DESC:
Field of Invention

The present invention relates to the field of pharmaceutical formulation of antibody. Specifically, the present invention relates to an anti-PD-1 antibody formulation and its pharmaceutical preparation.

Background of Invention

Programmed cell death 1 (PD-1) is a protein on the surface of activated T cells. Programmed cell death 1 ligand 1 (PDL1) and programmed cell death 1 ligand 2 (PDL2) are ligands of PD-1. After PDL1 or PDL2 binds to PD-1, the T cell becomes inactive, and that suppresses the autoimmune response. The same mechanism is observed in cancer cells. These cancer cells make PD-L1, which inhibits T-cells from attacking the tumor. The PD-1 antibody inhibits the interaction of PD-1 with PDL1 or PDL2 which results in T cell activation against tumor. Further, it has been a well-established fact that the anti-PD-1 antibody is useful in the treatment of various hyper-proliferative disorders.

Development of protein formulations can be challenging due to stability issues during manufacture and storage. Quality of protein formulation must be controlled throughout the shelf life. Controlling degradants or impurities is one of the major challenges in formulation development. Physical instability i.e. size variants (high 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-PD-1 antibody formulation to meet the medical requirement to treat PD1 mediated conditions.

Summary of the Invention

In an aspect of invention, the invention discloses a stable pharmaceutical formulation comprising anti-PD1 antibody; a buffering agent, wherein the buffering agent is selected from histidine, glycine, sodium succinate, and sodium phosphate.
In another aspect of invention, the invention discloses a stable pharmaceutical formulation comprising anti-PD1 antibody; a buffering agent, wherein the buffering agent is selected from histidine, glycine, sodium succinate, and sodium phosphate and a chelating agent.

In another aspect of invention, the invention discloses a stable pharmaceutical formulation comprising anti-PD1 antibody; a buffering agent, wherein the buffering agent is selected from histidine, glycine, sodium succinate, and sodium phosphate and a chelating agent, wherein the chelating agent is selected from pentetic acid (DTPA), and EDTA.

In another aspect of invention, the invention discloses a stable pharmaceutical formulation comprising anti-PD1 antibody; a buffering agent, wherein the buffering agent is selected from histidine, glycine, sodium succinate, and sodium phosphate and a chelating agent, wherein the chelating agent is selected from pentetic acid (DTPA), and EDTA; wherein said pharmaceutical formulation does not contain citrate.

In an aspect of invention, the invention discloses a stable pharmaceutical formulation comprising a nivolumab; a buffering agent, wherein the buffering agent is selected from histidine, glycine, sodium succinate, and sodium phosphate.

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 “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 “about” used herein would mean and include a variation of up to 10% from the particular value.

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 as buffering agent.

The term “chelating agent” 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 " 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 techniques known in the literatures involving steps such as freezing, primary drying, secondary drying and optionally annealing.

The term “stabilizer” as used herein refers to a compound which stabilizes an active ingredient(s) (herein anti-PD1 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.

The term "tonicity modifier " means a pharmaceutically acceptable agent suitable for providing isotonic formulations. The term "isotonic" relates to formulations that have essentially the same osmotic pressure as human blood. Examples of tonicity modifier include but are not limited to sugar, inorganic salt or polyols or combination thereof.

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 formulation of the present invention exhibits stability under stressed conditions.

The disclosed formulations of the invention reduce the degradation of antibody. In particular, the disclosed formulation lowers the impurity formation rate such as high molecular weight impurities or aggregates (HMWA) and charge variants over a period. This is useful, as charge variants (including acidic variants) and HMWA content are critical quality attributes, and any change in the content may influence the stability of the antibody molecule.

The invention discloses a formulation comprising an anti-PD-1 antibody and excipients capable of reducing the degradants in the formulation. It is demonstrated that formulations are effective at reducing the degradation rate (impurity formation rate). Advantageously, the formulation provided herein demonstrates lower degradation rates as compared to control formulation.

