Room temperature stable lyophilized protein

Abstract

Stable lyophilized therapeutic protein compositions and methods for their production are provided. Specifically, the use of water as a cake plasticizer and protein stabilizer is described. Also described is the inclusion of a multi-component stabilizer containing larger and smaller molecular entities. The inclusion of post-drying annealing under certain conditions also improves protein stability. Proteins are predicted to remain stable for up to 24 months at 25°C.

Description

[Technical field]

[0001] The present invention relates generally to the field of pharmaceutical formulations of biological molecules. In particular, the present invention relates to stable lyophilized therapeutic protein formulations. [Background technology]

[0002] Therapeutic macromolecules such as antibodies and receptor Fc fusion proteins must be formulated to not only make the molecules suitable for administration to patients, but also to maintain their stability during long-term storage. For example, therapeutic proteins (e.g., antibodies) in solution are prone to aggregation, chemical modification, or other forms of degradation unless the solution is properly formulated. The stability of a therapeutic protein in a liquid formulation depends not only on the types of excipients used in the formulation, and the amounts and ratios of those excipients relative to each other, but also on the concentration of the soluble protein and the method of manufacture. Considerations other than stability must also be taken into account when preparing a therapeutic protein formulation. These considerations include the viscosity of the solution and the concentration of the antibody that can be accommodated by a given formulation. Thus, great care must be taken when formulating a therapeutic protein to arrive at a formulation that remains stable over time at storage temperatures, contains an appropriate concentration of the antibody or other therapeutic protein, and has other properties that allow the formulation to be conveniently administered to patients.

[0003] Liquid formulations of therapeutic proteins are generally designed to provide long-term stability to proteins when frozen or refrigerated, but often fail to provide long-term stability at room temperature. One solution known in the art to maintain protein stability and retain the therapeutic activity of proteins is to lyophilize the molecules. Lyophilization (freeze-drying under controlled conditions) is commonly used for long-term storage of proteins. Lyophilized proteins are substantially resistant to degradation such as aggregation, oxidation, and other denaturing processes while in the freeze-dried state (see, for example, U.S. Patent No. 5,333,636). Lyophilization provides a dry "cake" that remains relatively stable at room temperature for a relatively long period of time. Room temperature stability is particularly important in storing and distributing therapeutic proteins around the world, especially in places where electricity and refrigeration are unreliable.

[0004] Lyophilization protection agents (also known as stabilizers), such as sucrose and trehalose, are often included in pre-lyophilization formulations to protect proteins from denaturation during the freeze-drying process.Plasticizers may also be included to reduce the overall relaxation time, and in some cases may help maintain the native structure of proteins.Plasticizers include sugar alcohols such as sorbitol and glycerol, other polyols, and small amounts of water.

[0005] In a study designed to optimize storage of lyophilized proteins at 5° C., Chang et al. investigated the effect of plasticizers on the stability of protein formulations. In lyophilized cakes containing a 1:1 sucrose-to-protein weight ratio (40 mg / mL protein before lyophilization) and no added plasticizer, the aggregation rate constant for one month at 50° C. was reportedly greater than 1.5% at a moisture content of 2.4% and approximately 2% at a moisture content of 3.3% (ibid., 1451, FIG. 4). (Non-Patent Document 1). Experiments were also performed at 40° C. and 25° C., and data were presented only for the most severe of the three stress conditions. Chang et al. noted rare documented cases of optimal storage stability at intermediate moisture contents and suggested that the residual moisture content should be optimized during formulation development, rather than something that must simply be minimized. (Id., 1451; see also Non-Patent Document 2). High moisture levels were shown to reduce the chemical stability of the formulations in all of the studies cited by Chang et al. (Non-Patent Document 3).

[0006] None of these studies suggest long-term stability (greater than 1, 2, or 3 years) at temperatures above 5°C, let alone 25°C, for the formulations tested.

[0007] Lyophilized formulations of biotherapeutics have demonstrated long-term stability under certain conditions. Kallmeyer et al., US Pat. No. 5,399,433, describes stable lyophilized antibody formulations at low concentrations (e.g., up to 8 mg / mL pre-lyophilized solution) containing, among other excipients, sugars (up to 200 mg / mL after reconstitution [e.g., sucrose, lactose, maltose, raffinose, trehalose]), amino acids (1-100 mg / mL before lyophilization [e.g., arginine, lysine, ornithine]), surfactants (0.05-0.5 mg / mL after reconstitution [e.g., polysorbates and polyoxyethylene-polyoxypropylene polymers]), and optionally buffers (10-20 mM after reconstitution [e.g., phosphate, acetate, citrate]), and / or tonicity agents (e.g., NaCl, 30 mM or less after reconstitution). Kallmeyer discloses that the lyophilizates can be stored at room temperature (i.e., 18-23° C.) for up to two years while remaining stable. Here, stability is indicated by very little or no particle formation in the reconstituted lyophilisate, i.e., particles greater than 10 microns in size but less than 6000, or particles greater than 25 microns in size but less than 600.

[0008] Dix et al., US Patent No. 5,399, 2003 describes a stable lyophilized VEGF-Trap (also known as aflibercept) formulation that maintains biological activity for at least 3 months. The application discloses a pre-lyophilization solution containing 5-75 mg / mL of trap molecule, 5-50 mM histidine buffer, 0.1-3% polyethylene glycol (PEG, stabilizer), 0.25-3% glycine (as a bulking agent), and 0.5-6% sucrose (as a stabilizer). Optionally, the pre-lyophilization solution contains citrate buffer (0.05 mM) and / or 0.003%-0.005% polysorbate.

[0009] In addition to lyophilized protein formulations, spray drying has also been used to create dry protein formulations. Chen and Walsh (Patent Document 4) disclose dry micronized protein particles with a diameter range of 2 to 30 microns, and a median diameter of about 10 to 12 microns, and in some cases about 6 to about 7 microns. These particles can then be coated with a polymer to further stabilize the protein and allow for sustained release of the protein over an extended period of time in an aqueous environment. The pretreated protein solutions from which the micronized particles were formed contained either (1) 25 mg / mL protein and 0.1% polysorbate, (2) 25 mg / mL protein, or (3) 50 mg / mL protein, 10 mM phosphate, and 2% sucrose. It was demonstrated that the protein contained within the polymer-coated micronized protein particles remained stable for at least 14 days.

[0010] Post-drying annealing of protein lyophilisates has been shown to result in greater than 4% initial antibody aggregation at 50° C., with an aggregation rate constant of approximately 2% aggregation per month. (See, e.g., J. Immunol. 2003, 143:1311-1320.) The US FDA may allow a formulation with 4% initial aggregation to be marketed, but the rate of aggregation over time would be unacceptable for a marketed pharmaceutical formulation. There should be no visible change in the pharmaceutical formulation during its shelf life.

[0011] There remains a need for a universal dry protein formulation that remains stable at room temperature for extended periods of time.The universal dry protein formulation is particularly suitable for use as a liquid formulation, for combining with polymers to produce sustained release formulations or devices, and for reconstitution for implantation or delivery via a myriad of other routes.Now, applicants have developed an improved stable protein lyophilisate and a method for making the same. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] U.S. Patent No. 6,436,897 [Patent Document 2] W01998 / 022136A2 [Patent Document 3] W02006 / 104852A2 [Patent Document 4] W02013 / 07506A1 [Non-patent literature]

[0013] [Non-Patent Document 1] “Effect of Sorbitol and Residual Moisture on the Stability of Lyophilized Antibodies: Implications for the Mechanism of Protein Stabilization in the Solid State,” J.Pharma.Sci.94(7):1445-1454, (2005) [Non-Patent Document 2] Breen et.al., “Effect of Moisture on the Stability of a Lyophilized Humanized Monoclonal Antibody Formulation,” Pharma.Res.18(9):1345-1353(2001) [Non-Patent Document 3] Hsu et al. “Determining the Optimum residual Moisture in Lyophilized Protein Pharmaceuticals,”Develop.Biol.Standard 74:255-271(1991) [Non-Patent Document 4] Wang et al., “The Impact of Thermal Treatment on the Stability of Freeze-Dried Amorphous Pharmaceuticals:II.Aggregation in an IgG1 Fusion Protein,” 99(2) J.Pharma.Sci.683-700(2010) Summary of the Invention

[0014] A relatively high content of residual moisture can serve as an effective plasticizer for lyophilized therapeutic proteins (aka therapeutic proteins in the "solid state"), which confers surprising stability to the proteins for at least 24 months while stored at room temperature. In one aspect, a room temperature stable lyophilized therapeutic protein formulation is provided in a low mobility solid state having 0.5-10 percent moisture. In another aspect, a method of making the room temperature stable lyophilized therapeutic protein formulation is provided. Specifically, a residual moisture level of more than 2%, more than 3%, more than 4%, more than 5%, or more than 6% in the lyophilized cake, but less than 10%, less than 8%, less than 7%, or less than 6%, allows annealing at relatively high temperatures for extended periods at room temperature. Without being limited by any mechanism of action, the residual moisture content during the lyophilization process appears to allow alpha relaxation to occur while preventing the formation of early protein aggregates in pharmaceutical formulations at relatively high concentrations, preferably 50-200 mg / mL, and even more preferably 100-150 mg / mL.

[0015] In a first aspect, a pharma- ceutically acceptable lyophilized cake is provided that comprises a stable protein, excipients, and about 0.5% to about 10% water, about 3% to about 6% water, about 4% to about 7% water, about 5% to about 8% water by weight, about 3% by weight, about 4% by weight, about 4.5% by weight, or about 6% by weight water. The protein of the pharma- ceutically acceptable lyophilized cake remains stable for at least one month at room temperature, which may be 17-25°C, 20-25°C, or about 25°C. What is meant by "stable" is generally that less than 5%, less than 4%, less than 3%, less than 2%, less than 1% of the protein degrades within 18 months of storage at room temperature, or there is little or no degradation. A common degradation pathway for proteins is the formation of aggregates and other high molecular weight (HMW) species. HMW species can be detected by many known methods, such as size exclusion chromatography and native or denaturing electrophoretic gel mobility, preferably size exclusion chromatography. Degradation also includes the formation of chemical products, such as deamidated residues, reduced disulfide bonds, hydrolysis, and peptide fragmentation, which can also be measured by methods known in the art.

[0016] In one embodiment, about 2% or less of the protein is degraded after 24 months of storage at about 25° C. Degradation is determined by the percent change in high molecular weight species as measured by size exclusion chromatography. The lower limit of detectable degradation by this method is about 0.5% degradation, with an assay variability of about 0.2-0.3%.

[0017] The pharma- ceutically acceptable lyophilized cake may include one or more excipients in addition to protein and moisture. In one embodiment, the excipient includes a buffering agent. The buffering agent may be any buffering agent that maintains an optimal pH for protein stability. Histidine is such a buffering agent, which has a pKa of about 6.0 and is capable of effectively buffering between pH 4.8 and 7.2. In some embodiments, the excipient is histidine. In a preferred embodiment, histidine is present in the pharma- ceutically acceptable lyophilized cake at about 0.34% to about 2.04% by weight.

[0018] In one embodiment, the excipient comprises a stabilizer. The stabilizer includes various molecules such as polyols, sugars, amino acids, salts, or any combination thereof. Examples of useful stabilizers include sorbitol, glycerol, mannitol, trehalose, sucrose, arginine, alanine, proline, glycine, sodium chloride, or any combination thereof. In one embodiment, the stabilizer comprises about 19.9% ​​to about 82.2% of the weight of the pharma-ceutically acceptable lyophilized cake.

[0019] The term "stabilizer" refers to at least one chemical that is not a buffer or a protein in a pharma- ceutically acceptable lyophilized cake. In some embodiments, the term stabilizer refers to a combination of chemicals (i.e., two or more chemicals) that work together to stabilize a protein or other macromolecule. For example, the stabilizer can be sucrose, or the stabilizer can be a combination of sucrose and arginine. In some embodiments, a larger molecular weight chemical, such as, for example, sucrose or trehalose, is paired with a smaller molecular weight chemical, such as, for example, arginine, proline, alanine, glycine, mannitol, sorbitol, and / or glycerol. Without wishing to be bound by theory, the smaller chemical increases mobility and allows the protein to relax to a lower energy state.

