EA043993B1 - ROOM TEMPERATURE STABLE LYOPHILIZED PROTEIN - Google Patents

Description

Field of technology

The invention generally relates to the field of pharmaceutical composition of biological molecules.

In particular, the invention relates to stable lyophilized therapeutic protein compositions.

State of the art

Therapeutic macromolecules, such as antibodies and receptor Fc fusion proteins, must be formulated in a manner that not only makes the molecules suitable for administration to patients, but also maintains their stability during long-term storage. For example, therapeutic proteins (such as antibodies) in a liquid solution are prone to aggregation, chemical modifications, or other forms of degradation if the solution is not properly formed. The stability of a therapeutic protein in a liquid composition depends not only on the types of excipients used in the composition and on the amount and proportions of these excipients relative to each other, but also on the concentration of soluble protein and the method of production. Considerations other than stability must also be taken into account when preparing a therapeutic protein composition. These considerations include the viscosity of the solution and the concentration of antibodies that may be included in the composition. Thus, when preparing a therapeutic protein, great care must be taken to provide a composition that remains stable over time at storage temperature, contains an adequate concentration of antibody or other therapeutic protein, and has other properties that allow the composition to be conveniently administered to patients.

Liquid formulations of therapeutic proteins are generally designed to provide long-term protein stability when frozen or refrigerated, but often do not provide long-term stability at room temperature. One solution known in the art for preserving the stability and maintaining the therapeutic activity of a protein is to lyophilize the molecule. Lyophilization (freeze drying under controlled conditions) is commonly used for long-term storage of proteins. Lyophilized protein is inherently resistant to degradation such as aggregation, oxidation and other degenerative processes when in the lyophilized state (see, for example, US Pat. No. 6,436,897). Lyophilization provides a dry residue that remains relatively stable at room temperature for a relatively long period of time. Stability at room temperature is especially important for the storage and distribution of therapeutic proteins around the world, especially in places where electricity and refrigeration are unreliable.

Lyoprotectants (also known as stabilizers) such as sucrose and trehalose are often included in the pre-lyophilization formulation to protect the protein from denaturation during the lyophilization process. Plasticizers may also be included to reduce overall relaxation time and in some cases may help maintain the native structure of proteins. Plasticizers include sugar alcohols such as sorbitol and glycerin, other polyols and small amounts of water.

In studies aimed at optimizing the storage of lyophilized proteins at 5°C, Chang et al. studied the effect of plasticizers on the stability of protein compositions. In lyophilized residues that contained a 1:1 weight ratio of sucrose to protein (pre-lyophilized protein at 40 mg/ml) and without additional plasticizers, the aggregation rate constant over a month at 50°C was reported to be greater than 1.5% at a water content of 2.4% and about 2% at a water content of 3.3% (Id. in 1451, Fig. 4) (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 94(7):1445-1454 (2005). The experiment was also conducted at 40°C and 25°C with data presented only for the most severe of the three stress conditions. Chang et al. noted rare examples of documented cases of optimal storage stability at intermediate moisture contents and suggested that residual moisture content should be optimized during formulation development rather than something that simply needs to be minimized (ibid., 1451; see also Breen et al., Effect of Moisture on the Stability of a Lyophilized Humanized Monoclonal Antibody Formulation, Pharma Res 18 (9):1345-1353 (2001)). High levels of moisture were shown in all studies cited by Chang et al. to reduce the chemical stability of the formulations; Hsu et al. Determining the Optimum residual Moisture in Lyophilized Protein Pharmaceuticals, Develop. Biol. Standard 74:255-271 (1991).

None of these studies suggest long-term stability (more than a year, two years, or three years or more) at temperatures above 5°C, let alone 25°C, even for the compositions tested.

Lyophilized biotherapeutic formulations have demonstrated long-term stability under certain conditions. Kallmeyer et al., WO1998022136A2, describes stable low concentration lyophilized antibody compositions (e.g., up to 8 mg/ml pre-lyophilized solution) that contain, among other excipients, sugar (up to 200 mg/ml after reconstitution [e.g., sucrose, lactose, maltose, raffinose, trehalose]), amino acid (1-100 mg/ml pre-lyophilized [e.g. arginine, lysine, ornithine]), surfactant (0.05 to 0.5 mg/ml after reconstitution [ e.g., polysorbates and polyoxyethylene-polyoxypropylene polymers]) and, optionally, a buffer (10-20 mM after reduction [e.g., phosphate, acetate, citrate]) and/or an isotonicizing agent [e.g., NaCl, not more than 30 mM after reduction). Kallmeyer describes that the lyophilisate can be stored at room temperature (i.e. 18-23°C) for up to two years while remaining stable. Herein, stability is demonstrated by the formation of very small or no particles in the reconstituted lyophilisate, that is, less than 6000 particles greater than 10 microns in size or less than 600 particles greater than 25 microns in size.

Dix et al., WO2006104852A2, describe a stable lyophilized formulation of VEGF-Trap (aka aflibercept) that maintains biological activity for at least three months. This application describes a pre-lyophilized solution containing 5-75 mg/ml decoy molecule, 5-50 mM histidine buffer, 0.1-3% polyethylene glycol (PEG; stabilizer), 0.25-3% glycine (as excipient) and 0.5-6% sucrose (as a stabilizer). Optionally, the pre-lyophilized solution contains citrate buffer (0.05 mM) and/or 0.003% to 0.005% polysorbate.

In addition to lyophilized protein compositions, spray drying has also been used to produce dry protein compositions. Chen and Walsh (WO201307506A1) describe dry micronized protein particles having a diameter range of two (2) to 30 microns and an average diameter of about 10 to 12 microns and in some cases from about 6 to about 7 microns. These particles can subsequently be coated with a polymer to further stabilize the protein and provide sustained release of the protein over time in an aqueous environment. The pretreated protein solution from which the micronized particles were prepared contained (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. The protein contained in polymer-coated micronized protein particles has been shown to remain stable for at least 14 days.

It has been shown that annealing after drying the protein lyophilisate at 50°C results in an initial aggregation of antibodies exceeding 4% and an aggregation rate constant of about 2% aggregation per month (see 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)). Although the US FDA may allow marketing of a composition with an initial aggregation of 4%, this rate of aggregation over time would be unacceptable for a marketed pharmaceutical product. There should be no visible changes during the shelf life of the pharmaceutical composition.

There remains a need for versatile dry protein compositions that remain stable over long periods of time at room temperature. The versatile dry protein compositions are, among other things, suitable for reconstitution for use as liquid compositions, for combining with polymers to form sustained release compositions or formulations, and for implantation or delivery through a variety of other routes. Herein, applicants have developed an improved stable protein lyophilisate and a method for its preparation.

The essence of the invention

The relatively high residual moisture content can serve as an effective plasticizer for lyophilized therapeutic proteins (so-called solid-state therapeutic proteins), which provides remarkable protein stability for at least 24 months of storage at room temperature. In one aspect, a room temperature stable lyophilized therapeutic protein composition is provided in a low mobility solid state having a moisture content of 0.5 to 10%. In another aspect, a method is provided for preparing this lyophilized therapeutic protein composition that is stable at room temperature. In particular, residual moisture levels greater than 2%, greater than 3%, greater than 4%, greater than 5%, or greater than 6% in the lyophilized residue, but less than 10%, less than 8%, less than 7%, or less than 6% allow annealing at relatively high temperatures for long periods of time at room temperature. Although not limited by any mechanism of action, the residual water content during the lyophilization process appears to allow alpha relaxation while preventing the formation of parent protein aggregates at relatively high concentrations of the pharmaceutical composition, preferably from 50 to 200 mg/ml, even more preferably 100 to 150 mg/ml.

In a first aspect, the pharmaceutically acceptable lyophilized residue contains a stable protein, an excipient, 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, about 3%, about 4%, about 4.5%, or about 6% water by weight. The protein of the pharmaceutically acceptable lyophilized residue remains stable for at least one month at room temperature, which may be 1725°C, 20-25°C, or about 25°C. In general, stable means that less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or very little or none of the proteins degrades during 18 months of storage at room temperature. temperature. A common pathway for protein degradation is

- 2 043993 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 denatured electrophoretic gel mobility, preferably size exclusion chromatography. Degradation also includes the formation of chemical products such as deamidated residues, reduced disulfide bonds, hydrolysis and fragmentation of peptides and the like, which can also be measured by methods known in the art.

In one embodiment, less than or about 2% of the protein is degraded after 24 months of storage at about 25°C. Degradation is determined by the percentage change in high molecular weight particles measured by size exclusion chromatography. The lower limit of detectable degradation using this method is about 0.5%, and the assay variability is about 0.2-0.3%.

The pharmaceutically acceptable lyophilized residue may include one or more excipients in addition to protein and moisture. In one embodiment, the excipients include a buffer. This buffer can be any buffer that maintains the optimal pH for protein stability. Histidine is such a buffer that 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 pharmaceutically acceptable lyophilized moiety in an amount of from about 0.34% to about 2.04% by weight.

In one embodiment, the fillers include a stabilizer. Stabilizers include various molecules such as polyols, sugars, amino acids, salts or any combination thereof. Examples of stabilizers used include sorbitol, glycerin, mannitol, trehalose, sucrose, arginine, alanine, proline, glycine, sodium chloride, or any combination thereof. In one embodiment, the stabilizer comprises from about 19.9% to about 82.2% by weight of the pharmaceutically acceptable lyophilized residue.

The term stabilizer means at least one chemical entity that is not a buffer or protein in a pharmaceutically acceptable lyophilized moiety. In some embodiments, the term stabilizer means a combination of chemical elements (ie, more than one chemical element) that together serve to stabilize a protein or other macromolecule. For example, the stabilizer may be sucrose, or the stabilizer may be a combination of sucrose and arginine. In some embodiments, a high molecular weight chemical, such as, for example, sucrose or trehalose, is combined with a low 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 entity increases the mobility, allowing the protein to relax to a lower energy state.