Moreover, provided herein are pharmaceutical formulations which are suitable for 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 therapeutic concentration of anti-PD1 antibody; and a buffering agent.
In another embodiment, the invention relates to, a stable pharmaceutical formulation comprising therapeutic concentration of anti-PD1 antibody; a buffering agent, and a chelating agent.

In an embodiment, the invention relates to a stable aqueous pharmaceutical formulation suitable to intravenous administration comprising a therapeutic concentration of anti-PD1 antibody; a buffering agent, and a chelating agent.

In an embodiment, the invention relates to a stable aqueous pharmaceutical formulation suitable to subcutaneous administration comprising a therapeutic concentration of anti-PD1 antibody; a buffering agent, a buffering agent, and a chelating agent.

In the above said embodiments, the concentration of anti-PD1 antibody is 10 mg/ml ‘or’ 20 mg/ml ‘or’ 30 mg/ml ‘or’ 40 mg/ml ‘or’ 50 mg/ml, ‘or’ 60 mg/ml, ‘or’ 70 mg/ml, ‘or’ 80 mg/ml, ‘or’ 90 mg/ml, ‘or’ 100 mg/ml.

In another embodiment the anti-PD1 antibody formulation is stable to a thermal stress of 25-40 °C for six months.

In another embodiment the anti-PD1 antibody formulation is stable to a thermal stress of 25-30 °C for six months.

In another embodiment the anti-PD1 antibody formulation is stable to a thermal stress of 40°C for a time period of up to four weeks.

In another embodiment the anti-PD1 antibody formulation is stable to a thermal stress of at 40°C for a time period of up to two weeks.

In an embodiment, the anti-PD1 antibody is nivolumab. In an embodiment, the concentration of the nivolumab used in the formulation is 8 mg/ml to 100 mg/ml. In another embodiment of the invention, the concentration of the nivolumab in the formulation is 10 mg/ml to 50 mg/ml. In yet another embodiment of the invention, the concentration of the nivolumab in the formulation is 50 mg/ml to 100 mg/ml.

In the above said embodiments, the concentration of nivolumab is 10 mg/ml ‘or’ 20 mg/ml ‘or’ 30 mg/ml ‘or’ 40 mg/ml ‘or’ 50 mg/ml, ‘or’ 60 mg/ml, ‘or’ 70 mg/ml, ‘or’ 80 mg/ml, ‘or’ 90 mg/ml, ‘or’ 100 mg/ml.

In another embodiment, the invention discloses a stable pharmaceutical formulation comprising a nivolumab, and a buffering agent.

In another embodiment the nivolumab formulation is stable to a thermal stress of 25-40 °C for six months.

In another embodiment the nivolumab formulation is stable to a thermal stress of 25-30 °C for six months.

In another embodiment the nivolumab formulation is stable to a thermal stress of 40°C for a period up to four weeks.

In another embodiment the nivolumab formulation is stable to a thermal stress of at 40°C for a period up to two weeks.

In another embodiment, the buffering agent is selected from the group of histidine, phosphate, glycine, maleate, succinate and combination thereof. In an embodiment, concentration of the buffering agent is 10 mM to 50 mM. In another embodiment, the concentration of the buffering agent is 20 mM to 50 mM. In another embodiment, the formulation buffer is citrate free.

In another embodiment, the buffering agent is histidine buffer, and the concentration of histidine buffer is 20 to 50 mM or 20 to 30 mM or 20mM.

In another embodiment, the buffering agent is glycine buffer, and the concentration of glycine buffer is 20 to 50 mM or 20 to 30 mM or 20mM.

In another embodiment, the buffering agent is sodium succinate buffer, and the concentration of sodium succinate buffer is 20 to 50 mM or 20 to 30 mM or 20mM.

In another embodiment, the buffering agent is sodium phosphate buffer, and the concentration of sodium phosphate buffer is 20 to 50 mM or 20 to 30 mM or 20mM.

In another embodiment the chelating agent is selected from EDTA and pentetic acid. In an embodiment, the concentration of the chelating agent is about 10 µM to about 30 µM. In an embodiment, the concentration of the pentetic acid is up to 20 µM.

