassays that measure the effect of the protein on a cell line in vitro or on an animal in vivo.

[0079] The protein formulations of the present invention can be administered to patients via a variety of routes appropriate for the indication and composition. The protein formulations of the present invention may be administered by any suitable technique, including, but not limited to, parenteral administration. If injected, the formulations can be administered, for example, via intraarticular, intravenous, intramuscular, intralesional, intraperitoneal, or subcutaneous routes, by bolus injection, or continuous infusion.

[0080] Dosage and frequency of administration may vary according to factors such as the route of administration, the particular protein used, the nature and severity of the disease being treated, whether the condition is acute or chronic, and the size and general condition of the subject. Appropriate dosages can be determined by procedures known in the relevant art, for example, in clinical trials, which may include dose escalation studies.

[0081] Kits are provided for use by a physician and / or subject, comprising one or more proteins in a formulation of the invention and a label or other instructions for use in treating any of the conditions discussed herein. In one embodiment, the kit comprises a sterile formulation of one or more proteins in a lyophilized formulation of the invention and a separate sterile reconstitution solution, which may be in one or more vials.

[0082] Having described the invention, the following examples are offered by way of illustration and not by way of limitation. [Example]

[0083] Example 1 1. Purpose The purpose of this study was to investigate the feasibility of developing a room-temperature stable formulation of Aranesp® with advantages in handling and storage. The current commercial formulation is a liquid formulation stored at 2-8°C. The lyophilized formulation is not a preferred formulation in the market because it must be reconstituted and is an injectable, two-step procedure. Since the LyoTip®, a device that provides a convenient method for delivering lyophilized formulations as liquid pre-filled syringes by combining both the reconstitution and injection processes into a seamless single step, the lyophilized formulation has been investigated for long-term storage studies in a room-temperature stable formulation in a 3 cc glass vial.

[0084] 2. Experimental Overview Aranesp® (2 mg / mL) was formulated into five lyophilized formulations by dialyzing Aranesp® bulk, and the shelf life was compared with that of a liquid formulation in 20 mM sodium phosphate, 140 mM sodium chloride, pH 6.2. Dialyzed samples (0.25 mL) were filled into 3 cc glass vials and lyophilized. The lyophilized samples were stored at 4°C, 29°C, 37°C, and 45°C for shelf life over 24 months. At each time point, the samples were reconstituted with 1 mL of one of three reconstitution solutions (water for P45MSu6 and S45MSu6, 4% sorbitol and 0.7% sodium chloride for P2MSu7, S2MSu7, and H2MSu7) to a final concentration of 500 μg / mL. Reconstituted samples were analyzed for SEC-HPLC (size exclusion HPLC), antibody 9G8A (to monitor relative protein unfolding), pH, % oxidation, RP-HPLC (impurities and truncations), subvisible particles, concentration, and osmolality.

[0085] 3. Substances and Equipment The following materials were used: Aranesp bulk material (2 mg / mL), polysorbate 80, glass 3 cc vials, Daikyo long stoppers for lyophilization. The following six formulation buffers were prepared: P2MSu7: 10 mM sodium phosphate, 2% mannitol, 0.5% sucrose, pH 7 (Lot #29070904-11) S2MSu7: 10 mM sodium succinate, 2% mannitol, 0.5% sucrose, pH 7 (Lot #29070904-21) H2MSu7: 10 mM histidine, 2% mannitol, 0.5% sucrose, pH 7 (Lot #29070904-19) P45MSu6: 10 mM sodium phosphate, 4.5% mannitol, 0.5% sucrose, pH 6 (Lot #29070627-18) S45MSu6: 10 mM sodium succinate, 4.5% mannitol, 0.5% sucrose, pH 6 (Lot #29070627-19) P62N: 20 mM sodium phosphate, 140 mM NaCl, pH 6.2 Additional equipment used included Slide-A Lyser dialysis cassettes (Pierce), Corning filters (0.22 μm), a refrigerated room for dialysis of samples over a 2-day period, and a Virtis freeze dryer was used for freeze-drying.

[0086] 4. Sample preparation procedure 4.1. Sample preparation Aranesp® (2 mg / mL) was dialyzed into five formulation buffers over two days in a refrigerator (2-8°C) using a Slide-A Lyser dialysis cassette. The buffers were checked for pH and osmolality at room temperature, and then the samples were dialyzed. After dialysis, the sample concentrations were checked by A280. Samples were prepared with and without 0.005% polysorbate-80. Samples containing polysorbate-80 were prepared by adding 10% (w / v) polysorbate-80 stock solution to the sample to make 0.005%. 10% polysorbate-80 solution was made by weighing 2.5 g of polysorbate-80 and dissolving it in 25 mL of each buffer. Samples were filtered using a Corning filter (0.22 μm) in a sterile hood. The filtered samples and placebo (0.25 mL) were filled into 3 cc glass vials and subjected to a lyophilization cycle using a Virtis freeze-dryer (Table 1). Liquid samples were prepared in 20 mM sodium phosphate, 140 mM sodium chloride, 0.005% polysorbate-80, pH 6.2, by adding polysorbate-80 to the bulk to achieve 0.005%. The liquid control sample, lyophilized sample, and placebo were stored at 4°C, 29°C, 37°C, and 45°C for shelf life over 24 months. At each time point, samples were reconstituted with 1 mL of one of three reconstitution solutions (water was used for P45MSu6 and S45MSu6, and 4% sorbitol and 0.7% sodium chloride was used for P2MSu7, S2MSu7, and H2MSu7) to a final concentration of 500 μg / mL. When samples (0.25 mL) containing 0.005% polysorbate-80 were reconstituted with 1 mL of the respective reconstitution buffer, the samples were diluted with 1 mL of reconstitution buffer and therefore contained 0.00125% polysorbate-80. Reconstituted samples were analyzed for SEC-HPLC (size-exclusion HPLC), 9G8A (to monitor relative protein unfolding), pH, % oxidation, and non-reducing RP-HPLC (impurities and truncations). The sample list, time points, and temperatures are shown in Appendix 1.