In one embodiment, the stabilizer is sucrose only, and the stabilizer constitutes from about 3% to about 15%, preferably about 5-11%, 4-7.5%, or 5-7.5% by weight of the pharmaceutically acceptable lyophilized residue in depending on the presence of other stabilizer components and the amount of protein, water and other excipients. In one embodiment, the protein to stabilizer weight ratio is from 1:1 to 3:1, preferably from 1.2:1 to 2:1, more preferably from 1.5:1.

In one embodiment, the stabilizer includes sucrose in combination with another stabilizing agent. These other stabilizing agents in combination 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 from about 4.83% to about 19.3% by weight of the pharmaceutically acceptable lyophilized residue. In some embodiments, the weight ratio of sucrose to weight of arginine is from about 3.2:1 to about 3.4:1. In another embodiment, the additional stabilizer comprises sorbitol, which may comprise from about 8.07% to about 22.4% by weight of the pharmaceutically acceptable lyophilized residue. In another embodiment, the additional stabilizer comprises mannitol, which may comprise from about 8.07% to about 22.4% by weight of the pharmaceutically acceptable lyophilized residue. In another embodiment, the additional stabilizer comprises glycerol, which may comprise from about 4.23% to about 12.7% by weight of the pharmaceutically acceptable lyophilized residue. In yet another embodiment, the additional stabilizer comprises alanine, which may comprise from about 4.11% to about 12.4% by weight of the pharmaceutically acceptable lyophilized residue.

In another embodiment, the stabilizer contains trehalose, which constitutes from about 15.8% to about 70.2% by weight of the pharmaceutically acceptable lyophilized residue, depending on the presence of other stabilizers and the amount of protein, water and other excipients.

In one embodiment, the stabilizer contains trehalose in combination with another stabilizer. These other stabilizers in combination with trehalose include any one or more of arginine, sorbitol, mannitol, glycerol and alanine. In one embodiment, the additional stabilizer contains arginine, which can range from about 0.81% to about 14.3% by weight of the headlights

- 3 043993 maceutically acceptable lyophilized residue. In another embodiment, the additional stabilizer comprises sorbitol, which may comprise from about 1.35 to about 22.4% by weight of the pharmaceutically acceptable lyophilized residue. In another embodiment, the additional stabilizer comprises glycerol, which may range from about 0.69% to about 12.7% by weight of the pharmaceutically acceptable lyophilized residue. In yet another embodiment, the additional stabilizer comprises alanine, which may range from about 0.69% to about 12.4% by weight of the pharmaceutically acceptable lyophilized residue.

In another embodiment, the fillers contain a surfactant. The surfactant may contain a nonionic detergent such as fatty acylated polyethoxylated sorbitan. In one embodiment, the surfactant typically includes polysorbate, or particularly polysorbate 80. In some embodiments, the pharmaceutically acceptable lyophilized residue contains from about 0.21% to about 0.96% surfactant, such as polysorbate 80. by weight.

In one embodiment, the protein is a therapeutic protein. In another embodiment, 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 various other fusion (recombinant or chimeric) proteins, such as receptor Fc fusion proteins, which include decoy molecules. In a specific embodiment, the protein is a therapeutic antibody, such as a recombinant human or humanized monoclonal antibody. Antibodies include hybrid antibodies as well as bispecific antibodies.

In one embodiment, protein comprises from about 6.27% to about 63.7% by weight of the pharmaceutically acceptable lyophilized residue. In some embodiments, the pharmaceutically acceptable lyophilized residue contains from about 6.27% to about 18.9% by weight. In other embodiments, the pharmaceutically acceptable lyophilized residue contains from about 33.4% to about 63.7% by weight.

Stabilizers are included in the pharmaceutically acceptable lyophilized residue to help maintain protein stability. Therefore, the pharmaceutically acceptable lyophilized residue contains specific ratios of stabilizer and protein. In some embodiments, the ratio of stabilizer to protein is from about 0.22:1 to about 6.6:1 by weight. Various embodiments include a ratio of stabilizer to protein selected from 0.44:1, 0.65:1, 0.87:1, 1.1:1 and 1.3:1, all by weight.

The pharmaceutically acceptable lyophilized residue is storable. In some embodiments, the pharmaceutically acceptable lyophilized residue is contained in a closed vial. The closure may be a cork. In other embodiments, the pharmaceutically acceptable lyophilized residue is contained in a syringe barrel. In yet other embodiments, the pharmaceutically acceptable lyophilized residue is contained in one chamber of a dual-chamber autoinjector.

In one embodiment, a pharmaceutically acceptable lyophilized residue is prepared by combining a protein, a buffer, a nonionic surfactant, and one or more stabilizers in water to form a pre-lyophilized aqueous solution. The solution is then freeze-dried to obtain a residue containing no more than 10% and no less than 0.5% moisture. Freeze-dried (lyophilized) protein is in a solid state. In a specific embodiment, the protein is a therapeutic recombinant human or humanized monoclonal antibody.

According to another aspect, a method is provided for preparing a composition comprising a therapeutic protein and no more than 10% water and no less than 0.5% water.

In one embodiment, the method includes the steps of obtaining an aqueous sample containing protein and excipient in a container. The container may be, but is not limited to, a vial, a syringe barrel, or the chamber of a dual chamber autoinjector. The container is open enough to allow the release of water vapor. The container containing the aqueous sample is placed in the chamber; heat is removed from the sample until the first temperature is reached at which ice crystals form in the sample. Air is removed from the chamber to achieve the first pressure. Thermal energy is then added to the sample to achieve a second temperature that allows water to be removed from the sample by sublimation. Residual water may remain trapped in the sample after sublimation, requiring an additional second drying step. This second stage of drying is accomplished by applying thermal energy to the sample while maintaining a first chamber pressure, thereby achieving a third temperature. At this temperature, water is desorbed from the sample until a moisture level of no more than 10% and no less than 0.5% is reached.

In one embodiment, during the initial stage of sublimation and primary drying, heat is removed from the aqueous sample at a rate of about 0.5°C/min. In one embodiment, the first temperature is about -45°C. In another embodiment, the first temperature is maintained for about 60 minutes. In yet another embodiment, the aqueous sample is held at 5°C for about 30 minutes until the first temperature is reached.

In one embodiment, the primary drying step is conducted at a second temperature of about 25°C. In one embodiment, the second temperature is achieved by increasing the temperature of the shelf at a rate of about 0.5°C/min. In one embodiment, the second temperature is maintained for about 50 hours. In one embodiment, the pressure in the chamber during primary drying is about 100 mTorr.

In one embodiment, the secondary drying step is carried out at a third temperature of about 35°C. In one embodiment, the rate of change is about 0.3°C/min. In one embodiment, the sample is held at the third temperature for about 6 hours.

After secondary drying, in one embodiment, the vial is sealed at a chamber pressure of approximately 608,000 mTorr. In one embodiment, the chamber is filled with N ₂ gas until sealed. In one embodiment, the vial is sealed with a 4432/50 Flurotec® coated butyl rubber lyophilization stopper.

In one embodiment, the dried sample is annealed to relax the protein into a lower energy state and improve its overall stability. To anneal a sample, thermal energy is applied to the sample to reach the fourth temperature. In some embodiments, the sample is maintained 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 reaches an optimal state of relaxation, the container is closed.

In one embodiment, the fourth temperature, that is, the annealing temperature, is below the glass transition temperature of the sample after the water desorption step. In a specific embodiment, the annealing temperature is about 70°C. In another specific embodiment, the annealing temperature is about 45°C. In one embodiment, the sample is held at the annealing temperature for about 72 hours. Because different proteins have different biophysical characteristics, in some embodiments the fourth temperature is determined using modulated differential scanning calorimetry (MDSC). In this document, a sample of a lyophilized protein composition that has undergone a secondary drying step is loaded into the calorimeter. The sample is then subjected to incremental heating through glass transition while the heat flow is controlled. Tg is determined. The samples are then kept at various temperatures below the T _g for various times to induce enthalpy relaxation of the molecules in the lyophilized residue. The relaxed samples are then subjected to DSC or MDSC and the peak area (heat capacity as a function of temperature) due to enthalpy reduction 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 optimal enthalpy relaxation are selected for the fourth (or annealing temperature) temperature and time; see also WQ, Calorimetric analysis of cryopreservation and freezedrying formulations , 1257 Methods Mol Biol 163-79 (2015).

In one embodiment, the pre-lyophilized aqueous solution (i.e., an aqueous sample; aka aqueous solution) contains several excipients, such as one or more stabilizers, one or more buffers, and optionally one or more surfactants.

In one embodiment, the pre-lyophilized aqueous solution as well as the reconstituted lyophilized liquid composition has a pH that helps maintain protein structure and function. In specific embodiments, the pre-lyophilized and subsequently reconstituted liquid compositions have a pH of about 6.0 ± 2. Molecules that buffer at a pH of about 6 are considered useful in this embodiment. Thus, in one embodiment, the buffer contains histidine. In a specific embodiment, the aqueous solution contains about 10 mM histidine.

In one embodiment, the pre-lyophilized 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.

In one embodiment, the stabilizer or combination of stabilizers includes sucrose. In a specific embodiment, the stabilizer contains sucrose at a concentration of about 10% (w/v) in an aqueous solution. In one case, sucrose is the only stabilizer. In another case, the aqueous solution contains about 3% ± 0.1% (w/v) arginine as a stabilizer in addition to 10% (w/v) sucrose.

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In other embodiments, the stabilizer comprises sucrose and at least one other molecular entity. In some embodiments, the aqueous solution contains about 5% sucrose and one other stabilizer. In one specific embodiment, the aqueous solution contains 5% sucrose and any 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 (f) about 2.66% ± 0.1% (w/v) sorbitol.

In some embodiments, the stabilizer contains sucrose and arginine in varying concentrations and proportions. In a specific embodiment, the aqueous solution contains about 7.5% sucrose and 2.3%±0.1% arginine. In another specific embodiment, the aqueous solution contains about 12.5% sucrose and 3.9%±0.1% arginine. In yet another specific embodiment, the aqueous solution contains about 15% sucrose and 4.6%±0.1% arginine.

