Formulations of monoclonal antibodies

Abstract

To provide a formulation for an antibody having a low or neutral pI with improved colloidal stability.SOLUTION: The present invention provides a formulation comprising: (i) a monoclonal antibody; and (ii) an ionic excipient; wherein the monoclonal antibody is present at a concentration of about 50 mg / ml or greater (e.g. about 50 mg / ml to about 200 mg / ml) and the ionic excipient is present at a concentration of about 50 to about 150 mM and the formulation has a pH of 5.5 to 6.5.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to antibody formulations, particularly monoclonal antibody formulations and uses thereof. The present invention is particularly concerned with providing improved colloidal stability of antibody formulations. [Background technology]

[0002] Due to the isoelectric point (pI) of some monoclonal antibodies being in the pharmaceutical pH range (pH 5.5-pH 7.5) for preferred formulations of proteins, these molecules present unique formulation challenges.

[0003] Colloidal instability at the pI of a molecule is due to the lack of electrostatic charge on the molecule, which allows for closer protein-protein interactions (so-called "self-association") that result in physical instability. For this reason, the pH of a protein formulation is typically selected to be at least one pH unit away from the protein pI. The aim is to provide colloidal stability and thus prevent physical instabilities such as aggregation, precipitation, opalescence, phase separation, and / or particle formation.

[0004] Thus, according to the "one pH unit away" rule, an antibody with a low or neutral pI, for example, a pI between pH 5.5 and pH 7.5, should be formulated in a formulation with a pH outside the range of 5.5 to 7.5. However, outside this range, additional instability may be observed. At more acidic pH, increased fragmentation rates, decreased conformational stability, and increased aggregation may be observed. At more basic pH, there is the potential for oxidation, deamidation, and increased fragmentation, as well as incompatibility with glass containers.

[0005] This instability is particularly problematic in antibody formulations where the antibody is present at commercially desirable concentrations, eg, 50 mg / ml or greater. Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there is a need to provide improved formulations for antibodies with low or neutral pI. In particular, there is a need to provide stable formulations for antibodies with low or neutral pI, especially such formulations having commercially desirable antibody concentrations. [Means for solving the problem]

[0007] The present invention provides novel antibody formulations, particularly novel monoclonal antibody formulations. In particular, the formulations provide a means for improving the colloidal stability of antibodies with low or neutral pI. Thus, the present invention provides an alternative to the "one pH away" rule for providing colloidal stability. Thus, the present invention allows for antibodies with low or neutral pI to be formulated within one pH unit of the antibody pI. Thus, the present invention allows for such antibodies to be formulated at commercially useful concentrations within the pH range of 5.5-7.5, while substantially avoiding the instability associated with more acidic or more basic pH.

[0008] The present invention relates to: i. monoclonal antibodies; and ii. ionic excipients; wherein the monoclonal antibody is present in a concentration of about 50 mg / ml or more (e.g., about 50 mg / ml to about 200 mg / ml), the ionic excipient is present in a concentration of about 50 to about 150 mM, and the formulation has a pH of 5.5 to 7.5.

[0009] Thus, the present invention provides: i. monoclonal antibodies; and ii. ionic excipients; wherein the monoclonal antibody is present at a concentration of about 50 mg / ml or more (e.g., about 50 mg / ml to about 200 mg / ml), the ionic excipient is present at a concentration of about 50 to about 150 mM, and the formulation has a pH of 5.5 to 7.5; and the aggregation rate of the monoclonal antibody in the formulation is reduced compared to the aggregation rate of the same antibody in the same formulation without the ionic excipient.

[0010] The formulations of the invention are particularly useful for antibodies with a low or neutral pI, for example in the range of pH 5.5 to pH 7.5. i. Monoclonal antibodies with a low or neutral pI (e.g., 5.5 to 7.5); and ii. ionic excipients; wherein the monoclonal antibody is present in a concentration of about 50 mg / ml or more (e.g., about 50 mg / ml to about 200 mg / ml), the ionic excipient is present in a concentration of about 50 to about 150 mM, and the formulation has a pH of 5.5 to 7.5.

[0011] Thus, the present invention relates to a formulation comprising: i. Monoclonal antibodies with a low or neutral pI (e.g., 5.5 to 7.5); and ii. ionic excipients; wherein the monoclonal antibody is present at a concentration of about 50 mg / ml or more (e.g., about 50 mg / ml to about 200 mg / ml), the ionic excipient is present at a concentration of about 50 to about 150 mM, and the formulation has a pH of 5.5 to 7.5; and the aggregation rate of the monoclonal antibody in the formulation is reduced compared to the aggregation rate of the same antibody formulated in the same formulation without the ionic excipient.

[0012] In one embodiment, the monoclonal antibody has a pI in the range of 5.5-7.5. In one embodiment, the monoclonal antibody has a pI in the range of 6.0-7.5. In one embodiment, the monoclonal antibody has a pI in the range of 6.3-7.5. In one embodiment, the monoclonal antibody has a pI in the range of 6.4-7.5. Without wishing to be bound by theory, low to neutral pIs can arise in proteins when there is a net balance of oppositely charged (positive amine groups and negative carboxylate groups) amino acid side chains on the protein or when different domains have overall opposite charges within the pH range of 5.5-7.5. Similarly, without wishing to be bound by theory, ionic excipients in the formulations of the invention can shield these opposing attractive charges and thus colloidally stabilize proteins with pIs in this range. Thus, the invention provides for the use of ionic excipients in antibody formulations to alter the charge state or distribution of the antibody in the formulation. The invention further provides for the use of an ionic excipient in an antibody formulation for the purpose of colloidally stabilizing the antibody in the formulation.

[0013] In one embodiment, the monoclonal antibody is present in the formulation described herein at a concentration of about 75 mg / ml or more (e.g., about 75 mg / ml to about 200 mg / ml). In one embodiment, the monoclonal antibody is present in the formulation described herein at a concentration of about 100 mg / ml or more (e.g., about 100 mg / ml to about 200 mg / ml). In one embodiment, the monoclonal antibody is present in the formulation described herein at a concentration of about 100 mg / ml to about 165 mg / ml. In one embodiment, the monoclonal antibody is present in a formulation described herein at a concentration of about 100 mg / ml.

[0014] In one embodiment, the ionic excipient is present at a concentration of about 75 mM to about 100 mM. In one embodiment, the ionic excipient is present at a concentration of about 75 mM. In one embodiment, the ionic excipient is present at a concentration of about 80 mM.

[0015] In one embodiment, the monoclonal antibody is an IgG1 or IgG4 monoclonal antibody. Most preferably, the monoclonal antibody is an IgG4 monoclonal antibody. IgG4 antibodies typically have a low or neutral pI. Thus, the present invention provides a method for the preparation of a method for the preparation of a monoclonal antibody comprising: i. IgG4 monoclonal antibody; and ii. ionic excipients; wherein the monoclonal antibody is present in a concentration of about 50 mg / ml or more (e.g., about 50 mg / ml to about 200 mg / ml), the ionic excipient is present in a concentration of about 50 to about 150 mM, and the formulation has a pH of 5.5 to 7.5.

[0016] Thus, the present invention relates to a formulation comprising: i. IgG4 monoclonal antibody; and ii. ionic excipients; wherein the monoclonal antibody is present at a concentration of about 50 mg / ml or more (e.g., about 50 mg / ml to about 200 mg / ml), the ionic excipient is present at a concentration of about 50 to about 150 mM, and the formulation has a pH of 5.5 to 7.5; and the aggregation rate of the monoclonal antibody in the formulation is reduced compared to the aggregation rate of the same antibody in the same formulation without the ionic excipient.

[0017] In one embodiment, the formulations described herein have a pH ranging from about pH 5.5 to about pH 6.5. In one embodiment, the formulations described herein have a pH ranging from about pH 5.7 to about pH 6.3. In one embodiment, the formulations described herein have a pH ranging from about pH 5.7 to about pH 6.1. A preferred formulation has a pH of about 5.8. Another preferred formulation has a pH of about 6.0.

[0018] In one embodiment, the ionizable excipient is a charged amino acid. In one embodiment, the ionizable excipient is lysine. In another embodiment, the ionizable excipient is arginine.

[0019] In one embodiment, the ionic excipient is a salt. Thus, the present invention provides a method for the preparation of a compound comprising: i. a monoclonal antibody as defined anywhere herein; and ii. salt; wherein the monoclonal antibody is present in a concentration of about 50 mg / ml or more (e.g., about 50 mg / ml to about 200 mg / ml), the salt is present in a concentration of about 50 to about 150 mM, and the formulation has a pH of 5.5 to 7.5.