[0087] 4.2. Freeze-drying procedure: The sample vials were loaded onto a pre-chilled (4°C) shelf of the freeze dryer. The samples underwent three steps: freezing, primary drying, and secondary drying. First, the samples were held at 4°C for 60 minutes, then cooled from 4°C to -50°C over 180 minutes. After holding at -45°C for 60 minutes, the shelf temperature was increased to -12°C over 70 minutes. Then, after holding at -12°C for 360 minutes, the temperature was reduced again to -50°C over 70 minutes. The samples were brought to -50°C over 60 minutes. For primary drying, the shelf temperature was increased to -10°C over 40 minutes and held for 1500 minutes with the chamber vacuum maintained at 50 mTorr. For secondary drying, the shelf temperature was increased to 25°C over 350 minutes and held for 720 minutes. The temperature was then reduced to 5°C over 140 minutes. After all cycles were completed, the vials were stoppered inside the drying chamber. [Table 1-1] [Table 1-2]

[0088] 5. Analysis method 5.1. 9G8A method 5.1.1. 9G8A method 1 Assays were analyzed using a Bioveris instrument for up to 12 months. Using serial dilutions, samples were diluted to 0.4 μg / mL in diluent (1% BSA and 0.1% PS-80 in PBS). Standard curve dilutions were also prepared using the diluent. 10 μL of each sample was loaded in quadruplicate onto a 96-well plate. The plate was then incubated at room temperature in the dark for 1 hour. 50 μL of TAG-labeled 9G8A antibody (5 μL of 9G8A per 3 mL of diluent) was added to each well, and the plate was incubated for an additional hour. 50 μL of biotin-goat polyclonal antibody (5 μL of biotin-goat per 3 mL of diluent) was added to the wells and incubated for an additional hour. 25 μL of streptavidin beads were added to the wells and incubated for 30 minutes. Finally, 115 μL of 1x PBS was added to the wells. The plate was analyzed using a Bioveris M-8 analyzer. ECL (Enzymatic-chemo-luminescent) values ​​were reported and converted to relative percent denaturation.

[0089] 5.1.2. 9G8A method 2 Because BioVeris was unable to support the instrument, we had to develop a manual method. Starting at month 18, samples were analyzed using the new 9G8A method. Diluted 9G8A antibody solution (100 μL) was added to each well required for the analysis set. The plate was incubated for 1 hour at room temperature. The plate was then washed three times with diluent (1% BSA and 0.1% PS-80 in PBS), blotting dry between washes. 100 μL of diluted standard, control, or sample was transferred to each well. After 1 hour of incubation, the liquid was decanted from the plate and washed three times. Secondary antibody solution was added and incubated for an additional hour. The liquid was decanted from the plate and washed again. 100 μL of HRP was added to each well and incubated for an additional 30 minutes. The liquid was decanted from the plate and washed three times with diluent. 100 μL of the prepared substrate reagent was added to each well and the plate was placed in a plate reader for analysis.

[0090] 5.2. Size Exclusion Chromatography (SE-HPLC) Size-exclusion chromatography was used as a stability-indicating assay. Separation was performed on an Agilent 1100 HPLC system equipped with Chromeleon software. The method used two columns (TosoHaas TSK gel G3000SWxl, 7.8 mm x 300 mm) mounted in series alongside a guard column (TSK gel Guard Column SWXL, 6.0 mm x 4.0 cm). 10 μg of protein was loaded onto the column and eluted isocratically with a mobile phase consisting of 20 mM sodium phosphate, 140 mM sodium chloride, pH 6.2, at a flow rate of 0.5 mL / min. Protein was monitored using UV detection at 215 nm and 280 nm. Peak area percentages relative to the total area counts in the chromatogram were used to quantify the amount of high-molecular-weight (HMW) and low-molecular-weight (LMW) species.

[0091] 5.3. Percent Oxidation Methionine 54 oxidation detection was performed via denatured peptide map analysis. Removal of sugars from the sample required filtration (Millipore Microcon Centrifugal Filter Devices Ultracel YM-10) via centrifugation (12,000 rpm for 20 minutes at 25°C). For trypsin digestion, the sample volume was reconstituted with water. The sample was diluted to 20 μg / mL with dilution buffer (20 mM sodium phosphate, 140 mM sodium chloride, 0.005% polysorbate 80, pH 6.2). Digestion buffer (10 mM methionine in 500 mM Tris-HCl, pH 8) was added at a digestion buffer to total volume ratio of 1:10. Trypsin (1 mg / mL) was then added at a trypsin to final protein concentration ratio of 1:5 (in μg). The sample was incubated at 29°C for 15 hours and then quenched with 10 μL of 25% TFA.

[0092] Oxidation detection was performed using reversed-phase chromatography. 2 μg (100 μL) of sample was loaded onto a C8 column (Phenomenex Develosil 5u 300C8-HG 300A 150 × 2.0 mm) and separated using a step gradient at a flow rate of 2 mL / min. (Mobile phase A consisted of 40% acetonitrile and 0.1% TFA for 5 min. Next, mobile phase B consisted of 46% acetonitrile and 0.1% TFA, ramped from 0% to 100% in 0.15 min, and run for 15 min. Finally, mobile phase A was run for 10 min.) The column temperature was set to 55 °C. Chromatographic analysis was performed on a Beckman HPLC Gold system coupled to a Thermo LCQ Deca mass spectrometer for ion quantification. Peak detection was at 214 nm (UV) and mass spectrometry (single ion mode) used a zoom scan with selected m / z: 1264 (unoxidized), 1272 (oxidized), 1342 (partially unoxidized), and 1350 (partially oxidized). Data were reported as % oxidation.

[0093] 5.4. Non-reducing reversed phase Because degradation products are easily detected using this technique, protein content and purity were determined using non-reducing reversed-phase (NRRP-HPLC). Non-reducing reversed-phase (NRRP) HPLC was used to separate truncated species from full-length erythropoietin monomers. Measurements were performed on a Shimadzu high-performance liquid chromatograph using a Phenomenex Jupiter 5 μm, 300A, C4 bonded-phase silica column (150 × 4.6 mm). Mobile phase A was 0.0651% trifluoroacetic acid (v / v) in water, and mobile phase B was 0.0651% trifluoroacetic acid in 90% acetonitrile. Samples were analyzed using a gradient system from 20% B to 100% B in 135 min at a flow rate of 1.0 mL / min with detection at 215 nm. Chromatogram overlays were used to compare the detection of degraded or truncated species.

[0094] pH Samples were monitored for pH using a Mettler Toledo MP200 pH meter. The instrument was calibrated using pH 4 and pH 7 standard buffers.

[0095] 5.6. Osmolality Osmolality data was used to determine the percentage of reconstitution buffer. Osmolality was measured with an Advanced Instruments, Inc. Micro Osmometer, Model 330. A 290 mOsm standard was used as a reference.

[0096] 6. Results and Discussion 6.1. 9G8A The 9G8A antibody binding assay (Elliott, Chang et al., 1996; Elliott, Lorenzini et al., 1996) was used to assess the conformational similarity of product samples. The 9G8A monoclonal antibody recognizes a linear epitope, ERYLL, consisting of amino acids 13-17 in both native and denatured Aranesp. These amino acids become more exposed after conformational changes or denaturation of Aranesp, resulting in increased 9G8A binding. Data were plotted as the ratio of sample reactivity to that of the darbepoetin alfa standard. A value of 1 indicates no difference in reactivity between the sample and standard.

[0097] Lyophilized formulations without (A) or with (B) polysorbate-80 were shown to be more stable than the liquid formulations up to 24 months of storage at 37°C. The liquid formulation, P62N, showed higher relative denaturation at higher temperatures (Figure 1).