In some embodiments, the stabilizer contains trehalose, either as the sole stabilizer or in combination with another stabilizer. In one embodiment, the stabilizer contains trehalose at a concentration of about 10% (w/v) in an aqueous solution. In another embodiment, the aqueous solution contains about 9.09% (w/v) trehalose and any of 0.48% (w/v) sorbitol, 0.24% (w/v) glycerol, 0. .28% (w/v) arginine and 0.24% (w/v) alanine. In another embodiment, the aqueous solution contains 8.33% (w/v) trehalose and any 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 contains 6.66% (w/v) trehalose and any 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 contains 5% (w/v) trehalose and any of 2.66% (w/v) sorbitol, 1.34% (w/v) glycerol, 1.54% (w/v) arginine and 1.3% (w/v) alanine.

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 about 150 kilodaltons or greater. The therapeutic protein included in the present invention may 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 cover any protein that binds to another molecular entity. For example, antigen binding proteins include antibodies, bispecific antibodies, antibody fragments, ScFv fusion proteins, complementarity determination region (CDR) containing proteins, ligands, receptors, ligand fragments, receptor fragments, fusion proteins containing ligand and/or receptor domains and receptor Fcs -fusion proteins, including decoy molecules.

In one embodiment, the therapeutic protein is an antibody. In a specific embodiment, the therapeutic antibody is a monoclonal antibody. In a more specific embodiment, the therapeutic protein is a recombinant human 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 decoy molecule, such as an aflibercept (VEGF decoy) molecule or a rilonacept (IL-1 decoy) molecule.

In some embodiments, the concentration of antibodies in the aqueous solution is low, which includes a concentration range greater than zero (i.e., low or less than 1 μg/ml) and less than or equal to 25 mg/ml. In one low concentration embodiment, the antibody concentration is about 2 mg/ml.

In other embodiments, the concentration of antibodies in the aqueous solution is an average, which includes a range of concentrations greater than 25 mg/ml and less than or equal to 100 mg/ml. In one medium concentration embodiment, the antibody concentration is about 50 mg/ml.

In other embodiments, the concentration of antibodies in the aqueous solution is high, which includes a concentration range greater than 100 mg/ml and less than or equal to 200 mg/ml. In one high-concentration embodiment, the antibody concentration is about 150 mg/ml.

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

As noted above, but without limitation to any mechanism of action, water can serve as a plasticizer for the lyophilized product, which unexpectedly improves protein stability. Thus, in one embodiment, the moisture content of the resulting composition (ie, the stable lyophilized composition) is >2% and <10% by weight. In another embodiment, the moisture content of the pharmaceutically acceptable lyophilized residue is >3% and <6% by weight. In another embodiment, the moisture content of the pharmaceutically acceptable lyophilized residue is >4% and <6% by weight. In one specific embodiment, the content

- 6 043993 moisture in the pharmaceutically acceptable lyophilized residue is about 6%. In another specific embodiment, the moisture content of the pharmaceutically acceptable lyophilized residue is about 4.5%. In yet another specific embodiment, the moisture content of the pharmaceutically acceptable lyophilized residue is about 3%.

In one embodiment, the lyophilized protein is stable for at least 24 months at room temperature. In a particular embodiment, < about 2% of the pharmaceutically acceptable lyophilized residue is degraded after 24 months of storage at 25°C. Although not limited to any mechanism, degradation may include proteolysis, chemical modification, aggregation, and the like. Aggregation is a common form of antibody degradation and is observed during the formation of high molecular weight (HMW) particles. Degradation can be determined using any protein assay known in the art or not yet invented. In a specific embodiment, antibody degradation is determined by measuring the change in percentage of high molecular weight (HMW) species using size exclusion (SE) chromatography.

Brief description of graphic materials

In fig. Figures 1 and 2 are histograms depicting the effects of temperature and humidity on protein stability over time. The X axis shows time in months and temperature. The Y axis shows the percentage change in the number of high molecular weight protein species. For each temperature, individual histograms represent increasing percentage water (w/w) from left to right along the x-axis, 0% water (open bars), 0.5% water (open bars), 1.5% water (descending filled filled bars). columns), 3.0% water (vertical filled bars), 4.5% water (horizontal filled bars), 6% water (open diamond bars), 8% water (checkered bars), and 10% water (dark dashed bars) .

Detailed description of the invention

Before reading the description of this invention, it should be understood that this invention is not limited to the specific methods described and the experimental conditions described, since such methods and conditions may vary. It should also be understood that the terminology used herein is intended solely to describe specific embodiments of the invention and is not limiting, since the scope of the present invention is limited only by the appended claims.

Absolute amounts and relative amounts of fillers, ingredients and other materials may be described by weight or moles. Units of mass can be expressed in grams, milligrams, micrograms, etc.). The term weight, such as w/v, means mass. Relative amounts may be expressed as percent by weight (i.e., percent by mass), where one (1) percent weight to volume (w/v) means 1 g of material per 100 ml of volume. Also, for example, one (1) component of ingredient A to one (1) component of ingredient B by weight means, for example, that for every (1) gram of ingredient A there is one (1) gram of ingredient B. Also, for example, one percent ( 1%) by weight of Ingredient A means, for example, that for every 100 g of total particle weight there is one (1) gram of Ingredient A. Relative quantities of an ingredient can also be expressed in terms of moles or number of molecules per given volume, e.g. millimoles per liter (millimoles (mM)), or per other ingredient, for example, X component of A per Y component of B in moles means that for every X moles of A there are Y moles of B.

Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of this invention, the preferred methods and materials are currently described. All publications mentioned in this document are incorporated herein by reference for their full description.

Fillers.

An excipient is an ingredient added along with the active drug substance to a pharmaceutical composition. Excipients help stabilize the drug substance and/or increase the volume of the composition. The term ingredient is used interchangeably with excipients.

Excipients include various substances for various purposes, such as buffering, swelling, solubilization, stabilization, plasticization and protection of the drug substance. Protectants protect against thermal stress and/or physical stress, such as agitation stress. Buffers are well known in the art.

Typically, a buffer is included in the aqueous protein solution before lyophilization to stabilize the protein before lyophilization and after reconstitution. The buffer may be included in the lyophilization solution at a concentration of 1 mM to 100 mM. In some specific embodiments, the buffer is included in the lyophilization solution at about 10 mM. In some embodiments, the buffer is present in the lyophilization solution at a concentration of 5 mM ± 0.75 mM to 15 mM ± 2.25 mM; from 6 mM±0.9 mM to 14 mM±2.1 mM; from 7 mM±1.05 mM to 13 mM±1.95 mM; from 8 mM±1.2 to 12 mM±1.8 mM; from 9 mM±1.35 mM to 11 mM±1.65 mM; 10 mM±1.5 mM; or about 10 mM. In some embodiments, the lyophilization solution buffer system contains histidine, phosphate, and/or acetate at 10 mM ± 1.5 mM.

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In some embodiments, the buffer is selected from a chemical capable of buffering anywhere in the pH range of about 3 to about 9 or in the pH range of about 3.7 to about 8.0. For example, the pre-lyophilized solution may have a pH of about 3.4, about 3.6, about 3.8, about 4.0, about 4.2, about 4.4, 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.

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 buffering range of about 3.5 to about 6, or about 3.7 to about 5.6, such as an acetate-buffered range. In one embodiment, the buffer has a buffering range of from about 5.5 to about 8.5, or from about 5.8 to about 8.0, such as a phosphate buffered range. In one embodiment, the buffer has a buffering range of from about 5.0 to about 8.0, or from about 5.5 to about 7.4, such as a histidine-buffered range.

Fillers include stabilizers. As used herein, a stabilizer is added to the lyophilization solution to stabilize the protein from aggregation or other degradation. Stabilization can occur by controlling the glass transition dynamics during lyophilization or by preserving the natural structure of the protein through specific interaction of the stabilizer with the protein. For a discussion of the biophysics 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).

Stabilizers for inclusion in the lyophilization solution include polyols, sugars, salts (eg, sodium chloride), amino acids, and the like. Various individual stabilizers can be used alone or in combination with one or more other stabilizers for optimal stabilizing effect. For example, a polyol may be combined with a sugar, a sugar with an amino acid, a polyol with an amino acid, a salt with a sugar, a salt with an amino acid, a salt with a polyol, and the like.

Polyols are organic molecules with more than one hydroxyl group (-OH). Polyols include monomers as well as polymers. Sugar alcohols are a subgroup of polyol sugar alcohols that can serve as useful stabilizers and include mannitol, xylitol, sorbitol, isomaltol, erythritol, maltitol and glycerin. Other monomeric polyols include ethylene glycol, propylene glycol and pentaerythritol. Polymer polyols can be polyesters or polyethers of polyol subunits. Exemplary polymeric polyols include polypropylene glycol, polyethylene glycol, and poly(tetramethylene ether) glycol.

Sugars are used as stabilizers (and also as fillers). Sugars can be classified as 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 protein lyophilization because reducing sugars can reduce proteins through 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 an amorphous glass during lyophilization. Trehalose or sucrose, alone or in combination with an amino acid or polyol, is used as a stabilizer in the practice of this invention.

In one embodiment, trehalose is used as the sole 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.

In another embodiment, sucrose is used as the sole stabilizer. In other embodiments, sucrose is combined with a polyol. In specific 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.

Amino acids are used as stabilizers. Glycine is a commonly used filler and stabilizer. Other amino acids used include arginine, alanine and proline. In some embodiments, arginine is used as a stabilizer. In certain specific embodiments, arginine is combined with sucrose or arginine is combined with trehalose. In other embodiments, alanine is used as a stabilizer. In certain specific embodiments, alanine is combined with sucrose or alanine is combined with trehalose.

In some cases, one or more surfactants may be used as a filler. It is assumed that surfactants provide additional

- 8 043993 greater stability due to a decrease in hydrophobic protein-protein interaction and, as a result, the formation of high molecular weight particles (i.e. aggregates). In some embodiments, one or more surfactants may be included in the pre-lyophilized protein-containing aqueous solution. In other embodiments, one or more surfactants may be included in the diluent solution for dilution. Surfactants include substances that reduce the surface tension of the liquid in which it is dissolved and/or reduce the interfacial tension between oil and water. Surfactants can be ionic or non-ionic. Typical nonionic surfactants that may be included in the pre-lyophilized or post-reconstitution solution 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 nonionic surfactants that may be included in the pre-lyophilized aqueous solution (or subsequently reconstituted solution) include, for example, polyoxyethylene sorbitan esters (or 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, poloxamer 407; polyethylene-polypropylene glycol; or polyethylene glycol (PEG). 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.