[0020] In one embodiment, the stabilizer is sucrose alone, and the stabilizer comprises about 3% to about 15%, preferably about 5-11%, 4-7.5%, or 5-7.5% of the weight of the pharma- ceutically acceptable lyophilized cake, depending on the presence of other stabilizer components, as well as the amount of protein, water, and other excipients. In one embodiment, the weight ratio of protein to stabilizer is 1:1 to 3:1, preferably 1.2:1 to 2:1, and more preferably about 1.5:1.

[0021] In one embodiment, the stabilizer comprises sucrose in combination with another stabilizer. Those other stabilizers combined with sucrose include any one or more of arginine, sorbitol, mannitol, glycerol, and alanine. In one embodiment, the additional stabilizer comprises arginine, which may comprise about 4.83% to about 19.3% of the weight of the pharma- ceutically acceptable lyophilized cake. In some embodiments, the weight-to-weight ratio of sucrose to arginine ranges from about 3.2:1 to about 3.4:1. In another embodiment, the additional stabilizer comprises sorbitol, which may comprise about 8.07% to about 22.4% of the weight of the pharma- ceutically acceptable lyophilized cake. In another embodiment, the additional stabilizer comprises mannitol, which may comprise about 8.07% to about 22.4% of the weight of the pharma- ceutically acceptable lyophilized cake. In another embodiment, the additional stabilizer comprises glycerol, which may comprise about 4.23% to about 12.7% by weight of the pharma- ceutically acceptable lyophilized cake, hi yet another embodiment, the additional stabilizer comprises alanine, which may comprise about 4.11% to about 12.4% by weight of the pharma-ceutically acceptable lyophilized cake.

[0022] In another embodiment, the stabilizer comprises trehalose, which comprises from about 15.8% to about 70.2% by weight of the pharma- ceutically acceptable lyophilized cake, depending on the presence of other stabilizers, as well as the amount of protein, water, and other excipients.

[0023] In one embodiment, the stabilizer comprises trehalose in combination with another stabilizer. The other stabilizers combined with trehalose comprise any one or more of arginine, sorbitol, mannitol, glycerol, and alanine. In one embodiment, the additional stabilizer comprises arginine, which may comprise about 0.81% to about 14.3% of the weight of the pharma- ceutically acceptable lyophilized cake. In another embodiment, the additional stabilizer comprises sorbitol, which may comprise about 1.35% to about 22.4% of the weight of the pharma- ceutically acceptable lyophilized cake. In another embodiment, the additional stabilizer comprises glycerol, which may comprise about 0.69% to about 12.7% of the weight of the pharma- ceutically acceptable lyophilized cake. In yet another embodiment, the additional stabilizer comprises alanine, which may comprise about 0.69% to about 12.4% of the weight of the pharma- ceutically acceptable lyophilized cake.

[0024] In another embodiment, the excipient comprises a surfactant. The surfactant may comprise a non-ionic detergent such as fatty acylated polyethoxylated sorbitan. In one embodiment, the surfactant comprises a polysorbate in general, or polysorbate 80 in particular. In some embodiments, the pharma-ceutically acceptable lyophilized cake comprises from about 0.21% to about 0.96% by weight of a surfactant such as polysorbate 80.

[0025] In one embodiment, the protein is a therapeutic protein. In other embodiments, the therapeutic protein is an antigen-binding protein. Antigen-binding proteins encompass a diverse group of molecules including antibodies, antibody fragments, receptors, ligands, recombinant molecules including complementarity determining regions, ligands, and receptor domains. Antigen-binding proteins include a variety of other fusion (recombinant or chimeric) proteins, such as receptor-Fc fusion proteins including trap molecules. In certain embodiments, the protein is a therapeutic antibody, such as a recombinant human-like or humanized monoclonal antibody. Antibodies include hybrid antibodies as well as bispecific antibodies.

[0026] In one embodiment, the protein represents about 6.27% to about 63.7% by weight of the pharma- ceutically acceptable lyophilized cake. In some embodiments, the pharma-ceutically acceptable lyophilized cake contains about 6.27% to about 18.9% by weight of protein. In other embodiments, the pharma-ceutically acceptable lyophilized cake contains about 33.4% to about 63.7% by weight of protein.

[0027] Stabilizers are included in the pharma- ceutically acceptable lyophilized cake to help maintain protein stability. Thus, the pharma-ceutically acceptable lyophilized cake contains a specific ratio of stabilizer to protein. In some embodiments, the ratio of stabilizer to protein is about 0.22:1 to about 6.6:1 by weight. Various embodiments include a stabilizer to protein ratio selected from 0.44:1, 0.65:1, 0.87:1, 1.1:1, and 1.3:1, all by weight.

[0028] A pharma- ceutically acceptable lyophilized cake is contained. In some embodiments, the pharma-ceutically acceptable lyophilized cake is contained in a closed vial. The closure may be a stopper. In other embodiments, the pharma-ceutically acceptable lyophilized cake is contained in a syringe barrel. In yet other embodiments, the pharma-ceutically acceptable lyophilized cake is contained in one chamber of a dual chamber autoinjector.

[0029] In one embodiment, a pharma- ceutically acceptable freeze-dried cake is produced by combining a protein, a buffer, a non-ionic surfactant, and one or more stabilizers in water to produce a pre-lyophilized aqueous solution. The solution is then freeze-dried to produce a cake that contains less than 10% and more than 0.5% moisture. The freeze-dried (lyophilized) protein is in a "solid state." In a particular embodiment, the protein is a therapeutic recombinant human-like or humanized monoclonal antibody.

[0030] According to another aspect, a process is provided for producing a composition comprising a therapeutic protein and 10% or less water and 0.5% or more water.

[0031] In one embodiment, the process includes obtaining an aqueous sample containing protein and excipients in a container. The container can be a vial, a syringe barrel, or a chamber of a dual chamber autoinjector, among others. The container is open enough to allow outgassing of water vapor. The container containing the aqueous sample is placed in the chamber and heat is removed from the sample to achieve a first temperature where ice crystals form in the sample. Air is removed from the chamber to achieve a first pressure. Thermal energy is then applied to the sample to achieve a second temperature to allow removal of water from the sample by sublimation. After sublimation, residual water may remain trapped in the sample, thereby necessitating an additional second drying step. The second drying step is achieved by applying thermal energy to the sample while maintaining the first pressure in the chamber, thereby achieving a third temperature. At that temperature, water desorbs from the sample to achieve a moisture level of 10% or less and 0.5% or more.

[0032] In one embodiment, heat is removed from the aqueous sample during the initial chilling and primary drying steps at a rate of about 0.5° C. per minute. In one embodiment, the first temperature is about −45° C. In another embodiment, the first temperature is held for about 60 minutes. In yet another embodiment, the aqueous sample is held at 5° C. for about 30 minutes before reaching the first temperature.

[0033] In one embodiment, the primary drying step is performed at a second temperature of about -25°C. In one embodiment, the second temperature is achieved by increasing the shelf temperature at a rate of about 0.5°C per minute. In one embodiment, the second temperature is held for about 50 hours. In one embodiment, the chamber pressure during primary drying is about 100 mTorr.

[0034] In one embodiment, the secondary drying step is performed at a third temperature of about 35° C. In one embodiment, the ramp rate for heating is about 0.3° C. per minute. In one embodiment, the sample is held at the third temperature for about 6 hours.

[0035] After secondary drying, in one embodiment, the vials are stoppered at a chamber pressure of about 608,000 mTorr. In one embodiment, the chamber is backfilled with N2 gas prior to stoppering. In one embodiment, the vials are stoppered with Flurotec® coated 4432 / 50 butyl rubber lyophilization stoppers.

[0036] In one embodiment, the dried sample is annealed to relax the protein to a lower energy state and improve its overall stability. To anneal the sample, thermal energy is applied to the sample to achieve a fourth temperature. In some embodiments, the sample is held at the fourth temperature for at least about 24 hours, at least about 48 hours, or at least about 60 hours to achieve optimal effective relaxation (alpha and beta relaxation) of the protein. Once the protein has reached its optimal relaxed state, the container is closed.

[0037] In one embodiment, the fourth temperature, i.e., the annealing temperature, is below the glass transition temperature of the sample after the water desorption step. In a particular embodiment, the annealing temperature is about 70° C. In another particular embodiment, the annealing temperature is about 45° C. In one embodiment, the sample is maintained at the annealing temperature for about 72 hours. Since different proteins have different biophysical characteristics, in some embodiments, the fourth temperature is determined via modulated differential scanning calorimetry (MDSC). Here, a calorimeter is loaded with a lyophilized protein formulation sample that has undergone a secondary drying step. The sample is then subjected to step heating through the glass transition while the heat flow is monitored. The Tg is determined. The sample is then held at various sub-Tg temperatures for various times to induce enthalpy relaxation of the molecules in the lyophilized cake. The "relaxed" samples are then subjected to DSC or MDSC and the peak area due to enthalpy recovery (heat capacity as a function of temperature) is determined (see Luthra et al., "Effects of annealing on enthalpy relaxation in lyophilized disaccharide formulations: mathematical modeling of DSC curves," 97(8) J Pharm Sci.3084-99, 2008, and L. Thomas, "Modulated DSC® Paper #5: Measurement of Glass Transitions and Enthalpy Recovery," TA Instruments Publication TP 010, New Castle DE (available for download on the World Wide Web at tainstruments.com (accessed May 13, 2016)). Those sub-Tg temperatures and times that provide the optimal enthalpy relaxation are selected for the fourth (aka annealing) temperature and time. See also WQ, “Calorimetric analysis of cryopreservation and freeze-drying formulations,” 1257 Methods Mol. Biol. 163-79 (2015).

[0038] In one embodiment, the pre-lyophilized aqueous solution (ie, aqueous sample, aka, aqueous solution) comprises multiple excipients, such as one or more stabilizers, one or more buffering agents, and optionally one or more surfactants.

[0039] In one embodiment, the pre-lyophilized aqueous solution and the reconstituted lyophilized liquid formulation have a pH that helps maintain protein structure and function.In a particular embodiment, the pre-lyophilized and reconstituted liquid formulation have a pH of about 6.0±2. Molecules that buffer around pH 6 are considered useful in this embodiment.Thus, in one embodiment, the buffering agent comprises histidine.In a particular embodiment, the aqueous solution contains about 10 mM histidine.

[0040] In one embodiment, the pre-lyophilization aqueous solution contains at least one stabilizer. In some embodiments, the stabilizer or combination of stabilizers includes one or more of trehalose, sorbitol, glycerol, arginine, alanine, mannitol, sucrose, proline, NaCl, and glycine.

[0041] In one embodiment, the stabilizer or combination of stabilizers comprises sucrose. In a particular embodiment, the stabilizer comprises sucrose at a concentration of about 10% (w / v) in an aqueous solution. In some cases, sucrose is the only stabilizer. In other cases, the aqueous solution comprises 10% sucrose (w / v) as a stabilizer, plus about 3%±0.1% (w / v) arginine.

[0042] In other embodiments, the stabilizer comprises sucrose and at least one other molecular entity. In some embodiments, the aqueous solution comprises about 5% sucrose and one other stabilizer. In one particular embodiment, the aqueous solution comprises 5% sucrose and any one of: (a) about 1.3%±0.1% (w / v) alanine, (b) about 1.5%±0.1% (w / v) arginine, (c) about 1.34%±0.1% (w / v) glycerol, (d) about 2.66%±0.1% (w / v) mannitol, and (e) about 2.66%±0.1% (w / v) sorbitol.

[0043] In some embodiments, the stabilizer comprises sucrose and arginine in various concentrations and ratios. In a particular embodiment, the aqueous solution comprises about 7.5% sucrose and 2.3%±0.1% arginine. In another particular embodiment, the aqueous solution comprises about 12.5% ​​sucrose and 3.9%±0.1% arginine. In yet another particular embodiment, the aqueous solution comprises about 15% sucrose and 4.6%±0.1% arginine.