[0098] 6.2. Aggregation detection by size exclusion chromatography (SEC) With the exception of the sample in P45MSu6, most of the Aranesp lyophilized formulations demonstrated greater stability at higher temperatures (37°C and 45°C) than the liquid formulations up to 24 months of storage (Figure 3). In addition, denaturation increases with temperature.

[0099] The SE-HPLC method used in this study does not separate the polysorbate peak from the HMW species. Therefore, a slight increase in the % HMW species was observed in the polysorbate-containing samples compared to the polysorbate-80-free samples (Figures 3 and 6). There were no significant differences between the lyophilized formulations at 4°C and 29°C; however, P45MSu6T was observed to have higher denaturation at 37°C and 45°C compared to the other lyophilized formulations (Figures 5 and 6). It was observed that H2MSu7 was more stable than the other formulations (Figure 4). Samples in the H2MSu7 formulation showed minimal dimer peaks (arrows) in the overlay after 24 months of storage at 45°C. Additionally, there were no significant differences between samples reconstituted with sorbitol or sodium chloride.

[0100] 6.3. pH measurement At 24 months of storage, there was less than 10% change in the majority of formulations at all temperatures (4°C, 29°C, 37°C, and 45°C).

[0101] 6.4. Percent Oxidation Methionine, located at position 54 of Aranesp, is prone to oxidation. This oxidation was quantified in trypsin-digested samples by LC / MS analysis. Data shown are % oxidation.

[0102] After 24 months of storage at 4°C, all lyophilized formulations showed less oxidation than the liquid formulations (Figure 8). Furthermore, no differences were observed among the three reconstitution buffers. The addition of polysorbate-80 had no significant effect on the stability of the lyophilized formulations.

[0103] Storage at elevated temperatures (29°C, 37°C, and 45°C) for up to 24 months resulted in all lyophilized formulations exhibiting significantly lower oxidation levels than the liquid formulations for all three reconstitution types, although oxidation increased with increasing temperature (Figures 9-11). However, the histidine-free polysorbate lyophilized formulation had significantly higher oxidation up to 12 months compared to all other lyophilized formulations. No differences were observed between sodium chloride and sorbitol reconstitution buffers. Two additional lyophilized formulations, histidine-containing polysorbate and phosphate, also exhibited higher oxidation when stored at 45°C for up to 24 months (although the phosphate formulation showed results with water reconstitution). Finally, the polysorbate-containing liquid formulations exhibited higher oxidation than the polysorbate-free liquid formulations, but no significant differences were observed between the lyophilized formulations.

[0104] 6.5. Detection of Degradation and Truncated Species by Non-Reducing Reversed-Phase HPLC Non-reducing reversed-phase HPLC was used to detect degradation and truncation species. A chromatogram overlay from non-reducing reversed-phase chromatography of Aranesp® after 24 months of storage at 45° C. is shown in FIG. 12. FIG. 13 shows a chromatogram overlay after 24 months of storage at 37° C. The three lyophilized samples did not show any degradation or truncation species in two different reconstitution diluents after 24 months of storage at 4° C., 29° C., 37° C., and 45° C. However, the liquid formulation sample degraded significantly at both temperatures. The main peak of the sample in the liquid formulation was nearly eliminated at 45° C.

[0105] 7. Conclusion Lyophilized formulations of Aranesp were evaluated for stability from room temperature to 45°C and at 24 months. The data obtained suggest no significant changes in the assessed metrics, including aggregation, truncation, unfolded species, and oxidation. The formulation containing the relatively low amount of mannitol, 2%, demonstrated a better stability profile compared to the 4.5% mannitol formulation. The data obtained from this feasibility study suggest that the Aranesp lyophilized formulation is stable from room temperature to at least 45°C and at least 24 months.

[0106] Appendix 1. Title: Feasibility of developing a room temperature stable lyophilized Aranesp formulation using LyoTip Objective: To develop and optimize NESP lyophilized formulations for LyoTips, 0.25 mL of each formulation (2 mg / mL) was lyophilized and prepared for stability studies at four temperatures. The samples were diluted with 1 mL of reconstitution solution to 500 μg / mL. Temperature and storage conditions: 4℃, 29℃, 37℃, 45℃ Interval: 0, 1, 3, 6, 12, 18, 24 months Buffers: sodium succinate, sodium phosphate, histidine Compound concentration: Starting concentration: approx. 2 mg / mL, reconstituted sample concentration: approx. 500 μg / mL Polysorbate concentration: 0.005% ARANESP: Concentration: 2mg / mL Vials: 3cc vial type, the vials were washed and sterilized. Stopper: Freeze-dried stopper Sample preparation:

[0107] 1. Various buffer solutions: 1. P2MSu7: 10 mM sodium phosphate, 2% mannitol, 0.5% sucrose, pH 7–11) 2. S2MSu7: 10 mM sodium succinate, 2% mannitol, 0.5% sucrose, pH 7 3. H2MSu7: 10 mM histidine, 2% mannitol, 0.5% sucrose, pH 7 4. P45MSu6: 10 mM sodium phosphate, 4.5% mannitol, 0.5% sucrose, pH 6 5. S45MSu6: 10 mM sodium succinate, 4.5% mannitol, 0.5% sucrose, pH 6

[0108] 2. Lyophilized formulation: Aranesp bulk (100 mL per formulation) was dialyzed into formulation buffer in a refrigerator for 2 days using a Slide-A Lyser for dialysis. After dialysis, the concentration was measured by UV-Vis.

[0109] 3. Sample Preparation: To each sample, a 10% solution of polysorbate in water was added to give a 0.005% polysorbate formulation. Each sample was filtered in a sterile hood using a 0.22 μm syringe filter. a. Polysorbate-free sample Fill 0.25 mL of sample into 3 cc vials and use lyophilization stoppers for the lyophilized samples. The P62N500 sample will remain as a liquid solution for the control. [Table 1-3] b. Samples containing polysorbates [Table 1-4] Fill 0.25 mL of sample into 3 cc vials and use lyophilization stoppers for the lyophilized samples. The P62N500 sample will remain as a liquid solution for the control. c. Polysorbate-free placebo [Table 1-5] d. Placebo containing polysorbate [Table 1-6] 4. Sample details [Table 1-7] [Table 1-8] [Table 1-9] 5. Sample preparation for analysis: At each time point, samples were reconstituted with 1 mL of the following solutions: a. P45MSu6 and S45MSu6 are water For P2MSu7, S2MSu7, and H2MSu7, use the following solutions: b. 3% sorbitol: 1 mL c. 0.45% sodium chloride: 1 mL Analysis and time points: 1 mL per sample vial [Table 1-10]

