DESC:
Field of Invention
The present invention relates to the field of pharmaceutical formulation of antibodies. Specifically, the present invention relates to an anti-IL-5 antibody formulation and its pharmaceutical preparation.

Background of Invention
Eosinophils are terminally differentiated granulocytes found in most mammals. It has been observed in several allergic diseases (i.e. allergic rhinitis and asthma) that there is an increase in the number of circulating eosinophils. Eosinophils have an important role in the pathology of airway disorders. Eosinophils are IL-5Ra-expressing cells that respond to IL-5. IL-5 is a major regulator of eosinophil accumulation in tissues and is responsible for the maturation and survival of eosinophils. Therefore, several therapies are being developed for targeting IL-5 such as mepolizumab, and reslizumab.

The development of antibody formulations can be challenging due to stability issues during manufacture and storage. Various factors, like cell culture medium, purification process, freeze-thaw, and storage conditions are responsible for aggregate formation. Controlling degradants or impurities is one of the major challenges in formulation development. Physical instability, i.e. size variants (high or low molecular weight impurities), and chemical instability, i.e. charge variants (acidic and basic variants), are the most common degradants observed in protein formulations. Therefore, there is a need for a stable anti-IL-5 antibody formulation. US11299541B2 discloses an anti-IL-5 antibody formulation with excipients having citrate.

Therefore, there is a need for a stable anti-IL-5 antibody formulation to meet the medical requirement to treat IL-5 mediated conditions.

Summary of the Invention
In an aspect of invention, the invention discloses a stable pharmaceutical formulation comprising anti-IL-5 antibody; a buffering agent, wherein the buffering agent is selected from adipic acid buffer, lysine buffer, and maleic acid buffer.

In another aspect of invention, the invention discloses a stable pharmaceutical formulation comprising anti-IL-5 antibody; a buffering agent, wherein the buffering agent is selected from adipic acid buffer, lysine buffer, and maleic acid buffer; and one or more amino acid.

In another aspect of invention, the invention discloses a stable pharmaceutical formulation comprising anti-IL-5 antibody; a buffering agent, wherein the buffering agent is selected from adipic acid buffer, lysine buffer, and maleic acid buffer; and one or more amino acid selected from proline, methionine, alanine.

In another aspect of invention, the invention discloses a stable pharmaceutical formulation comprising anti-IL-5 antibody; a buffering agent, wherein the buffering agent is selected from adipic acid buffer, lysine buffer, and maleic acid buffer; and one or more amino acid selected from proline, methionine, alanine; wherein said pharmaceutical formulation does not contain citrate.

The details of one or more embodiments of the invention set forth below are illustrative in nature only and not intended to limit the scope of the invention. Other features, objects and advantages of the inventions will be apparent from the description and claims.

Detail Description of Invention
Definitions
The term “about” used herein would mean and include a variation of up to 10% from the particular value.

The term “aqueous formulation” or “aqueous pharmaceutical composition” refers to a preparation that utilizes water as the solvent, which aids in improving both the solubility and stability of a drug.

The term “antioxidant” refers to an agent that inhibits the oxidation of other molecules. Examples of antioxidants herein include citrate, methionine, lipoic acid, uric acid, glutathione, tocopherol, carotene, lycopene, cysteine, and malate.

The term “buffer” or “buffering agent” used herein refers to an agent which resists any change in pH of a solution, near a chosen value, up on addition of acid or base. The buffer herein includes buffering agents, or its' derivative, or salts and combination thereof. In some example antibody and water also act a buffering agent.

The term “chelators/chelating agents” refers to a compound which can form at least one bond with a metal atom. A chelating agent is typically a multidentate ligand that can be used in formulations as a stabilizer to complex with species, which might otherwise promote instability. Exemplary chelating agents include Ethylenediaminetetraacetic acid (EDTA), diethylene triamine pentaaceticacid (DTPA), nitrilotriacetic acid (NTA), trans-diaminocyclohexane tetraacetic acid (DCTA), aspartic acid, and methionine.