[0044] In some embodiments, the stabilizer comprises trehalose, either as the sole stabilizer or in combination with another stabilizer. In one embodiment, the stabilizer comprises trehalose at a concentration of about 10% (w / v) in an aqueous solution. In another embodiment, the aqueous solution comprises about 9.09% (w / v) trehalose and any one of 0.48% (w / v) sorbitol, 0.24% (w / v) glycerol, 0.28% (w / v) arginine, and 0.24% alanine % (w / v). In another embodiment, the aqueous solution comprises 8.33% (w / v) trehalose and any one of 0.89% (w / v) sorbitol, 0.45% (w / v) glycerol, 0.51% (w / v) arginine, and 0.43% (w / v) alanine. In another embodiment, the aqueous sample comprises 6.66% (w / v) trehalose and any one of 1.77% (w / v) sorbitol, 0.9% (w / v) glycerol, 1.03% (w / v) arginine, and 0.87% (w / v) alanine. In another embodiment, the aqueous sample comprises 5% (w / v) trehalose and any one of 2.66% (w / v) sorbitol, 1.34% (w / v) glycerol, 1.54% (w / v) arginine, and 1.3% (w / v) alanine.

[0045] The therapeutic protein of this process can be any therapeutic protein, including smaller peptides as well as larger proteins. In one embodiment, the therapeutic protein of the invention is greater than 100 kilodaltons, or greater than about 150 kilodaltons. The therapeutic protein included in the present invention can be an isolated endogenous protein, a heterologously expressed protein, and / or a recombinant protein, such as a chimeric fusion protein or a variant of an endogenous polypeptide. In one embodiment, the therapeutic protein is an antigen-binding protein. Antigen-binding proteins encompass any protein that binds to another molecular entity. For example, antigen-binding proteins include antibodies, bispecific antibodies, antibody fragments, ScFv fusion proteins, complementarity determining region (CDR)-containing proteins, ligands, receptors, ligand fragments, receptor fragments, fusion proteins comprising ligands and / or receptor domains, and receptor-Fc fusion proteins comprising trap molecules.

[0046] In one embodiment, the therapeutic protein is an antibody. In a particular embodiment, the therapeutic antibody is a monoclonal antibody. In a more specific embodiment, the therapeutic protein is a recombinant human-like or humanized antibody produced in a heterologous cell line. In another embodiment, the therapeutic protein is a receptor-Fc fusion protein. In another more specific embodiment, the therapeutic protein is a trap molecule, such as an aflibercept molecule (VEGF trap) or a rilonacept molecule (IL-1 trap).

[0047] In some embodiments, the antibody concentration in the aqueous solution is low, which is greater than zero (i.e., as low as or lower than 1 μg / mL), including a concentration range of 25 mg / mL or less. In one low concentration embodiment, the antibody concentration is about 2 mg / mL.

[0048] In other embodiments, the antibody concentration in the aqueous solution is moderate, which includes a concentration range of greater than 25 mg / mL and up to 100 mg / mL. In one embodiment of a moderate concentration, the antibody concentration is about 50 mg / mL.

[0049] In other embodiments, the antibody concentration in the aqueous solution is high, including concentrations ranging from greater than 100 mg / mL to up to 200 mg / mL. In one embodiment of a high concentration, the antibody concentration is about 150 mg / mL.

[0050] In yet other embodiments, the antibody concentration in the aqueous solution is ultra-high, which includes antibody in a concentration range of greater than 200 mg / mL. In one ultra-high embodiment, the antibody concentration is about 205 mg / mL.

[0051] As mentioned above, but not limited by any mechanism of action, water can serve as a plasticizer for the lyophilized product, which surprisingly improves protein stability. Thus, in one embodiment, the moisture content of the produced composition (i.e., stable lyophilized formulation) is 2% by weight or more and 10% by weight or less. In another embodiment, the moisture content of the pharma-ceutically acceptable lyophilized cake is 3% by weight or more and 6% by weight or less. In another embodiment, the moisture content of the pharma-ceutically acceptable lyophilized cake is 4% by weight or more and 6% by weight or less. In a particular embodiment, the moisture content of the pharma-ceutically acceptable lyophilized cake is about 6%. In another particular embodiment, the moisture content of the pharma-ceutically acceptable lyophilized cake is about 4.5%. In yet another particular embodiment, the moisture content of the pharma-ceutically acceptable lyophilized cake is about 3%.

[0052] In one embodiment, the lyophilized protein is stable at room temperature for at least 24 months. In certain embodiments, about 2% or less of the pharma- ceutically acceptable lyophilized cake degrades after 24 months of storage at 25° C. Without being limited by any mechanism, degradation may involve proteolysis, chemical modification, aggregation, and the like. Aggregation is a common form of antibody degradation and is noted by the formation of high molecular weight (HMW) species. Degradation may be determined using any protein analysis known in the art or yet to be invented. In certain embodiments, antibody degradation is determined by measuring the percent change in high molecular weight (HMW) species by size exclusion (SE) chromatography. [Brief description of the drawings]

[0053] [Figure 1] 1 is a bar graph showing the effect of temperature and moisture on protein stability over time. The X-axis represents time and temperature in months. The Y-axis represents the percent change in the amount of high molecular weight species of protein. For each temperature, individual histograms represent the percent moisture content (w / w) increasing from left to right along the X-axis: 0% water (open bars), 0.5% water (light grey bars), 1.5% water (downward crosshatch bars), 3.0% water (vertical bars), 4.5% water (horizontal bars), 6% water (open diamond bars), 8% water (checkered), and 10% water (dark grey bars). [Diagram 2] 1 is a bar graph showing the effect of temperature and moisture on protein stability over time. The X-axis represents time and temperature in months. The Y-axis represents the percent change in the amount of high molecular weight species of protein. For each temperature, individual histograms represent the percent moisture content (w / w) increasing from left to right along the X-axis: 0% water (open bars), 0.5% water (light grey bars), 1.5% water (downward crosshatch bars), 3.0% water (vertical bars), 4.5% water (horizontal bars), 6% water (open diamond bars), 8% water (checkered), and 10% water (dark grey bars). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] Before the present invention is described, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention is limited only by the appended claims.

[0055] Absolute and relative amounts of excipients, ingredients, and other materials may be expressed by mass or moles. Mass units may be expressed as grams, milligrams, micrograms, etc.). The term "weight" in "weight / volume" or "w / v" means "mass." Relative amounts may be expressed as weight percent (i.e., mass percent), with 1 percent weight / volume (w / v) meaning 1 gram of material per 100 milliliters of volume. Also, for example, 1 part component "A" per 1 part component "B" by weight means, for example, that there is 1 gram of component "B" for every 1 gram of component "A." Also, for example, 1 weight percent (1%) of component "A" means, for example, that there is 1 gram of component "A" for every 100 grams of total mass of the particle. The relative amounts of components may also be expressed in terms of moles or molecules per given volume, such as millimoles (mM) per liter, or other per component units, e.g., X moiety component "A" per Y moiety component "B" by moles means that there are Y moles of "B" for every X moles of "A."

[0056] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described. All publications mentioned herein are incorporated by reference herein as though set forth in their entirety.

[0057] Excipients An excipient is an ingredient that is added along with the active drug substance in a pharmaceutical formulation. Excipients help stabilize the drug substance and / or add bulk to the formulation. The term ingredient is used interchangeably with excipient.

[0058] Excipients include a variety of substances for various purposes such as buffering, bulking, solubilizing, stabilizing, plasticizing, and protecting the drug substance. Protective agents protect against physical stresses such as heat stress and / or agitation. Buffering agents are well known in the art.

[0059] Generally, a buffering agent is included in the pre-lyophilized aqueous protein solution to stabilize the protein before lyophilization and after reconstitution. The buffering agent may be included in the pre-lyophilization solution at a concentration of 1 mM to 100 mM. In some particular embodiments, the buffering agent is included in the pre-lyophilization solution at about 10 mM. In certain embodiments, the buffering agent is present in the pre-lyophilization solution at a concentration of 5 mM ± 0.75 mM to 15 mM ± 2.25 mM, 6 mM ± 0.9 mM to 14 mM ± 2.1 mM, 7 mM ± 1.05 mM to 13 mM ± 1.95 mM, 8 mM ± 1.2 mM to 12 mM ± 1.8 mM, 9 mM ± 1.35 mM to 11 mM ± 1.65 mM, 10 mM ± 1.5 mM, or about 10 mM. In certain embodiments, the buffer system of the pre-lyophilization solution comprises 10 mM ± 1.5 mM histidine, phosphate, and / or acetate.

[0060] In some embodiments, the buffering agent is selected from chemicals capable of buffering anywhere within a pH range of about 3 to about 9, or about 3.7 to about 8.0. For example, the pre-lyophilization solution can have a pH of about 3.4, about 3.6, about 3.8, about 4.0, about 4.2, about 4.6, about 4.8, about 5.0, about 5.2, about 5.4, about 5.6, about 5.8, about 6.0, about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, or about 8.0.

[0061] The buffer may be a combination of individual buffers, such as, for example, a combination of histidine and acetate (His-acetate buffer). In one embodiment, the buffer has a buffer range of about 3.5 to about 6, or about 3.7 to about 5.6, such as the range buffered by acetate. In one embodiment, the buffer has a buffer range of about 5.5 to about 8.5, or about 5.8 to about 8.0, such as the range buffered by phosphate. In one embodiment, the buffer has a buffer range of about 5.0 to about 8.0, or about 5.5 to about 7.4, such as the range buffered by histidine.

[0062] Excipients include stabilizers. As used herein, stabilizers are added to pre-lyophilization solutions to stabilize proteins against aggregation or other degradation. Stabilization can occur by controlling glass dynamics during the lyophilization process or by helping to preserve the native structure of proteins through specific interactions between the stabilizer and the protein. For biophysical considerations of stabilizers during lyophilization, see Chang et al., "Mechanism of protein stabilization by sugars during freeze-drying and storage: native structure preservation, specific interaction, and / or immobilization in a glassy matrix?" 94(7) J.Pharm.Sci.1427-44(2005).

[0063] Stabilizing agents for inclusion in the pre-lyophilization solution include polyols, sugars, salts (e.g., sodium chloride), amino acids, etc. For optimal stabilizing effect, various individual stabilizers may be used alone or in combination with one or more other stabilizers. For example, sugars and polyols, amino acids and sugars, amino acids and polyols, sugars and salts, amino acids and salts, polyols and salts, etc. may be combined.

[0064] A polyol is an organic molecule with two or more hydroxyl groups (-OH). Polyols include polymers as well as monomers. Sugar alcohols are a subgroup of polyols. Sugar alcohols that may serve as useful stabilizers include mannitol, xylitol, sorbitol, isomalt, erythritol, maltitol, and glycerol. Other monomeric polyols include ethylene glycol, propylene glycol, and pentaerythritol. Polymeric polyols may be polyesters or polyethers of polyol subunits. Useful exemplary polymeric polyols include polypropylene glycol, polyethylene glycol, and poly(tetramethylene ether) glycol.

[0065] Sugars are used as stabilizers (as well as bulking agents). Sugars can be classified as either reducing or non-reducing sugars. Non-reducing sugars include the disaccharides sucrose and trehalose. Reducing sugars include glucose, maltose, and lactose. In general, non-reducing sugars are preferred for lyophilization of proteins because reducing sugars can reduce proteins via the Maillard reaction. See generally Lavakumar et al., “Lyophilization / Freeze Drying-A Review,” 3(4) Int. J. Novel Trends in Pharm. Sci. 2277-2782 (2013). The disaccharides trehalose and sucrose are relatively inert and tend to form amorphous glasses during lyophilization. Trehalose or sucrose, either alone or in combination with amino acids or polyols, are used as stabilizers in the practice of the present invention.

[0066] In one embodiment, trehalose is used as the only stabilizer. In other embodiments, trehalose is combined with a polyol. In some embodiments, the stabilizer is a combination of trehalose and sorbitol, or trehalose and glycerol. In other embodiments, trehalose is combined with an amino acid. Specifically, trehalose is combined with alanine, or trehalose is combined with arginine.

[0067] In other embodiments, sucrose is used as the only stabilizer. In other embodiments, sucrose is combined with a polyol. In certain embodiments, the stabilizer is a combination of sucrose and mannitol, sucrose and sorbitol, or sucrose and glycerol. In other embodiments, sucrose is combined with an amino acid. Specifically, sucrose is combined with arginine, or sucrose is combined with alanine.