The amount of surfactant contained in the pre-lyophilization solution or reconstitution solution may vary depending on the specific properties and purposes desired for the lyophilized composition. In some embodiments, the pre-lyophilization solution or reconstitution solution may contain from about 0.001% (w/v) to about 0.5% (w/v) surfactant (e.g., polysorbate 20 or polysorbate 80 ). For example, the pre-lyophilization solution may contain 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%; about 0.024%; about 0.025%; about 0.026%; about 0.027%; about 0.028%; about 0.029%; about 0.03%; about 0.031%; about 0.032%; about 0.033%; about 0.034%; about 0.035%; about 0.036%; about 0.037%; about 0.038%; about 0.039%; about 0.04%; about 0.041%; about 0.042%; about 0.043%; about 0.044%; about 0.045%; about 0.046%; about 0.047%; about 0.048%; about 0.049%; about 0.05%; about 0.051%; about 0.052%; about 0.053%; about 0.054%; about 0.055%; about 0.056%; about 0.057%; about 0.058%; about 0.059%; about 0.06%; about 0.061%; about 0.062%; about 0.063%; about 0.064%; about 0.065%; about 0.066%; about 0.067%; about 0.068%; about 0.069%; about 0.07%; about 0.071%; about 0.072%; about 0.073%; about 0.074%; about 0.075%; about 0.076%; about 0.077%; about 0.078%; about 0.079%; about 0.08%; about 0.081%; about 0.082%; 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 (eg, polysorbate 20 or polysorbate 80).

One or more plasticizers are included in the lyophilized protein composition. Plasticizers are typically used to increase the flow or flexibility of a system. The increased fluidity is believed to result from the plasticizer increasing the free volume of the system and lowering the glass transition temperature. The addition of plasticizers modifies both alpha-relaxation and beta-relaxation in the lyophilized residue. Alpha relaxation is also known as primal or glass relaxation and is a global relaxation process. Beta relaxation is a more local process involving the movement of a protein polymer chain that can be better modified by smaller molecules (i.e. plasticizers). Both relaxation processes reduce the overall energy of the system and, especially in the case of beta relaxation, are believed to affect protein stability. It is well known in the field of protein biophysics that plasticizers decrease beta relaxation time and can simultaneously reduce protein stability (see, for example, Cicerone and Douglas, β-Relaxation governs protein stability in sugar-glass matrices, Soft Matter 8:2983-2991, 2012 ).

The invention also relates to one embodiment of lyophilized protein compositions containing one or more Sugars in combination with one or more plasticizers in a certain ratio to create a stabilizer that provides sufficient flexibility and alpha-relaxation of the residue, maintaining or increasing the stability of the protein. In some embodiments, the stabilizer contains a weight ratio of sugar to plasticizer (ie, weight to weight) of about 19:1 to about 1:1. In some embodiments, the stabilizer includes a sugar to plasticizer weight ratio 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.

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Suitable plasticizers include polyols such as sorbitol, glycerol (glycerol), mannitol and xylitol, amino acids such as glycine, arginine, proline and alanine, and salts such as NaCl. Interestingly, water can also act as a plasticizer. However, water is generally frowned upon because it is both a chemical reagent and a plasticizer, resulting in increased mobility and decreased Tg. Increased mobility correlates with increased reactivity and hydrolysis within the system.

Increased reactivity and hydrolysis destroy 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).

As described above, mannitol, glycerin, sorbitol, and glycine in combination with water are used in some embodiments as a plasticizer. Without being bound by theory, it is believed that these molecules, in particular embodiments of the composition, reduce beta relaxation time as well as increase protein stability.

In some specific embodiments, the sugar sucrose or trehalose is combined with the plasticizer sorbitol in a 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 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.

The invention also provides the aspect that water serves as a stabilizing plasticizer in the lyophilized composition and helps reduce the rate of protein aggregation in lyophilized compositions stored at room temperature. The amount of moisture required to effectively stabilize the protein in the lyophilized residue at room temperature increases with increasing protein content and decreases with decreasing protein content. Thus, a lyophilized composition prepared from a pre-lyophilized solution containing 50 mg/ml protein may require a lower moisture content than a lyophilized composition prepared from a pre-lyophilized solution containing 150 mg/ml protein. In addition, the optimal amount of moisture required to effectively stabilize the protein in the lyophilized residue varies with storage temperature. For example, an intermediate amount of moisture used for long-term storage of lyophilisate protein at room temperature. At higher storage temperatures (eg 37°C) less moisture is required.

For example, in FIG. 1 shows the effect of moisture on the stability of a typical antibody lyophilization composition stored at 25, 37 or 50°C. Herein, the pre-lyophilized composition contains 150 mg/ml IgG, 5% sucrose and 1.54% arginine. The final lyophilized residue contained moisture (w/w) at 0%, 0.5%, 1.5%, 3.0%, 4.5%, 6%, 8% or 10%. As shown in FIG. 1, Higher storage temperature resulted in decreased protein stability within 2-3 months. Interestingly, the optimal amount of moisture to ensure maximum stability was higher at room temperature than at 37 or 50°C. In this particular example, the optimal moisture content for protein stability at room temperature was about 4.5% (w/w) and from about 1.5% to about 3% when stored at 50°C for 2 months. Longer term stability data is illustrated in FIG. 2, which shows an exemplary composition for lyophilizing antibodies stored at 25 and 37°C for 12 and 6 months, respectively. As shown in FIG. 2, a moisture content of 3.0% to 4.5% (w/w) provided the best stability at both temperatures tested and a moisture content of 4.5% provided the greatest stability after 12 months at room temperature (25°C).

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

In some embodiments, the lyophilized composition contains >0.5 wt% water, >0.6 wt% water, >0.7 wt% water, >0.8 wt% water, >0.9 wt% water water, >1 wt.% water>1.5 wt.% water, >2

- 10 043993 wt.% water, >2.5 wt.% water, >3 wt.% water, >3.5 wt.% water, >4 wt.% water, >4.5 wt.% water, > 5 wt.% water, >5.5 wt.% water, >6 wt.% water, >6.5 wt.% water, >7 wt.% water, >7.5 wt.% water, >8 wt. .% water, >8.5 wt.% water, >9 wt.% water or >9.5 wt.% water, but not more than 10 wt.% water. However, extra-optimal amounts of water may increase protein instability. Thus, in some embodiments, the lyophilized composition contains <10 wt% water, <9.5 wt% water, <9 wt% water, <8.5 wt% water, <8 wt% water, < 7.5 wt.% water, <7 wt.% water, <6.5 wt.% water, <6 wt.% water, <5.5 wt.% water, <5 wt.% water, <4, 5 wt% water, <4 wt% water, <3.5 wt% water, <3 wt% water, <2.5 wt% water, <2 wt% water, <1.5 wt .% water, <1 wt.% water, <0.9 wt.% water, <0.8 wt.% water, <0.7 wt.% water, <0.6 wt.% water, but not less 0.5 wt.% water.

The phrase moisture content can be used interchangeably with water content. However, moisture content is used to describe the amount of water in a lyophilized residue, while water content is used to describe the amount of water in an aqueous solution, gel, other liquid, gas, ice or solid form of a composition such as a lyophilized residue, spray-dried particles, and the like. In some embodiments, the moisture content of the lyophilized composition is from 0.5% to 10 wt%, from 1% to 10 wt%, from 2% to 10 wt%, from 3% to 10 wt%, from 4 % to 10 wt.%, from 5% to 10 wt.%, from 6% to 10 wt.%, from 7% to 10 wt.%, from 8% to 10 wt.%, from 9% to 10 wt. %, from 0.5% to 9 wt.%, from 0.5% to 8 wt.%, from 0.5% to 7 wt.%, from 0.5% to 6 wt.%, from 0.5 % to 5 wt.%, from 0.5% to 4 wt.%, from 0.5% to 3 wt.%, from 0.5% to 2 wt.%, from 0.5% to 1 wt.% , from 1% to 2 wt.%, from 1.5% to 2.5 wt.%, from 2% to 3 wt.%, from 2.5% to 3.5 wt.%, from 3% to 4 wt.%, from 3.5% to 4.5 wt.%, from 4% to 5 wt.%, from 4.5% to 5.5 wt.%, from 5% to 6 wt.%, from 5 .5% to 6.5 wt.%, from 6% to 7 wt.%, from 6.5% to 7.5 wt.%, from 7% to 8 wt.%, from 7.5% to 8, 5 wt%, 8% to 9 wt%, 8.5% to 9.5 wt%, 9% to 10 wt%, 9.5% to 10 wt% or about 1% , 2%, 3%, 4%, 5%, 6%, 7% or 8 wt.%. In some embodiments, the moisture content of the lyophilized composition is from 3% to 6% by weight; from 3.1% to 5.9 wt.%, from 3.2% to 5.8 wt.%, from 3.3% to 5.7 wt.%, from 3.4% to 5.6 wt. %, from 3.5% to 5.5 wt.%, from 3.6% to 5.4 wt.%, from 3.7% to 5.3 wt.%, from 3.8% to 5.2 wt.%, from 3.9% to 5.1 wt.%, from 4% to 5 wt.%, from 4.1% to 4.9 wt.%, from 4.2% to 4.8 wt.% %, 4.3% to 4.7% by weight, 4.4% to 4.6% by weight, or about 4.5% by weight. In some embodiments, the moisture content of the lyophilized composition is from 2% to 4% by weight; from 2.1% to 3.9 wt.%, from 2.2% to 3.8 wt.%, from 2.3% to 3.7 wt.%, from 2.4% to 3.6 wt. %, from 2.5% to 3.5 wt.%, from 2.6% to 3.4 wt.%, from 2.7% to 3.3 wt.%, from 2.8% to 3.2 wt.%, from 2.9% to 3.1 wt.% or about 3 wt.%.