[0110] Analysis method: C4 Jupiter Column 300A; 250 x 4.6 mm; S. / No.: 202394, Batch No. 5267-4 Mobile phase: A - 0.065% TFA in water, B - 0.065% TFA in 95% acetonitrile, Run time: 145 min, flow rate 0.75 ml / min, Detection: 215nm, 230nm, 280nm Gradient: 20% B (5 min); 20% to 70% B (100 min); wash and equilibration (40 min) Sample loading: 3 μg SEC HPLC: Columns: Two TSK G3000swxl columns, isocratic, flow rate 0.5 mL / min, run time 80 min, buffer 100 mM sodium phosphate, 0.5 M sodium chloride (pH 6.9) Detection: 215 nm, 230 nm, 280 nm, buffer injection: 100 μL, HSA and Tween sample: 3 μg. Total area and % of main peak to total area reported at 215 nm

[0111] Sample list per time point [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15]

[0112] Example 2 1. Purpose The Aranesp lyophilized formulation was selected from Example 1 above, but because the LyoTip® is made from Crystal Zenith plastic material, a compatibility study of the CZ plastic vial (Diakyo Crystal Zenith) with the glass vial was required. This study examined the compatibility of the container (Daikyo Crystal Zenith) with the glass vial to assess the effect of Aranesp® storage stability and the potential for a sting-free formulation of Aranesp®. For the sting-free formulation, a 1% benzyl alcohol solution containing 0.7% sodium chloride at pH 7.0 was used to reconstitute the lyophilized sample.

[0113] 2. Experimental Overview Aranesp® (1.84 mg / mL) was formulated in 10 mM histidine, 2% mannitol, 0.5% sucrose, 0.005% polysorbate 80, pH 7 by dialysis against Aranesp formulation buffer (20 mM sodium phosphate, 140 mM sodium chloride, pH 6.2). The dialyzed sample (0.25 mL) was transferred to a 2 cc Crystal The product was filled into Zenith vials and 3 cc glass vials and lyophilized. The lyophilized samples were stored at 4°C, 29°C, 37°C, and 45°C for 12 months for shelf stability. At each time point, the sample was reconstituted with 1 mL of one of three reconstitution solutions (0.70% NaCl, pH 7; 0.70% NaCl + 1% benzyl alcohol, pH 7.0; 0.70% NaCl, pH 7 containing 0.004% PS-80) to a final concentration of 500 μg / mL. The reconstituted samples were analyzed by SE-HPLC (size exclusion HPLC), 9G8A (to monitor relative protein unfolding), pH, % oxidation, RP-HPLC (impurities and truncations), subvisible particles, concentration, and osmolality.

[0114] 3. Substances and Equipment 3.1. Aranesp Bulk (1.84 mg / mL) Polysorbate-80 3.3. Blowback Glass 3cc Vial (Type 1 Glass EP) 3.4. Diakyo Crystal Zenith Vials (West. Daikyo DS CZ Vials 2mL 13mm, long stoppers for lyophilization were used for the stoppers.) 3.5. 10 mM histidine, 2% mannitol, 0.5% sucrose, pH 7 (H2MSu7) 3.6. Slide-A Lyser dialysis cassettes were obtained from Pierce. 3.7. Corning filters (0.22 μm) were used for filtration. 3.8. The sample was dialyzed in a refrigerator for 2 days. 3.9. A Virtis freeze dryer was used for freeze drying.

[0115] 4. Sample preparation procedure Aranesp® (1.84 mg / mL) was formulated in 10 mM histidine, 2% mannitol, 0.5% sucrose, 0.005% polysorbate 80, pH 7, by dialysis in Aranesp formulation buffer (20 mM sodium phosphate, 140 mM sodium chloride, pH 6.2) over 2 days in a refrigerator (2-8°C) using a Slide-A Lyser dialysis cassette. Prior to dialysis, the buffer was checked for pH and osmolality at room temperature. After dialysis, the sample concentration was checked. A 1% (w / v) stock solution of polysorbate-80 was added to the sample to achieve 0.005%. A 1% polysorbate-80 solution was made by adding 0.25 g of polysorbate 80 to 25 mL of HM2MSu7 buffer. In a sterile hood, the sample was filtered using a Corning filter (0.22 μm). Filtered samples (0.25 mL) were filled into two different types of vials (Crystal Zenith and glass vials) and subjected to a lyophilization cycle in Building 8 using a Virtis freeze dryer (Table 2). 0.25 mL of placebo and protein-containing formulations were lyophilized and stored at 4°C, 29°C, 37°C, and 45°C for 12 months for shelf life. At each time point, samples were reconstituted with 1 mL of one of three reconstitution solutions (0.70% NaCl, pH 7; 0.70% NaCl + 1% benzyl alcohol, pH 7.0; 0.70% NaCl, pH 7 containing 0.004% PS-80) to a final concentration of 500 μg / mL. Reconstituted samples were analyzed for SEC-HPLC (size-exclusion HPLC), 9G8A (to monitor relative protein unfolding), pH, % oxidation, non-reducing RP-HPLC (impurities and truncations), subvisible particles, concentration, and osmolality. The sample list, time points, and temperatures are shown in Appendix 1 and 2. [Table 2-1] [Table 2-2]

[0116] 5. Analysis method 5.1. 9G8A method If necessary, samples were diluted to 0.34 to 4.2 μg / mL in 1x PBS, 1% BSA, and 0.1% polysorbate-80 buffer and distributed to a 96-well plate. 50 μL of TAG (BioVeris)-labeled 9G8A (2 μg / mL) anti-rHuEPO antibody was added, and the samples were incubated at room temperature for 1 hour on a plate shaker. Next, 50 μL of affinity-purified biotinylated rabbit anti-rHuEPO antibody (stock concentration: 0.5 μg / mL) was added, and the samples were incubated at room temperature for 1 hour on a plate shaker. Next, 25 μL of streptavidin-coated beads (stock concentration: 0.566 μg / mL) diluted to 0.6 μg / mL in 1x phosphate-buffered saline (PBS), 1% bovine serum albumin, and 0.1% polysorbate-80 was added, and the samples were incubated at room temperature for 30 minutes on a plate shaker. The plate was transferred to an M8 analyzer (IGEN International, Gaithersburg, MD) and the chemiluminescent signal was measured according to the manufacturer's instructions. The chemiluminescent signal was converted to a ratio of the sample to the Aranesp bulk standard and reported as relative reactivity, where 1 is equal to the standard and a value >1 indicates protein denaturation.

[0117] 5.2. Size Exclusion Chromatography (SE-HPLC) Size-exclusion chromatography was used as a stability-indicating assay. Separation was performed on an Agilent 1100 HPLC system equipped with Chromeleon software. The method used two columns (TosoHaas TSK gel G3000SWxl, 7.8 mm x 300 mm) mounted in series alongside a guard column (TSK gel Guard Column SWXL, 6.0 mm x 4.0 cm). 10 μg of protein was loaded onto the column and eluted isocratically with a mobile phase consisting of 20 mM sodium phosphate, 140 mM sodium chloride, pH 6.2, at a flow rate of 0.5 mL / min. Protein was monitored using UV detection at 215 nm and 280 nm. The peak area percentage relative to the total area count in the chromatogram was used to quantify the amount of high-molecular-weight (HMW) and low-molecular-weight (LMW) species.