In the present invention, the term "lyophilized formulation " refers to a solid injectable preparation that can be dissolved in water (preferably water for injection) when in use, and refers to a pharmaceutical formulation obtained by freeze-drying an aqueous solution containing a predetermined amount of medicinal ingredients. The lyophilization was performed with conventional lyophilization technique known in the literatures involving steps such as freezing, primary drying, secondary drying and optionally annealing.

The term “pharmaceutical formulation” refers to preparations which are in such form as to permit the biological activity of the active ingredients to be unequivocally effective, and which contain no additional components which are significantly toxic to the subjects to which the formulation would be administered. The terms “pharmaceutical formulation”, “formulation”, “pharmaceutical composition”, or “composition” are used herein interchangeably.

The term “stable” formulation refers to the formulation wherein the antibody therein retains its physical stability and/or chemical stability and/or biological activity upon storage.

The term “stabilizer” as used herein refers to a compound which stabilizes an active ingredient(s) (herein anti-IL-5 antibody) under various storage conditions. Storage conditions such as- various temperature condition (for example, at 25°C or at 40 °C, or at 50 °C; or during freezing or thawing condition); various pH condition (under acidic pH condition or neutral or basic pH condition); under real-time storage condition; etc. Examples of stabilizers include but not limited to buffer(s), carbohydrate(s) such as sugar(s) or sugar alcohol (polyols), surfactant(s), salt(s), amino acid(s) either alone or in suitable combination.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

Detailed Description of the Embodiments
The pharmaceutical formulations of the present invention exhibit stability under stressed condition.

The disclosed formulations of the invention specifically reduce degradation of drug product. In particular, the disclosed formulation lowers the impurity formation rate with respect to high molecular weight impurities or aggregates (HMWA), low molecular weight impurities or aggregates (LMWA), and charge variants over a period. This is predominantly useful as the charge variants (including acidic variants) and the aggregates content is a critical quality attribute and any change in the content may influence the stability of the antibody molecule.

The antibody pharmaceutical formulation disclosed in the instant invention comprising an anti-IL-5 antibody and excipients capable of reducing the degradants in the formulation. It is demonstrated that formulations are effective at reducing degradation rate (impurity formation rate). Advantageously, the formulations provided herein demonstrate lower degradation rates as compared to control formulation.

Moreover, provided herein are pharmaceutical formulations which are suitable to parenteral administration.

Provided herein are pharmaceutical formulations which provide stable formulations of antibody in solution and are suitable for subcutaneous or intravenous administration. Further the present invention relates to a stable pharmaceutical formulation in a pre-filled syringe, pre-filled pen, vial, or cartridge.

Antibodies may be formulated in accordance with the present invention in either aqueous or lyophilized form, the latter being capable if being reconstituted into an aqueous form.

In an embodiment, the invention relates to a stable pharmaceutical formulation comprising a therapeutic concentration of anti-IL-5 antibody, a buffering agent, an amino acid; wherein said pharmaceutical formulation does not contain citrate.

In an embodiment, the invention relates to a stable pharmaceutical formulation suitable for subcutaneous administration comprising a therapeutic concentration of anti-IL-5 antibody, a buffering agent, an amino acid; wherein said pharmaceutical formulation does not contain citrate.

In an embodiment, the invention relates to a stable pharmaceutical formulation suitable for intravenous administration comprising a therapeutic concentration of anti-IL-5 antibody, a buffering agent, an amino acid; wherein said pharmaceutical formulation does not contain citrate.

In an embodiment of the invention, the anti-IL-5 antibody used is mepolizumab or reslizumab. The concentration of the anti-IL-5 antibody used in the formulation is about 10 mg/ml to 150 mg/ml.