[0068] Amino acids are used as stabilizers. Glycine is a commonly used bulking agent and stabilizer. Other useful amino acids include arginine, alanine, and proline. In some embodiments, arginine is used as stabilizer. In some specific embodiments, arginine is combined with sucrose or arginine is combined with trehalose. In other embodiments, alanine is used as stabilizer. In some specific embodiments, alanine is combined with sucrose or alanine is combined with trehalose.

[0069] In some cases, one or more surfactants may be used as excipients. Surfactants are believed to provide additional stability by reducing protein-protein hydrophobic interactions and the resulting formation of high molecular weight species (i.e., aggregates). In some embodiments, one or more surfactant(s) may be included in the pre-lyophilized protein-containing aqueous solution. In other embodiments, one or more surfactant(s) may be included in the reconstituted diluent solution. Surfactants include substances that reduce the surface tension of the fluid in which they are dissolved and / or reduce the interfacial tension between oil and water. Surfactants may be ionic or non-ionic. Exemplary non-ionic surfactants that may be included in the pre-lyophilized or reconstituted solutions include, for example, alkyl poly(ethylene oxide), alkyl polyglucosides (e.g., octyl glucoside and decyl maltoside), fatty alcohols such as cetyl alcohol and oleyl alcohol, cocamide MEA, cocamide DEA, and cocamide TEA. Specific non-ionic surfactants that may be included in the pre-lyophilized aqueous solution (or reconstituted solution) include, for example, polyoxyethylene sorbitan esters (also known as polysorbates), such as polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85; poloxamers, such as poloxamer 188 and poloxamer 407; polyethylene-polypropylene glycols; or polyethylene glycols (PEGs). Polysorbate 20 is also known as TWEEN 20, sorbitan monolaurate, and polyoxyethylene sorbitan monolaurate. Polysorbate 80 is also known as TWEEN 80, sorbitan monooleate, and polyoxyethylene sorbitan monooleate.

[0070] The amount of surfactant contained in the pre-lyophilization solution or reconstitution solution can vary depending on the particular properties and purpose desired for the lyophilized formulation. In certain embodiments, the pre-lyophilization solution or reconstitution solution can contain about 0.001% (w / v) to about 0.5% (w / v) of a surfactant (e.g., polysorbate 20 or polysorbate 80).For example, before freeze-drying, the concentration was about 0.001%, about 0.0015%, about 0.002%, about 0.0025%, about 0.003%, about 0.0035%, about 0.004%, about 0.0045%, about 0.005%, about 0.0055%, about 0.006%, about 0.0065%, about 0.007%, about 0.0075%, about 0.008%, about 0.0085%, about 0.009%, about 0.0095%, about 0.01%, about 0.015%, about 0.016%, about 0.017%, about 0.018%, about 0.019%, about 0.02%, about 0.021%, about 0.022%, about 0. 023%, approx. 0.024%, approx. 0.025%, approx. 0.026%, approx. 0.027%, approx. 0.028%, approx. 0.029%, approx. 0.03%, approx. 0.031%, approx. 0.032%, approx. 0.033%, approx. 0.034%, approx. 0.035%, approx. 0.036%, approx. 0.037%, approx. 0.038%, approx. 0.039%, approx. 0.04%, approx. 0.041%, approx. 0.042%, approx. 0.043%, approx. 0.044%, approx. 0.045%, approx. 0.046%, approx. 0.047%, approx. 0.048%, approx. 0.049%, approx. 0.05%, approx. 0.051%, approx. 0.052%, approx. 053%, approx. 0.054%, approx. 0.055%, approx. 0.056%, approx. 0.057%, approx. 0.058%, approx. 0.059%, approx. 0.06%, approx. 0.061%, approx. 0.062%, approx. 0.063%, approx. 0.064%, approx. 0.065%, approx. 0.066%, approx. 0.067%, approx. 0.068%, approx. 0.069%, approx. 0.07%, approx. 0.071%, approx. 0.072%, approx. 0.073%, approx. 0.074%, approx. 0.075%, approx. 0.076%, approx. 0.077%, approx. 0.078%, approx. 0.079%, approx. 0.08%, approx. 0.081%, approx. 0.082%, approx. In some embodiments, the composition may contain about 0.083%, about 0.084%, about 0.085%, about 0.086%, about 0.087%, about 0.088%, about 0.089%, about 0.09%, about 0.091%, about 0.092%, about 0.093%, about 0.094%, about 0.095%, about 0.096%, about 0.097%, about 0.098%, about 0.099%, about 0.10%, about 0.15%, about 0.20%, about 0.25%, about 0.30%, about 0.35%, about 0.40%, about 0.45%, or about 0.50% surfactant (e.g., polysorbate 20 or polysorbate 80).

[0071] One or more plasticizers are included in the lyophilized protein composition. Plasticizers are generally used to increase the fluidity or flexibility of a system. The increase in fluidity is believed to be the result of the plasticizer increasing the free volume of the system and decreasing the glass transition temperature. The addition of plasticizers modifies both alpha and beta relaxations in the lyophilized cake. Alpha relaxation, also known as first order relaxation or glass relaxation, is a global relaxation process. Beta relaxation is a more localized process related to protein polymer backbone motion that can be better modified by smaller molecules (i.e., plasticizers). Both relaxation processes are believed to reduce the overall energy of the system and, especially in the case of beta relaxation, affect protein stability. It is commonly known in the field of protein biophysics that plasticizers can reduce beta relaxation times and concomitantly reduce protein stability (see, for example, Cicerone and Douglas, “β-Relaxation governs protein stability in sugar-glass matrices,” Soft Matter 8:2983-2991, 2012).

[0072] The present invention also provides, in one aspect, embodiments of lyophilized protein formulations that contain one or more sugars combined with one or more plasticizers in a specific ratio to create a "stabilizer" that provides sufficient flexibility and alpha relaxation to the cake while maintaining or enhancing protein stability. In some embodiments, the stabilizer comprises a weight-to-weight (i.e., mass-to-mass) ratio of sugar to plasticizer of about 19:1 to about 1:1. In some embodiments, the stabilizer comprises a weight-to-weight ratio of sugar to plasticizer of about 19:1, about 18:1, about 17:1, about 16:1, about 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1.

[0073] Useful plasticizers include polyols such as sorbitol, glycerol (glycerin), mannitol, and xylitol, amino acids such as glycine, arginine, proline, and alanine, and salts such as NaCl. Interestingly, water can also function as a plasticizer. However, water is generally not preferred because it is both a chemical reactant and a plasticizer, which leads to increased mobility and decreased Tg. Increased mobility correlates with increased reactivity and hydrolysis in the system. Increased reactivity and hydrolysis compromise protein stability. See Terakita et al., “The Influence of Water on the Stability of Lyophilized Formulations with Inositol and Mannitol as Excipients,” 57(5) Chem.Pharm.Bull.459-463 (2009).

[0074] As noted above, mannitol, glycerol, sorbitol, and glycine in combination with water are used as plasticizers in some embodiments. Without being bound by theory, it is believed that these molecules in certain formulation embodiments shorten beta relaxation times while also enhancing protein stability.

[0075] In some particular embodiments, the sugar sucrose or trehalose is combined with the plasticizer sorbitol in a weight to weight ratio of about 4:1, about 3.9:1, about 3.8:1, about 3.7:1, about 3.6:1, about 3.5:1, about 3.4:1, about 3.3:1, about 3.2:1, about 3.1:1, about 3:1, about 2.9:1, about 2.8:1, about 2.7:1, about 2.6:1, about 2.5:1, about 2.4:1, about 2.3:1, about 2.2:1, about 2.1:1, about 2:1, about 2:1, about 1.9:1, about 1.8:1, about 1.7:1, about 1.6:1, about 1.5:1, about 1.4:1, about 1.3:1, about 1.2:1, about 1.1:1, or about 1:1. In other specific embodiments, the sugar sucrose or trehalose is combined with the plasticizer arginine in a weight to weight ratio of about 33:1, about 32:1, about 31:1, about 30:1, about 29:1, about 28:1, about 27:1, about 26:1, about 25:1, about 24:1, about 23:1, about 22:1, about 21:1, about 20:1, about 19:1, about 18:1, about 17:1, about 16:1, about 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4.5:1, about 4:1, about 3.5:1, or about 3:1.

[0076] The present invention also shows that, in one embodiment, water serves as a stabilizing plasticizer in the lyophilized composition, helping to reduce the rate of protein aggregation in the lyophilized composition stored at room temperature. The amount of moisture required to effectively stabilize the protein in the lyophilized cake at room temperature increases with increasing protein content and decreases with decreasing protein content. Thus, a lyophilized composition obtained from a pre-lyophilized solution containing 50 mg / mL of protein may require a lower moisture content than a lyophilized composition obtained from a pre-lyophilized solution containing 150 mg / mL of protein. Also, the optimal amount of moisture required to effectively stabilize the protein in the lyophilized cake varies with storage temperature. For example, an intermediate amount of moisture is beneficial for long-term storage of protein lyophilisates at room temperature. At higher storage temperatures (e.g., 37°C), less moisture is required.

[0077] For example, FIG. 1 shows the effect of moisture on the stability of an exemplary antibody lyophilized formulation stored at 25° C., 37° C., or 50° C., where the pre-lyophilized formulation contained 150 mg / mL IgG, 5% sucrose, and 1.54% arginine. The final lyophilized cake contained 0%, 0.5%, 1.5%, 3.0%, 4.5%, 6%, 8%, or 10% moisture (w / w). As shown in FIG. 1, higher storage temperatures resulted in lower protein stability over 2-3 months. Interestingly, the optimal amount of moisture resulting in maximum stability was higher at room temperature than at 37° C. or 50° C. In this particular example, the optimal moisture content for protein stability at room temperature was about 4.5% (w / w) and about 1.5% to about 3% for 2 months storage at 50° C. Longer term stability data is presented in Figure 2, which shows an exemplary antibody lyophilized formulation stored at 25° C. and 37° C. for 12 and 6 months, respectively. As shown in Figure 2, moisture contents of 3.0% to 4.5% (w / w) provided the best stability at both temperatures tested, with a moisture content of 4.5% providing the greatest stability at 12 months under room temperature (25° C.) conditions.

[0078] In one embodiment, the invention provides a stable lyophilized protein (e.g., antibody) formulation comprising 1.5%-8% water (w / w) that remains stable at room temperature (25° C.) for at least 12 months, where stability refers to less than a 2% increase in high molecular weight species over a 12 month storage period. In another embodiment, the invention provides a stable lyophilized protein (e.g., antibody) formulation comprising 3.0%-4.5% water (w / w) that remains stable at room temperature (25° C.) for at least 12 months, where stability refers to less than a 1.5% increase in high molecular weight species over a 12 month storage period.

[0079] In some embodiments, the lyophilized composition contains 0.5% or more water by weight, 0.6% or more water by weight, 0.7% or more water by weight, 0.8% or more water by weight, 0.9% or more water by weight, 1% or more water by weight, 1.5% or more water by weight, 2% or more water by weight, 2.5% or more water by weight, 3% or more water by weight, 3.5% or more water by weight, 4% or more water by weight, 4.5% or more water by weight, 5% or more water by weight, 5.5% or more water by weight, 6% or more water by weight, 6.5% or more water by weight, 7% or more water by weight, 7.5% or more water by weight, 8% or more water by weight, 8.5% or more water by weight, 9% or more water by weight, or 9.5% or more water by weight, but not more than 10% by weight. However, water in excess of the optimal amount may increase protein instability. Thus, in some embodiments, the lyophilized composition contains 10% or less by weight water, 9.5% or less by weight water, 9% or less by weight water, 8.5% or less by weight water, 8% or less by weight water, 7.5% or less by weight, 7% or less by weight water, 6.5% or less by weight water, 6% or less by weight water, 5.5% or less by weight water, 5% or less by weight water, 4.5% or less by weight water, 4% or less by weight water, 3.5% or less by weight water, 3% or less by weight water, 2.5% or less by weight water, 2% or less by weight water, 1.5% or less by weight water, 1% or less by weight water, 0.9% or less by weight water, 0.8% or less by weight water, 0.7% or less by weight water, 0.6% or less by weight water, but more than 0.5% by weight water.