[0020] Thus, the present invention provides: i. a monoclonal antibody as defined anywhere herein; and ii. salt; wherein the monoclonal antibody is present in a concentration of about 50 mg / ml or more (e.g., about 50 mg / ml to about 200 mg / ml), the salt is present in a concentration of about 50 to about 150 mM, and the formulation has a pH of 5.5 to 7.5; and the aggregation rate of the monoclonal antibody in the formulation is reduced compared to the aggregation rate of the same antibody in the same formulation without the salt.

[0021] In one embodiment, the salt is present at a concentration of about 75 mM to about 100 mM, hi one embodiment, the salt is present at a concentration of about 75 mM or about 80 mM.

[0022] In one embodiment, the salt is NaCl, for example at a concentration of about 75 mM to about 100 mM, suitably at a concentration of about 75 mM.

[0023] In one embodiment, the salt is arginine hydrochloride, for example at a concentration of about 75 mM to about 100 mM, suitably at a concentration of about 80 mM.

[0024] In one embodiment, the formulation further comprises a sugar. Among other known advantages, the presence of a sugar can improve the tonicity of the formulation. This is desirable because preferred formulations are isotonic or near isotonic (e.g., having an osmolality of 240-500 mOsm / kg). In one embodiment, the ionic excipient is a salt and the formulation further comprises a sugar.

[0025] In one embodiment, the formulation further comprises a sugar, and the ionic excipient is present at a concentration ranging from about 75 mM to about 150 mM. In one embodiment, the formulation further comprises a sugar, and the ionic excipient is present at a concentration ranging from about 75 mM to about 100 mM. In one embodiment, the formulation further comprises a sugar, and the sugar is present at a concentration ranging from about 100 mM to 140 mM, and the ionic excipient is present at a concentration ranging from about 75 mM to 100 mM.

[0026] Thus, the present invention provides: i. A monoclonal antibody as defined anywhere herein; ii. An ionic excipient (e.g., a salt) as defined anywhere herein; iii. Sugar as defined anywhere herein; The monoclonal antibody is present at a concentration of about 50 mg / ml or more (e.g., about 50 mg / ml to about 200 mg / ml), the ionic excipient is present at a concentration of about 50 to about 150 mM, and the formulation has a pH of 5.5 to 7.5. The ionic excipient is preferably present at a concentration in the range of about 75 mM to about 150 mM, more preferably about 75 mM to about 100 mM.

[0027] Thus, the present invention provides: i. a monoclonal antibody as defined anywhere herein; and ii. An ionic excipient (e.g., a salt) as defined anywhere herein; iii. Sugar as defined anywhere herein; wherein the monoclonal antibody is present at a concentration of about 50 mg / ml or more (e.g., about 50 mg / ml to about 200 mg / ml), the ionic excipient is present at a concentration of about 50 to about 150 mM, and the formulation has a pH of 5.5 to 7.5; the aggregation rate of the monoclonal antibody in the formulation is lower compared to the aggregation rate of the same antibody in the same formulation without the ionic excipient. The ionic excipient is preferably present at a concentration in the range of about 75 mM to about 150 mM, more preferably about 75 mM to about 100 mM.

[0028] In one embodiment, the sugar is trehalose. In another embodiment, the sugar is sucrose. For example, the sugar is present at a concentration of about 100 mM to about 140 mM, suitably at a concentration of about 120 mM.

[0029] In one embodiment, the formulation further comprises one or more buffering agents. In one embodiment, the one or more buffering agents are buffering agents comprising histidine. In one embodiment, the one or more buffering agents are selected from buffering agents comprising histidine succinate, histidine acetate, histidine citrate, histidine chloride, or histidine sulfate. In one embodiment, the one or more buffering agents are histidine, histidine hydrochloride, or a combination thereof (histidine / histidine hydrochloride). In one embodiment, the one or more buffering agents are L-histidine / L-histidine hydrochloride monohydrate. For example, the buffering agent may be at a concentration of about 10 mM to about 50 mM, suitably at a concentration of about 30 mM. It should be understood that the buffering agent may itself be an ionic excipient. Thus, in one embodiment, the buffering agent is an ionic excipient. In this embodiment, the concentration of the buffering agent should be greater than 50 mM, i.e., consistent with the concentration of the ionic excipients disclosed herein. In other words, in one embodiment, the ionic excipient also acts as a buffer in the formulation, in which an additional buffer may or may not be present.

[0030] In one embodiment, the formulation further comprises a surfactant, hi one embodiment, the surfactant is a polysorbate, including, for example, polysorbate 80.

[0031] In one embodiment, the formulation further comprises a sugar and one or more buffering agents. In one embodiment, the ionic excipient is a salt and the formulation further comprises a sugar and one or more buffering agents.

[0032] In one embodiment, the formulation further comprises a surfactant, a sugar, and one or more buffering agents. In one embodiment, the ionic excipient is a salt, and the formulation further comprises a surfactant, a sugar, and one or more buffering agents.

[0033] Thus, the present invention provides: i. A monoclonal antibody as defined anywhere herein; ii. An ionic excipient (e.g., a salt) as defined anywhere herein; iii. Sugar as defined anywhere herein; iv. one or more buffering agents as defined anywhere herein; and v. optionally a surfactant, as defined anywhere herein; wherein the monoclonal antibody is present in a concentration of about 50 mg / ml or more (e.g., about 50 mg / ml to about 200 mg / ml), the ionic excipient is present in a concentration of about 50 to about 150 mM, and the formulation has a pH of 5.5 to 7.5.

[0034] Thus, the present invention provides: i. a monoclonal antibody as defined anywhere herein; and ii. An ionic excipient (e.g., a salt) as defined anywhere herein; iii. Sugar as defined anywhere herein; iv. one or more buffering agents as defined anywhere herein; and v. an optional surfactant as defined anywhere herein; wherein the monoclonal antibody is present at a concentration of about 50 mg / ml or more (e.g., about 50 mg / ml to about 200 mg / ml), the ionic excipient is present at a concentration of about 50 to about 150 mM, and the formulation has a pH of 5.5 to 7.5; and the aggregation rate of the monoclonal antibody in the formulation is reduced compared to the aggregation rate of the same antibody in the same formulation without the ionic excipient.

[0035] The formulations described herein may also include one or more additional excipients including, for example, one or more sugars, salts, amino acids, polyols, chelating agents, emulsifying agents, and / or preservatives.

[0036] One preferred formulation provided by the present invention comprises 150 mg / ml antibody, 50 mM sodium acetate / acetic acid, 106 mM trehalose dehydrate, 70 mM sodium chloride, 0.05% (w / v) polysorbate 80, the formulation having a pH of pH 5.8. The antibody in this formulation is preferably an anti-GM-CSF-Rα IgG4 antibody, more preferably an anti-GM-CSF-Rα IgG4 antibody having the VH and VL sequences of the CAM-3001 antibody as described herein. The antibody in this formulation is preferably an anti-GM-CSF-Rα IgG4 antibody having the same six CDRs as the CAM-3001 antibody as described herein.

[0037] One preferred formulation provided by the present invention comprises 150 mg / ml antibody, 50 mM sodium acetate, 85 mM sodium chloride, and 0.01% polysorbate 80, the formulation having a pH of pH 5.5. The antibody in this formulation is preferably an anti-IL-13 IgG4 antibody, more preferably an anti-IL-13 IgG4 antibody having the VH and VL sequences of the CAT-354 antibody as described herein. The antibody in this formulation is preferably an anti-IL-13 IgG4 antibody having the same six CDRs as the CAT-354 antibody as described herein.

[0038] The formulation of the present invention is preferably a pharmaceutical formulation.

[0039] The present invention provides a pharmaceutical formulation as described anywhere herein for use as a medicament.

[0040] The present invention provides a pharmaceutical formulation as described anywhere herein for use in the treatment of a disease.

[0041] The present invention provides a method of treating a disease in a subject comprising administering to the subject a pharmaceutical formulation as described anywhere herein. Also provided herein is a method of treating a subject by administering to the subject a therapeutically effective amount of a pharmaceutical formulation as described anywhere herein.

[0042] In one embodiment, the subject is a human.

[0043] The disease to be treated depends on the specific antibody contained in the formulation. In one embodiment, the disease is cancer. The disease can be selected from the group consisting of diabetes, cardiovascular disease, infectious diseases, rheumatoid arthritis, vasculitis, giant cell arthritis, glomerular nephropathy, lupus nephritis, uveitis, atopic dermatitis, liver cirrhosis, psoriatic arthritis, chronic obstructive pulmonary disease, severe asthma, neutrophilic asthma, and myeloid leukemia.