In some embodiments, the weight percentage of water in the lyophilized residue 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 water content of the lyophilized residue is greater than 3% but less than 10% by weight.

The water content of the lyophilized residue can be determined by any one or more methods known in the art. These methods include gravimetric methods including thermogravimetry, gas chromatography, near-infrared spectroscopy, coulometry and the Karl-Fischer or relative humidity sensor method. Some of these methods are discussed in JK Townes, 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. ed. August 1, 2010; and references cited in these documents. For example, the loss on drying (LOD) (gravimetric) method can be used, in which the lyophilized residue is weighed, subjected to additional heating to completely remove all water and other volatiles, and then weighed again. The loss of mass is attributed 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 state of SO2 using 1 ₂ , where one mole of 1 ₂ is a sink per mole of H2O. Near-infrared spectroscopy measures reflectance from 1100 to 2500 nm through a glass vial (glass surface) containing protein to determine moisture content without destroying the sample; see United States Pharmacopoeia, 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.

Lyophilized residue.

The lyophilized composition containing the protein and stabilizer forms a solid matrix, also known as a residue or lyophilized residue. Pharmaceutically acceptable osta

- 11 043993 current (used interchangeably or as a pharmaceutically acceptable lyophilized residue) is amorphous (glassy rather than crystalline) and has an aesthetically elegant appearance. The pharmaceutically acceptable residue must not exhibit shrinkage, cracking, partial or complete breakdown, melting, or discoloration. Red, black, brown, yellow or other colored residue is discolored and is unacceptable. The ideal residue is mechanically strong and resistant to breakage during processing, porous and sponge-like, has a uniform texture and forms a single unit and is uniformly white in color. The residue should be uniformly attached to the walls of the vial and not show peeling or other signs of shrinkage; see Carpenter et al., Rational design of stable lyophilized protein formulations: Some practical advice, Pharmaceutical Research, 14 (8):969-975, 1997.

The residue must be free of visual defects due to freezing problems, including a tube-like structure; dry foam on the top surface; crusting or glazing on the surface of the residue; and horizontal layering or ring formation. The residue must also be free of visible defects due to drying problems, including shrinkage, where the volume of the residue is less than that of the frozen matrix and signs of wall delamination are obvious; cracking, when the residue shows cracks in the dry matrix and the residue does not form a single entity; varying degrees of loss of residue structure, such as complete or partial destruction of the residue; melting, where the residue contains a ring of dissolved material in the lower region; partial melting, where only a small area at the base of the residue contains dissolved material; and browning, which is the yellow or brown discoloration of the residue due to the inclusion of a reducing sugar that has undergone the Maillard reaction. Melting is particularly problematic as it can lead to delayed dissolution time, protein aggregation, degradation and loss of activity; see FDA, Guide to Inspections of Lyophilization of Parenterals (7/93). Finished product inspection. Last update 2009 2009, last accessed 7/8/2016 at http://www.fda.gov/ICECI/Inspections/InspectionGuides/ucm074909.htm.

Protein medicinal substance.

The term protein means 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. A protein may contain one or more polypeptides to form a single functioning biomolecule. Polypeptides typically contain more than 50 amino acids, while peptides typically contain 50 amino acids or less. Proteins may contain one or more covalent and non-covalent modifications. Disulfide bridges (that is, between cysteine residues to form cystine) may be present in some proteins. These covalent bonds can be within a single polypeptide chain or between two separate polypeptide chains. For example, disulfide bridges are essential for the proper structure and function of insulin, immunoglobulins, protamine and the like. 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).

In addition to disulfide bond formation, proteins can undergo other post-translational modifications. These modifications include lipidation (e.g., myristoylation, palmitoylation, farnesoylation, geranylgeranylation, and formation of glycosylphosphatidylinositol (GPI) anchors), alkylation (e.g., methylation), acylation, amidation, glycosylation (e.g., adding glycosyl groups to arginine, aspargine, cysteine, hydroxylysine, serine, threonine, tyrosine and/or tryptophan) and phosphorylation (i.e. the addition of a phosphate group to serine, threonine, tyrosine and/or histidine). For a recent review of posttranslational modification of proteins produced in eukaryotes, see Mowen and David, Unconventional posttranslational 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).

Immunoglobulins (or antibodies) are examples of proteins that have multiple polypeptide chains and extensive post-translational modifications. A canonical immunoglobulin protein (eg, IgG) contains four polypeptide chains, two light chains and two heavy chains. Each light chain is linked to one heavy chain through a cystine disulfide bond, and two heavy chains are linked to each other through two cystine disulfide bonds. Immunoglobulins produced in mammalian systems are also glycosylated at different residues (e.g., asparagine residues) by different polysaccharides and may differ between species, which may influence 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)).

As used herein, the term protein includes therapeutic proteins, recombinant proteins used in research or therapy, decoy proteins and other receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, human antibodies, bispecific antibodies, antibody fragments, nanobodies, recombinant chimeric antibodies, cytokines, chemokines, peptide hormones and the like. Proteins can be produced using recombinant cell production systems such as the insect baculovirus system, yeast systems (e.g.

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Pichia sp.), mammalian systems (e.g. CHO cells and CHO derivatives such as

SNO-K1). 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).

Some recombinant Fc-containing proteins contain receptors or receptor fragments, ligands or ligand fragments that have related binding partners in biological systems. Receptor Fc fusion proteins refer to recombinant molecules that contain a soluble receptor fused to the Fc domain of an immunoglobulin. Some receptor Fc fusion proteins may contain the ligand binding domains of multiple different receptors. These receptor Fc fusion proteins are known as decoys or decoy molecules. Rilonocept and aflibercept are examples of market decoys that antagonize IL1R (see US Pat. No. 7,927,583) and VEGF (see US Pat. No. 7,087,411), respectively. Other recombinant Fc-containing proteins include those recombinant proteins that contain a peptide fused to an Fc domain, such as Centocor MIMETIBODYTM 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).

Fc fusion proteins include part or all of two or more proteins, one of which is part of an immunoglobulin Fc molecule, that are not fused in their natural state. For example, the Fc fusion protein is a decoy protein, such as, for example, an IL-1 decoy (e.g., rhinoalcept, which contains the IL-1RAcP ligand binding region fused to the extracellular region of IL1R1 fused to the hIgG1 Fc; see US Pat. No. 6,927,004, which incorporated herein by reference in its entirety), or a VEGF decoy (e.g., aflibercept, which contains VEGF Fltl receptor Ig domain 2 fused to VEGF Flkl receptor Ig domain 3 fused to hIgG1 Fc; e.g., SEQ ID NO: 1; see US Pat. Nos. 7,087,411 and 7,279,159, which are incorporated herein by reference in their entirety).

In some embodiments, the protein is included in the pre-lyophilized aqueous solution at a concentration greater than 40 mg/mL. In some embodiments, the pre-lyophilized aqueous solution contains protein at a concentration of from about 50 mg/ml to about 250 mg/ml; from about 100 mg/ml to about 200 mg/ml; from about 125 mg/ml to about 175 mg/ml. In some embodiments, the pre-lyophilized aqueous solution contains 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 125 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 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.

Containers.

In some embodiments, a pre-lyophilized aqueous solution containing the therapeutic protein is contained in a container. The freeze-drying process is applied to the solution in a ventilated container, which is subsequently sealed to store the lyophilized composition. The term container is used very broadly in this document. The container, for example, may be a bulk solids container such as a bottle, jar or canister holding from 2 ml to four liters or more, ampoule, vial (glass or plastic), syringe (glass or plastic), cartridge or auto-injector. The vial can be no larger than 0.2 ml or no larger than 100 ml. Typical vials may be made from clear or amber glass, Type I borosilicate glass, Type II sodosilicate glass, or Type III sodosilicate glass. The vial may be closed with a stopper, cap, flip cap, or screw cap.

SCHOTT® style tubular glass is especially useful for freeze drying.

With the advent of biologics and patient self-administration of injectable drugs, auto-injectors have become a more important drug product container. Self-administration of a lyophilized drug product requires the patient to reconstitute the lyophilized residue with sterile water for injection or other sterile solvent. To provide sterile reconstitution, volume control, ease of handling, and overall simplification, prefilled syringes with two or more chambers can be used. Dual-chamber auto-injectors 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. An example of dual chamber sample injectors is described in US 6,149,626 A, issued November 21, 2006, and US 7,959,600 B2, issued June 14, 2011.

Protein stability.

The lyophilized form of the protein provides several benefits, one of which is maintaining protein stability over time, especially for at least 18 months

- 13 043993 at room temperature. Room temperature refers to the temperature of the normal working environment. Room temperature includes temperatures in 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 so 25°C. The phrase room temperature can be used interchangeably with the phrase ambient temperature. Room temperature includes controlled room temperature, which indicates a typical work environment temperature of 20 to 25°C, with short-term variations of 15° to 30°, as may be found in pharmacies, hospitals and warehouses. Controlled room temperature includes a calculated average kinetic temperature of not more than 25° (see The Pharmacopeia of the United States of America, ThirtyThird 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).

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 an appropriate environment or under specified conditions. A protein can be stable even if it does not retain 100% of its physical structure, chemical structure, or biological function after storage for a certain period of time. As used herein, for example, a lyophilized protein is considered stable when no more than 2% of the amount of protein is present in high molecular weight form after storage at room temperature for up to 24 months. In some embodiments, the lyophilized or other solid form of the protein is considered stable when 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 of the protein is in high molecular weight form after storage at room temperature 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 more 18 months.

As used herein, a lyophilized or other solid form of a protein is considered unstable at room temperature if the percentage change in high molecular weight particles is greater than about 0.5%, greater than about 0.6%, greater than about 0.7%, greater than about 0.8%, or more than about 0.9% during the first month of storage at room temperature. Thus, a lyophilized or other solid form of protein with a percentage increase in high molecular weight species of <0.5% during the first month of storage at room temperature can be considered stable.

