AN IMPROVED PROCESS OF STORING AND PREPARING THE PROTEIN
The present invention provides a method for reducing the protein aggregation by adjusting the pH below 6.0 of liquid formulation comprising the antibody or fusion protein. The present invention also provides methods for storing the pre-formulation for longer period without using any sugar or additives which can be utilized for preparation of liquid or lyophilized formulation.
1 . A process for the preparation of stable formulation comprising:
a. Pre-formulation capable to formulate in lyophilized or liquid formulation comprising:
i. An antibody of interest,
ii. Suitable buffer,
iii. Suitable pH;
b. Adjusting the pH of pre-formulation to about pH 5.0 to about 5.9;
c. Optionally performing ultrafiltration;
d. Storing the pre-formulation at freezing temperature for suitable period of time;
e. Performing the freeze thaw cycle of pre-formulation;
f. Mixing the suitable excipients in pre-formulation to formulate final formulation;
Wherein the pre-formulation comprises substantially low aggregates or High molecular weight impurities after the storage at frozen temperature.
2 . The process according to claim 1 , wherein the pre-formulation is stored for suitable period selected from at least by 12 hours, 24 hours, 30 hours, 40 hours, 50 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours, 7 days, 10 days, 15 days, 20 days, 25 days, 30 days, 40 days, 50 days, 60 days, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, and 12 months.
3 . The process according to claim 1 , wherein the pre-formulation freeze thaw cycle is selected from first cycle, second cycle, third cycle, four cycle, five cycle, six cycle, seven cycle, eight cycle, nine cycle, and ten cycle.
4 . The process according to claim 1 , wherein the pre-formulation is free of any sugar or sugar alcohol, sucrose, mannitol, trehalose, raffinose, and sorbitol.
5 . The process according to claim 1 , wherein the pre-formulation is free of any amino acid selected from arginine, lysine, and glycine.
6 . The process according to claim 1 , wherein the adjusted pH of pre-formulation is selected from about 5.5 to about 5.8.
7 . The process according to claim 1 , wherein the pre-formulation is obtained from chromatographic steps.
8 . The process according to claim 7 , wherein the chromatographic steps are selected from ion exchange, anion exchange, cation exchange, mixed-mode chromatography, hydrophobic exchange chromatography, and ceramic hydroxyapatite chromatography (CHT).
9 . The process according to claim 1 , wherein the pre-formulation suitable pH is identical to pH of the buffer used for the elution of antibody of interest from chromatographic column.
10 . The process according to claim 9 , wherein the pre-formulation suitable pH is about from 6 to about 7.
11 . The process according to claim 1 , wherein the pre-formulation comprises a buffer selected from phosphate, citrate, phosphate-citrate, histidine, and acetate, and salt thereof.
12 . The process according to claim 1 , wherein the pre-formulation is stored at freezing temperature selected from 0° C. to −80° C., −10° C., −20° C., −30° C., −40° C., −50° C., −60° C., −70° C., or −80° C.
13 . The process according to claim 12 , wherein the pre-formulation is stored at freezing temperature is 0° C. to −20° C.
14 . The process according to claim 1 , wherein the pre-formulation comprises substantially low aggregates or High molecular weight impurities after the storage at frozen temperature compared to pre-formulation storage at pH 7.
15 . The process according to claim 1 , wherein the pre-formulation comprises substantially low aggregates or high molecular weight 0.1% or less compared to pre-formulation storage at pH 7.
16 . The process according to claim 14 , wherein the pre-formulation comprises substantially low aggregates or high molecular weight selected from about 0.09% or less, 0.08% or less, 0.07% or less, 0.06% or less, 0.05% or less, 0.04%, or less and 0.03% or less compared to pre-formulation kept at pH 7.
17 . The process according to claim 1 , wherein the pre-formulation is formulated into final formulation by mixing suitable excipients in pre-formulation, the suitable excipients comprises:
a. buffer selected from phosphate, citrate, phosphate-citrate, histidine, and acetate, and salt thereof;
b. optionally suitable aggregation inhibitor selected from Arginine or arginine HCl, Lysine or Lysine HCl, glycine, and proline;
c. optionally sugar, sugar alcohol;
d. suitable surfactant selected from polysorbate and poloxamer;
e. pH 6.0 to 7.0; and
f. an antibody of interest.
18 . The process according to claim 17 , wherein the final formulation is a liquid formulation.
19 . The process according to claim 17 , wherein the final formulation is lyophilized formulation.
20 . The process according to claim 1 , wherein the antibody is selected from IgG1, IgG2, IgG3, IgG4, and fusion proteins.
21 . The process according to claim 1 , wherein the antibody is IgG1 capable to bind IgE.
22 . The process according to claim 1 , wherein the antibody is Omalizumab or ligelizumab.