[0080] The phrase "moisture content" may be used interchangeably with "water content." However, "moisture content" is used to describe the water content of a lyophilized cake, whereas "water content" is used to describe the amount of water in an aqueous solution, gel, other liquid, gas, ice, or solid form formulation such as a lyophilized cake, spray-dried particles, etc. In some embodiments, the water content of the lyophilized composition is 0.5% to 10% by weight, 1% to 10% by weight, 2% to 10% by weight, 3% to 10% by weight, 4% to 10% by weight, 5% to 10% by weight, 6% to 10% by weight, 7% to 10% by weight, 8% to 10% by weight, 9% to 10% by weight, 0.5% to 9% by weight, 0.5% to 8% by weight, 0.5% to 7% by weight, 0.5% to 6% by weight, 0.5% to 5% by weight, 0.5% to 4% by weight, 0.5% to 3% by weight, 0.5% to 2% by weight, 0.5% to 1% by weight, 1% to 2 ... 0.5% to 2.5% by weight, for example, 2% to 3%, 2.5% to 3.5%, 3% to 4%, 3.5% to 4.5%, 4% to 5%, 4.5% to 5.5%, 5% to 6%, 5.5% to 6.5%, 6% to 7%, 6.5% to 7.5%, 7% to 8%, 7.5% to 8.5%, 8% to 9%, 8.5% to 9.5%, 9% to 10%, 9.5% to 10% by weight, or about 1%, 2%, 3%, 4%, 5%, 6%, 7% or 8% by weight. In some embodiments, the water content of the lyophilized composition is between 3% and 6%, between 3.1% and 5.9%, between 3.2% and 5.8%, between 3.3% and 5.7%, between 3.4% and 5.6%, between 3.5% and 5.5%, between 3.6% and 5.4%, between 3.7% and 5.3%, between 3.8% and 5.2%, between 3.9% and 5.1%, between 4% and 5%, between 4.1% and 4.9%, between 4.2% and 4.8%, between 4.3% and 4.7%, between 4.4% and 4.6%, or about 4.5% by weight.In some embodiments, the water content of the lyophilized composition is between 2% and 4% by weight, between 2.1% and 3.9% by weight, between 2.2% and 3.8% by weight, between 2.3% and 3.7% by weight, between 2.4% and 3.6% by weight, between 2.5% and 3.5% by weight, between 2.6% and 3.4% by weight, between 2.7% and 3.3% by weight, between 2.8% and 3.2% by weight, between 2.9% and 3.1% by weight, or about 3% by weight.

[0081] In some embodiments, the weight percent water content in the lyophilized cake is about 3%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 5.6%, about 5.7%, about 5.8%, about 5.9%, about 6%, about 6.1%, about 6.2%, about 6.3%, about 6.4%, about 6.5%, about 6.6%, about 6.7%, about 6.8%, about 6.9%, about 7%, about 7.1%, about 7.2%, about 7.3%, about 7.4%, about 7.5%, about 7.6%, about 7.7%, about 7.8%, about 7.9%, or about 8%. In some embodiments, the moisture content of the lyophilized cake is greater than 3% and less than 10% by weight.

[0082] The moisture content of the lyophilized cake can be determined by any one or more methods known in the art. These methods include thermogravimetric analysis, gas chromatography, near infrared spectroscopy, coulometry, and gravimetric methods including Karl Fischer method or relative humidity sensor method. Some of these methods are reviewed in JKTownes, "Moisture content in proteins: its effects and measurement," 705 J. Chromatography A 115-127, 1995, and Malik et al., "Analytical Options for the Measurement of Residual Moisture Content in Lyophilized Biological Materials," Am. Pharma. Rev. August 01, 2010, and references cited therein. For example, the loss on drying (LOD) method (weight) can be used, in which the lyophilized cake is weighed, subjected to additional heating to completely remove all water and other volatiles, and then weighed again. The mass loss is due to the water (and other volatiles) contained in the starting material. Another method for determining water content is the Karl Fischer method (volumetric or coulometric), which determines the amount of H2O by measuring the oxidation of SO2 by I2: one mole of I2 consumed per mole of H2O. Near-infrared spectroscopy measures the reflectance from 1100 nm to 2500 nm through a glass vial containing the protein (a glass surface) to determine the water content without destroying the sample.See United States Pharmacopeia, XXIII Revision, USP Convention, Rockville, MD 1995, pp. 1801-1802, and Savage et al., “Determination of Adequate Moisture Content for Efficient Dry-Heat Viral Inactivation in Lyophilized Factor VIII by Loss on Drying and by Near Infrared Spectroscopy,” 26 Biologicals 119-124, 1998.

[0083] Freeze-dried cake The lyophilized composition containing the protein and stabilizer forms a solid matrix, also known as a "cake" or "lyophilized cake." A "pharmacologically acceptable cake" (used interchangeably with "pharmacologically acceptable lyophilized cake") is amorphous (not glassy, ​​crystalline) and has an aesthetically elegant appearance. A pharma- ceutically acceptable cake should not exhibit shrinkage, cracking, partial or total collapse, meltback, or discoloration. Red, black, brown, yellow, or other lightly colored cakes are discolored and unacceptable. An ideal cake is mechanically strong, resistant to breakage during handling, porous and spongy, uniform to the touch, and forms a single entity, and is uniformly white. The cake should adhere uniformly to the walls of the vial and should not show any signs of delamination or other shrinkage. See Carpenter et al., "Rational design of stable lyophilized protein formulations: Some practical advice," Pharmaceutical Research, 14(8):969-975, 1997.

[0084] The cake should be free of visual defects due to freezing problems, including chimney-like structures, dried foam on the top region, crusting or glazing on the cake surface, and horizontal stratification or ring formation. The cake should be free of visual defects due to drying problems, including shrinkage, where the cake volume is smaller than the frozen matrix and signs of wall delamination are evident, cracking, where the cake shows cracks in the drying matrix and the cake does not form a single entity, loss of cake structure to different degrees, such as total or partial collapse of the cake, meltback, where the cake contains a ring of dissolved material in the lower region, partial meltback, where only a small region at the base of the cake contains dissolved material, and browning, which is a yellow or brown discoloration of the cake due to the inclusion of reducing sugars that have undergone the Maillard reaction. Meltback is particularly problematic as it can lead to slow dissolution times, protein aggregation, degradation, and reduced potency. See FDA, “Guide to Inspections of Lyophilization of Parenterals (7 / 93). Finished product inspection. Last update 2009,” 2009 (last accessed July 8, 2016 from http: / / www.fda.gov / ICECI / Inspection- tions / InspectionGuides / ucm074909.htm).

[0085] Protein Drug Substances The term "protein" refers to any amino acid polymer having more than about 50 amino acids covalently linked through amide bonds. Proteins contain one or more amino acid polymer chains, commonly known in the art as "polypeptides". Proteins may contain one or more polypeptides to form a single functional biomolecule. "Polypeptides" generally contain more than 50 amino acids, while "peptides" generally contain 50 or fewer amino acids. Proteins may contain one or more covalent and non-covalent modifications. Disulfide bridges (i.e., forming cystine between cysteine ​​residues) may be present in some proteins. These covalent bonds may be within a single polypeptide chain or between two individual polypeptide chains. For example, disulfide bridges are essential for the proper structure and function of insulin, immunoglobulins, protamines, etc. For a recent review of disulfide bond formation, see Oka and Bulleid, “Forming disulfides in the endoplasmic reticulum,” 1833(11) Biochim Biophys Acta 2425-9 (2013).

[0086] In addition to disulfide bond formation, proteins can be subject to other post-translational modifications, including lipidation (e.g., myristoylation, palmitoylation, farnesoylation, geranylgeranylation, and glycosylphosphatidylinositol (GPI) anchor formation), alkylation (e.g., methylation), acylation, amidation, glycosylation (e.g., addition of glycosyl groups to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, and / or tryptophan), and phosphorylation (i.e., addition of phosphate groups to serine, threonine, tyrosine, and / or histidine). For recent reviews of post-translational modifications of proteins produced in eukaryotes, see Mowen and David, “Unconventional post-translational modifications in immunological signaling,” 15(6) Nat Immunol 512-20 (2014), and Blixt and Westerlind, “Arraying the post-translational glycoproteome (PTG),” 18 Curr Opin Chem Biol. 62-9 (2014).

[0087] Immunoglobulins (also known as "antibodies") are examples of proteins with multiple polypeptide chains and extensive post-translational modifications. A standard immunoglobulin protein (e.g., IgG) contains four polypeptide chains, two light chains and two heavy chains. Each light chain is linked to one heavy chain via a cystine disulfide bond, and the two heavy chains are linked to each other via two cystine disulfide bonds. Immunoglobulins produced in mammalian systems are also glycosylated at various residues (e.g., asparagine residues) with various polysaccharides and can vary between species, which can affect the antigenicity of therapeutic antibodies (see Butler and Spearman, "The choice of mammalian cell host and possibilities for glycosylation engineering", 30 Curr Opin Biotech 107-112 (2014)).

[0088] As used herein, "protein" includes therapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, human antibodies, bispecific antibodies, antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. Proteins can be produced using recombinant cell-based production systems such as insect baculovirus systems, yeast systems (e.g., Pichia species), mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells). For a recent review discussing therapeutic proteins and their production, see Ghaderi et al., "Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation," 28 Biotechnol Genet Eng Rev. 147-75 (2012).

[0089] Some recombinant Fc-containing proteins contain receptors or receptor fragments, ligands or ligand fragments with cognate binding partners in biological systems. "Receptor Fc fusion protein" refers to a recombinant molecule containing a soluble receptor fused to an immunoglobulin Fc domain. Some receptor Fc fusion proteins may contain the ligand binding domains of multiple different receptors. These receptor Fc fusion proteins are known as "traps" or "trap molecules." Rilonocept and aflibercept are examples of commercially available traps that antagonize IL1R (see U.S. Pat. No. 7,927,583) and VEGF (see U.S. Pat. No. 7,087,411), respectively. Other recombinant Fc-containing proteins include recombinant proteins containing peptides fused to Fc domains, such as Centocor's MIMETIBODY™ technology. Recombinant Fc-containing proteins are described in C. Huang, “Receptor-Fc fusion therapeutics, traps, and MIMETIBODY technology,” 20(6) Curr. Opin. Biotechnol. 692-9 (2009).

[0090] An "Fc fusion protein" comprises part or all of two or more proteins (one of which is the Fc portion of an immunoglobulin molecule) that are not fused in their natural state. For example, an Fc fusion protein is a trap such as, for example, an IL-1 trap (e.g., Rilonacept, which contains the IL-1RAcP ligand binding domain fused to the IL-1R1 extracellular domain fused to the Fc of hIgG1; see U.S. Patent No. 6,927,004, which is incorporated herein by reference in its entirety), or a VEGF trap (e.g., Aflibercept, which contains the Ig domain 2 of the VEGF receptor Flt1, e.g., SEQ ID NO: 1, fused to the Ig domain 3 of the VEGF receptor Flk1, which is fused to the Fc of hIgG1; see U.S. Patent Nos. 7,087,411 and 7,279,159, which are incorporated herein by reference in their entirety).

[0091] In some embodiments, the protein is contained in the pre-lyophilization aqueous solution at a concentration of more than 40 mg / mL. In some embodiments, the pre-lyophilization aqueous solution contains a protein at a concentration of about 50 mg / mL to about 250 mg / mL, about 100 mg / mL to about 200 mg / mL, about 125 mg / mL to about 175 mg / mL. In some embodiments, the pre-lyophilization aqueous solution contains a protein at a concentration of about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, about 100 mg / mL, about 105 mg / mL, about 110 mg / mL, about 115 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, about 200 mg / mL, about 210 mg / mL, about 220 mg / mL, about 230 mg / mL, about 240 mg / mL, about 250 mg / mL, about 260 mg / mL, about 270 mg / mL, about 280 mg / mL, about 290 mg / mL, about 300 mg / mL, about 310 mg / mL, about 320 mg / mL, about 330 mg / mL, about 340 mg / mL, about 350 mg / mL, about 360 mg / mL, about 370 mg / mL, about 380 mg / mL, about 390 mg / mL, about 400 mg / mL, about 410 Contains a protein concentration of about 25 mg / mL, about 130 mg / mL, about 135 mg / mL, about 140 mg / mL, about 145 mg / mL, about 150 mg / mL, about 155 mg / mL, about 160 mg / mL, about 165 mg / mL, about 170 mg / mL, about 175 mg / mL, about 180 mg / mL, about 185 mg / mL, about 190 mg / mL, about 195 mg / mL, or about 200 mg / mL.