In an embodiment, the pH of the formulation is 5.2 to 6.8. In another the pH of the formulation is 5.5 to 6.5. In yet another embodiment the pH of the formulation is 5.7 to 6.3.

In another embodiment, the stabilized formulation further comprises a parenterally acceptable stabilizer. The stabilizer is selected from amino acids or salts or sugars or combinations thereof. In an embodiment, the stabilizer is arginine or sodium chloride or mannitol or sucrose or combination thereof. In an embodiment, the concentration of the stabilizer is 25 mM to 200 mM. In another embodiment, the concentration of the stabilizer is 50 mM to 100 mM.

In an embodiment, the stabilized formulation further comprises a tonicity modifier. The tonicity modifier is sugar or inorganic salt or polyols or combination thereof. In another embodiment, the tonicity modifier is sodium chloride or mannitol or sucrose or trehalose or combination thereof. In another embodiment, the tonicity modifier is mannitol and/or sucrose. In an embodiment, the concentration of tonicity modifier is 2 to 7 % W/V or 3 to 5% W/V.

In an embodiment, the formulation further comprises a non-ionic surfactant such as but not limited to polysorbate 80.

In another embodiment, stable pharmaceutical formulation comprises; 10-100 mg/mL nivolumab, 20-50 mM histidine, 2 to 5 % W/V mannitol, 25-200 mM NaCl, 0.02 % (W/V) PS80; 0 to 20 µM pentetic acid; wherein the formulation is stable to temperature stress up to 40°C.

In another embodiment, stable pharmaceutical formulation comprises; 10-100 mg/mL nivolumab, 20-50 mM glycine, 2 to 5 % W/V mannitol, 25 mM NaCl, 0.02 % (W/V) PS80; 0 to 20 µM pentetic acid; wherein the formulation is stable to temperature stress up to 40°C.

In another embodiment, stable pharmaceutical formulation comprises 10-100 mg/mL nivolumab, 20-50 mM sodium succinate, 2 to 5 % W/V mannitol, 25-200 mM NaCl, 0.02 % (W/V) PS80; 0 to 20 µM pentetic acid; wherein the formulation is stable to temperature stress up to 40°C.

In another embodiment, stable pharmaceutical formulation comprises 10-100 mg/mL nivolumab, 20-50 mM sodium phosphate, 2 to 5 % W/V mannitol, 25-200 mM NaCl, 0.02 % (W/V) PS80; 0 to 20 µM pentetic acid; wherein the formulation is stable to temperature stress up to 40°C.

In another embodiment, stable pharmaceutical formulation comprises 10-100 mg/mL nivolumab, 20-50 mM histidine, 2 to 5 % W/V mannitol, 25-100 mM NaCl, 0.02 % (W/V) PS80; 25 mM to 200 mM arginine.

In an embodiment, the invention relates to a process of preparation of a stable pharmaceutical formulation of the present invention comprising a biologically active nivolumab, a buffering agent, wherein the buffering agent is selected from histidine, glycine, sodium succinate, and sodium phosphate; and a chelating agent, wherein the chelating agent is selected from pentetic acid (DTPA), and EDTA.

In an embodiment, the invention relates to a process of preparation of a stable pharmaceutical formulation of the present invention comprising a biologically active nivolumab, a buffering agent, wherein the buffering agent is selected from histidine, glycine, sodium succinate, and sodium phosphate; and a chelating agent, wherein the chelating agent is selected from pentetic acid (DTPA), and EDTA; wherein the pharmaceutical formulation further comprises tonicity modifier, stabilizer and non-ionic surfactant.

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

Experimental Section
Example 1
Nivolumab 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 nivolumab was harvested and purified using different techniques of downstream processing. Purified drug substance at a concentration of 10 mg/ml was formulated with excipients to obtain five different formulations as given in table 1. In another experiment the nivolumab at a high concentration of 100 mg/ml was formulated with excipients to obtain five different formulations (Table 2).