[0044] The present invention provides a lyophilized cake that can be reconstituted into a formulation as defined herein or into a pharmaceutical formulation as defined herein using only sterile water. Also provided herein is a formulation that can be lyophilized to form a lyophilized cake, which can be reconstituted into a formulation as defined herein or into a pharmaceutical formulation as defined herein using only sterile water. [Brief description of the drawings]

[0045] [Figure 1] Figure 1 shows the conversion of an IgG4 monoclonal antibody into half molecules. Figure 1-1 and Figure 1-2 show the sequences for the CAT-354 antibody. The VH and VL sequences are labeled and the six CDRs are underlined. [Diagram 2] FIG. 2 shows the effect of salt on the aggregation rate of IgG4 molecules. [Figure 3A] FIG. 3A shows the results of varying pH versus kD. [Figure 3B] FIG. 3B shows the results of varying pH on Kd in the presence of 100 mM salt. [Figure 4] Figure 4 shows the effect of salt on kD. Note that in general, colloidal stability decreases at pH 5, but increases at pH 6 and pH 7. [Diagram 5] FIG. 5 shows the aggregation rate data at 40° C. for formulations with and without 100 mM salt. [Figure 6] FIG. 6 shows the change in aggregation rate with pH for MEDI7814 formulations. [Figure 7] FIG. 7 shows the effect of ionic excipients on antibody aggregation rate at pH 6 (MEDI7814). [Figure 8] Figure 8 shows the MEDI8897 heavy chain nucleotide sequence and translation. The CDRs are underlined, the amino acid differences that form the allelic constant regions are circled, and the boundaries between the variable and constant regions are indicated by "|"). [Figure 9] Figure 9 shows the MEDI8897 light chain nucleotide sequence and translation. The CDRs are underlined and the boundaries between the variable and constant regions are indicated by "|"). [Figure 10] FIG. 10 shows the stability of MEDI8897 formulations at 5° C., 25° C., and 40° C. for 3 months. [Figure 11] FIG. 11 shows the effect of ionic excipients on antibody aggregation rate at pH 6 (MEDI578). [Figure 12] FIG. 12 shows the purity by size exclusion chromatography. [Figure 13] Figure 13 shows the reduction of sub-visible particles in formulations containing ionic excipients under thermal stress conditions. Colloidal stability is improved. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] Difficulties in formulating some monoclonal antibodies, particularly IgG4 antibodies, arise from the fact that they have a pI close to physiological pH, i.e., the pH generally desired for human administration. These difficulties may relate in particular to colloidal stability.

[0047] IgG4 monoclonal antibodies are characterized by a structure that prevents complement activation and also allows for the exchange of half molecules in vivo (one heavy and one light chain) between two IgG4 molecules, generating new IgG4 with bivalent reactivity. See Figure 1. The IgG4 hinge is three amino acids shorter than the IgG1 hinge, and IgG4 has two cysteines available for covalent interactions between the H chains. [Aalberse and Schuurman, “IgG4 breaking the rules,” Immunology 2002 105:9-19].

[0048] The present invention provides novel monoclonal antibody formulations, in particular novel IgG4 monoclonal antibody formulations and novel IgG1 monoclonal antibody formulations.

[0049] The invention provides a formulation comprising: (i) a monoclonal antibody; and (ii) an ionic excipient (e.g., a salt); wherein the monoclonal antibody is present at a concentration of about 50 mg / ml or more (e.g., about 50 mg / ml to about 200 mg / ml), the ionic excipient is present at a concentration of about 50 to about 150 mM, and the formulation has a pH of 5.5 to 7.5.

[0050] The invention further provides a formulation comprising: (i) a monoclonal antibody; and (ii) an ionic excipient (e.g., a salt); wherein the monoclonal antibody is present in a concentration of about 50 mg / ml or more (e.g., about 50 mg / ml to about 200 mg / ml), the ionic excipient is present in a concentration of about 50 to about 150 mM, and the formulation has a pH of 5.5 to 7.5; and wherein the aggregation rate of the monoclonal antibody in the formulation is reduced compared to the aggregation rate of the same antibody in the same formulation without the ionic excipient.

[0051] The aggregation rate can be measured according to standard techniques as described herein. Surprisingly, the formulation according to the invention has been shown to have good stability and reduced self-aggregation, e.g., ≦2.0% aggregation when stored at room temperature for 3 months. Thus, the present invention provides the use of an ionic excipient in an antibody formulation to increase the stability of the antibody in the formulation. The present invention further provides the use of an ionic excipient in an antibody formulation to reduce self-aggregation of the antibody in the formulation.

[0052] The formulations of the invention are particularly useful for antibodies with a low or neutral pI, e.g., antibodies with a pI in the range of pH 5.5 to pH 7.5, pH 6.0 to pH 7.5, pH 6.3 to pH 7.5, pH 6.4 to pH 7.5, or pH 6.5 to pH 7.5. The pI of an antibody can be measured according to standard techniques, e.g., by capillary isoelectric focusing (cIEF). Thus, the invention provides a formulation comprising: (i) a monoclonal antibody with a low or neutral pI (e.g., 5.5 to 7.5); and (ii) an ionic excipient; the monoclonal antibody is present at a concentration of about 50 mg / ml or greater (e.g., about 50 mg / ml to about 200 mg / ml), the ionic excipient is present at a concentration of about 50 to about 150 mM, and the formulation has a pH of 5.5 to 7.5. Accordingly, the present invention further provides a formulation comprising: (i) a monoclonal antibody having a low or neutral pI (e.g., 5.5 to 7.5); and (ii) an ionic excipient; wherein the monoclonal antibody is present at a concentration of about 50 mg / ml or more (e.g., about 50 mg / ml to about 200 mg / ml), the ionic excipient is present at a concentration of about 50 to about 150 mM, and the formulation has a pH of 5.5 to 7.5; and wherein the aggregation rate of the monoclonal antibody in the formulation is reduced compared to the aggregation rate of the same antibody in the same formulation without the ionic excipient.

[0053] In one embodiment, the monoclonal antibodies have a pI in the range of pH 6.4 to pH 7.5. In another embodiment, the monoclonal antibodies described herein have a pI in the range of about pH 5.5 to about pH 6.0, about pH 5.7 to about pH 6.0, or about pH 5.5, about pH 5.6, about pH 5.7, about pH 5.8, about pH 5.9, about pH 6.0, about pH 6.1, about pH 6.2, about pH 6.3, about pH 6.4, or about pH 6.5. In an embodiment, the pI of the monoclonal antibodies provided herein is 5.7 to 6.0, more suitably about 5.8 or 6.0.

[0054] In one embodiment, the monoclonal antibody is an IgG1 or IgG4 monoclonal antibody. Most preferably, the monoclonal antibody is an IgG4 monoclonal antibody. Thus, the present invention provides a formulation comprising: (i) an IgG4 monoclonal antibody having a low or neutral pI (e.g., 5.5 to 7.5); and (ii) an ionic excipient; the monoclonal antibody is present at a concentration of about 50 mg / ml or more (e.g., about 50 mg / ml to about 200 mg / ml), the ionic excipient is present at a concentration of about 50 to about 150 mM, and the formulation has a pH of 5.5 to 7.5. Accordingly, the invention further provides a formulation comprising: (i) an IgG4 monoclonal antibody having a low or neutral pI (e.g., 5.5 to 7.5); and (ii) an ionic excipient; the monoclonal antibody is present in a concentration of about 50 mg / ml or more (e.g., about 50 mg / ml to about 200 mg / ml), the ionic excipient is present in a concentration of about 50 to about 150 mM, and the formulation has a pH of 5.5 to 7.5; and the aggregation rate of the monoclonal antibody in the formulation is reduced compared to the aggregation rate of the same antibody in the same formulation without the ionic excipient.

[0055] In one embodiment, the monoclonal antibody is an anti-GM-CSF-Rα monoclonal antibody, an anti-IL-13 monoclonal antibody, an anti-RSV monoclonal antibody, or an anti-C5 / C5a monoclonal antibody. In an exemplary embodiment, the monoclonal antibody is an IgG4 monoclonal antibody, such as an anti-GM-CSF-Rα monoclonal antibody. In one embodiment, the monoclonal antibody is CAM-3001. In one embodiment, the monoclonal antibody is MEDI8897.