In another embodiment of the invention, the buffering agent is selected from a group of adipic acid buffer, lysine buffer, maleic acid buffer and combination thereof. The buffering agent of the invention are adipic acid-sodium hydroxide, lysine HCL-hydrochloric acid, maleic acid-sodium maleate. In an embodiment of the invention, the concentration of the buffering agent in the formulation is 10mM to 50mM. In another embodiment of the invention, the concentration of the buffering agent in the formulation is 20mM to 50mM.

In another embodiment of the invention the pH of the formulation is 5.5 to 7.0. In another embodiment the pH of the formulation is 6.0 to 6.8. In yet another embodiment the pH of the formulation is 6.1 to 6.5.

In an embodiment of the invention, the stabilized formulation further comprises a parenterally acceptable stabilizer. The stabilizer is selected from the group of amino acids. In an embodiment of the invention, the stabilizer is proline and/or methionine. In another embodiment of the invention, the concentration of the stabilizer in the formulation is 1 mM to 500 mM. In yet another embodiment of the invention, the concentration of the stabilizer in the formulation is 50 mM to 300 mM.

In another embodiment of the invention, the stabilized formulation further comprises a parenterally acceptable chelating agent and/or anti-oxidant. In an embodiment of the invention, the chelating agent and/or anti-oxidant is selected form methionine, aspartic acid, pentetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA). In an embodiment of the invention, the concentration of the chelating agent and/or anti-oxidant in the formulation is 0.01 mM to 500 mM. In yet another embodiment of the invention, the concentration of the chelating agent and/or anti-oxidant in the formulation is 1 mM to 100 mM. In yet another embodiment of the invention, the concentration of the chelating agent and/or anti-oxidant in the formulation is 30 mM to 80 mM. In an embodiment, the concentration of aspartic acid or methionine is 1 mM to 100 mM. In an embodiment, the concentration of pentetic acid is 0.01 mM to 1.0 mM.

In an embodiment of the invention, methionine may act as a chelating agent or anti-oxidant or stabilizer or in combination thereof. The concentration of methionine may vary as per single or combined function.

In an embodiment of the invention, the stabilized formulation comprises anti-IL-5 antibody; adipic acid buffer, and one or more amino acid.

In another embodiment of the invention, the stabilized formulation comprises anti-IL-5 antibody; adipic acid buffer, and one or more amino acid; wherein the amino acid is selected from proline, methionine, alanine.

In another embodiment of the invention, the stabilized formulation comprises anti-IL-5 antibody; adipic acid buffer, and one or more amino acid; wherein the amino acid is selected from proline, methionine, alanine; wherein said pharmaceutical formulation does not contain citrate.

In an embodiment of the invention, the stabilized formulation comprises mepolizumab; adipic acid buffer, and one or more amino acid.

In an embodiment of the invention, the stabilized formulation comprises anti-IL-5 antibody; lysine buffer, and one or more amino acid.

In another embodiment of the invention, the stabilized formulation comprises anti-IL-5 antibody; lysine buffer, and one or more amino acid; wherein the amino acid is selected from proline, methionine, alanine.

In another embodiment of the invention, the stabilized formulation comprises anti-IL-5 antibody; lysine buffer, and one or more amino acid; wherein the amino acid is selected from proline, methionine, alanine; wherein said pharmaceutical formulation does not contain citrate.

In an embodiment of the invention, the stabilized formulation comprises mepolizumab; lysine buffer, and one or more amino acid.

In an embodiment of the invention, the stabilized formulation comprises anti-IL-5 antibody; maleic acid buffer, and one or more amino acid.

In another embodiment of the invention, the stabilized formulation comprises anti-IL-5 antibody; maleic acid buffer, and one or more amino acid; wherein the amino acid is selected from proline, methionine, alanine.

In another embodiment of the invention, the stabilized formulation comprises anti-IL-5 antibody; maleic acid buffer, and one or more amino acid; wherein the amino acid is selected from proline, methionine, alanine; wherein said pharmaceutical formulation does not contain citrate.

In an embodiment of the invention, the stabilized formulation comprises mepolizumab; maleic acid buffer, and one or more amino acid.