[0118] 5.3. Concentration Protein concentrations were determined by UV / VIS spectroscopy using an Agilent UV / Vis spectrophotometer model 8453 system using Chemstation software. Absorbance at 280 nm was determined using Beer's law with an extinction coefficient of 0.98 in a cuvette with a 1 cm path length.

[0119] 5.4. Analysis of particles invisible to the naked eye using HIAC Subvisible particle counts were monitored by HIAC in the 2-25 μm range. Prior to analysis, samples were degassed under vacuum for 1 hour with the caps open. Four 0.2 mL measurements were taken for each sample, and Millipore water was used to blank the system before measuring the sample. The first run was discarded, and the last three were averaged to obtain cumulative counts per milliliter. Particle sizes of 2, 5, 7.5, 10, 15, 20, and 25 μm were monitored under USP guidelines.

[0120] 5.5. Oxidation % An oxidation detection assay was performed, involving digestion of the sample with trypsin, followed by reverse-phase chromatography with mass spectrometry detection (RP-HPLC / MS). The LC / MS oxidation assay was used to determine the percentage of oxidized methionine 54. Solutions of all samples were prepared at 500 μl or 10 μg of 0.02 mg / mL sample in 20 mM sodium phosphate, 140 mM sodium chloride, 0.005% polysorbate 80, pH 6.2. Then, 50 μl of 0.5 M Tris-HCl, 10 mM methionine (pH 8.0) was added to the sample. Trypsin (2 μl of 1 mg / mL) was added to the sample and incubated overnight at 29°C. The sample was quenched with 10 μl of 25% TFA. Sample analysis was accomplished using a Beckman HPLC System Gold (Osiris, System ID #408774) coupled to an LCQ Deca mass spectrometer (Promasseous). The mobile phase was 42% acetonitrile, 0.1% TFA using an isocratic method on a Zorbax 300SB-C8 column (150 × 2.1 mm, 5 μm, 300 Å, Part Number: 883750-906) at 60 °C with a flow rate of 0.20 ml / min. Detection was by UV at 215 and 280 nm using a zoom scan with selected m / z: 1264 (unoxidized), 1272 (oxidized), 1342 (partially unoxidized), and 1350 (partially oxidized). The ratio of specific peptide peaks was used to estimate the percentage of oxidation products.

[0121] 5.6. Non-reducing reversed phase Nonreducing reversed-phase high-performance liquid chromatography (NRRP-HPLC) was used to determine protein content and purity, as degradation products are easily detected using this technique. Nonreducing reversed-phase (NRRP) HPLC was used to separate truncated species from full-length erythropoietin monomers. Measurements were performed on a Shimadzu high-performance liquid chromatograph using a Phenomenex Jupiter 5 μm, 300A, C4 bonded-phase silica column (150 × 4.6 mm, 00F-4167-E0). Mobile phase A was 0.0651% trifluoroacetic acid (v / v) in water, and mobile phase B was 0.0651% trifluoroacetic acid in 90% acetonitrile. Samples were analyzed using a gradient system from 20% B to 100% B in 135 min at a flow rate of 1.0 mL / min, with detection at 215 nm. Chromatogram overlays were used to compare the detection of degraded or truncated species.

[0122] 5.7. pH measurement Samples were monitored for pH using a Mettler Toledo MP200 pH meter. Standard buffers of pH 4 and pH 7 were used to calibrate the instrument.

[0123] 5.8. Osmolality Determination Osmolality was measured using a Micro Osmometer, Model 330, manufactured by Advanced Instruments, Inc. A 290 mOsm standard was used as a reference.

[0124] 6. Results and Discussion 6.1. 9G8A The 9G8A antibody binding assay was used to evaluate the conformational similarity of product samples (Elliott, Chang et al., 1996; Elliott, Lorenzini et al., 1996). The 9G8A monoclonal antibody recognizes a linear epitope, ERYLL, consisting of amino acids 13-17 in both native and denatured Aranesp. These amino acids become more exposed after conformational changes or denaturation of Aranesp, resulting in increased 9G8A binding. Data were plotted as the ratio of sample reactivity to that of the darbepoetin alfa standard. A value of 1 indicates no difference in reactivity between the sample and standard.

[0125] There were no significant differences when comparing different reconstitution diluents containing benzyl alcohol and polysorbate-80. However, there was a slight increase in relative denaturation at higher temperatures for samples in Crystal Zenith (CZ) vials compared to samples in glass vials. The results are summarized in Figure 22.

[0126] 6.2. Detection of aggregates by size exclusion chromatography (SEC) Up to 37°C, there was no significant difference in the % HMW species for Aranesp® when comparing the three different reconstitution dilutions containing benzyl alcohol and polysorbate-80 (Figure 23). However, at 45°C, there was a slight increase in high molecular weight species for samples in glass vials when compared to samples in Crystal Zenith (CZ) vials (Figure 23). Aranesp® in CZ vials showed a higher % main peak at 45°C than samples in glass vials (Figure 24).

[0127] 6.3. Analysis of particles invisible to the naked eye; No trends in particle counts were observed between samples in glass and cz vials. Particle counts remained below the USP guidelines for 10 μm and 25 μm particles in both protein and placebo samples at all four temperatures (Figure 25). The USP guidelines are ≦6,000 particles per container for particles ≧10 μm and ≦600 particles per container for particles ≧25 μm.

[0128] 6.4. Visual Inspection Samples in CZ vials stored at 4°C for 12 months showed that the lyophilized cakes had melted for both the protein and placebo in the CZ vials (Figure 26), indicating that moisture penetrates the CZ vials.

[0129] 6.5. Concentration measurement There was a slight decrease in protein concentration after samples were stored in CZ vials at 4° C. for 12 months, however, there was no difference in protein concentration between samples and containers at other temperatures and time points.

[0130] 6.6. Oxidation % The percent oxidation for Aranesp stored over 12 months at four different temperatures (4°C, 29°C, 37°C, and 45°C) is shown in Figure 28. A rapid rate of oxidation was observed for samples in CZ vials compared to samples in glass vials at 29°C, 37°C, and 45°C. After 12 months of incubation, at the 29°C storage temperature, samples in CZ vials showed approximately 25% oxidation, while samples in glass vials showed approximately 5% oxidation. The difference was more significant at higher storage temperatures. There were no differences among the three reconstitution buffers (0.7% sodium chloride, 1% benzyl alcohol, or 0.004% polysorbate-80) for up to 12 months of storage at the four temperatures (4°C, 29°C, 37°C, and 45°C).

[0131] 6.7. Detection of Degradation and Truncated Species by Non-Reducing Reversed-Phase HPLC Non-reducing reversed-phase HPLC was used to detect degradation and truncated species. An overlay of chromatograms from non-reducing reversed-phase chromatography of Aranesp® after 12 months of storage at four different temperatures is shown in Figure 29. Lyophilized samples in CZ vials or glass vials showed no degradation or truncated species after 12 months of storage at 4°C, 29°C, 37°C, and 45°C in three different reconstitution dilutions.

