Lyophilized pharmaceutical composition of Fc-peptide fusion protein

Abstract

A novel and thermostable lyophilized pharmaceutical composition of Romiplostim (Fcpeptide fusion protein) along with buffer, bulking agent, stabilizer, and surfactant at pH range of 4.0-6.0.

Claims

1. A lyophilized pharmaceutical composition comprising Romiplostim, citro-phosphate buffer, Polysorbate 20 as surfactant and bulking agent selected from sucrose, trehalose or a combination thereof.

2. The lyophilized pharmaceutical composition of claim 1, wherein the concentration of Romiplostim is between 0.1 mg/mL to 1.0 mg/mL.

3. The lyophilized pharmaceutical composition of claim 1, wherein the Polysorbate 20 is present in the concentration of 0.001% w/v to 1% w/v.

4. The lyophilized pharmaceutical composition of claim 1, wherein the bulking agent is present in the concentration of 5.0% w/v to 15% w/v.

5. The lyophilized pharmaceutical composition as claimed in claim 1 comprising: a) 0.5 mg/mL Romiplostim, b) 9 mM-11 mM citro-phosphate buffer, c) 8.0% w/v-10% w/v sucrose, and d) 0.003% w/v-0.005% w/v Polysorbate 20 at pH 5.0.

6. The lyophilized pharmaceutical composition as claimed in claim 1 comprising: a) 0.5 mg/mL Romiplostim, b) 9 mM-11 mM citro-phosphate buffer, c) 7.0% w/v-9.0% w/v trehalose, d) 1.0% w/v-3.0% w/v sucrose, and e) 0.003% w/v-0.005% w/v Polysorbate 20 at pH 5.0.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the comparative CEX-HPLC profile of Romiplostim Formulations 1, 2, 3 (Table-4), & Generic DP at 0 D, 3 D, 7 D & 15 D.

(2) FIG. 2 shows the comparative SEC-HPLC profile of Romiplostim Formulations 1, 2, 3 (Table-5), & Generic DP at 0 D, 3 D, 7 D & 15 D.

(3) FIG. 3 shows the comparative CEX-HPLC profile of Romiplostim Formulations 4, 5, 6, 7 (Table-8), & Generic DP at 0 D, 3 D, 7 D & 15 D.

(4) FIG. 4 shows the comparative SEC-HPLC profile of Romiplostim Formulations 4, 5, 6, 7 (Table-9), & Generic DP at 0 D, 3 D, 7 D & 15 D.

(5) FIG. 5 shows the comparative CEX-HPLC profile of Romiplostim Formulations 2, 4, 5, 7, 8 (Table-11), & Generic DS at 0 D, 3 D, 7 D & 15 D & 21 D.

(6) FIG. 6 shows the comparative SEC-HPLC profile of Romiplostim Formulations 2, 4, 5, 7, 8 (Table-12), & Generic DS at 0 D, 3 D, 7 D & 15 D & 21 D.

(7) FIG. 7 shows the comparative CEX-HPLC profile of Romiplostim Formulations 4, 8 (Table-17) charged at 40 C. on OD, 3 D, 7 D & 15 D & 30 D.

(8) FIG. 8 shows the comparative SEC-HPLC profile of Romiplostim Formulations 4, 8 (Table-18) charged at 40 C. on OD, 3 D, 7 D & 15 D & 30 D.

DESCRIPTION OF THE INVENTION

(9) The present invention relates to a lyophilized pharmaceutical composition comprising a Fc-peptide fusion protein, buffer, bulking agent, stabilizer and surfactant.

(10) The present invention relates to a novel, stable, lyophilized pharmaceutical composition comprising Romiplostim (Fc-peptide fusion protein), buffer, bulking agent, stabilizer, and surfactant at a pH range of 4.0-6.0.

(11) As used herein, buffer refers to a buffered solution that resists changes in pH by the action of its acid-base conjugate components. Buffer is used in the present invention to maintain the pH in the range of about 4.0 to 6.0, more preferably in the range of 4.5-5.5 and the buffer is selected from the group consisting of citrate, citro-phosphate, alanine, glycine, arginine, acetate, succinate, histidine either alone or a combination thereof.