Table 1: Anti-PD-1 antibody formulations
Formulation Nivolumab and excipients
F1 10 mg/mL nivolumab, 20 mM histidine, 165 mM mannitol, 50 mM NaCl, 0.02 % (W/V) PS80; 0.02 mM pentetic acid; pH 6 ±0.3
F2 10 mg/mL nivolumab, 20 mM glycine, 165 mM mannitol, 50 mM NaCl, 0.02 % (W/V) PS80; 0.02 mM pentetic acid; pH 6 ±0.3
F3 10 mg/mL nivolumab, 20 mM sodium succinate, 165 mM mannitol, 50 mM NaCl, 0.02 % (W/V) PS80; 0.02 mM pentetic acid; pH 6 ±0.3
F4 10 mg/mL nivolumab, 20mM sodium phosphate, 165 mM mannitol, 50 mM NaCl, 0.02 % (W/V) PS80; 0.02 mM pentetic acid; pH 6 ±0.3
F5 10 mg/mL nivolumab, 20 mM histidine, 165 mM mannitol, 50 mM NaCl, 0.02 % (W/V) PS80; 200 mM arginine; pH 6 ±0.3
Control formulation 10 mg/mL nivolumab, 5.88 mg/mL sodium citrate dehydrate, mannitol 30 mg/mL, NaCl 2.92 mg/mL, 0.2 mg/mL PS80, 0.008 mg/mL pentetic acid; pH 6

Table 2: Anti-PD-1 antibody formulations (High concentration)
Formulation Nivolumab and excipients
F6 100 mg/ml nivolumab, 20 mM histidine buffer pH 6 ±0.3, 3% mannitol, 50 mM NaCl, 0.02 % (w/v) PS80; 0.02 mM pentetic acid
F7 100 mg/ml nivolumab, 20 mM histidine buffer pH 6 ±0.3; 100 mM NaCl; 3% mannitol; 0.2 mg/ml PS80; 20 µM pentetic acid
F8 100 mg/ml nivolumab, 20 mM histidine buffer pH 6 ±0.3; 75 mM NaCl; 3% mannitol; 0.2 mg/ml PS80; 2% sucrose ; 20 µM pentetic acid
F9 100 mg/ml nivolumab, 20 mM histidine buffer pH 6 ±0.3; 100 mM NaCl; 3% mannitol; 0.2 mg/ml PS80; 50 mM methionine; 20 µM pentetic acid
F10 100mg/ml nivolumab, 20 mM histidine buffer pH 6 ±0.3; 50 mM NaCl; 3% mannitol; 0.2 mg/ml PS80; 100 mM arginine-cl; 20 µM pentetic acid
F11 100mg/ml nivolumab, 20 mm histidine buffer pH 6 ±0.3, 50 mM NaCl, 3% mannitol, 0.2 mg/ml PS80, 100 mM Cacl2, 20 µM pentetic acid

Example 2: Thermal stress condition stability data
Stability study of the aforesaid formulations was conducted by means of the following parameters. The stability of 10 mg/mL nivolumab formulations F1, F2, F3, F4, and F5 were determined by placing samples at 40° C and humidity 75% ± 5%. Protein stability was assessed for aggregation, charge isoforms (acidic variants). Samples were taken out after 7, 14, 21 and 28 days to assess stability. The sample analysis was conducted by CEX-HPLC and SE-HPLC techniques known in the art. Stability data for each formulation was compared with control formulation. Table 3 summarizes the comparative HMWP formation rate at thermal stress conditions for formulation F1, F2, F3, F4, and F5. Table 4 provides the comparative acidic variant formation rate at thermal stress conditions for formulation F1, F2, F3, F4, and F5.

Similar stability study was carried out for high concentration (100 mg/mL) nivolumab formulations (provided in table 2). Table 5 summarizes comparative HMWP formation rate at thermal stress conditions for formulation F6, F7, F8, F9, F10 and F11. Table 6 provides comparative acidic variant formation rate at thermal stress conditions for formulation F6, F7, F8, F9, F10 and F11.