[0132] 6.8. pH measurement Over the storage period and at the four different temperatures, the initial pH did not change (Figure 30).

[0133] 6.9. Osmolality Measurement The sample reconstituted with benzyl alcohol exhibits a higher osmolality than the other samples at all four temperatures. There was no difference between samples in different containers. There was an outlier in the 4°C sample, but no trend. The results are summarized in Figure 31.

[0134] 7. Conclusion The inventors have investigated a potential reformulation of Aranesp that can be stored at room temperature for at least two years and may be sting-free. A preliminary container comparison study was conducted on Aranesp stability in Daikyo Crystal Zenith (CZ) vials versus glass vials using the Aranesp lyophilized formulation (10 mM histidine, 2% mannitol, 0.5% sucrose, 0.005% polysorbate 80, pH 7) and three reconstitution buffers (0.7% sodium chloride, 1% benzyl alcohol, 0.004% polysorbate-80). Physical and chemical degradation was monitored using SE-HPLC, % oxidation, pH, RP-HPLC, and the 9G8A immunoassay. Results showed no differences between the reconstitution buffer (0.7% sodium chloride, 1% benzyl alcohol, 0.004% polysorbate-80) and the different containers (CZ and glass vials) based on 9G8A (unfolding), subvisible particles, RP-HPLC (truncation), and SE-HPLC (aggregation) analyses. However, a reduced cake structure and a higher percentage of oxidation were observed for samples in CZ vials compared to samples in glass vials. This indicated that CZ vials are susceptible to moisture and oxygen penetration. This study showed that 1% benzyl alcohol can be used as a reconstitution buffer for non-stinging purposes. Also, a lyophilized Aranesp formulation at pH 7, which can be stored at room temperature for at least two years, may reduce the stinging effect.

[0135] 8. Additional Notes [Table 2-3] [Table 2-4] [Table 2-5]

[0136] Appendix 2. Time and Date [Table 2-6]

[0137] Example 3 overview A lyophilized formulation of anti-EPO monoclonal antibody was developed for room temperature stability. Two lyophilized formulations and two concentrations (100 μg / mL and 500 μg / mL) were investigated. 1. GMST: 10 mM Na-glutamate (from glutamic acid), 4% mannitol, 2% sucrose, 0.01% polysorbate 20, pH 5.2 2. HMST: 10 mM histidine, 4% mannitol, 2% sucrose, 0.01% polysorbate 20, pH 6.0 The sample in the histidine formulation showed a slightly higher % main peak than in the glutamate buffer based on the SE-HPLC results. Overall, samples from the two formulations did not show any significant changes in aggregation and overall structure and thermal stability after incubation at 37°C for up to 12 weeks.

[0138] Materials, methods, and devices material: Anti-EPO mAb 8C10 (see U.S. Patent Application Publication No. 20130295113A1, Application No. 13 / 888,777) Formulated buffer: GMST: 10 mM sodium glutamate (from glutamic acid), 4% mannitol, 2% sucrose, 0.01% polysorbate 20, pH 5.2 HMST: 10 mM histidine, 4% mannitol, 2% sucrose, 0.01% polysorbate 20, pH 6.0 [Table 3] method: Size Exclusion Chromatography (SEC) Size exclusion (SEC) was used to monitor monomers and high molecular weight species (HMWS), such as dimers and aggregates. The method employed an Agilent 1100 (Name: KGB) HPLC with two columns (Tosoh G3000SWxl, 7.8 mm x 300 mm) mounted in series alongside a guard column (TSKgel Guard Column SWXL, 6.0 mm x 4.0 cm). The flow rate was 0.5 mL / min isocratic with a mobile phase consisting of 50 mM sodium phosphate; 300 mM sodium chloride, pH 6.8. Protein detection was monitored using UV detection at 215 nm. The percentage of area counts relative to total area counts was used for the % main peak and % HMW (high molecular weight) peak.

[0139] Reduced CE-SDS method The CE-SDS method was performed using a Beckman Coulter PA-800 (double-sided) with a 20 cm (working length) and 30 cm (total length) and 50 μm inner diameter, equipped with a 100×800 orifice and bare fused silica, along with a Beckman SDS-MW kit. Data was collected using Beckman Karat 32 software. Peak integration was performed using Chromeleon, and % purity was calculated based on the corrected area using Excel. Using a 30K MWCO Nanospin, 1.0 mL of 100 mcg / mL samples of GMST100 and HMST100, and 200 μL of 500 mcg / mL samples of GMST500 and HMST500, respectively, were concentrated to a final volume of 45 μL. 50 μL of sample buffer and 5 μL of 2-mercaptoethanol were added, heated at 70°C for 10 minutes, and then loaded into PCR sample tubes for the CE-SDS assay.

[0140] DSC The thermal stability of the samples was assessed by DSC on a MicroCal VP-Capillary DSC system, which continuously measures the temperature difference between the reference and sample cells and calibrates it to power units. This data channel is called the DP signal, or differential power between the reference and sample cells. Unfolding of the protein molecule appears as an endothermic transition on the DSC thermogram and corresponds to the thermal transition (melting) temperature (T m The DSC data can be characterized by the following: (1) The sample was heated from 4°C to 110°C at a heating rate of 60°C / h. The pre-scan time was 15 min and the filter period was 10 s. The concentrations used in the DSC experiments were 0.1 and 0.5 mg / mL. Data analysis was performed using MicroCal Origin 7 software.

[0141] Analytical Ultracentrifugation (AUC) Analysis Samples were analyzed using a Beckman Coulter ProteomeLab XL-I instrument. Anti-EPO mAb samples were stored at 4°C prior to analysis and analyzed undiluted. Sedimentation velocity experiments were performed at 45,000 rpm, and absorbance at 280 nm was monitored. Experiments were performed in a double-sector centerpiece cell assembly equipped with quartz windows. Scans were collected at 20°C with 120 scans per sample, with no delay between scans. AUC-SV data were analyzed using Sedfit v11.8 using the "Continuous c(s) distribution" model. Parameters for the c(s) analysis of variance were: s range 2-30, resolution 200, and data selection for analysis 6.4-6.8. Friction ratio, time-invariant noise, and meniscus position were allowed to emerge in the AUC-SV analysis during nonlinear least-squares fitting. For the AUC-SV distribution, the following parameters were used: partial specific volume 0.73 mL / g, density and viscosity of both Glu and His buffers were 1.02100 g / mL and 0.01210 Pa.