[0056] Monoclonal antibodies include antibody functional portions, such as antibodies, or antigen-binding fragments, variants, or derivatives thereof. Monoclonal antibodies further include, but are not limited to, human, humanized, or chimeric antibodies, single-chain antibodies, bispecific antibodies, epitope-binding fragments, such as Fab, Fab', and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments containing either the VL domain or the VH domain, fragments produced by a Fab expression library. ScFv molecules are known in the art and are described, for example, in U.S. Pat. No. 5,892,019. Immunoglobulin or antibody molecules encompassed by this disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. In preferred embodiments, the monoclonal antibody is an IgG1 or IgG4 monoclonal antibody.

[0057] antibody concentration Suitably, the monoclonal antibody is present in the formulations described herein at a concentration of, for example, about 50 mg / ml to about 300 mg / ml, about 50 mg / ml to about 200 mg / ml, about 100 mg / ml to about 200 mg / ml, about 100 mg / ml to about 165 mg / ml, about 100 mg / ml to about 150 mg / ml, or about 50 mg / ml, about 75 mg / ml, about 100 mg / ml, about 105 mg / ml, about 110 mg / ml, about 115 mg / ml, about 120 mg / ml, or about 130 mg / ml. 1, 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, 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 (including values ​​and ranges within these ranges).

[0058] Suitably, the monoclonal antibody is present in the formulations described herein at a concentration of about 100 mg / ml to about 165 mg / ml. Suitably, the monoclonal antibody is present in the formulations described herein at a concentration of about 100 mg / ml.

[0059] pH Suitably, the formulations described herein have a pH in the range of about pH 5.5 to about pH 7.5 to provide near-optimal or optimal chemical stability (hydrolysis, deamidation, isomerization). In one embodiment, the formulations described herein have a pH in the range of about pH 5.7 to about pH 6.3. In one embodiment, the formulations described herein have a pH in the range of about 5.5 to about 6.5. In one embodiment, the formulations described herein have a pH in the range of about pH 5.7 to about pH 6.1. A preferred formulation has a pH of about 5.8. Another preferred formulation has a pH of about 6.0.

[0060] Suitably, the formulations described herein have a pH in the range of about pH 5.5 to about pH 6.0, about pH 5.7 to about pH 6.0, or about pH 5.5, about pH 5.6, about pH 5.7, about pH 5.8, about pH 5.9, about pH 6.0, about pH 6.1, about pH 6.2, about pH 6.3, about pH 6.4, or about pH 6.5. In embodiments, the pH of the formulations provided herein is 5.7 to 6.0, more suitably the formulations have a pH of about 5.8 or 6.0.

[0061] A formulation pH closer to about pH 7.4 may also be desirable for injection site tolerance.

[0062] Ionic excipients Exemplary ionic excipients for use in the formulation include salts and charged amino acids. The ionic excipients can include a combination of a salt and a charged amino acid.

[0063] Exemplary charged amino acids include arginine and lysine.

[0064] Exemplary salts include the salts of charged amino acids, such as the succinate, acetate, and sulfate salts of arginine and lysine.

[0065] Further exemplary salts are those described herein, including, but not limited to, sodium chloride and other salts with sodium, potassium, calcium, magnesium, and the like, such as chlorides, carbonates, sulfates, acetates, gluconates, lactates, malates, and other auxiliaries commonly used in the art of parenteral administration.

[0066] Suitably, the salt is selected from sodium chloride (NaCl), lysine hydrochloride, and arginine hydrochloride. In one embodiment, the salt is NaCl. In another embodiment, the salt is arginine hydrochloride. In another embodiment, the salt is lysine hydrochloride.

[0067] The concentration of the ionic excipient, suitably the salt, in the pharmaceutical formulations described herein will generally be in the range of about 50 mM to about 150 mM, more suitably about 50 mM to about 100 mM, about 60 mM to about 80 mM, or about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM (including any range or value within these ranges).

[0068] In one embodiment, the ionic excipient is present at a concentration of about 50 mM to about 125 mM.

[0069] In one embodiment, the ionic excipient is present at a concentration of about 50 mM to about 100 mM.

[0070] In one embodiment, the ionic excipient is present at a concentration of about 75 mM to about 100 mM.

[0071] In a suitable embodiment, the salt is NaCl, for example at a concentration of about 50 mM to about 100 mM, suitably at a concentration of about 70 mM.

[0072] In a suitable embodiment, the salt is arginine hydrochloride, for example at a concentration of about 50 mM to about 100 mM, suitably at a concentration of about 80 mM.

[0073] Buffer The formulations described herein suitably include one or more buffering agents. As used herein, "buffering agent" refers to an excipient for maintaining the pH of the formulation. Exemplary buffering agents for use in the formulations provided herein include, but are not limited to, histidine, histidine hydrochloride (histidine HCl), sodium succinate, sodium acetate, sodium acetate / acetic acid, sodium phosphate, citrate, phosphate, succinate, glycine, and acetate. In one embodiment, the buffering agent for use in the formulations described herein is sodium acetate / acetic acid. In one embodiment, the one or more buffering agents are buffering agents that include histidine. In one embodiment, the one or more buffering agents are selected from buffering agents that include histidine succinate, histidine acetate, histidine citrate, histidine chloride, or histidine sulfate. In one embodiment, the one or more buffering agents are histidine, histidine hydrochloride, or a combination thereof (histidine / histidine hydrochloride). In one embodiment, the one or more buffering agents is L-histidine / L-histidine hydrochloride monohydrate.

[0074] The concentration of the buffering agent, suitably sodium acetate / acetic acid, in the pharmaceutical formulations described herein is generally in the range of about 10 mM to about 100 mM, more suitably about 15 mM to about 80 mM, about 25 mM to about 75 mM, about 30 mM to about 60 mM, about 40 mM to about 60 mM, about 40 mM to about 50 mM, or about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, or about 75 mM (including any range or value within these ranges).

[0075] In one embodiment, the one or more buffering agents is L-histidine / L-histidine hydrochloride monohydrate, for example at a concentration of about 10 mM to about 50 mM, suitably at a concentration of about 30 mM.

[0076] The pH of the buffer is preferably in the range of pH 5.5 to pH 6.0.

[0077] It should be understood that the buffering agent may itself be an ionic excipient. Thus, in one embodiment, the buffering agent is an ionic excipient. In this embodiment, the concentration of the buffering agent should be higher than 50 mM, i.e., consistent with the concentration of the ionic excipient disclosed herein. The preferred concentration of the buffering agent in this embodiment is as discussed elsewhere herein with respect to the ionic excipient.

[0078] Stated another way, in one embodiment, the ionic excipient also acts as a buffer in the formulation, in which an additional buffer may or may not be present.

[0079] Sugars and surfactants Sugars, such as, but not limited to, trehalose, lactose, mannitol, mellibiose, melezitose, raffinose, mannotriose, stachyose, and sucrose, are suitable for inclusion in the formulations described herein. In other embodiments, trivalent or higher molecular weight sugar alcohols, such as polyols, can be used, such as glycerin, dextran, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol. Examples of reducing sugars include, but are not limited to, glucose, maltose, maltulose, isomaltulose, and lactulose. Examples of non-reducing sugars include, but are not limited to, trehalose, which is a non-reducing glycoside of a polyhydroxy compound selected from sugar alcohols and other linear polyalcohols. Examples of sugar alcohols include, but are not limited to, monoglycosides, which are compounds obtained by reduction of disaccharides, such as lactose, maltose, lactulose, and maltulose. The glycosidic side group can be either a glucoside or a galactoside. Further examples of sugar alcohols include, but are not limited to, glucitol, maltitol, lactitol, and isomaltulose. In one embodiment, the sugar is selected from the group consisting of trehalose, lactose, mannitol, raffinose, and sucrose. In certain embodiments, trehalose is used as the sugar in the formulations described herein. In certain embodiments, sucrose is used as the sugar in the formulations described herein.

[0080] Suitably, the amount of sugar, e.g., trehalose, in the formulations described herein is about 1% (w / v) to about 10% (w / v).Unless otherwise specified, percentages (%) of ingredients used herein refer to weight / volume (w / v) %. In exemplary embodiments, the amount of sugar in the pharmaceutical formulations described herein is from about 1% (w / v) to about 8% (w / v), or from about 2% (w / v) to about 6% (w / v), from about 2% (w / v) to about 5% (w / v), from about 3% (w / v) to about 5% (w / v), or about 1% (w / v), about 2% (w / v), about 3% (w / v), about 4% (w / v), about 5% (w / v), about 6% (w / v), about 7% (w / v), about 8% (w / v), about 9% (w / v), or about 10% (w / v), including all values ​​and ranges therein.

[0081] The formulations described herein suitably include a surfactant.