(12) In yet another embodiment of the present invention, an aforementioned composition is provided wherein the bulking agent selected from the group consisting of trehalose, mannitol, glycine, sucrose, dextran, polyvinylpyrolidone, carboxymethylcellulose, lactose, sorbitol, or xylitol.

(13) In yet another embodiment of the present invention, stabilizer used is selected from the group consisting of monosaccharide such as glucose and mannose; dissacharides such as sucrose, trehalose, and maltose; sugar alcohols such as mannitol and xylitol, polyols such as glycerol, propylene glycol and polyethylene glycol and the like either alone or in combination thereof.

(14) In yet another embodiment of the present invention, surfactant is used in order to prevent adsorption of Fc-peptide fusion protein on the surface of the vial, ampoule, carpoule, cartridge or syringe. Surfactants lower surface tension of a protein solution, thereby, preventing its adsorption or aggregation on to a hydrophobic surface. Preferred surfactants of the present invention include a polysorbate-based non-ionic surfactant and a poloxamer-based non-ionic surfactant or a combination thereof.

(15) In yet another embodiment of the present invention, a novel, stable, lyophilized pharmaceutical composition of a Fc-peptide fusion protein is provided wherein the formulation is maintained at a pH of about 4.0 to 6.0, more preferably at pH 4.5 to 5.5, in a buffer system selected from the group consisting of citrate, citro-phosphate, alanine, glycine, arginine, acetate, succinate, histidine either alone or a combination thereof.

(16) In yet another embodiment of the present invention, a novel, stable, lyophilized pharmaceutical composition of a Fc-peptide fusion protein is provided which encompasses romiplostim as a Fc-peptide fusion protein comprising citrate, citro-phospahte, alanine, arginine as buffer either alone or in combination thereof, trehalose, mannitol either alone or in combination thereof as bulking agent, optionally use of sucrose, PEG, glycerol as stabilizer either alone or in combination thereof, polysorbate 20 as surfactant and formulation is maintained at pH of about 5.0.

(17) In yet another embodiment, the present invention provides a novel & stable lyophilized pharmaceutical composition of romiplostim buffer, bulking agent, stabilizer, surfactant; wherein buffer is at concentration of 5 mM to 25 mM and wherein the pH of the composition is in a range of about 4.0-6.0; wherein bulking agent is at concentration of 5.0% to 15.0%; wherein stabilizer is at concentration of 0.1% to 20% w/v; wherein surfactant is at concentration of 0.004% to 0.4% w/v.

(18) The novel & thermostable lyophilized pharmaceutical composition of Fc-peptide fusion protein described in the present invention has the following advantages: 1. Involves use of a buffer system selected from the group consisting of citrate, citro-phosphate, alanine, glycine, arginine, acetate, succinate and histidine which maintains the pH of the formulation between 4.0 to 6.0, more preferably between 4.5 to 5.5 and also maintains the purity of the formulation at elevated temperature. 2. Involves use of bulking agent to maintain the stability of composition during and after lyophilization. 3. Involves use of surfactant to prevent adsorption of Fc-peptide fusion protein on container. 4. Optionally use of a stabilizer which provides better stability. 5. The pharmaceutical composition of present invention is maintained at pH between 4.5 to 5.5 which is critical in maintaining the purity and stability of the composition at elevated temperatures during storage. 6. Involves operational simplicity.

(19) The following example illustrate the pharmaceutical compositions described in the present invention and the means of carrying out the invention to obtain a thermostable lyophilized pharmaceutical composition comprising Romiplostim.