[0142] Sample preparation method The samples were dialyzed against two formulation buffers at 2-8°C for 3 days. After sample dialysis, the sample concentrations were measured using A280nm. The samples were diluted or concentrated to 100 μg / mL and 500 μg / mL with the two formulation buffers. The samples were filtered through a 0.22 μm filter, and 1 mL of sample was dispensed into 3 cc vials, which were then stoppered. A portion of the sample was used for pre-freeze time-zero testing. The samples were lyophilized according to the lyophilization process. After lyophilization, the samples were stored in a refrigerator until each time point. At each time point, the samples were reconstituted with 1 mL of water.

[0143] Freeze-drying procedure Sample vials were loaded onto a "pre-chilled" (4°C) shelf of the freeze dryer. The samples underwent three steps: freezing, primary drying, and secondary drying. First, the samples were held at 4°C for 30 minutes, cooled from 4°C to -45°C over 123 minutes, and held at 45°C for 180 minutes. After the 180-minute hold at -45°C, the shelf temperature was increased to -12°C over 150 minutes. Next, after a 240-minute hold at -12°C, the temperature was reduced again to -45°C over 150 minutes. The samples were brought to -45°C over 120 minutes. For primary drying, the shelf temperature was increased to -10°C, the condenser temperature was reduced to less than -50°C, and the chamber vacuum was maintained at 120 mTorr for 25 hours. For secondary drying, the shelf temperature was increased to 25°C over 234 minutes while the chamber pressure was reduced to 100 mTorr. The shelf temperature was then increased to 25°C over 11 hours while the chamber pressure was maintained at 100 mTorr. After all cycles were completed, the vials were stoppered inside the drying chamber.

[0144] Results and Discussion Size Exclusion Chromatography (SEC) Both lyophilized samples showed a slight increase in the % low molecular weight species (LMW%) up to 12 weeks of storage for both 4°C and 37°C storage (Figure 32). Based on the 7-week data at the higher temperature (37°C), samples in the histidine formulation showed a lower % LMW at 100 μg / mL. The histidine formulation also showed slightly lower % high molecular weight species (HMW%) than samples in the glutamate formulation at the higher temperature (Figure 33). However, there was no difference before and after lyophilization. Overall, samples in the histidine formulation showed a higher % main peak than in the glutamate formulation (Figure 34). Due to sample availability, the histidine formulation was not evaluated at 12 weeks.

[0145] Decomposition by capillary electrophoresis (CE-SDS) This analytical method is used to analyze heavy chains (HC), light chains (LC), nonglycosylated HC (NGHC), and other minor peak species under reducing and denaturing conditions. Reduced CE-SDS separates proteins based on differences in their hydrodynamic size under reducing and denaturing conditions. Protein species are bound to the anionic surfactant SDS and electrokinetically injected into a bare fused silica capillary filled with SDS gel buffer. Under a voltage applied across the capillary, the SDS-coated proteins are separated by their differential migration in the hydrophilic polymer-based solution. Proteins are detected by a photodiode array (PDA) detector as they pass through a UV detection window. Purity is assessed by determining the calibrated peak area percent of each component. Using the reduced CE-SDS method, there were no significant differences between the preparations for % heavy chain (Figure 35), % light chain (Figure 36), and % nonglycosylated heavy chain (Figure 37). The overlaid electropherograms shown in Figure 38 demonstrated that there were no detectable degradation species observed in all of the conditions tested at 7 weeks.

[0146] Thermal stability by differential scanning calorimetry (DSC) Differential scanning calorimetry (DSC) was used to analyze anti-EPO mAb samples in two different buffers. This technique allows the relative thermal stability of each sample to be compared. DSC scans of four pre-frozen samples in GMST and HMST buffers are shown in Figure 39, and the thermal transition temperatures are listed in Table 3. Typical standard deviations for thermal transition temperature measurements were within ±0.5°C. The DSC data suggested that the thermal stability of the pre-frozen samples was similar in both buffers and at both concentrations. Comparisons of DSC scans of each anti-EPO mAb sample at different conditions and time points are shown in Figures 40-43, and the thermal transition temperatures are also listed in Table 4. Once again, the DSC data suggested that the thermal stability of the samples remained similar in both buffers and at both concentrations and temperatures over time periods up to 12 weeks. After 7 weeks, the C of each sample H The two domains appeared to unfold at a relatively low temperature compared to that at time 0. Further studies suggested that the difference could be due to analyzing the samples at different times. [Table 4-1] [Table 4-2]

[0147] Analytical ultracentrifugation (AUC) analysis of aggregates in anti-EPO mAb The analytical ultracentrifugation sedimentation velocity (AUC-SV) method was used to determine the size distribution of anti-EPO mAb samples. The main advantage of AUC-SV is that the analysis is performed in the actual formulation buffer and there is no concern about aggregate loss to the column matrix or filter. Therefore, AUC-SV can also detect weakly associated reversible high molecular weight species (HMWS). AUC-SV can characterize, size, and quantify aggregate content of both covalently and noncovalently bound dimers and larger aggregates.

[0148] AUC-SV is an inherently highly variable technique. Standard AUC-SV analysis was performed in triplicate at a protein concentration of 0.5 mg / mL. Some of the anti-EPO mAb samples were available only at 0.1 mg / mL, but analysis at concentrations below 0.5 mg / mL typically results in higher variability. Samples formulated at 0.5 mg / mL were available in small quantities, and therefore, analysis was performed in only one replicate. After Biacore analysis, only three of the 0.5 mg / mL samples were available, which were analyzed in duplicate. Additionally, only the Glu sample at T=12 weeks was available.

[0149] High-resolution sedimentation coefficient distributions c(s) of anti-EPO mAb samples are illustrated in Figure 44. The vertical axis of the graph shows the concentration distribution, and the horizontal axis shows the separation of species based on their sedimentation coefficients. The graph has been magnified 10x to better visualize the small fraction of higher molecular weight species. All analyzed samples had very similar distribution patterns, with the major HMWS detected in all replicate measurements being dimers, and minor amounts of trimers were detected in some replicates. The size distribution patterns (not shown) after T = 7 weeks and T = 12 weeks were similar to those at T = 0 weeks illustrated in Figure 13. Trace amounts of LMWS species were detected in many replicates.

[0150] All results of the AUC-SV analysis are summarized in Table 5 and also represented graphically in Figure 45 for easier interpretation. As can be seen from the data, HMWS content in all samples varied within a range of 2-5%, and due to the fairly high variability of the method (for reasons discussed above), no conclusions can be drawn about the effect of buffer composition (Glu vs. His) or protein concentration (0.1 vs. 0.5 mg / mL). The only observable effect was an increase in LMWS content in the His buffer samples; pre-freeze samples contained trace amounts of LMWS below 0.1%, which increased to 0.7 ± 0.4% in the post-freeze samples. There is also indication of an increase in HMWS content in the Glu buffer (7.1%) after 12 weeks at 37°C; unfortunately, the corresponding His-37°C-12-week sample was not available. A recommendation from this set of experiments would be to perform a much longer-term experiment (at least several times longer than this experiment) at higher temperatures (37°C) to observe the possible effects of buffer composition and protein concentration on long-term stability.