[0082] The term "surfactant" as used herein refers to an organic substance with an amphiphilic structure; that is, the amphiphilic structure consists of groups of opposite solubility tendencies, typically an oil-soluble hydrocarbon chain group and a water-soluble ionic group. Depending on the charge of the surface-active moiety, surfactants can be classified as anionic, cationic, and nonionic surfactants. Surfactants are often used as wetting agents, emulsifiers, solubilizers, and dispersants for the preparation of various pharmaceutical formulations and biological materials. Polysorbates (e.g., polysorbate 20, 40, 60, or 80); poloxamers (e.g., poloxamer 188); triton; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; pharma- ceutical acceptable surfactants such as sodium methyl cocoyl taurate or disodium methyl oleyl taurate; and the MONAQUA® series (Mona Polysorbate 80 (Polysorbate 20, Polysorbate 40, Polysorbate 60, Polysorbate 80, Polysorbate 81, Polysorbate 82, Polysorbate 83, Polysorbate 84, Polysorbate 85, Polysorbate 86, Polysorbate 87, Polysorbate 88, Polysorbate 89, Polysorbate 90, Polysorbate 91, Polysorbate 92, Polysorbate 93, Polysorbate 94, Polysorbate 95, Polysorbate 96, Polysorbate 97, Polysorbate 98, Polysorbate 9 ...

[0083] Suitably, the formulations described herein contain from about 0.001% to about 0.5% (w / v) surfactant (suitably polysorbate 80), more suitably from about 0.002% to about 0.1% surfactant, for example from about 0.01% to about 0.2%, from about 0.02% to about 0.01%, from about 0.02% to about 0.07%, from about 0.03% to about 0.06%, from about 0.04% to about 0.06%, or from about 0.02%, about 0.025%, about 0.03%, about 0.035%, about 0.04%, about 0.045%, about 0.05%, about 0.055%, about 0.060%, about 0.065%, about 0.07%, about 0.075%, about 0.08%, about 0.085%, about 0.09%, about 0.095%, or about 0.1% (including any range or value within these ranges) of a surfactant.

[0084] The formulations described herein suitably include a surfactant and a sugar. The formulations described herein suitably include a surfactant and one or more buffering agents. The formulations described herein suitably include a sugar and one or more buffering agents. The formulations described herein suitably include a surfactant, a sugar, and one or more buffering agents.

[0085] The formulations described herein may also include one or more additional excipients including, for example, one or more sugars, salts, amino acids, polyols, chelating agents, emulsifying agents, and / or preservatives.

[0086] Medicinal Use The preparation of the present invention is preferably a pharmaceutical preparation.Suitably, the pharmaceutical preparation described herein is "pharmaceutical acceptable" and therefore meets the necessary approval requirements required by the regulatory agency of the Federal or State Government or as described in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias, and therefore can be used in animals, particularly humans.

[0087] The present invention provides a pharmaceutical formulation as described anywhere herein for use as a medicament.The present invention provides a pharmaceutical formulation as described anywhere herein for use in treating a disease.The present invention provides a method of treating a disease in a subject, comprising administering to a subject a pharmaceutical formulation as described anywhere herein.Also provided herein is a method of treating a subject by administering to the subject a therapeutically effective amount of a pharmaceutical formulation as described anywhere herein.

[0088] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, including, but not limited to, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. In one embodiment, the subject is a human.

[0089] The disease may be selected from the group consisting of diabetes, cardiovascular disease, infectious diseases, rheumatoid arthritis, vasculitis, giant cell arteritis, glomerular nephropathy, lupus nephritis, uveitis, atopic dermatitis, liver cirrhosis, psoriatic arthritis, chronic obstructive pulmonary disease, severe asthma, neutrophilic asthma, and myeloid leukemia.

[0090] In embodiments, the formulation is administered to the subject by subcutaneous administration or injection.

[0091] Suitably the formulation is a liquid formulation or a frozen formulation.

[0092] Also provided herein is a method of preparing a pharmaceutical formulation comprising preparing a pharmaceutical formulation as described herein and suitably filling the pharmaceutical formulation into a syringe to provide a pre-filled syringe.

[0093] Suitably, the pharmaceutical formulations described herein are prepared in sterile water or resuspended in a desired amount in sterile water for injection.

[0094] In exemplary embodiments, the pharmaceutical formulation comprises from about 0.1 mL to about 20.0 mL, more suitably from about 0.5 mL to about 15.0 mL, from about 0.5 mL to about 12.0 mL, from about 1.0 mL to about 10.0 mL, from about 1.0 mL to about 5.0 mL, from about 1.0 mL to about 2.0 mL, or from about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL, about 1.0 mL, about 1.1 mL, about 1.0 mL, about 1.5 mL, about 1.6 mL, about 1.7 mL, about 1.8 mL, about 1.9 mL, about 2.0 mL, or about 2.5 mL. 0.2mL, about 1.3mL, about 1.4mL, about 1.5mL, about 1.6mL, about 1.7mL, about 1.8mL, about 1.9mL, about 2.0mL, about 2.1mL, about 2.2mL, about 2.3mL, about 2.4mL, about 2.5mL, about 2.6mL, about 2.7mL, about 2.8mL, about 2.9mL, or about 3.0mL (including any range or value within these ranges).

[0095] In suitable embodiments, the pharmaceutical formulations described herein are liquid formulations, i.e., pharmaceutical formulations prepared in sterile water or water for injection (WFI), although the pharmaceutical formulations may also be frozen formulations or pre-lyophilized formulations.

[0096] The present invention also provides a lyophilized cake that can be reconstituted into a formulation according to the present invention as described herein using only sterile water. It is to be understood that the ratio of antibody:ionic excipients is the same in the lyophilized cake as in the formulation after lyophilization. In one embodiment, the molar ratio of ionic excipient:antibody is in the range of 450:1 to 40:1. When the formulation is lyophilized, the concentration of the formulation provided herein is the concentration after reconstitution, and thus the so-called "drug drug" concentration. As an example, when a half-reconstitution method is used (where half of the amount of water removed during lyophilization is added back during reconstitution), after reconstitution, the concentration of the antibody is twice as high as it was before lyophilization, i.e. twice as high as it was in the so-called "drug-substance" composition before lyophilization. Thus, the present invention further provides a composition that can be lyophilized to form a lyophilized cake, which can be reconstituted into a formulation according to the present invention as described herein using only sterile water. Suitable reconstitution methods will be known to those skilled in the art.

[0097] In embodiments, it is desirable to provide a liquid pharmaceutical formulation as described herein and prepare a frozen formulation by freezing the formulation under suitable conditions.For example, a frozen formulation can be provided by freezing the liquid formulation below 0°C, more suitably at about -20°C, about -40°C, about -60°C, or suitably at about -80°C.The pharmaceutical formulation is also suitably prepared as a liquid formulation and stored at about 2°C to about 8°C, or at about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, or about 8°C.

[0098] Suitable protocols and methods for preparing lyophilized pharmaceutical formulations from liquid and / or frozen formulations are known in the art.

[0099] Stability of the formulation In an exemplary embodiment, the formulations described herein are stable for long-term storage at room temperature or a temperature range of about 2°C to about 8°C, suitably at about 5°C. Room temperature as used herein is generally within the range of about 22°C to about 25°C. Suitably, the pharmaceutical formulation is stable after storage at about 2°C to about 8°C (e.g., 5°C) for at least 6 months. As used herein, the term "stable" (or "stability") during storage is used to indicate that the monoclonal antibody, suitably an IgG4 monoclonal antibody, pharmaceutical formulation resists aggregation, degradation, half-antibody formation, and / or fragmentation. The stability of the monoclonal antibody can be assessed by the degree of aggregation, degradation, half-antibody formation, or fragmentation as measured by high performance size exclusion chromatography (HPSEC), static light scattering (SLS), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), urea unfolding technique, intrinsic tryptophan fluorescence, differential scanning calorimetry, and / or ANS binding techniques, in comparison to a standard.

[0100] The overall stability of pharmaceutical formulations containing monoclonal antibodies can be assessed by various immunological assays including, for example, ELISA and radioimmunoassays using isolated antigen molecules.

[0101] The phrase "low to undetectable levels of aggregation" as used herein refers to a pharmaceutical formulation containing about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, or about 0.5% or less by weight of protein aggregation as measured by high performance size exclusion chromatography (HPSEC) or static light scattering (SLS) techniques. Suitably, the pharmaceutical formulation exhibits ≦5.0% aggregation, more suitably ≦4.0% aggregation, ≦3.0% aggregation, ≦2.0% aggregation, ≦1.0% aggregation, or 0.5% aggregation. Suitably, the liquid pharmaceutical formulation and / or the frozen pharmaceutical formulation exhibits ≦5.0% aggregation, more suitably ≦4.0% aggregation, ≦3.0% aggregation, ≦2.0% aggregation, ≦1.0% aggregation, or 0.5% aggregation.