[0092] container In some embodiments, the pre-lyophilized aqueous solution containing the therapeutic protein is contained in a container. The freeze-drying process is applied to the solution in a semi-closed container, which is subsequently closed to contain the freeze-dried composition. The term container is applied very broadly herein. The container may be, for example, a bulk container such as a bottle, jar, or canister that can contain from 2 mL up to 4 liters or more, an ampoule, a vial (glass or plastic), a syringe (glass or plastic), a cartridge, or an auto-injector. A vial may be as small as 0.2 mL or less or as large as 100 mL. Exemplary vials may be made of clear or amber glass, type I borosilicate glass, type II sodium silicate glass, or type III sodium silicate glass. The vial may be closed with a stopper, cap, flip cap, or screw cap. SCHOTT® style tubular glass is particularly useful in freeze-drying applications.

[0093] With the advent of biological agents and patient self-administration of injectable drugs, autoinjectors have become a more important container for drug products. Self-administration of lyophilized drug products requires the patient to reconstitute the lyophilized cake with sterile water for injection or other sterile solvent. To help ensure sterile reconstitution, volume control, ease of handling, and overall simplification, dual or multi-chamber pre-filled syringes may be used. Dual-chamber autoinjectors or other pre-filled syringes contain a lyophilized drug product in one chamber and a pre-measured amount of diluent or liquid pharmaceutical composition in the other chamber. Exemplary dual-chamber injectors are described in U.S. Pat. No. 6,149,626A, issued Nov. 21, 2006, and U.S. Pat. No. 7,959,600B2, issued Jun. 14, 2011.

[0094] Protein stability Proteins in lyophilized form offer several advantages, one of which is maintaining protein stability for extended periods of time, particularly at least 18 months at room temperature. "Room temperature" refers to the temperature of a normal working environment. Room temperature includes temperatures within the range of 10-40°C, 17-27°C, 20-24°C, 25°C±3°C, 25°C±2°C, 25°C±1°C, or about 25°C. The phrase "room temperature" may be used interchangeably with the phrase "ambient temperature." Room temperature encompasses "controlled room temperature," which refers to the temperature of a normal working environment of 20°C to 25°C with transient deviations (deviations) of 15° to 30° that may be experienced in pharmacies, hospitals, and warehouses. "Controlled room temperature" includes calculated average kinetic temperatures of 25° C. or less (see The Pharmacopeia of the United States of America, Thirty-Third Revision and the National Formulary, Twenty-Eighth Edition, USP 33-NF 28 Reissue, General Notices and Requirements, "Applying to Standards, Tests, Assays, and Other Specifications of the United States Pharmacopeia", § 10.30 Storage Temperature and Humidity (May 1, 2010), available at http: / / www.usp.org / sites / default / files / usp_pdf / EN / USPNF / USP33-NF28-ReissueGeneralNotices.pdf (last accessed July 8, 2016)).

[0095] The term "stability" refers to the retention of an acceptable degree of physical structure (thermodynamic and colloidal stability), chemical structure (kinetic stability), or biological function (functional stability) of a protein after storage in a relevant environment or under certain conditions. A protein can be stable even if it does not maintain 100% of its physical structure, chemical structure, or biological function after a period of storage. Here, for example, a lyophilized protein is considered stable when no more than 2% of the protein population is present in high molecular weight form after storage for up to 24 months at room temperature. In some embodiments, the protein is about 3%, about 2.9%, about 2.8%, about 2.7%, about 2.6%, about 2.5%, about 2.4%, about 2.3%, about 2.2%, about 2.1%, about 2%, about 1.9%, about 1.8%, about 1.7%, about 1.6%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1% or less. A lyophilized or other solid form of a protein is considered stable if it is in high molecular weight form after storage at room temperature for about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 36 months, or 18 months or more.

[0096] Here, a lyophilized or other solid form of a protein is considered "unstable" at room temperature if the percent change in high molecular weight species is greater than about 0.5%, greater than about 0.6%, greater than about 0.7%, greater than about 0.8%, or greater than about 0.9% during the first month of storage at room temperature. Thus, a lyophilized or other solid form of a protein having a percent increase in high molecular weight species of 0.5% or less during the first month of storage at room temperature can be considered stable.

[0097] Stability can be measured, among other things, by determining the percentage of native molecules remaining in the formulation after storage at a specified temperature for a specified time or after delivery to a patient. The percentage of protein that retains its native form (e.g., the percentage of native species relative to total protein, including high and low molecular weight species) can be determined, among other things, by size-exclusion chromatography (e.g., size-exclusion high performance liquid chromatography [SE-HPLC]). In the case of lyophilized proteins, the cake is first solubilized and then the protein is subjected to testing. Native proteins include proteins that are not aggregated or otherwise degraded. In certain embodiments, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the native form of the protein can be detected in the lyophilized cake after storage at a specified temperature for a specified period of time. More than 80%, preferably more than 90%, of the protein in the lyophilized cake should be in its native form. The predefined period for which stability is measured can be at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least 22 months, at least 23 months, at least 24 months, or more. The temperature at which samples may be held when assessing stability may be any temperature between about -80°C to about 50°C, e.g., about -80°C, about -30°C, about -20°C, about 0°C, about 4°C to 8°C, about 5°C, about 25°C, or other room temperature, about 35°C, about 37°C, or other physiological temperature, about 45°C, or about 50°C.

[0098] Stability can be measured, among other things, by determining the percentage of protein that forms aggregates (i.e., high molecular weight species, also known as HMW species) in the lyophilized cake after a specified period of time at a specified temperature, with stability being inversely proportional to the percentage of high molecular weight (HMW) species formed. The percentage of HMW species of protein can be determined, among other things, by size exclusion chromatography after solubilization, as described above. A lyophilized protein composition can also be considered stable if less than about 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the protein is detected in HMW form after 3 months at room temperature.

[0099] Stability can be measured by determining the percentage of protein that is degraded or otherwise found as low molecular weight (LMW) species in the lyophilized cake after a specified period at a specified temperature, with stability being inversely proportional to the percentage of LMW species detected in the solubilized lyophilized cake. The percentage of LMW species of protein can be determined by size exclusion chromatography, as described above. A lyophilized cake of protein can also be considered stable if less than about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the first molecules are detected in LMW form after 3 months at room temperature.

[0100] Other methods such as differential scanning calorimetry (DSC) to determine thermal stability, controlled stirring to determine mechanical stability, and absorbance at about 350 nm or about 405 nm to determine solution turbidity can be used to assess the stability of lyophilized proteins. For example, formulations of the invention have been shown to exhibit a 50% or higher OD of the formulation after storage at about 5° C. to about 25° C. for 6 months or more. 405 The change in OD of the formulation at time 0 405 to less than about 0.05 (e.g., 0.04, 0.03, 0.02, 0.01, or less).

[0101] Stability can also be assessed by measuring the biological activity, physiological activity, or binding affinity of an antibody or other protein to its target. For example, a lyophilized antibody can be considered stable if, after storage for a predefined period (e.g., up to 1 month, 12 months, 18 months, 24 months, etc.), e.g., at 5°C, 25°C, 37°C, 45°C, 50°C, etc., the antibody contained within the lyophilized formulation binds to its cognate epitope-containing antigen with an affinity that is at least 50%, 95%, or more than the binding affinity of the antibody before said lyophilization and storage. Binding affinity can be determined, for example, by ELISA or plasmon resonance. Biological activity can be determined by antibody, soluble receptor, or ligand activity assays, e.g., by contacting cells expressing the cognate binding partner with a reconstituted formulation containing the antibody, soluble receptor, or ligand. Binding of the antibody, soluble receptor, or ligand to such cells can be measured directly, e.g., via FACS analysis, etc. A protein may be considered "stable" if the specific biological or physiological activity (i.e., potency) of the protein is at least 50% of its initial (T0) potency after storage at room temperature for at least 18 months.A stable protein has at least 51% potency, at least 52% potency, at least 53% potency, at least 54% potency, at least 55% potency, at least 56% potency, at least 57% potency, at least 58% potency, at least 59% potency, at least 60% potency, at least 61% potency, at least 62% potency, at least 63% potency, at least 64% potency, at least 65% potency, at least 66% potency, at least 67% potency, at least 68% potency, at least 69% potency, at least 70% potency, at least 72% potency, at least 74% potency, at least 76% potency, at least 78% potency, at least 79% potency, at least 80% potency, at least 81% potency, at least 82% potency, at least 83% potency, at least 84% potency, at least 85% potency, at least 86% potency, at least 87% potency, at least 88% potency, at least 89% potency, at least 90% potency, at least 95% potency, at least 96% potency, at least 97% potency, at least 98% potency, at least 99% potency, at least 100% potency, at least 105% potency, at least 106% potency, at least 107% potency, at least 108% potency, at least 109% potency, at least 110% potency, at least 111% potency, at least 112% potency, at least 113% potency, at least 114% potency, at least 115% potency, at least 116% potency, at least 117% pot The compound may retain at least 71% efficacy, at least 72% efficacy, at least 73% efficacy, at least 74% efficacy, at least 75% efficacy, at least 76% efficacy, at least 77% efficacy, at least 78% efficacy, at least 79% efficacy, at least 80% efficacy, at least 81% efficacy, at least 82% efficacy, at least 83% efficacy, at least 84% efficacy, at least 85% efficacy, at least 86% efficacy, at least 87% efficacy, at least 88% efficacy, at least 89% efficacy, at least 90% efficacy, at least 91% efficacy, at least 92% efficacy, at least 93% efficacy, at least 94% efficacy, at least 95% efficacy, at least 96% efficacy, at least 97% efficacy, at least 98% efficacy, or at least 99% efficacy.

[0102] A "stable" protein undergoes little or no change in structure or specific activity after storage at room temperature for extended periods of time, such as up to 18 months. Structural changes include the formation of aggregates or other high molecular weight forms of the protein, protein degradation, such as hydrolysis of peptide bonds, and chemical degradation, such as deamidation, asialylation, etc. For example, a common form of antibody degradation is the formation of aggregates, including reversible dimers and trimers, as well as more stable and less reversible tetramers and higher order multimers. "Irreversible aggregates" are a subset of aggregates that do not readily solubilize or dissociate upon reconstitution of the lyophilized cake. A "stable" protein undergoes an increase in the formation of high molecular weight species that is less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% after storage at room temperature for up to 7 months, up to 8 months, up to 9 months, up to 10 months, up to 11 months, up to 12 months, up to 13 months, up to 14 months, up to 15 months, up to 16 months, up to 17 months, up to 18 months, up to 19 months, up to 20 months, up to 21 months, up to 22 months, up to 23 months, or up to 24 months. Methods that detect high molecular weight species may have a detection variability of 0.2-0.3%.

[0103] The temperature-dependent rate of protein aggregation or other high molecular weight species formation follows Arrhenius or modified Arrhenius kinetics (slightly curved Arrhenius plot) (see, e.g., Chakroun et al., “Mapping the Aggregation Kinetics of a Therapeutic Antibody Fragment,” Mol. Pharmaceut. 13:307-319 (2016)). Thus, the aggregation rate and percent change of high molecular weight species over time at a given temperature for a given formulation can be predicted based on the time-square root relationship and Arrhenius or modified Arrhenius kinetics. For example, lyophilized samples are incubated at given temperatures (e.g., 5° C., 25° C., 37° C., 50° C.) in sealed glass vials. Aliquots are taken at regular intervals to remove insoluble aggregates and then subjected to SE-HPLC. The observed aggregation rate is determined directly from a linear fit of the time-dependent main peak vs. HMW peak area data. Predictions of the percent change of the high molecular weight species are made by applying the time-root rate law and Arrhenius kinetics to the data. For example, the observed rate constants at various temperatures are fitted to the Arrhenius equation, ln k=ln A-(E / RT), where E is the activation energy (cal / mol), R is the Universal Gas Constant (1.987 cal / mol / K), T is the absolute temperature (in Kelvin), and ln A is a temperature constant that includes factors such as collision frequency. The value of k is extrapolated from the Arrhenius fit to predict the change over time (e.g., 24 months) of the high molecular weight species at a given temperature (e.g., 25°C).