Example 1

(20) a) Selection of Buffer

(21) TABLE-US-00001 TABLE 1 Formulation composition Formulation No. Generic Components Ingredient F1 F2 F3 F4 F5 F6 F7 F8 formulation Active Romiplostim 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 Protein DS mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL Buffer L-Histidine 10.3 mM Na-Citrate 10 mM 10 mM 2.2 mM 10 mM 2.2 mM dihydrate Glycine 10 mM Monobasic 2.5 mM 2.5 mM sodium phosphate dihydrate dibasic 2.5 mM 2.5 mM sodium phosphate dihydrate Citrate 2.8 mM 2.8 mM monohydrate L-Arginine 15 mM Alanine 15 mM Bulking Trehalose 10% 8% 8% 6% 9% Agent dihydrate Mannitol 4% 4% 4% Sucrose 2% 2% 2% 2% 2% 9% 2% Cryoprotectant/ PEG 4% Stabilizer Nonionic Polysorbate 0.004% 0.004% 0.004% 0.004% 0.004% 0.004% 0.004% 0.004% 0.004% surfactant 20 Vehicle Water for q.s. to q.s. to q.s. to q.s. to q.s. to q.s. to q.s. to q.s. to q.s. to injection 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL pH modifier 10% w/v q.s. to q.s. to q.s. to citrate pH 5.0 pH 5.0 pH 5.0 monohydyate 10% v/v HCl q.s. to q.s. to q.s. to q.s. to pH 5.0 pH 5.0 pH 5.0 pH 5.0
Method of Preparation:

(22) Romiplostim formulation was prepared in formulation composition given in the above table by dissolving the excipients in water for injection. The protein concentration was set to 0.5 mg/mL and the pH of the formulation is set to 5.0 similar to the reference formulation. 0.75 mL solution filled in 5 mL USP type 1 vial and half stoppered with bromobutyl rubber stopper. After completion of lyophilization cycle, vials are sealed with flip off seals and stored at 2 C.-8 C. Filled vial were charged at 40 C. for 15 days stress stability study. During stability study following test were done:

(23) TABLE-US-00002 TABLE 2 Purpose of the tests Tests Purpose of the tests SE-HPLC To monitor aggregates (H.M.W. impurities) CEX HPLC To monitor charge related impurities Potency To monitor effect on in vitro bioassay pH To monitor effect on pH Physical appearance To monitor physical appearance

Example 2

(24) Stress Stability Data of Formulation 1, 2, 3 (Lyophilized):

(25) a) Physical Appearance:

(26) All the samples were observed to be white to pale yellow lyophilized cake.

(27) b) Physical Appearance after Reconstitution:

(28) All the samples were observed to be clear and colorless till 15 D ST

(29) c) pH

(30) TABLE-US-00003 TABLE 3 pH of Formulation 1, 2 & 3 Buffers pH Time point 0 D 15 D Formulation 1 5.04 5.02 Formulation 2 5.07 5.04 Formulation 3 5.05 5.10 Generic DP 5.11 5.13
d) CEX-HPLC

(31) TABLE-US-00004 TABLE 4 CEX data of Formulation 1, 2 & 3 % Purity Sample ID 0 day 3 days 7 days 15 days Formulation 1 97.3 96.5 95.8 95.8 Formulation 2 97.3 96.0 94.4 93.0 Formulation 3 96.3 94.4 93.6 92.4 GENERIC DP 96.3 ND 94.6 94.2
Observation:

(32) Based on 15 days stress data the purity of the formulation 1, 2 and 3 was comparable with the reference formulation (generic DP). (FIG. 1)

(33) e) SEC-HPLC

(34) TABLE-US-00005 TABLE 5 SEC data of Formulation 1, 2 & 3 % Purity Sample ID 0 day 3 days 7 days 15 days Formulation 1 99.0 99.1 99.0 99.0 Formulation 2 99.1 99.0 98.7 98.5 Formulation 3 99.1 99.1 99.0 98.8 GENERIC DP 99.7 ND 99.6 99.4
Observation:

(35) Based on 15 days stress data the purity of the formulation 1, 2 and 3 was comparable with the reference formulation (generic DP). (FIG. 2)

(36) f) Potency

(37) The biological activity of Romiplostim is determined by cell based in-vitro bio-assay. The assay is based on the proliferation of meghkarocytes on UT-7 cell line (acute myeloid leukemia) cell line expressing TPO receptor. Romiplostim specifically proliferate activity of UT-7 cell line in a dose dependent manner

(38) TABLE-US-00006 TABLE 6 % Potency data of Formulation 1, 2 & 3 % Purity Sample ID 0 day 3 days 7 days 15 days Formulation 1 80 91 83 97 Formulation 2 88 81 104 127 Formulation 3 95 91 113 165 GENERIC DP 82 114 135
Observation:

(39) There is no change in potency at 40 C. after 15 days as compared to initial in all Formulation.

Example 3

(40) a) Physical Appearance

(41) All the samples were observed to be white to pale yellow lyophilized cake.