[0102] The phrase "low to undetectable levels of fragmentation" as used herein refers to a pharmaceutical formulation containing about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 98% or more, or about 99% or more of the total monoclonal antibody in a single peak as determined, for example, by HPSEC or reduced capillary gel electrophoresis (rCGE), representing the non-degraded monoclonal antibody or non-degraded fragments thereof, and not including other single peaks having more than about 5%, more than about 4%, more than about 3%, more than about 2%, more than about 1%, or more than about 0.5% of the total monoclonal antibody. Fragmentation can be suitably measured in IgG4 monoclonal antibodies. Without wishing to be bound by theory, it is believed that the reduction in self-aggregation is due to improved colloidal stability, as evidenced by increased kD values. In exemplary embodiments, the formulations described herein have reduced opalescence and reduced phase separation as detected by visual observation, light scattering, nephelometry, or turbidimetry.

[0103] Further embodiments, features, and advantages of the embodiments, as well as the structure and operation of the various embodiments are described in detail below with reference to the accompanying drawings.

[0104] Working Example Example 1 - IgG4 formulation The challenge of formulating antibodies with low or neutral pI values ​​is that additional instability is observed when these antibodies are formulated at pHs outside the range of 5.5-7.5 (which are generally required to increase protein charge and colloidal stability). At acidic pH, increased fragmentation rates, decreased conformational stability, and increased aggregation are observed. At basic pH, there is the potential for increased oxidation, deamidation, and fragmentation, as well as incompatibility with glass containers. IgG4 antibodies are particularly useful for examining such instability, as they typically have low or neutral pI values.

[0105] The following details the methods used to develop the formulations described herein that optimize the storage stability of monoclonal antibodies with neutral pI, while also overcoming the physical instability of neutral pH discussed above.

[0106] As described herein, the formulation has a neutral pH in combination with an ionic excipient such as a salt. Optionally, sugars can also be used in the formulation, which has been shown to provide further improvements in some cases.

[0107] Sugars (sucrose and trehalose are examples) can further enhance conformational stability (in combination with ionic excipients); ionic excipients (e.g., sodium chloride, lysine hydrochloride, and arginine hydrochloride) enhance the colloidal stability of monoclonal antibody molecules. Furthermore, the neutral pH of the formulations (approximately pH 6) also minimizes the acidic and basic degradation pathways mentioned above. These formulations provide excellent overall storage stability for these monoclonal antibodies. Other suitable components of the formulations are buffers with neutral pKa (e.g., NaAC, histidine HCl, sodium phosphate).

[0108] As described herein and listed in Table 1, CAM-3001 (an anti-GM-CSFRα monoclonal antibody), two anti-IL-13 monoclonal antibodies, and an anti-C5 / C5a monoclonal antibody were selected for this study because they are IgG4 with neutral pI values.

[0109] [Table 1]

[0110] CAT-354 is a human antibody of the IgG4 subclass that specifically binds human interleukin 13 (IL-13) and blocks its interaction with the IL-13 receptor. The DNA and derived amino acid sequences of the light and heavy chains of CAT-354 are provided at Error! Reference source not found and Error! Reference source not found, respectively. Error! Reference source not found and Error! Reference source not found are provided at Error! Reference source not found. H and V L The sequence is labeled and the six CDRs are underlined. The CAT-354 molecule is a human monoclonal IgG4 (lambda light chain) antibody (including oligosaccharides) with a molecular weight of about 147,000 Daltons (Da). The antibody consists of two identical heavy chains of about 49,500 Da each and two identical light chains of about 22,500 Da each. CAT-354 contains a fucosylated biantennary complex and a high mannose N-linked carbohydrate attached to each heavy chain at Asn-299. The average size of the oligosaccharide moieties is about 1,650 Da per heavy chain.

[0111] CAM-3001 is an antibody that binds to granulocyte-macrophage colony-stimulating factor receptor alpha (GM-CSFRα). CAM-3001 is disclosed in WO 2007 / 110631, the disclosure of which is incorporated herein by reference. The heavy chain CDRs (HCDRs) of CAM-3001 are disclosed in WO 2007 / 110631 as SEQ ID NOs: 53-55. The light chain CDRs (LCDRs) of CAM-3001 are disclosed in WO 2007 / 110631 as SEQ ID NOs: 58-60. The heavy chain variable region (VH) of CAM-3001 is disclosed in WO 2007 / 110631 as SEQ ID NO: 52. The light chain variable region (VL) of CAM-3001 is disclosed in WO 2007 / 110631 as SEQ ID NO: 218.

[0112] As shown in FIG. 2, in the presence of salt at a concentration of 100 mM, the rate of aggregation increases when the molecule is at pH 5, but decreases when the molecule is at pH 6 and pH 7.

[0113] To achieve the most stable formulations, the solution properties and diffusion interaction parameters (k D The correlation between the D was measured as an indicator of colloidal stability, and differential scanning calorimetry (DSC) was examined as a measure of conformational stability. Figure 3 shows the k at various pH values ​​for the monoclonal antibodies examined. D FIG. 3B shows the k at various pH values ​​for the monoclonal antibodies examined after adding salt at a concentration of 100 mM. D At low ionic strength, k D Generally, k becomes more negative with increasing pH. Addition of salt increases k for all molecules examined. D becomes less likely to become negative.

[0114] Salt was added to the formulation at a concentration of 100 mM to examine colloidal stability, with results shown in Figure 4. Note that in general, colloidal stability decreases at pH 5, but increases at pH 6 and pH 7.

[0115] In the results shown in Figure 5, percent aggregation data at 40°C was obtained for formulations with and without 100 mM salt. Formulations with salt closer to the pI (pH 6 or 7) show reduced aggregation compared to formulations without salt further from the pI.

[0116] The aggregation rate results shown in Figures 6 and 7 indicate that charged amino acids can also act as ionic stabilizers near the pI of the molecule, in some cases providing more stabilization than NaCl.

[0117] The addition of salt to histidine-based buffers was investigated to determine the effect on IgG4 stability. The following buffer systems were investigated: control buffer (25 mM histidine, 7% sucrose, pH 6); test buffers (25 mM histidine, 7% sucrose; 100 mM NaCl, pH 6) and the effect of salt on appearance is summarized in Table 2 below.

[0118] [Table 2]

[0119] As demonstrated above, the addition of salt reduced the rate of aggregation and monomer loss and improved the appearance of the formulation.

[0120] In summary, formulations containing salt with or without sugar are less opalescent, in some cases have reduced phase separation, and provide equal or better stability compared to formulations containing only sucrose.

[0121] Freeze-thaw studies showed that there was no significant change in product quality, such as subvisible particles, after three freeze-thaw cycles. In summary, the addition of both salt and sugar improved the stability of IgG4 molecules, even when formulated at a pH close to their pI.

[0122] Example 2 - IgG1 formulation MEDI8897 is a human IgG1κ-YTE monoclonal antibody against the RSV-F protein. Three amino acid substitutions (M252Y / S254T / T256E; termed YTE) in the CH2 region of the Fc domain were introduced to extend the serum half-life of MEDI8897. Sequence information for MEDI8897 is shown in Figures 8 and 9. The pI of MEDI8897 was measured by cIEF to be 6.4-6.7 with a major peak at 6.4. The pI overlaps with the range of the formulation buffer (5.5-6.5) suggesting potential issues with manufacturing, formulation, and storage stability.

[0123] The thermal stability of MEDI8897 was measured by differential scanning calorimetry. The Tml was found to be 61° C., whereas the Tm2 was 82° C. A Tml of greater than 50° C. suggests that the molecule has acceptable colloidal stability.

[0124] Stability Summary When MEDI8897 was placed in standard buffer (25 mM histidine, 7% sucrose, pH 6.0), phase separation was observed at 2-8°C. The top layer had a protein concentration of 75 mg / ml while the bottom layer was 125 mg / ml. Upon equilibration at 25°C, the two distinct phases disappeared and only one single phase was observed. Phase separation at 2-8°C is believed to be due to the pI of MEDI8897 being close to the formulation pH of 6.0. Exploratory studies were initiated to find a more suitable formulation buffer for MEDI8897 stability evaluation, targeting conditions that would maintain solubility and prevent phase separation of MEDI8897 at 100 mg / ml.