Stability can be measured, in particular, by determining the percentage of native molecule that remains in the composition after storage for a certain period of time at a certain temperature or after delivery to the patient. The percentage of a protein that retains its native form (e.g., the portion of the native species relative to the 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 protein, the residue is first solubilized and then the protein is tested. A native protein includes a protein that is 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 residue after storage for a certain amount of time at a certain temperature. More than 80% of the protein in the lyophilized residue should be in its native form and preferably more than 90%. The specified amount of time after which stability is measured may 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, according to at least 22 months, at least 23 months, at least 24 months or more. The temperature at which samples may be stored when assessing stability can be any temperature from about -80°C to about 50°C, for example, storage at about -80°C, about -30°C, about -20°C, about 0°C, about 4-8°C, about 5°C, about 25°C or other room temperatures, about 35°C, about 37°C or other physiological temperatures, about 45°C or about 50°C.

- 14 043993

Stability can be measured, among other things, by determining the percentage of protein that forms an aggregate (i.e. high molecular weight species, i.e. HMW species) in the lyophilized residue after a certain amount of time at a certain temperature, where stability is inversely proportional to the percentage of high molecular weight (HMW) species. which are formed. The percentage of HMW protein species can be determined, among other things, by size exclusion chromatography after solubilization as described above. A lyophilized protein composition may also be considered stable if after three months at room temperature it is 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 was found in the HMW form.

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 residue after a certain amount of time at a certain temperature, where stability is inversely proportional to the percentage of LMW species found in the solubilized lyophilized residue. The percentage of LMW protein species can be determined by size exclusion chromatography as described above. The protein lyophilized residue can also be considered stable if after three months at room temperature it is less than 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% the first molecule discovered in the LMW form.

Other methods can be used to assess the stability of a lyophilized protein, such as, for example, 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. For example, a composition of the present invention may be considered stable if, after 6 months or more of storage at a temperature of from about 5° C. to about 25° C., the change in OD ₄₀₅ of the composition is less than about 0.05 (e.g., 0.04, 0.03, 0.02, 0.01 or less) from the OD ₄₀₅ of the composition at time zero.

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 may be considered stable if, after storage at, for example, 5°C, 25°C, 37°C, 45°C, 50°C, etc. over a period of time (e.g., up to 1 month, 12 months, 18 months, 24 months, etc.), the antibody contained in the lyophilized composition binds to its cognate epitope-containing antigen with an affinity that is at least 50% , 95% or more of the binding affinity of the antibody prior to specified lyophilization and storage. Binding affinity can be determined, for example, using ELISA or plasmon resonance. Biological activity can be determined using an antibody, soluble receptor or ligand activity assay, such as, for example, contacting a cell that expresses a cognate binding partner with a reconstituted composition containing the antibody, soluble receptor or ligand. The binding of an antibody, soluble receptor or ligand to such a cell can be measured directly, for example, using FACS analysis. A protein may be considered stable when the biological or physiological specific activity (ie, potency) of the protein is at least 50% of its initial ( _T0 ) potency after storage at room temperature for at least 18 months. The stable protein maintains an efficiency of at least 51%, an efficiency of at least 52%, an efficiency of at least 53%, an efficiency of at least 54%, an efficiency of at least 55%, an efficiency of at least 56%, an efficiency of at least 57%, an efficiency of at least 58% , efficiency not less than 59%, efficiency not less than 60%, efficiency not less than 61%, efficiency not less than 62%, efficiency not less than 63%, efficiency not less than 64%, efficiency not less than 65%, efficiency not less than 66%, efficiency not less than 67%, efficiency not less than 68%, efficiency not less than 69%, efficiency not less than 70%, efficiency not less than 71%, efficiency not less than 72%, efficiency not less than 73%, efficiency not less than 74%, efficiency not less than 75 %, efficiency not less than 76%, efficiency not less than 77%, efficiency not less than 78%, efficiency not less than 79%, efficiency not less than 80%, efficiency not less than 81%, efficiency not less than 82%, efficiency not less than 83%, efficiency not less than 84%, efficiency not less than 85%, efficiency not less than 86%, efficiency not less than 87%, efficiency not less than 88%, efficiency not less than 89%, efficiency not less than 90%, efficiency not less than 91%, efficiency not less 92%, efficiency not less than 93%, efficiency not less than 94%, efficiency not less than 95%, efficiency not less than 96%, efficiency not less than 97%, efficiency not less than 98% or efficiency not less than 99% after storage 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 at room temperature.

A stable protein undergoes virtually no change in structure or specific activity after storage at room temperature for an extended period of time, for example up to 18 months. Changes in structure 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 and the like. For example, a common form of antibody degradation is the formation of aggregates, which include reversible dimers and trimers, as well as more stable and less reversible tetramers and higher order multimers. Irreversible aggregate

- 15 043993 you represent a subgroup of aggregates that are difficult to solubilize or do not bind when reconstituting the lyophilized residue. The stable protein undergoes an increase in high molecular weight particle formation 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% during storage at room temperature 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 for detecting high molecular weight species can have a detection variability of 0.2 to 0.3%.

The temperature-dependent rate of protein aggregation or other formation of high molecular weight species follows Arrhenius kinetics or modified Arrhenius kinetics (slightly curved Arrhenius plots) (see, for example, Chakroun et al., Mapping the Aggregation Kinetics of a Therapeutic Antibody Fragment, Mol. Pharmaceut. 13: 307-319 (2016)). Thus, the aggregation rates and percentage change in high molecular weight species at a given temperature for a given composition over time can be predicted based on the square root of the time dependence and Arrhenius or modified Arrhenius kinetics. For example, lyophilized samples are incubated at a specified temperature (eg, 5°C, 25°C, 37°C, 50°C) in sealed glass vials. Aliquots were removed at regular intervals, insoluble aggregates were removed, and then subjected to SE-HPLC. The observed aggregation rates are determined directly from the time-dependent linear fit of the main peak versus the HMW peak area. Predictions of percentage change for high molecular weight species are then made by applying the square root of time rate law and Arrhenius kinetics to the data. For example, the observed rate constant at different temperatures is then fitted to the Arrhenius equation: In k=ln A (E/RT), where E is the activation energy (in cal/mol), R is the universal gas constant (1.987 cal/mol/K) , T is the absolute temperature (in Kelvin) and ln A is the temperature constant, which includes factors such as collision frequency. The value of k is extrapolated from the Arrhenius approximation to predict the change in macromolecular species at a given temperature (eg 25°C) over time (eg 24 months).

Lyophilization.

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-lyophilized aqueous solution containing drug substance and excipients as described above. The pre-lyophilized aqueous solution is placed in an open container (eg, a vial) and the open container is placed in a lyophilization chamber on a shelf. The lyophilization process involves three main steps: (1) freezing with optional annealing cycles, (2) primary drying, and (3) secondary drying with an optional post-drying annealing step. The first stage is freezing. At this stage, the shelf temperature is lowered to cool the drug in the vial. Ice crystals form inside the composition and the rest of the composition becomes more concentrated and viscous. The concentrated residue solidifies to form an amorphous, crystalline, or combined crystalline/amorphous state. In one embodiment, the residue solidifies into an amorphous glassy state.

In some lyophilization techniques, the frozen composition is annealed to enhance crystallization. The solid residue is kept above the final freezing point for a period of time to crystallize certain components, such as excipients such as mannitol and glycine.

The ice is then removed by sublimation. The pressure in the lyophilization chamber (for example, 40-400 Torr) and the shelf temperature (-30 ° C - + 10 ° C) are set below the triple point of water. The temperature is maintained below the glass transition temperature ( _Tg ) for amorphous residues to prevent degradation of the residue structure.

Some water may remain in the matrix after the initial drying step. The remaining water is removed through the desorption process at the secondary drying stage. At this stage, the shelf temperature is increased to accelerate desorption and achieve optimal moisture content in the lyophilized product. In one embodiment, the final moisture content is no less than about 0.5% and no more than about 10%. In another embodiment, the final moisture content is no less than about 3% and no more than about 6%. In a specific embodiment, the moisture content is greater than 3% but less than 4%. In another specific embodiment, the moisture content is about 3%. In another specific embodiment, the moisture content is about 3.5%. In another specific embodiment, the moisture content is about 4%. In another specific embodiment, the moisture content is about 4.5%. In yet another specific embodiment, the moisture content is about 6%.

In one embodiment, the lyophilized product (lyophilized residue) is subjected to an annealing step after a secondary drying step. Annealing after the drying stage is also called fi

- 16 043993 zymic aging or structural relaxation. This annealing step after drying promotes the relaxation of the amorphous matrix to an equilibrium glassy state (alpha relaxation), increasing the structural relaxation time at storage temperature, decreasing the mobility in the glassy state, likely lowering the protein to a more stable energetic state, thereby optimizing protein stability. Here, the annealing step transiently accelerates molecular mobility to provide an overall reduction in the entropy of the glassy state and rotational forms of the protein.

After the annealing step, the number of random thermodynamic molecular glass states is minimized and glass equilibrium is maximized. In some embodiments, the annealing temperature is below the Tg of the product. In some embodiments, the annealing temperature is from 25°C to about 90°C, from 25°C to about 80°C, from 25°C to about 75°C, from about 25°C to about 70°C, from 35 °C to about 70°C, from 40°C to about 70°C, from 30°C to about 55°C, from 50°C to about 60°C, from 55°C to about 65°C, from 60 °C to about 70°C, from 65°C to about 75°C, from 70°C to about 80°C, or from 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 a specific embodiment, the annealing temperature is above 25°C. In another specific embodiment, the annealing temperature is above 50°C.