(42) b) Physical Appearance after Reconstitution

(43) All the samples were observed to be clear and colorless till 15 D ST

(44) c) pH

(45) TABLE-US-00007 TABLE 7 pH of Formulation 4, 5, 6 & 7 pH Time point Buffers 0 D 15 D Formulation 4 4.9 5.0 Formulation 5 5.9 6.0 Formulation 6 6.0 6.0 Formulation 7 5.0 5.0 Generic DP 5.1 5.1
d) CEX-HPLC

(46) TABLE-US-00008 TABLE 8 CEX data of Formulation 4, 5, 6 & 7 % Purity Sample ID 0 day 3 days 7 days 15 days Formulation 4 93.3 93.4 93.4 93.5 Formulation 5 96.1 95.0 94.5 95.1 Formulation 6 89.9 84.4 79.0 77.2 Formulation 7 96.6 96.0 95.4 95.1 GENERIC DP 96.3 ND 94.6 94.2
Observation:

(47) Based on 15 days stress data the purity of the formulation 4, 5 and 7 was comparable with the reference formulation (generic DP). (FIG. 3)

(48) e) SEC-HPLC

(49) TABLE-US-00009 TABLE 9 SEC data of Formulation 4, 5, 6 & 7 % Purity Sample ID 0 day 3 days 7 days 15 days Formulation 4 99.3 99.3 99.2 99.1 Formulation 5 99.3 99.3 99.2 98.8 Formulation 6 98.3 96.1 94.2 92.4 Formulation 7 99.6 99.6 99.5 99.0 GENERIC DP 99.7 ND 99.6 99.4
Observation:

(50) Based on 15 days stress data the purity of the formulation 4, 5 and 7 was comparable with the reference formulation (generic DP). (FIG. 4)

(51) f) Potency

(52) The biological activity of Romiplostim is determined by cell based in-vitro bio-assay. The assay is based on the proliferation of meghkarocytes on UT-7 cell line (acute myeloid leukemia) cell line expressing TPO receptor. Romiplostim specifically proliferate activity of UT-7 cell line in a dose dependent manner

(53) TABLE-US-00010 TABLE 10 % Potency data of Formulation 4, 5, 6 & 7 DP % relative potency 15 D ST Sample ID 0 day 3 days 7 days 15 days Formulation 4 80.0 83.0 91.0 77.0 Formulation 5 106.0 111.0 118.0 Formulation 6 91.0 85.0 93.0 86.0 Formulation 7 67.0 93.0 72.0 78.0 GENERIC DP 82 ND 114 135
Observation:

(54) There is no change in potency at 40 C. after 15 days as compared to initial in all formulation.

Example 4

(55) Accelerated Stability Data Formulation 2, 4, 5, 7 & 8 (Liquid State):

(56) a) Physical Appearance

(57) All the samples were observed to be clear and colorless till 21 D AT

(58) b) CEX-HPLC

(59) TABLE-US-00011 TABLE 11 CEX data of Formulation 2, 4, 5, 7 & 8 % Purity Sample ID 0 day 3 days 7 days 15 days 21 days Formulation 2 95.6 96.8 96.4 96.4 96.4 Formulation 4 96.0 96.3 96.0 95.1 94.8 Formulation 5 92.4 89.1 82.9 72.4 58.8 Formulation 7 93.3 88.4 84.5 75.5 68.5 Formulation 8 96.4 96.1 94.2 90.9 90.7 Generic DS 96.1 95.2 93.1 87.8 85.4
Observation:

(60) Based on 21 days stress data the purity of the formulation 4 and 8 was comparable with the reference formulation (generic DS). (FIG. 5)

(61) c) SEC-HPLC

(62) TABLE-US-00012 TABLE 12 SEC data of Formulation 2, 4, 5, 7 & 8 % Purity Sample ID 0 day 3 days 7 days 15 days 21 days Formulation 2 98.3 99.6 99.6 99.6 99.7 Formulation 4 98.7 99.5 99.5 99.5 99.7 Formulation 5 99.6 99.1 99.2 99.5 99.5 Formulation 7 99.5 99.5 99.6 99.3 99.5 Formulation 8 98.1 99.6 99.5 99.5 99.8 Generic DS 99.2 99.4 99.4 99.3 99.4
Observation:

(63) Based on 21 days stress data the purity of the formulation 4 and 8 was comparable with the reference formulation (generic DS). (FIG. 6)

(64) d) Potency

(65) The biological activity of Romiplostim is determined by cell based in-vitro bio-assay. The assay is based on the proliferation of meghkarocytes on UT-7 cell line (acute myeloid leukemia) cell line expressing TPO receptor. Romiplostim specifically proliferate activity of UT-7 cell line in a dose dependent manner

(66) TABLE-US-00013 TABLE 13 % Potency data of Formulation 2, 4, 5, 7 & 8 % Relative Potency Sample ID 0 day 3 days 7 days 15 days 21 days Formulation 2 100 98 119 111 88 Formulation 4 103 114 100 103 86 Formulation 5 89 72 81 70 71 Formulation 7 87 103 87 78 81 Formulation 8 118 97 100 109 89 Generic DP 115 115 118 125 117
Observation:

(67) There is no change in potency at 40 C. after 21 days as compared to initial in all Formulations.

Example 5

(68) Rationale:

(69) Based on the above data, all three buffers show a good buffering capacity, and thermo stability profile. To confirm the results obtained during the initial screening, another set of stability study were carried out with the formulation 4 and 8.

(70) TABLE-US-00014 TABLE 14 Study condition and Time points for Formulation 4 & 8 Sr. No. Condition Temperature Time points 1 Stress 40 C. 2 C. 0 D, 3 D, 7 D, 15 D & 30 D
Method of Preparation:

(71) Romiplostim formulation was prepared in formulation composition given in the above table by dissolving the excipients in water for injection. The protein concentration was set to 0.5 mg/mL and the pH of the formulation is set to 5.0 similar to the reference formulation. 0.75 mL solution filled in 5 mL USP type 1 vial and half stoppered with bromobutyl rubber stopper. After completion of lyophilization cycle, vials are sealed with flip off seals and stored at 2 C.-8 C. Filled vial were charged at 40 C. for 30 days stress stability study. During stability study following test were done:

(72) TABLE-US-00015 TABLE 15 Purpose of the tests Tests Purpose of the tests SE-HPLC To monitor aggregates (H.M.W. impurities) CEX HPLC To monitor charge related impurities Potency To monitor effect on in vitro bioassay pH To monitor effect on pH Physical appearance To monitor physical appearance
a) Physical Appearance

(73) All the samples were observed to be white to pale yellow lyophilized cake till 28 D ST.

(74) b) Physical Appearance after Reconstitution

(75) All the samples were observed to be clear and colorless till 28 D ST.

(76) c) pH

(77) TABLE-US-00016 TABLE 16 pH data of Formulation 4 & 8 pH Time point Buffers 0 D 3 D 7 D 15 D 30 D Formulation 4 5.0 5.0 5.0 5.0 5.0 Formulation 8 5.1 5.1 5.1 5.0 5.0
d) CEX-HPLC

(78) TABLE-US-00017 TABLE 17 CEX data of Formulation 4 & 8 buffers % Purity Sample ID 0 day 3 days 7 days 15 days 30 days Formulation 4 94.5 96.6 96.0 95.9 95.0 Formulation 8 94.3 94.4 94.2 93.9 94.2
Observation:

(79) Formulation 4 & 8 are showing good stability and comparable (figure-7).

(80) e) SEC-HPLC

(81) TABLE-US-00018 TABLE 18 SEC data of Formulation 4 & 8 buffers % Purity Sample ID 0 day 3 days 7 days 15 days 30 days Formulation 4 99.2 99.5 99.5 99.5 99.4 Formulation 8 99.6 99.6 99.6 99.6 99.5
Observation:

(82) Formulation 4 & 8 are showing good stability and comparable in SEC profile (FIG. 8).

(83) f) Potency

(84) TABLE-US-00019 TABLE 19 % Potency data of Formulation 4 & 8 buffers % relative potency (By cell based assay) Sample ID 0 day 3 days 7 days 15 days 30 days Formulation 4 95 82 99 93 103 Formulation 8 99 93 96 109 106
Observation:

(85) There is no change in potency at 40 C. after 30 days as compared to initial in all Formulations.