[0151] In summary, no detectable effect of buffer composition (Glu vs. His) or protein concentration (0.1 vs. 0.5 mg / mL) was observed by AUC-SV during the 7-week experiment. [Table 5-1] [Table 5-2] [Table 5-3]

[0152] conclusion In conclusion, using SEC, AUC, reduced CE-SDS, and DSC, the two lyophilized formulations maintained antibody stability for at least 12 weeks of storage at 4°C and 37°C.

[0153] Each reference cited herein is incorporated by reference in its entirety for all that it teaches and for all purposes.

[0154] The present invention is not limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention; functionally equivalent methods and components are also inventive. Indeed, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Examples of embodiments of the present invention include the following: (Item 1) 1. A solid protein formulation comprising a stabilizer, a sugar alcohol, a sugar, and a surfactant, wherein the stabilizer is selected from the group consisting of histidine, tryptophan, methionine, leucine, phenylalanine, serine, glutamic acid, arginine, or lysine; the sugar alcohol is selected from the group consisting of mannitol, xylitol, sorbitol, maltitol, lactitol, glycerol, erythritol, or arabitol; the sugar is selected from the group consisting of sucrose, maltose, lactose, glucose, fructose, or galactose; and the surfactant is selected from the group consisting of polysorbate-80, polysorbate-20, polysorbate-21, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-81, or polysorbate-85. (Item 2) 2. The formulation of item 1, wherein the stabilizer is histidine. (Item 3) 3. The formulation according to item 2, wherein the concentration of histidine is about 0.1 mM to about 100 mM before solidification. (Item 4) 4. The formulation of item 3, wherein the concentration of histidine is about 10 mM before solidification. (Item 5) 2. The formulation of item 1, wherein the sugar alcohol is mannitol. (Item 6) 6. The formulation according to item 5, wherein the concentration of mannitol is about 0.1% to about 10% before solidification. (Item 7) 7. The formulation according to item 6, wherein the concentration of mannitol is 2% before solidification. (Item 8) 2. The formulation of item 1, wherein the sugar is sucrose. (Item 9) 9. The formulation of item 8, wherein the concentration of sucrose is about 0.05% to about 5% before solidification. (Item 10) 10. The formulation of item 9, wherein the concentration of sucrose is 0.5% before solidification. (Item 11) 2. The formulation of item 1, wherein the surfactant is polysorbate-80. (Item 12) Item 12. The formulation of item 11, wherein the concentration of polysorbate-80 is about 0.001% to about 0.01% before solidification. (Item 13) 13. The formulation of item 12, wherein the concentration of polysorbate-80 is 0.005% before solidification. (Item 14) 2. The formulation according to item 1, wherein the pH is about 6.0 to about 8.0 before solidification. (Item 15) 15. The formulation according to item 14, wherein the pH is 7.0 before solidification. (Item 16) 2. The formulation of item 1, wherein the concentration of the protein before solidification is from about 0.1 mg / ml to about 100 mg / ml. (Item 17) 17. The formulation of item 16, wherein the concentration of the protein is about 2 mg / ml before solidification. (Item 18) 2. The formulation of item 1, wherein the protein is a glycoprotein. (Item 19) 19. The formulation of item 18, wherein the protein is darbepoetin alfa. (Item 20) 19. The formulation of any of items 18, wherein the protein is an antibody. (Item 21) 21. The formulation of item 20, wherein the antibody is a monoclonal antibody. (Item 22) 21. The formulation of item 20, wherein the antibody is a polyclonal antibody. (Item 23) Item 1. The formulation according to item 1, which is lyophilized. (Item 25) 24. The formulation of item 23, which is reconstituted with a liquid. (Item 26) A solid protein formulation containing darbepoetin alfa, histidine, mannitol, sucrose, and polysorbate-80. (Item 27) 27. The formulation of item 26, wherein prior to solidification, the histidine concentration is 10 mM, the mannitol concentration is 2%, the sucrose concentration is 0.5%, and the polysorbate-80 concentration is 0.005%. (Item 28) 27. The formulation of items 1 or 26, wherein the protein is stable. (Item 29) 29. The formulation of item 28, wherein the protein is stable for at least one month. (Item 30) 29. The formulation of item 28, wherein the protein is stable for at least 3 months. (Item 31) 29. The formulation of item 28, wherein the protein is stable for at least 6 months. (Item 32) 29. The formulation of item 28, wherein the protein is stable for at least 12 months. (Item 33) 29. The formulation of item 28, wherein the protein is stable for at least 18 months. (Item 34) 29. The formulation of item 28, wherein the protein is stable for at least 24 months. (Item 35) 29. The formulation of item 28, wherein the protein is stable at about 4 to 45°C. (Item 36) 29. The formulation of item 28, wherein the protein is stable at about 4°C. (Item 37) 29. The formulation of claim 28, wherein the protein is stable at about 29°C. (Item 38) 29. The formulation of item 28, wherein the protein is stable at about 37°C. (Item 39) 29. The formulation of item 28, wherein the protein is stable at about 45°C. (Item 40) 27. The formulation of item 1 or 26, wherein the percent oxidation of the protein is about 5% to about 10%. (Item 41) 27. The formulation of item 1 or 26, wherein the percent oxidation of the protein is less than about 10%. (Item 42) 27. The formulation of item 1 or 26, wherein the percent oxidation of the protein is less than about 5%. (Item 43) 27. A method for preparing a solid protein formulation according to item 1 or 26, comprising: a) diluting the protein with lyophilization buffer; and b) lyophilizing the diluted protein The method comprising: (Item 44) 27. A kit comprising the solid protein formulation according to item 1 or 26 and a reconstitution buffer. (Item 45) A solid protein formulation comprising a monoclonal antibody, monosodium glutamate, mannitol, sucrose, and polysorbate 20. (Item 46) A solid protein formulation comprising a monoclonal antibody, histidine, mannitol, sucrose, and polysorbate 20. (Item 47) 47. The protein formulation according to item 45 or 46, wherein the concentration of the monoclonal antibody before solidification is about 100 μg / ml to about 500 μg / ml. (Item 48) 46. ​​The protein formulation of item 45, wherein the pH is about 5.2 before solidification. (Item 49) 47. The protein formulation of item 46, wherein the pH is about 6.0 before solidification. (Item 50) 46. ​​The protein formulation of item 45, wherein the concentration of monosodium glutamate is about 10 mM before solidification. (Item 51) 47. The protein formulation of item 46, wherein the concentration of histidine is about 10 mM before solidification. (Item 52) 47. The protein formulation of item 45 or 46, wherein the concentration of mannitol is about 4% before solidification. (Item 53) 47. The protein formulation of item 45 or 46, wherein the concentration of sucrose is about 2% before solidification. (Item 54) 47. The protein formulation of item 45 or 46, wherein the concentration of polysorbate 20 is about 0.01% before solidification.

Claims

[Claim 1] The method described in the specification.

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