The lyophilized residue is maintained at the annealing temperature for a period of time sufficient to allow relaxation of the structure of the residue, as determined by entropy recovery from DSC or isothermal calorimetry. In some embodiments, the annealing temperature is maintained for about 12 hours to about two weeks, about 12 hours to one week, about 12 hours to about several days, about 18 hours to about 72 hours, about 24 hours to about 36 hours , from about 30 hours to about 42 hours, from about 36 hours to about 48 hours, from about 42 hours to about 54 hours, from about 48 hours to about 60 hours, from about 54 hours to about 66 hours, from about 60 hours to about 72 hours, from about 66 hours to about 78 hours, or from about 72 hours to about 84 hours. In some embodiments, the annealing temperature is maintained for about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 h, about 17 h, about 18 h, about 19 h, about 20 h, about 21 h, about 22 h, about 23 h, about 24 h, about 25 h, about 26 h, about 27 h, about 28 h, 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 h, about 42 h, about 43 h, about 44 h, about 45 h, about 46 h, about 47 h, about 48 h, about 49 h, about 50 h, about 51 h, about 52 h, about 53 h, 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.

In a specific embodiment, the annealing temperature is above 50°C, which is maintained for about 72 hours. In another specific embodiment, the annealing temperature is about 25°C, which is maintained for about 72 hours.

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 usually 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

The following examples are provided to provide those skilled in the art with a complete description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of the invention, which the inventors believe to be their invention. Efforts have been made to ensure accuracy in terms of numbers used (e.g. quantities, sizes, etc.), but some experimental errors and deviations must be taken into account.

Example 1. Lyophilization procedure.

An aqueous pre-lyophilization solution containing the antibody and excipients (as described herein) was loaded into a Type I borosilicate glass vial. The filled vial was placed in a LYOSTAR 3 lyophilizer (SP Scientific, Warminster, PA). The chamber was closed and the shelf temperature was reduced to 5°C. The sample was kept at 5°C for 30 min before freezing. The rate of change during freezing was 0.5°C/min. The shelf temperature was maintained at -45°C for 60 min.

- 17 043993

Primary drying was performed at a set vacuum value of about 100 mTorr and a shelf temperature of about -25°C, which was achieved at a heating rate of about 0.5°C/min for about 50 hours.

Secondary drying was carried out at 35°C, which was achieved with a heating ramp rate of about 0.3°C/min. Secondary drying lasted about 6 hours.

After secondary drying, the chamber was filled with nitrogen gas to a pressure of about 0.8 atmospheres (about 608,000 mTorr) and the vial was sealed with a 4432/50 Flurotec® coated butyl rubber lyophilization stopper.

Example 2 Protein stability as a function of moisture content.

Water molecules can serve as a plasticizer and stabilizer in a lyophilized product. Water is useful as a plasticizer because of 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 composition. The moisture content can be controlled by designing the lyophilization process. The moisture content of the lyophilized residues was determined with a Computrac® Vapor Pro® moisture analyzer (Arizona Instrument LLC, Chandler, AZ).

Recombinant monoclonal antibodies (mAb1, mAb2, mAb3) were produced in EESYR® cells (see US patent No. 7771997 B2, issued August 10, 2010) and were formed 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 composition). The liquid composition was lyophilized as described above to specific moisture content levels (w/w%) and stored at 50°C, 37°C or 25°C for specified times. After storage, the lyophilized compositions were reconstituted with water and subjected to size exclusion high performance liquid chromatography (SE-HPLC). High molecular weight species (HMW) were detected, integrated and compared with controls at T=0. The percentage change in species HMW as a fraction of total protein was calculated and presented in Table. 1. Lyophilized 150 mg/ml mAb1 was most stable with a moisture content of about 4.5% when stored at 25°C.

Lyophilized mAb1 compositions having a moisture content of 3-10% were predicted to degrade <~2.0% after 24 months of storage at 25°C. Arrhenius kinetics algorithms were applied to the measured degradation rates and predictions based on the square root of the time rate law were at least 24 months. The percentage change in HMW of species for each moisture content point is given in Table. 2. The pre-lyophilized aqueous composition contains 150 mg/ml mAb1, 10 mM histidine, pH 6.0, 0.1% polysorbate 80, 5% sucrose, 1.54% arginine.

Example 3. Effect of annealing after drying.

Protein stability was improved by annealing the lyophilized drug product. It is believed that annealing the lyophilized composition below the glass transition temperature causes the amorphous molecules to relax to a lower energy state, yielding a more stable product. Annealing the lyophilized composition for 72 hours at 70°C resulted in a lower observed increase in %HMW for 150 mg/ml mAb1 lyophilized with 10% sucrose and 3.08% arginine (pre-lyophilized composition: 150 mg/ml mAb1, 10 mM histidine, pH 6.0, 0.1% polysorbate 80, 10% sucrose, 3.08% arginine). The percentage change in HMW antibody types is given in Table. 3 for annealed and unannealed compositions stored for 6 months at 25°C, 37°C and 50°C.

Table 1 ______Percentage increase in high molecular weight species______

Temperature °C 50 37 25 Time (months) 0.5 1 2 1 3 6 1 3 6 H ₂ O 0 3, 6 5.16 7.79 1.72 3.37 5.0 0.55 1.19 1.8 0.5 3, 1 4.42 6, 84 1, 48 2.91 4.3 0.51 1.02 1.6 1.5 2.4 3.56 5.54 1, 07 2.19 3.3 0.31 0.7 1.1 3.0 2.3 3.38 5.46 0.88 1.77 2.9 0.26 0.55 0.9 4.5 2.7 3.77 6, 18 0.91 1.86 2.9 0.22 0.46 0.7 6, 0 2.1 4.6 7.75 0.9 2.33 BUT 0, 1 0.63 BUT 8.0 2.9 5.8 10, 67 14 2.98 BUT 0.2 0.92 BUT 10 3, 6 7.0 13.29 18 4.14 But 0, 4 1.16 BUT

- 18 043993

table 2

Degradation prediction at 25°C of 150 mg/ml lyophilized mAbl - A%HMW species Time H ₂ O 1 month 3 months 6 months 9 months 12 months 18 months 2 4 months 0% 0.77 1.33 1, 89 2.31 2, 67 3.24 3.77 0.5% 0.66 1, 14 1, 61 1.97 2.27 2.78 3.21 1.5% 0.47 0.82 1.16 1.42 1, 64 2.01 2.32 thirty% 0.36 0.63 0.89 1.09 1.26 1.54 1.78 4.5% 0.31 0.55 0.77 0.94 1, 09 1.34 1.54 6.0% 0.34 0.60 0.84 1, 03 1, 19 1.46 1, 69 8.0% 0.49 0.85 1.20 1.47 1.70 2.08 2.41 10% 0.64 1.11 1.58 1.93 2.23 2.73 3, 15

Arrhenius kinetics algorithms were applied to the measured degradation rates and projections, based on the square root value of the rate versus time law, were at least 24 months for annealed samples compared to unannealed samples stored at 37°C. Annealing is predicted to produce approximately 20% less HMW species than the unannealed sample after 24 months (i.e., 5.76% vs. 6.05% A%HMW).

Table 3

A%HMW with annealing vs without annealing Temperature Annealing No annealing 25°C 0.2% 0.6% 37°C 0.5% 1.5% 50°С 0.8% 2.1%

Example 4. Effect of individual fillers on stability.

Specific stabilizers, including sugars, polyols, salts and amino acids, were evaluated for their individual ability to stabilize the lyophilized antibody.

Lyophilized 150 mg/ml mAbl was most stable when formulated with sucrose (see Table 4). The T _g of the sucrose composition is high enough (~110°C) for storage at room temperature and provides sufficient latitude for adding a plasticizer if necessary.

Example 5 Effect of trehalose combination on stability.

Combining trehalose or sucrose with plasticizers such as sorbitol or mannitol unexpectedly resulted in a more stable composition at room temperature. Combinations of sorbitol and trehalose stabilized lyophilized 150 mg/ml shAA more than trehalose alone. The base pre-lyophilized formulation contained mAbl 150 mg/ml, 10 mM histidine, pH 6.0, 0.1% polysorbate 80, to which various combinations of trehalose and sorbitol were added prior to lyophilization. The stability of the lyophilized protein in combination with trehalose and/or sorbitol was tabulated as A%HMW in Table. 5.

Table 4

Effect of individual excipients on mAbl A%HMW

50°C @0.5 months. 50°C @ 1 month 50°C @ 2 months 25°C @ 4 months No stabilizer 23.74 32.33 43.76 11.04 10% sucrose 2, 68 4.12 6, 24 0.87 10% trehalose 4, 02 5, 98 8.9 1, 66 3.36% proline 9, 08 13.48 19, 52 3.79 5.32% sorbitol 8.29 12.45 18.32 1.83 5.32% mannitol 4.98 7, 61 11.58 1.4 2.19% glycine 8.57 12.3 18, 63 3.28 0.85% NaCl 8.43 11.54 17.95 2.28 2.69% glycerol 12.86 17.34 25, 91 4.49 3.08% arginine 6, 66 10, 06 14.86 2.95 2.60% alanine 9, 84 14.58 22.23 4.03

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Table 5

Effect of trehalose and/or sorbitol on mAbl Δ%HMW

Stabilizer combination 10% trehalose 6, 66% trehalose, 1.77% sorbitol 5% trehalose, 2.66% sorbitol 3, 88% trehalose, 3.55% sorbitol 25°C (6 months) 2.0% 1.8% 1.8% 1.8% 37°C (6 months) 5.4% 50% 5.1% 5.6% 50°C (6 months) 8.1% 7.9% 8.5% 9.8% 25°C (24 months)* 4.06% 3.43% 3.26% 3.26%

* Projections are based on the square root of the velocity versus time law and Arrhenius kinetics.

Example 6 Effect of sucrose combination on stability.

Combinations of mannitol and sucrose stabilized lyophilized 150 mg/ml mAb1 more than sucrose alone. The base pre-lyophilized composition contains 150 mg/ml mAb1, 10 mM histidine, pH 6.0, 0.1% polysorbate 80, to which various combinations of sucrose and mannitol have been added prior to lyophilization. The stability of the lyophilized protein in combination with sucrose and/or mannitol was tabulated as Δ%HMW in Table. 6. The combination of mannitol and sucrose stabilized lyophilized 150 mg/ml mAb1 more than sucrose alone.