[0104] Freeze drying Methods for lyophilizing proteins in general, and therapeutic antibodies or other antigen-binding proteins in particular, are well known in the art. Briefly, in one embodiment, lyophilization begins with a pre-lyophilization aqueous solution containing the drug substance and excipients, as described above. The pre-lyophilization aqueous solution is placed in an open container (e.g., a vial), which is placed in a lyophilization chamber on a shelf. The lyophilization process includes three basic steps: (1) freezing with optional annealing cycles, (2) primary drying, and (3) secondary drying with optional post-drying annealing steps. The first step is freezing. Here, the shelf temperature is lowered to cool the formulation in the vial. Ice crystals form in the formulation, and the remainder of the formulation becomes more concentrated and viscous. The concentrated remainder solidifies to form an amorphous, crystalline, or combined crystalline / amorphous state. In one embodiment, the remainder solidifies in an amorphous glass state.

[0105] In some freeze-drying protocols, the frozen composition is subjected to annealing to promote crystallization: the solid remainder is held above the final freezing temperature for a period of time to crystallize some components, such as bulking agents like mannitol and glycine.

[0106] The ice is then removed by sublimation. The pressure of the freeze-drying chamber (e.g., 40 to 400 Torr) and shelf temperature (-30 °C to +10 °C) are adjusted below the triple point of water. The temperature is adjusted to below the glass transition temperature (T g ) is maintained.

[0107] After the primary drying step, some water may remain trapped within the matrix. The remaining water is removed through a desorption process during the secondary drying step, where the shelf temperature is increased to facilitate desorption and achieve optimal moisture content in the lyophilized product. In one embodiment, the final moisture content is about 0.5% or more and about 10% or less. In another embodiment, the final moisture content is about 3% or more and about 6% or less. In a particular embodiment, the moisture content is greater than 3% but less than 4%. In another particular embodiment, the moisture content is about 3%. In another particular embodiment, the moisture content is about 3.5%. In another particular embodiment, the moisture content is about 4%. In another particular embodiment, the moisture content is about 4.5%. In yet another particular embodiment, the moisture content is about 6%.

[0108] In one embodiment, the lyophilized product ("lyophilized cake") is subjected to an annealing step after the secondary drying step. Post-drying annealing is also referred to as physical aging or structural relaxation. This post-drying annealing step promotes the relaxation of the amorphous matrix to an equilibrium glassy state (alpha relaxation), increases the structural relaxation time at the storage temperature, reduces the mobility in the glassy state, and reduces the protein to a more stable energy state, which is likely to optimize protein stability. Here, the annealing step promotes molecular mobility for a short period of time, allowing an overall reduction in the entropy of the glassy state and the rotational conformation of the protein.

[0109] After the annealing step, the number of random thermodynamic molecular states of the glass is minimized and the glass equilibrium is maximized. In some embodiments, the annealing temperature is below the Tg of the product. In some embodiments, the annealing temperature is 25°C to about 90°C, 25°C to about 80°C, 25°C to about 75°C, about 25°C to about 70°C, 35°C to about 70°C, 40°C to about 70°C, 30°C to about 55°C, 50°C to about 60°C, 55°C to about 65°C, 60°C to about 70°C, 65°C to about 75°C, 70°C to about 80°C, or 75°C to about 85°C. In some embodiments, the annealing temperature is about 90°C, about 80°C, about 79°C, about 78°C, about 77°C, about 76°C, about 75°C, about 74°C, about 73°C, about 72°C, about 71°C, about 70°C, about 69°C, about 68°C, about 67°C, about 66°C, about 65°C, about 64°C, about 63°C, about 62°C, about 61°C, about 60°C, about 59°C, about 58°C, about 57°C, about 56°C, about 57°C, about 56°C. , about 55°C, about 54°C, about 53°C, about 52°C, about 51°C, about 50°C, about 49°C, about 48°C, about 47°C, about 46°C, about 45°C, about 44°C, about 43°C, about 42°C, about 41°C, about 40°C, about 39°C, about 38°C, about 37°C, about 36°C, about 35°C, about 34°C, about 33°C, about 32°C, about 31°C, about 30°C, about 29°C, about 28°C, about 27°C, about 26°C, or 25°C. In certain embodiments, the annealing temperature is greater than 25°C. In another particular embodiment, the annealing temperature is greater than 50°C.

[0110] The lyophilized cake is held at the annealing temperature for a period of time sufficient to allow relaxation of the cake structure as defined by entropy recovery from DSC or isothermal calorimetry. In some embodiments, the annealing temperature is held for about 12 hours to about 2 weeks, about 12 hours to 1 week, about 12 hours to about several days, about 18 hours to about 72 hours, about 24 hours to about 36 hours, about 30 hours to about 42 hours, about 36 hours to about 48 hours, about 42 hours to about 54 hours, about 48 hours to about 60 hours, about 54 hours to about 66 hours, about 60 hours to about 72 hours, about 66 hours to about 78 hours, or about 72 hours to about 84 hours. In some embodiments, the annealing temperature is about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours, about 78 hours, about 79 hours, about 80 hours, about 81 hours, about 82 hours, about 83 hours, about 84 hours, about 85 hours, about 86 hours, about 87 hours, about 88 hours, about 89 hours, about 90 hours, about 91 hours, about 92 hours, about 93 hours, about 94 hours, about 95 hours, about The retention time is about 7 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours, about 78 hours, about 79 hours, about 80 hours, about 81 hours, about 82 hours, about 83 hours, or about 84 hours.

[0111] In a particular embodiment, the annealing temperature is above 50° C., which is held for about 72 hours. In another particular embodiment, the annealing temperature is about 25° C., which is held for about 72 hours.

[0112] In one embodiment, the annealing conditions are determined experimentally by performing a post-drying annealing step in a differential scanning calorimeter. The endothermic region of the temperature curve generally occurs immediately after the annealing effect, allowing the practitioner to select the annealing temperature. See Sartor et al., "Calorimetric Studies of the Kinetic Unfreezing of Molecular Motions in Hydrated Lysozyme, Hemoglobin, and Myoglobin," 66 Biophysical J. 249-258 (1994). EXAMPLES

[0113] The following examples are presented to provide those of ordinary skill in the art with a description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, sizes, etc.), but some experimental error and deviation should be accounted for.

[0114] Example 1: Freeze-drying procedure Aqueous pre-lyophilization solutions containing antibodies (as described herein) and excipients were loaded into Type I borosilicate glass vials. The filled vials were placed in a LYOSTAR3 freeze dryer (SP Scientific, Warminster, PA). The chamber was closed and the shelf temperature was reduced to 5°C. Samples were held at 5°C for 30 minutes before freezing. The heating rate for freezing was 0.5°C / min. The shelf temperature was held at -45°C for 60 minutes.

[0115] Primary drying was carried out at a vacuum set point of about 100 mTorr and a shelf temperature of about -25°C, which was achieved for about 50 hours with a ramp rate for heating of about 0.5°C / min.

[0116] Secondary drying was carried out at 35° C. and was achieved with a ramp rate for heating of about 0.3° C. / min. Secondary drying lasted for about 6 hours.

[0117] After secondary drying, the chamber was backfilled with nitrogen gas to a pressure of about 0.8 atmospheres (about 608,000 mTorr) and the vials were stoppered with Flurotec® coated 4432 / 50 butyl rubber lyophilization stoppers.

[0118] Example 2: Moisture Content vs. Protein Stability Water molecules can serve as plasticizers and stabilizers in lyophilized products. Water serves as a plasticizer due to its small size and ability to form hydrogen bonds with other water molecules and other molecules (such as protein molecules). The advantage of using water as a stabilizer / plasticizer is that the ratio of stabilizer (water) to protein can be increased without affecting the tonicity of the reconstituted formulation. The moisture content can be adjusted through the design of the lyophilization process. The moisture content of the lyophilized cake was determined by a Computrac® Vapor Pro® moisture meter (Arizona Instrument LLC, Chandler, AZ).

[0119] Recombinant monoclonal antibodies (mAb1, mAb2, mAb3) were produced in EESYR® cells (see U.S. Patent No. 7,771,997B2 issued August 10, 2010) and formulated at a concentration of 150 mg / mL in 10 mM histidine, pH 6.0, 0.1% polysorbate 80, 5% sucrose, and 1.54% arginine (all % w / v in pre-lyophilized liquid formulations). The liquid formulations were lyophilized as described above to specific levels of water content (% w / w) and stored at 50°C, 37°C, or 25°C for specific times. After storage, the lyophilized formulations were reconstituted with water and subjected to size-exclusion high performance liquid chromatography (SE-HPLC). High molecular weight (HMW) species were detected, integrated, and compared to a T=0 control. The percent change of HMW species as a fraction of total protein was calculated and reported in Table 1. Lyophilized mAb1 at 150 mg / mL was the most stable with a moisture content of approximately 4.5% when stored at 25°C.

[0120] Lyophilized formulations of mAb1 with moisture contents between 3 and 10% were predicted to degrade by approximately 2.0% or less after 24 months of storage at 25 °C. An Arrhenius kinetic algorithm was applied to the measured degradation rates, with predictions based on the time-square root rate law out to at least 24 months. The percent change in HMW species for each moisture content point is reported in Table 2. The pre-lyophilized aqueous formulation was 150 mg / mL mAb1, 10 mM histidine, pH 6.0, 0.1% polysorbate 80, 5% sucrose, 1.54% arginine.

[0121] Example 3: Effect of Post-Dry Annealing Annealing the lyophilized drug product improved protein stability. It is hypothesized that annealing the lyophilized formulation below the glass transition temperature relaxes the amorphous molecules to a lower energy state, resulting in a more stable product. Annealing the lyophilized formulation at 70° C. for 72 hours resulted in a lower observed increase in % HMW for 150 mg / mL mAb1 lyophilized in 10% sucrose and 3.08% arginine (pre-lyophilized formulation: 150 mg / mL mAb1, 10 mM histidine, pH 6.0, 0.1% polysorbate 80, 10% sucrose, 3.08% arginine). The percent change in antibody HMW species is reported in Table 3 for annealed and non-annealed formulations stored at 25° C., 37° C., and 50° C. for 6 months.

[0122] [Table 1]

[0123] [Table 2]

[0124] An Arrhenius kinetic algorithm was applied to the measured degradation rates and predictions based on the time-root rate law were made out to at least 24 months for annealed versus unannealed samples stored at 37° C. Annealing was predicted to result in approximately 20% fewer HMW species than the unannealed samples at 24 months (i.e., △% HMW of 5.76% versus 6.05%).

[0125] [Table 3]

[0126] Example 4: Effect of Individual Excipients on Stability Certain stabilizers, including sugars, polyols, salts, and amino acids, were evaluated for their individual ability to stabilize lyophilized antibodies. Lyophilized 150 mg / mL mAb1 was most stable when formulated with sucrose (see Table 4). The T of the sucrose formulation g is high enough for storage at room temperature (about 110° C.) and provides sufficient tolerance for the addition of plasticizers, if required.

[0127] Example 5: Combination effect of trehalose on stability Combining trehalose or sucrose with plasticizers such as sorbitol or mannitol surprisingly resulted in more stable room temperature formulations. The combination of sorbitol and trehalose stabilized lyophilized 150 mg / mL mAb1 more than trehalose alone. The base pre-lyophilized formulation was 150 mg / mL mAb1, 10 mM histidine, pH 6.0, 0.1% polysorbate 80, with various combinations of trehalose and sorbitol added prior to lyophilization. The stability of lyophilized proteins in combination with trehalose and / or sorbitol is shown in Table 5 as Δ% HMW.

[0128] [Table 4]

[0129] [Table 5]

[0130] Example 6: Combination effect of sucrose on stability The combination of mannitol and sucrose stabilized 150 mg / mL lyophilized mAb1 more than sucrose alone. The base pre-lyophilized formulation was 150 mg / mL mAb1, 10 mM histidine, pH 6.0, 0.1% polysorbate 80, with various combinations of sucrose and mannitol added prior to lyophilization. The stability of lyophilized protein in combination with sucrose and / or mannitol is shown as Δ% HMW in Table 6. The combination of mannitol and sucrose stabilized 150 mg / mL lyophilized mAb1 more than sucrose alone.