Table 3: Comparative HMWP formation rate at thermal stress conditions
Rate of formation of HMWP (%) at thermal stress (40°C)
Time (week) Control formulation F1 F2 F3 F4 F5
0 0.59 0.48 0.52 0.50 0.50 1.42
1 0.71 0.51 0.67 0.61 0.60 1.41
3 0.83 0.61 0.76 0.73 0.70 2.04
4 1.02 0.74 0.89 0.90 0.83 1.78
Rate of HMWP formation (?/week) 0.10 0.06 0.08 0.09 0.08 0.14

Table 4: Comparative acidic variant formation rate at thermal stress conditions
Rate of formation of acidic variants (%) at thermal stress (40°C)
Time (week) Control formulation F1 F2 F3 F4 F5
0 14.4 14.0 14.5 14.2 14.8 25.2
1 20.1 18.5 19.9 19.6 19.7 28.1
3 30.8 28.1 30.8 29.5 29.6 36.2
4 34.2 31.2 33.8 34.0 33.0 40.2
Rate of acidic variant formation (?/week) 5.05 4.39 4.96 4.95 4.63 3.81

Table 5: Comparative HMWP formation rate at thermal stress conditions
Rate of formation of HMWP (%) at thermal stress (40°C)
Time (week) F6 F7 F8 F9 F10 F11
0 0.68 0.70 0.71 0.61 0.69 0.73
1 0.41 0.96 1.17 0.5 0.57 0.69
3 1.26 1.32 1.56 1.21 1.31 2.14
4 2.21 2.1 2.13 1.75 1.55 2.66
Rate of HMWP formation (?/week) 0.39 0.32 0.32 0.30 0.25 0.53

Table 6: Comparative acidic variant formation rate at thermal stress conditions
Rate of formation of acidic variant (%) at thermal stress (40°C)
Time (week) F6 F7 F8 F9 F10 F11
0 16.26 16.23 16.4 16.3 16.26 16.39
1 18.03 17.82 17.26 17.62 16.5 17.08
3 20.40 20.21 21.16 20.47 19.95 20.30
4 24.38 24.3 22.75 23.67 21.94 22.52
Rate of acidic variant formation (?/week) 1.86 1.85 1.66 1.76 1.48 1.55

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 10 to about 100 mg/ml anti-PD1 antibody;
b) about 20 mM to 50 mM buffering agent, wherein the buffering agent is selected from the group consisting of histidine, glycine, sodium succinate, sodium phosphate, or combination thereof;
c) about 20 µM chelating agent selected from pentetic acid (DTPA), and ethylenediaminetetraacetic acid (EDTA);
and pH of 6.0 ± 0.3.

2. A stable pharmaceutical formulation comprising:
a) about 10 to about 100 mg/mL Nivolumab;
b) about 20 mM to 50 mM buffering agent, wherein the buffering agent is selected from the group consisting of histidine, glycine, sodium succinate, sodium phosphate, or combination thereof;
and a pH of about 5.7 to 6.3 (6.0 ± 0.3).

3. The pharmaceutical formulation of claim 1, wherein the anti-PD1 antibody is nivolumab.

4. The pharmaceutical formulation of claim 1 or 2, the pharmaceutical formulation further comprises one or more stabilizer and the concentration of the one or more stabilizer is about 50 mM to about 100 mM.

5. The pharmaceutical formulation of claim 4, the one or more stabilizer is selected form sodium chloride, mannitol, arginine, sucrose or combination thereof.

6. The pharmaceutical formulation of claim 1 or 2, the pharmaceutical formulation further comprises tonicity modifier and the concentration of the tonicity modifier is about 2 % to about 5 % W/V; wherein the tonicity modifier is selected from sodium chloride, mannitol, sucrose, trehalose and combination thereof.

7. The pharmaceutical formulation of claim 2, the pharmaceutical formulation further comprises chelating agent and the concentration of the chelating agent is about 20 µM; wherein the chelating agent selected from pentetic acid (DTPA), and ethylenediaminetetraacetic acid (EDTA).

8. The pharmaceutical formulation of claim 1 or 2, the pharmaceutical formulation further comprises non-ionic surfactant and the concentration of the non-ionic surfactant is about 0.02 % (w/v), wherein non-ionic surfactant is polysorbate 80 (PS80).

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 or lyophilized.