[0125] Formulation in standard buffers (25 mM histidine, 7% sucrose) with pH below 5.9 or above 6.7 mitigated phase separation. Addition of 75 mM NaCl to standard buffers between pH 5.0-6.7 also mitigated phase separation. Finally, acetate and phosphate buffers at pH values ​​away from the pI also mitigated phase separation. Based on these screening studies and prior knowledge of mAbs with pIs within the formulation space, an alternative buffer (25 mM His / HisHCl, 75 mM NaCl, 4% sucrose, 0.02% PS80, pH 6.0) was selected for evaluation.

[0126] kD Test In the initial kD screening, all samples were evaluated in a 25 mM histidine (pH 5.5) based buffer at 2–10 mg / ml at 25 °C. This buffer was chosen instead of pH 6.0 because MEDI8897 is more soluble at pH 5.5, facilitating DLS measurements that are sensitive to insoluble particles. Ionic excipients including arginine-HCl, lysine-HCl, and NaCl were evaluated at concentrations of 10 mM, 25 mM, 50 mM, 75 mM, and 100 mM. Additionally, proline, alanine, Na2SO4, and histidine were evaluated at a concentration of 100 mM only. Finally, 2%, 4%, and 6% sucrose were evaluated to determine whether sucrose affects protein-protein interactions. All conditions were compared to a buffer control (25 mM histidine, pH 5.5).

[0127] The control sample showed distinct protein-protein interactions, with hydrodynamic radii increasing from 6.2 to 7.8 nm at 2 to 10 mg / ml. Arginine-HCl, Lys-HCl, and NaCl showed a decrease in protein-protein interactions (PPI) starting at a concentration of 25 mM, as evidenced by the lack of an increase in hydrodynamic size over the concentration range of 2 to 10 mg / ml. No further effect was seen between 25 and 100 mM. At a concentration of 100 mM, proline and alanine showed similar PPI to the control, while Na2SO4 and histidine reduced PPI. Finally, sucrose concentration had no effect on PPI. The data illustrates that charged excipients (Arg-HCl, Lys-HCl, histidine, and Na2SO4) reduce protein-protein interactions, whereas neutral excipients (sucrose, proline, alanine) do not reduce PPI. Thus, the addition of ionic excipients at pH 5.5 reduced phase separation at 100 mg / ml.

[0128] Stability evaluation at 40℃ Based on the kD screening, several conditions were selected for stability evaluation at 40° C. Table 3 summarizes the formulation conditions and the one-month degradation rates observed at 40° C.

[0129] [Table 3]

[0130] This study demonstrates that arginine and lysine are more stabilizing than NaCl. Furthermore, 75 mM and above appear to stabilize against aggregation. Based on this study, arginine was selected as the most stabilizing lyo-friendly excipient and was carried forward into the next series of studies.

[0131] Drug stability against last lyophilization cycle / representative material The stability of the drug product was evaluated. Three months of data was collected for the formulation after reconstitution at 100 mg / ml in 30 mM L-histidine / L-histidine hydrochloride monohydrate, 80 mM L-arginine hydrochloride, 120 mM sucrose, 0.04% (w / v) polysorbate 80, pH 6.0. The results are shown in Figure 10. Storage at 2-8 °C showed virtually no change over the three month period, supporting the suitability of the formulation and freeze-dry cycle for clinical use. Thus, these data demonstrate that the formulation provides adequate stability and solubility and is suitable for the first time in human clinical use.

[0132] [Table 4]

[0133] Example 3 - IgG4 formulation Formulations of antibody MEDI578 were tested at pH 6. MEDI578 is an IgG4 monoclonal antibody with a pI of 6.3-6.8.

[0134] The results are shown in Figure 11.

[0135] Example 4 - IgG4 formulation All formulations tested were polysorbate-free and contained 100 mg / ml MEDI578, 20 mM histidine, pH 6.5. MEDI578 is an IgG4 monoclonal antibody with a pI of 6.3-6.8.

[0136] [Table 5]

[0137] FIG. 12 shows the purity by size exclusion chromatography.

[0138] Figure 13 shows the reduction of subvisible particles in formulations containing ionic excipients under thermal stress conditions. Colloidal stability is improved.

[0139] Embodiments of the present invention are further described in the following sections: [Section 1] A formulation comprising: i. monoclonal antibodies; and ii. ionic excipients; wherein said monoclonal antibody is present at a concentration of about 50 mg / ml or greater, said ionic excipient is present at a concentration of about 50 to about 150 mM, and said formulation has a pH of 5.5 to 7.5. [Section 2] A formulation comprising: i. monoclonal antibodies; and ii. ionic excipients; wherein said monoclonal antibody is present at a concentration of about 50 mg / ml or greater, said ionic excipient is present at a concentration of about 50 to about 150 mM, said formulation having a pH of 5.5 to 7.5; and wherein the aggregation rate of said monoclonal antibody in said formulation is reduced compared to the aggregation rate of the same antibody in the same formulation without the ionic excipient. [Section 3] 3. The formulation according to item 1 or 2, wherein the monoclonal antibody has a pI in the range of pH 5.5 to pH 7.5. [Section 4] 4. The formulation according to any one of items 1 to 3, wherein the monoclonal antibody has a pI in the range of pH 6.0 to pH 7.5. [Section 5] 4. The formulation according to any one of items 1 to 3, wherein the monoclonal antibody has a pI in the range of pH 6.4 to pH 7.5. [Section 6] 6. The formulation according to any one of items 1 to 5, wherein the monoclonal antibody is an IgG4 monoclonal antibody. [Section 7] 7. The formulation according to any one of items 1 to 6, wherein the monoclonal antibody is present in the formulation at a concentration of about 100 mg / ml to about 200 mg / ml. [Section 8] 8. The formulation of claim 7, wherein the monoclonal antibody is present in the formulation at a concentration of about 100 mg / ml. [Section 9] The formulation according to any one of items 1 to 8, which has a pH in the range of about pH 5.5 to about pH 6.5. [Section 10] The formulation according to any one of items 1 to 8, which has a pH in the range of about pH 5.7 to about pH 6.3. [Section 11] The formulation according to any one of items 1 to 8, which has a pH in the range of about pH 5.7 to about pH 6.1. [Section 12] 12. The formulation according to claim 11, having a pH of about pH 6.0. [Section 13] 13. The formulation according to any one of items 1 to 12, wherein the ionic excipient is a salt. [Section 14] Item 14. The formulation according to item 13, wherein the salt is NaCl. [Section 15] Item 14. The formulation according to item 13, wherein the salt is arginine hydrochloride. [Section 16] Item 14. The formulation according to item 13, wherein the salt is lysine hydrochloride. [Section 17] 17. The formulation according to any one of items 1 to 16, wherein the ionic excipient is present at a concentration of about 75 mM to about 100 mM. [Section 18] 18. The formulation of claim 17, wherein the ionic excipient is present in a concentration of about 80 mM. [Section 19] 19. The formulation according to any one of items 1 to 18, further comprising a sugar. [Section 20] 20. The formulation according to item 19, wherein the sugar is trehalose. [Section 21] 20. The formulation according to claim 19, wherein the sugar is sucrose. [Section 22] 22. The formulation according to any one of items 19 to 21, wherein the sugar is present at a concentration of about 100 mM to about 140 mM. [Section 23] 23. The formulation of claim 22, wherein the sugar is present at a concentration of about 120 mM. [Section 24] 24. The formulation according to any one of items 1 to 23, further comprising one or more buffering agents. [Section 25] 25. The formulation according to claim 24, wherein the one or more buffering agents are selected from histidine, histidine hydrochloride, and histidine / histidine hydrochloride. [Section 26] 26. The formulation according to claim 25, wherein the one or more buffering agents is L-histidine / L-histidine hydrochloride monohydrate. [Section 27] 27. The formulation according to any one of items 24 to 26, wherein the one or more buffering agents are present in a concentration of about 10 mM to about 50 mM. [Section 28] 28. The formulation of claim 27, wherein the one or more buffering agents are present in a concentration of about 30 mM. [Section 29] 29. The formulation according to any one of items 1 to 28, further comprising a surfactant. [Section 30] 30. The formulation according to item 29, wherein the surfactant is a polysorbate. [Section 31] Item 31. The formulation according to item 30, wherein the surfactant is polysorbate 80. [Section 32] 32. The formulation according to any one of items 29 to 31, wherein the surfactant is present in the formulation at a concentration of about 0.02% (w / v) to about 0.07% (w / v). [Section 33] 33. The formulation of claim 32, wherein the surfactant is present in the formulation at a concentration of about 0.04% (w / v). [Section 34] 34. The formulation of any one of claims 1 to 33, further comprising one or more additional excipients, for example, one or more sugars, salts, amino acids, polyols, chelating agents, emulsifiers, and / or preservatives. [Section 35] Item 2. The formulation according to item 1, comprising 150 mg / ml of anti-IL-13 IgG4 antibody, 50 mM sodium acetate, 85 mM sodium chloride, and 0.01% polysorbate 80, wherein the formulation has a pH of pH 5.5. [Section 36] 36. The formulation according to item 35, wherein the antibody has the six CDR sequences of CAT-354 as shown in Figure 1-1 and Figure 1-2. [Section 37] 36. The formulation of claim 35, wherein the antibody has the VH and VL sequences of CAT-354 shown in Figures 1-1 and 1-2. [Section 38] Item 2. The formulation according to item 1, comprising 150 mg / ml of anti-GM-CSF-Rα IgG4 antibody, 50 mM sodium acetate / acetic acid, 106 mM trehalose dihydrate, 70 mM sodium chloride, and 0.05% (w / v) polysorbate 80, wherein the formulation has a pH of pH 5.8. [Section 39] 39. The formulation of claim 38, wherein the antibody has six CDR sequences of CAM-3001. [Section 40] 39. The formulation of claim 38, wherein the antibody has the VH and VL sequences of CAM-3001. [Section 41] 41. The formulation according to any one of items 1 to 40, which is a pharmaceutical formulation. [Section 42] 42. The pharmaceutical preparation according to item 41, for use as a medicament. [Section 43] 42. The pharmaceutical preparation according to item 41 for use in treating a disease. [Section 44] 42. A method for treating a disease in a subject, comprising administering to the subject the pharmaceutical preparation according to item 41. [Section 45] A lyophilized cake that can be reconstituted using only sterile water to give the formulation according to any one of items 1 to 40 above or the pharmaceutical formulation according to any one of items 41 to 43 above. [Section 46] 41. A formulation which can be lyophilized to form a lyophilized cake, and which can be reconstituted using only sterile water to the formulation according to any one of items 1 to 40 above or the pharmaceutical formulation according to any one of items 41 to 43 above. All publications, patents, journal articles, and other materials cited in this application are hereby incorporated by reference.