[0131] [Table 6]

[0132] Sucrose was combined with polyols and amino acids to evaluate its ability to stabilize lyophilized antibodies. 150 mg / mL mAb1 was combined with sucrose (Suc) and any one of sorbitol (Sor), glycerol (Gly), arginine (Arg), and alanine (Ala) in different ratios. The change in HMW species was evaluated at 25°C and 5°C at various storage times. The results (Δ% HMW) are shown in Table 7. None of the combinations of excipients tested stabilized 150 mg / mL lyophilized mAb1 more than sucrose alone when stored at 25°C. Surprisingly, the combination of sorbitol and sucrose is predicted to improve stability over sucrose alone when stored at 5°C for 120 months and is more stable than sucrose alone.

[0133] Example 7: Combined Effect of Sucrose and Arginine on Drug Substance Stability Lyophilized mAb1 150 mg / mL and lyophilized mAb2 150 mg / mL have comparable stability when formulated with 10% sucrose and 3.08% arginine. The % change in HMW species was calculated at 1 month storage at 25° C., 37° C., and 50° C. Lyophilized mAb1 150 mg / mL and lyophilized mAb2 150 mg / mL were predicted to have comparable stability for up to 24 months or more at 25° C. when formulated with 10% sucrose and 3.08% arginine. The results of the Δ% HMW analysis are shown in Table 8.

[0134] [Table 7]

[0135] [Table 8]

[0136] Example 8: Effect of Stabilizer to Drug Product Ratio on Drug Product Stability Combinations of sucrose and arginine were formulated with the antibody at various weight ratios and the Δ% HMW was determined at various time points at 50° C. and 25° C. Lyophilized 150 mg / mL antibody formulations with a stabilizer to protein ratio of 0.87:1 or greater are predicted to degrade ≦1% after 24 months of storage at 25° C. (based on time root rate law and Arrhenius kinetics). The results are listed in Table 9.

[0137] [Table 9]

[0138] [Table 10]

[0139] Example 9: Stabilizing effect of excipients on lyophilized proteins at low protein concentrations in the pre-lyophilized liquid formulation Certain stabilizers, including sugars, polyols, salts, and amino acids, were evaluated for their individual ability to stabilize lyophilized antibodies. Lyophilized 2 mg / mL mAb3 showed no noticeable degradation when sucrose, trehalose, or arginine were included as stabilizers. The results are shown in Table 10. Glycerol was also tested, but significant degradation was observed after lyophilization.

[0140] Trehalose was combined with polyols and amino acids to evaluate its ability to stabilize lyophilized antibodies. 2 mg / mL mAb3 was combined with trehalose (Tre) and any one of sorbitol (Sor), glycerol (Gly), arginine (Arg), and alanine (Ala) in different ratios. The changes in HMW species were evaluated at various storage times at 25°C. The results (Δ% HMW) are shown in Table 11. No significant degradation was observed in any of the 2 mg / mL mAb3 lyophilized formulations tested after 3 months of storage at 25°C. [Table 11]

Claims

1. 1. A lyophilized cake comprising: (a) a protein at a concentration of 50 mg / mL or greater in the pre-lyophilized formulation; (b) a stabilizer comprising between about 4% and about 7.5% sucrose by weight of the pre-lyophilized formulation and arginine in a weight to weight ratio of sucrose to arginine of 3.2:1 to 3.4:1; (c) a buffering agent; and (d) a moisture content between about 3% and about 6% by weight; and the freeze-dried cake does not disintegrate.

2. 3. The freeze-dried cake comprising: (a) is free of yellow, brown, black, and red; (b) does not melt and does not involve density changes; and / or (c) comprises a glass transition temperature greater than 25°C; The freeze-dried cake of claim 1.

3. 3. The freeze-dried cake of claim 2 comprising a moisture content of about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, or about 6% water by weight.

4. (a) the increase in the amount of aggregated protein in the lyophilized cake after storage at room temperature for about two months is less than about 0.6%; (b) the amount of aggregated protein in the lyophilized cake stored at room temperature for about 18 months or about 24 months increases by less than about 2%; and / or (c) the aggregated proteins are detected by size exclusion chromatography or electrophoresis; The freeze-dried cake of claim 1.

5. 2. The lyophilized cake of claim 1, wherein the buffering agent comprises from about 0.34% to about 2.04% by weight of histidine.

6. 2. The freeze-dried cake of claim 1, further comprising a stabilizer selected from the group consisting of a polyol, a sugar, an amino acid, a salt, and combinations thereof, wherein (i) the polyol is selected from the group consisting of sorbitol, glycerol, mannitol, and combinations thereof; (ii) the sugar is selected from the group consisting of trehalose, and combinations thereof; (iii) the amino acid is selected from the group consisting of alanine, proline, glycine, and combinations thereof; and (iv) the salt is sodium chloride.

7. 2. The freeze-dried cake of claim 1, wherein the stabilizer comprises one or more polyols present at about 19% to about 83% by weight.

8. the stabilizer further comprises mannitol, the mannitol being present at about 8% to about 23% by weight; The freeze-dried cake of claim 6, comprising:

9. the stabilizer further comprises sorbitol, the sorbitol being present at about 1.3% to about 23% by weight of sorbitol; The freeze-dried cake of claim 6, comprising:

10. Further comprising a surfactant: (a) the surfactant is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, polyethylene glycol (PEG), PEG 3350, and combinations thereof; and / or (b) the surfactant is present from about 0.02% to about 1% by weight; The freeze-dried cake of claim 1.

11. 2. The lyophilized cake of claim 1, wherein the protein is present at about 6% to about 64% by weight, about 6% to about 19% by weight, or about 33% to about 64% by weight.

12. 12. The lyophilized cake of claim 11, wherein the protein is an antigen binding protein, optionally wherein the antigen binding protein is selected from the group consisting of an antibody, an antibody fragment, and a receptor-Fc fusion protein.

13. 2. The lyophilized cake of claim 1, wherein the lyophilized cake is contained within a vial, the vial comprising Type I borosilicate glass and closed with a polytetrafluoroethylene coated butyl rubber stopper.

14. 1. A lyophilized cake comprising: (a) a protein at a concentration of 50 mg / mL or greater in the pre-lyophilized formulation; (b) a stabilizer in a ratio of about 0.2 parts by weight of stabilizer to 1 part by weight of protein (0.2:1) to about 1.5 parts by weight of stabilizer to 1 part by weight of protein (1.5:1), wherein the stabilizer comprises sucrose and further comprises arginine, sorbitol, glycerol, and / or alanine; (c) water at a concentration of about 3% by weight or more and about 6% by weight or less; and wherein the freeze-dried cake is not discolored.

15. The freeze-dried cake of claim 14, further comprising a stabilizer selected from the group consisting of a non-reducing sugar, a sugar alcohol, an amino acid, and combinations thereof, wherein the non-reducing sugar is trehalose; the sugar alcohol is mannitol; and the amino acid is selected from the group consisting of proline and glycine.

16. arginine, mannitol or sorbitol, and optionally: (a) the weight ratio of sucrose to arginine is from about 3.2:1 to about 3.4:1; (b) the weight ratio of sucrose to mannitol is from about 1.8:1 to about 2:1; (c) the weight ratio of trehalose to arginine is from about 3.2:1 to about 3.4:1; or (d) the weight ratio of trehalose to sorbitol is from about 1:1 to about 2:1; The freeze-dried cake of claim 15.

17. 15. The freeze-dried cake of claim 14, wherein the weight ratio of stabilizer to protein is selected from the group consisting of about 0.22:1, 0.44:1, 0.65:1, 0.87:1, 1.1:1, and 1.3:

1.

18. The lyophilized cake of claim 14, wherein the protein is selected from the group consisting of an antibody, an antibody fragment, and a receptor-Fc fusion protein.

19. 15. The freeze-dried cake of claim 14, further comprising a buffering agent, optionally wherein the buffering agent is selected from the group consisting of citrate, phosphate, histidine, succinate, carbonate, acetate, and combinations thereof.

20. The lyophilized cake of claim 14, wherein the lyophilized cake is contained within a syringe.

21. 15. The freeze-dried cake of claim 14, wherein the discoloration is selected from the group consisting of red, yellow, brown, black, and combinations thereof.

22. 1. A method for making a pharma- ceutically acceptable lyophilized cake, comprising: (a) drying to remove water from a sample comprising protein, stabilizer, and water to obtain a pharma- ceutically acceptable lyophilized cake comprising a moisture content of about 3% to about 6% by weight, wherein the protein has a concentration of 50 mg / mL or greater in the pre-lyophilized formulation, the stabilizer comprises sucrose, and the stabilizer further comprises arginine, sorbitol, glycerol, and / or alanine; (b) heating the lyophilized cake at a temperature below the glass transition temperature of the pharma- ceutically acceptable lyophilized cake; The above method.

23. The temperature below the glass transition temperature is: (a) from about 25°C to about 90°C; (b) maintained for about 72 hours; and / or (c) after step (a) of claim 22 and before step (b) of claim 22, by subjecting the sample to differential scanning calorimetry; 23. The method of claim 22.

24. 23. The method of claim 22, wherein the protein is selected from the group consisting of an antibody, an antibody fragment, and a receptor-Fc fusion protein.

25. 23. The method of claim 22, wherein the protein is present in the sample at a concentration of about 50 mg / mL to about 200 mg / mL.

26. 23. The method of claim 22, wherein the sample further comprises a buffer, optionally comprising histidine.

27. The stabilizer comprises: (a) further selected from the group consisting of trehalose, sorbitol, glycerol, arginine, alanine, mannitol, sucrose, proline, NaCl, glycine, and combinations thereof; (b) containing sucrose at a concentration of less than about 10% weight / volume (w / v); and / or (c) further comprising trehalose, optionally wherein said trehalose is present in said sample at a concentration of less than about 10% weight / volume (w / v); 23. The method of claim 22.

28. 28. The method of claim 27, wherein the sample further comprises about 3%±0.1% arginine.

29. 28. The method of claim 27, wherein the sucrose is present in the sample at a concentration of about 5% (w / v), and optionally the sample further comprises: (i) about 1.3%±0.1% (w / v) alanine; (ii) about 1.5%±0.1% (w / v) arginine; (iii) about 1.34%±0.1% (w / v) glycerol; (iv) about 2.66%±0.1% (w / v) mannitol; or (v) about 2.66%±0.1% (w / v) sorbitol.

30. 28. The method of claim 27, wherein the stabilizer comprises: (i) about 7.5% (w / v) sucrose and 2.3%±0.1% (w / v) arginine; (ii) about 12.5% ​​(w / v) sucrose and 3.9%±0.1% (w / v) arginine; or (iii) about 15% (w / v) sucrose and 4.6%±0.1% (w / v) arginine.

31. The trehalose comprises: (a) trehalose is present in the sample at a concentration of about 9.09% (w / v), and the sample further comprises about 0.48% (w / v) sorbitol, about 0.24% (w / v) glycerol, about 0.28% (w / v) arginine, or about 0.24% (w / v) alanine; (b) trehalose is present in the sample at a concentration of about 8.33% (w / v), and the sample further comprises about 0.89% (w / v) sorbitol, about 0.45% (w / v) glycerol, about 0.51% (w / v) arginine, or about 0.43% (w / v) alanine; (c) trehalose is present in the sample at a concentration of about 6.66% (w / v), the sample further comprising about 1.77% (w / v) sorbitol, about 0.9% (w / v) glycerol, about 1.03% (w / v) arginine, or about 0.87% (w / v) alanine; or (d) trehalose is present in the sample at a concentration of about 5% (w / v), and the sample further comprises about 2.66% (w / v) sorbitol, about 1.34% (w / v) glycerol, about 1.54% (w / v) arginine, or about 1.3% (w / v) alanine; 28. The method of claim 27.

32. 23. The method of claim 22, wherein the moisture content is about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, or about 6% by weight.

33. (a) the amount of degraded protein in the lyophilized cake stored at room temperature for about two months increases by less than about 0.6%; (b) the amount of degraded protein in the lyophilized cake stored at room temperature for about 18 months or about 24 months increases by less than about 2%; and, optionally, (c) degradation of the protein is determined by measuring the percent change in high molecular weight species by size exclusion chromatography; 23. The method of claim 22.

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