In yet another embodiment of the invention, the stabilized formulation may further comprise tonicity modifying agent. The tonicity modifying agent is sodium chloride. In an embodiment, the concentration of sodium chloride is 1 mM to 500 mM.

In yet another embodiment of the invention, the stabilized formulation may further comprise surfactant. The surfactant is selected from polysorbate 20, and poloxomer 188. In an embodiment, the concentration of surfactant is 0.01 % to 0.05 %.

In another embodiment, the invention relates to a process of preparation of a stable pharmaceutical formulation of the present invention comprising a biologically active mepolizumab, a buffering agent, and an amino acid; wherein said pharmaceutical formulation does not contain citrate.

In another embodiment, the invention relates to a process of preparation of a stable pharmaceutical formulation of the present invention comprising a biologically active mepolizumab, a buffering agent, and an amino acid.

In yet another embodiment, the invention relates to a process of preparation of a stable pharmaceutical formulation of the present invention comprising a biologically active mepolizumab, a buffering agent, an amino acid; wherein said pharmaceutical formulation further comprises a tonicity modifying agent and/or a surfactant, and/or a chelating agent and/or anti-oxidant; wherein said pharmaceutical formulation does not contain citrate.

In yet another embodiment, the invention relates to a process of preparation of a stable pharmaceutical formulation of the present invention comprising a biologically active mepolizumab, a buffering agent, an amino acid; wherein said pharmaceutical formulation further comprises a tonicity modifying agent and/or a surfactant, and/or a chelating agent and/or anti-oxidant.

The examples which follow are illustrative of the invention and are not intended to be limiting.

Experimental Section
Example 1
Mepolizumab was prepared by recombinant expression of immunoglobulin light and heavy chain genes in a mammalian host cell such as Chinese Hamster Ovary cells. Further, the expressed mepolizumab antibody was harvested and purified using different techniques of downstream processing. Purified drug substance was formulated with following excipients to obtain 6 different formulations as given in table 1.

Table 1: Anti-IL-5 antibody formulations
Formulation Mepolizumab and excipients
F1 100 mg/mL Mepolizumab; 20 mM Adipic acid-Sodium hydroxide; 250 mM Proline; 50 mM methionine; pH 6.3 ± 0.2
F2 100 mg/mL Mepolizumab; 20 mM lysine hydrocloride; 250 mM Proline; 50 mM methionine; pH 6.3 ± 0.2
F3 100 mg/mL Mepolizumab; 20 mM Maleic acid-sodium maleate; 250 mM Proline; 50 mM methionine; pH 6.3 ± 0.2
F4 100 mg/mL Mepolizumab; 20 mM Maleic acid-sodium maleate; 250 mM Proline; 50 mM methionine; 50 mM Aspartic acid; 0.05 mM DTPA; 0.02 % polysorbate 20; pH 6.3 ± 0.2
F5 100 mg/mL Mepolizumab; 20 mM lysine hydrocloride; 250 mM Proline; 50 mM methionine; 50 mM Aspartic acid; 0.05 mM DTPA; 0.02 % polysorbate 20; pH 6.3 ± 0.2
F6 100 mg/mL Mepolizumab; 20 mM Adipic acid-Sodium hydroxide; 250 mM Proline; 50 mM methionine; 50 mM Aspartic acid; 0.05 mM DTPA; 0.02 % polysorbate 20; pH 6.3 ± 0.2
Control formulation 100 mg / mL of mepolizumab, 0.95 mg / mL of citric acid monohydrate, 4.16 mg / mL of sodium phosphate dibasic heptahydrate, 120 mg / mL of sucrose, 0.019 mg / mL of EDTA disodium dihydrate and 0.20 mg / mL of polysorbate 80 with a pH of 6.3.