[0140] Although the present invention has been fully described in connection with several embodiments thereof with reference to the accompanying drawings, it should be understood that various changes and modifications may be apparent to those skilled in the art. Such changes and modifications should be understood to be included within the scope of the present invention as defined by the appended claims, unless they depart from the scope of the present invention.

Claims

1. 1. A formulation comprising: i. monoclonal antibodies; and ii. ionic excipients; wherein said monoclonal antibody is present at a concentration of about 50 mg / ml or greater, said ionic excipient is present at a concentration of about 50 to about 150 mM, and said formulation has a pH of 5.5 to 7.

5.

2. 1. A formulation comprising: i. monoclonal antibodies; and ii. ionic excipients; wherein said monoclonal antibody is present at a concentration of about 50 mg / ml or greater, said ionic excipient is present at a concentration of about 50 to about 150 mM, and said formulation has a pH of 5.5 to 7.5; and wherein the aggregation rate of said monoclonal antibody in said formulation is reduced compared to the aggregation rate of the same antibody in the same formulation without the ionic excipient.

3. 3. The formulation of claim 1 or 2, wherein the monoclonal antibody has a pI in the range of pH 5.5 to pH 7.

5.

4. The formulation of any one of claims 1 to 3, wherein the monoclonal antibody has a pI in the range of pH 6.0 to pH 7.

5.

5. The formulation of any one of claims 1 to 3, wherein the monoclonal antibody has a pI in the range of pH 6.4 to pH 7.

5.

6. The formulation of any one of claims 1 to 5, wherein the monoclonal antibody is an IgG4 monoclonal antibody.

7. The formulation of any one of claims 1 to 6, wherein the monoclonal antibody is present in the formulation at a concentration of about 100 mg / ml to about 200 mg / ml.

8. 8. The formulation of claim 7, wherein the monoclonal antibody is present in the formulation at a concentration of about 100 mg / ml.

9. The formulation of any one of claims 1 to 8, having a pH in the range of about pH 5.5 to about pH 6.

5.

10. 9. The formulation of any one of claims 1 to 8, having a pH in the range of about pH 5.7 to about pH 6.

3.

11. 9. The formulation of any one of claims 1 to 8, having a pH in the range of about pH 5.7 to about pH 6.

1.

12. 12. The formulation of claim 11 having a pH of about pH 6.

0.

13. The formulation according to any one of claims 1 to 12, wherein the ionic excipient is a salt.

14. 14. The formulation of claim 13, wherein the salt is NaCl.

15. 14. The formulation of claim 13, wherein the salt is arginine hydrochloride.

16. 14. The formulation of claim 13, wherein the salt is lysine hydrochloride.

17. 17. The formulation of any one of claims 1 to 16, wherein the ionic excipient is present at a concentration of about 75 mM to about 100 mM.

18. 18. The formulation of claim 17, wherein the ionic excipient is present at a concentration of about 80 mM.

19. The formulation of any one of claims 1 to 18, further comprising a sugar.

20. 20. The formulation of claim 19, wherein the sugar is trehalose.

21. 20. The formulation of claim 19, wherein the sugar is sucrose.

22. 22. The formulation of any one of claims 19 to 21, wherein the sugar is present at a concentration of about 100 mM to about 140 mM.

23. 23. The formulation of claim 22, wherein the sugar is present at a concentration of about 120 mM.

24. The formulation of any one of claims 1 to 23, further comprising one or more buffering agents.

25. 25. The formulation of claim 24, wherein the one or more buffering agents are selected from histidine, histidine hydrochloride, and histidine / histidine hydrochloride.

26. 26. The formulation of claim 25, wherein the one or more buffering agents is L-histidine / L-histidine hydrochloride monohydrate.

27. The formulation of any one of claims 24 to 26, wherein the one or more buffering agents are present at a concentration of about 10 mM to about 50 mM.

28. 28. The formulation of claim 27, wherein the one or more buffering agents are present at a concentration of about 30 mM.

29. A formulation according to any one of claims 1 to 28, further comprising a surfactant.

30. 30. The formulation of claim 29, wherein the surfactant is a polysorbate.

31. 31. The formulation of claim 30, wherein the surfactant is polysorbate 80.

32. 32. The formulation of any one of claims 29 to 31, wherein the surfactant is present in the formulation at a concentration of about 0.02% (w / v) to about 0.07% (w / v).

33. 33. The formulation of claim 32, wherein the surfactant is present in the formulation at a concentration of about 0.04% (w / v).

34. 34. The formulation of any one of claims 1 to 33, further comprising one or more additional excipients including, for example, one or more sugars, salts, amino acids, polyols, chelating agents, emulsifiers, and / or preservatives.

35. 10. The formulation of claim 1 comprising 150 mg / ml anti-IL-13 IgG4 antibody, 50 mM sodium acetate, 85 mM sodium chloride, and 0.01% polysorbate 80, said formulation having a pH of pH 5.

5.

36. The formulation of claim 35, wherein the antibody has the six CDR sequences of CAT-354 as shown in Figures 1-1 and 1-2.

37. The formulation of claim 35, wherein the antibody has the VH and VL sequences of CAT-354 shown in Figures 1-1 and 1-2.

38. 2. The formulation of claim 1 comprising 150 mg / ml anti-GM-CSF-Rα IgG4 antibody, 50 mM sodium acetate / acetic acid, 106 mM trehalose dihydrate, 70 mM sodium chloride, and 0.05% (w / v) polysorbate 80, said formulation having a pH of pH 5.

8.

39. The formulation of claim 38, wherein the antibody has the six CDR sequences of CAM-3001.

40. The formulation of claim 38, wherein the antibody has the VH and VL sequences of CAM-3001.

41. The formulation according to any one of claims 1 to 40, which is a pharmaceutical formulation.

42. 42. A pharmaceutical formulation according to claim 41 for use as a medicament.

43. 42. A pharmaceutical formulation according to claim 41 for use in the treatment of a disease.

44. 42. A method for treating a disease in a subject comprising administering to the subject the pharmaceutical formulation of claim 41.

45. A lyophilized cake which can be reconstituted into a formulation according to any one of claims 1 to 40 or a pharmaceutical formulation according to any one of claims 41 to 43 using only sterile water.

46. 44. A formulation that can be lyophilized to form a lyophilized cake and that can be reconstituted using only sterile water to the formulation of any one of claims 1 to 40 or the pharmaceutical formulation of any one of claims 41 to 43.

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