Table 6

Effect of sucrose and mannitol on mAb1 A%HMW

Stabilizer combination 10% sucrose 5% sucrose, 2.66% sorbitol 25°C (6 months) 16% 1.3% 37°C (6 months) 4.0% 3.7% 50°C (2 months) 5.9% 7.2% 25°C (24 months)* 2.86% 2.52%

* Projections are based on the square root of the velocity versus time law and Arrhenius kinetics.

Sucrose was combined with polyols and amino acids to evaluate the ability to stabilize the lyophilized antibody. MAb1 150 mg/ml was combined with sucrose (Suc) and any of sorbitol (Sor), glycerol (Gly), arginine (Arg) and alanine (Ala) in various proportions. The change in HMW of the species was assessed at 25°C and 5°C for different storage periods. The results (Δ%HMW) are presented in table. 7. None of the excipient combinations tested stabilized lyophilized mAb1 150 mg/mL more than sucrose alone when stored at 25°C. Surprisingly, the combination of sorbitol and sucrose was predicted to improve stability compared to sucrose alone when stored at 5°C for 120 months and to be more stable than sucrose alone.

Example 7: Effects of a combination of sucrose and arginine on drug stability.

Lyophilized 150 mg/ml mAb1 and lyophilized 150 mg/ml mAb2 have comparable stability when mixed with 10% sucrose and 3.08% arginine. The change in %HMW of species was calculated after 1 month of storage at 25°C, 37°C and 50°C. Lyophilized mAb1 150 mg/mL and lyophilized mAb2 150 mg/mL (based on the square root of Arrhenius' rate law and kinetics) were predicted to have comparable stability when formed with 10% sucrose and 3.08% arginine at 25°C to 24 months and beyond. The results of the Δ%HMW analysis are presented in table. 8.

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Table 7

Effect of other excipients in combination with sucrose on mAbl Δ%ΗΜ W

25°C/1 month 25°C/3 months 25°C/6 months * 25°C/24 months * 5°C/120 months 100% sucrose 0.47% 0.96% 1.55% 3.08% 1.18% 9.09% Sor Suc/0.48% 0.53% 1.04% 1.60% 8.33% Sor Suc/0.89% 0.47% 0.65% 1.52% 6.66% Sor Suc/1.77% 0.46% 0.98% 1.53% 5.00% Sor Suc/2.66% 0.48% 1.02% 1.55% 3.08% 0.90% 9.09%Gly Suc/0.24% 0.52% 1.06% 1.66% 8.33% Gly Suc/0.45% 0.55% 1.11% 1.72% 6.66%Gly Suc/0.90% 0.65% 1.27% 2.05% 5.00% Gly Suc/1.34% 0.71% 1.48% 2.32% 4.68% 1.48% 9.09% Arg Suc/0.28% 0.51% 1.02% 1.61% 8.33% Suc/0.51% 0.50% 1.03% 1.61% Arg 6.66% Arg Suc/1.03% 0.57% 1.14% 1.83% 5.00%Arg Suc/1.54% 0.65% 1.35% 2.08% 4.21% 1.68% 9.09% Ala Suc/0.24% 0.50% 0.99% 1.58% 8.33% Ala Suc/0.43% 0.54% 1.05% 1.64% 6.66% Ala Suc/0.87% 0.65% 1.30% 1.91% 5.00% Ala Suc/1.30% 0.72% 1.54% 2.45% 4.90% 1.79%

* Projections are based on the square root of the velocity versus time law and Arrhenius kinetics.

Table 8

Effect of 10% sucrose/3.08% arginine on mAb1 and mAb2 Δ%HMW

mAbl mAb2 25°C 37°C 50°C * 25°C 25°C 37°C 50°C * 25°C (1 (1 (1 (24 (1 (1 (1 (24 months ) months ) months ) months ) months ) months ) months ) months ) 0.2% 0.5% 1.5% 0.97% 0.1% 0.5% 1.5% 0.71%

* Projections are based on the square root of the velocity versus time law and Arrhenius kinetics.

Example 8. Effect of the ratio of stabilizer to drug product on the stability of the drug product.

Combinations of sucrose and arginine were formed with antibodies in different proportions by weight, and the Δ%HMW value was determined at different times at 50°C and 25°C. Lyophilized antibody formulations of 150 mg/mL with stabilizer >0.87:1 (based on the square root of the rate-time law and Arrhenius kinetics) are expected to degrade <1% after 24 months of storage at 25°C. The results are shown in table. 9.

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Table 9

Effect of stabilizer to protein ratio on stability (A%HMW)

Sucrose/ Stabilizer: antibody 50°С 50°С 50°С * 25°С Arginine (0.5 months) (1 month) (2 months) (24 months) 5%/1.5% 0.44:1 3.7% 5.4% 8.2% 3.74% 7.5%/2.3% 0.65:1 1.9% 2.7% 4.1% 1.77% 10%/3.1% 0.87:1 10% 1.5% 2.3% 0.97% 12.5%/3.9% 1.1:1 0.6% 0.9% 1.3% 0.43% 15%/4.6% 1.3:1 0.4% 0.6% 0.9% 0.35%

* Projections are based on the square root of the velocity versus time law and Arrhenius kinetics.

Table 10

The influence of individual fillers on wb3 A%HMW

50°C @0.5 months. 50°C @ 1 month 50°C @ 2 months 25°C @ 7 months No stabilizer 1.4% 2.31% 3.34% 0.78% 10% sucrose 0.03% 0.03% 0.04% 0.04% 10% trehalose 0.09% 0.08% 0.16% 0.23% 3.36% proline 2.0% 2.92% 12.37% 3.25% 5.32% sorbitol 3.5% 6.24% 8.85% 1.47% 5.32% mannitol 21.55% 26.87% 30.78% 14.44% 2.19% glycine 9.67% 16.22% 24.39% 3.77% 0.85% NaCl 4.51% 6.4% 7.83% 6.27% 3.08% arginine 0.05% 0.14% 0.09% 0.04% 2.60% alanine 18.04% 22.18% 23.68% 14.74%

Example 9: Stabilizing effect of lyophilized protein excipients on low protein concentrations in a pre-lyophilized liquid composition.

Specific stabilizers, including sugars, polyols, salts and amino acids, were evaluated for their individual ability to stabilize the lyophilized antibody.

Lyophilized 2 mg/mL mAl3 showed no noticeable degradation when sucrose, trehalose, or arginine were included as a stabilizer. The results are presented in table. 10. Glycerol was also tested, but significant degradation was observed after lyophilization.

Trehalose was combined with polyols and amino acids to evaluate the ability to stabilize the lyophilized antibody. 2 mg/ml mAl3 was combined with trehalose (Tre) and any of sorbitol (Sor), glycerol (Gly), arginine (Arg) and alanine (Ala) in different proportions. Changes in HMW of species were assessed at 25°C at different storage times. The results (A%HMW) are presented in table. I. No noticeable degradation was observed in any of the 2 mg/mL lyophilized combinations of mLab3 tested after 3 months of storage at 25°C.

Claims (9)

Table 11 Effect of trehalose and other excipients on the stability of low concentration mAb3 (Δ%HMW) 25°C/1 month 25°C/3 months *25°С/120 months. 10.0% trehalose 0.01% 0.007% 9.09% Tre/0.48% Sor 0.03% 6.66% Tre/1.77% Sor 0.01% 5.00% Tre/2.66% Sor 0.01% 5.00% Tre/1.34% Sor 0.007% 9.09% Tre/0.24% Gly 0.01% 5.00% Tre/1.34% Gly 0.02% 5.00% Tre/0.90% Gly 0.10% 9.09% Tre/0.28% Apr 0.01% 8.33% Tre/0.51% Apr 0.04% 0.01% 6.66% Tre/1.03% Apr 0.03% 0.04% 5.00% Tre/1.54% Apr 0.05% 0.05% 0.33% 9.09% Tre/0.24% Ala 0.01% 8.33% Tre/0.43% Ala 0.08% 0.08% 6.66% Tre/0.87% Ala 0.06% 0.12% 5.00% Tre/1.30% Ala 0.04% 0.2% 1.03% * Projections are based on the square root of the velocity versus time law and Arrhenius kinetics. CLAIM 1. A pharmaceutically acceptable lyophilized residue containing: (a) protein; (b) a stabilizer containing about 5% sucrose by weight of the pre-lyophilized residue and arginine in a weight ratio of sucrose to arginine from about 3.2:1 to about 3.4:1; (c) a buffer; and (d) a moisture content of from about 3 to about 6 wt.% water. 2. The lyophilized residue according to claim 1, wherein said lyophilized residue has a glass transition temperature above 25°C. 3. The lyophilized residue according to claim 1 or 2, containing about 3, about 3.5, about 4, about 4.5, about 5, about 5.5 or about 6 wt.% water. 4. Lyophilized residue according to any one of claims 1 to 3, containing approximately 4.5 wt.% water. 5. The lyophilized residue according to any one of claims 1 to 4, wherein said buffer contains histidine, optionally said histidine being present in an amount of from about 0.34 to about 2.04 weight percent. 6. Lyophilized residue according to any one of claims 1-5, additionally containing: (a) a stabilizer selected from the group consisting of a polyol, sugar, amino acid, salt, and combinations thereof, and optionally: (i) said polyol selected from the group consisting of sorbitol, glycerol, mannitol, and combinations thereof; (ii) said sugar is trehalose; (iii) said amino acid is selected from the group consisting of alanine, proline, glycine, sodium chloride and combinations thereof; and (iv) said salt is sodium chloride; and/or (b) the stabilizer is present in an amount of from about 19 to about 83 weight percent. 7. The lyophilized residue of claim 6, wherein said stabilizer further comprises mannitol, optionally mannitol being present in an amount of from 8 to about 23 weight percent. 8. The lyophilized residue of claim 6, wherein said stabilizer further comprises sorbitol, wherein the sorbitol is optionally present in an amount of from about 1.3 to about 23 weight percent sorbitol. 9. Lyophilized residue according to any one of claims 1 to 8, additionally containing a surfactant, and optionally: (a) said surfactant is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, polyethylene glycol (PEG), PEG 3350 and combinations thereof; or (b) said surfactant is present in an amount of from about 0.02 to about 1 weight percent. -