Example 2: Thermal stress condition stability data
The stability study of the aforesaid formulation was conducted by means of the following parameters. The stability of formulations F1, F2, F3, F4, F5, and F6 at 100 mg/mL mepolizumab was determined by placing samples at 40°C. Protein stability was assessed with regard to aggregation and charge isoforms. Samples were withdrawn after 3, 7, 13, 20, and 28 days to assess stability. The stability assessment was carried out by SE-HPLC. The stability data of all the 6 formulations was compared with control formulation in table 2 and table 3. Table 2 summarizes the comparative High molecular weight (HMW) impurity formation rate at thermal stress conditions of the formulation F1, F2, F3, F4, F5 and F6 for day1 and day 28. Table 2 summarizes the comparative Low molecular weight (LMW) impurity formation rate at thermal stress conditions of the formulation F1, F2, F3, F4, F5 and F6 for day1 and day 28.

Table 2: Comparative data of HMW impurity formation rate at thermal stress conditions
Time points in Days SE HPLC Data at 40°C: % High Molecular weight (HMW) impurities
Control F1 F2 F3 F4 F5 F6
T0 0.38 0.61 0.51 0.69 0.50 0.54 0.54
T28 1.69 1.41 1.34 3.31 2.13 1.27 1.87

Table 3: Comparative data of LMW impurity formation rate at thermal stress conditions
Time points in Days SE HPLC Data at 40°C: % Low Molecular Weight Impurities
Control F1 F2 F3 F4 F5 F6
T0 0.5 0.60 0.47 0.83 0.50 0.46 0.56
T28 3.91 3.41 3.67 3.79 4.56 4.05 3.78

Table 4: Comparative data of main peak at thermal stress conditions
Time points in Days SE HPLC Data at 40°C: % Main peak
Control F1 F2 F3 F4 F5 F6
T0 99.13 98.80 99.02 98.48 99.01 99.00 98.90
T28 94.41 95.18 94.99 92.91 93.31 94.67 94.35

Further, the formulations of the current invention were kept for stability studies up to 12 months at 5 ± 3°C. The pharmaceutical formulations of the current invention found to be stable up to 12 months.
,CLAIMS:
1. A stable pharmaceutical formulation comprising:
a) about 100 mg/mL anti-IL-5 antibody;
b) about 10 mM to about 50 mM buffering agent, wherein the buffering agent is selected from adipic acid buffer, lysine buffer, and maleic acid buffer;
c) about 1 mM to about 500 mM amino acid;
and pH of 6.3 ± 0.2.

2. A stable pharmaceutical formulation comprising:
a) about 100 mg/mL mepolizumab;
b) about 10 mM to about 50 mM buffering agent, wherein the buffering agent is selected from adipic acid buffer, lysine buffer, and maleic acid buffer;
c) about 1 mM to about 500 mM amino acid;
and pH of 6.3 ± 0.2.

3. The pharmaceutical formulation of claim 1 or 2, wherein the amino acid is selected from proline, methionine, and alanine.

4. The pharmaceutical formulation of claim 1, wherein the anti-IL-5 antibody is mepolizumab or reslizumab.

5. The pharmaceutical formulation of claim 1 or 2, the pharmaceutical formulation further comprises a surfactant and/or an anti-oxidant and/or a chelating agent.

6. The pharmaceutical formulation of claim 5, wherein the surfactant is selected from polysorbate 20 or poloxomer 188 and the concentration of the surfactant is about 0.01 % to about 0.05 %.

7. The pharmaceutical formulation of claim 5, wherein the anti-oxidant is selected from aspartic acid or methionine and the concentration of the anti-oxidant is about 1 mM to about 100 mM.

8. The pharmaceutical formulation of claim 5, wherein the chelating agent is selected form pentetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), methinonine and combination thereof and the concentration of the chelating agent is about 0.01 mM to about 100 mM.

9. The pharmaceutical formulation of any of the preceding claims wherein the pharmaceutical formulation does not contain citrate.

10. The pharmaceutical formulation of any of the preceding claims wherein the pharmaceutical formulation is aqueous formulation or lyophilized formulation.