PHARMACEUTICAL LIQUID COMPOSITION HAVING INCREASED STABILITY

Abstract

Provided is a liquid pharmaceutical composition having increased stability, and more particularly, a stable liquid pharmaceutical composition including a protein.

Claims

1.-46. (canceled)

47. A pharmaceutical composition comprising: an anti-TNFα antibody, histidine, and polysorbate 20, wherein pH is 5.0 to 5.5, and the pharmaceutical composition has one or more selected from the following characteristics; (i) improvement in photostability, as compared with a histidine-free composition; and (ii) improvement in thermal stability, freeze-thaw stability, or both thermal stability and freeze-thaw stability, as compared with a composition comprising polysorbate 80 instead of polysorbate 20.

48. The pharmaceutical composition of claim 47, wherein the improvement in photostability is reduction in an oxidation rate of an amino acid residue and/or an aggregate content (% High Molecular Weight; % HMW) in the antibody under light-exposed conditions.

49. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition has a low oxidation rate of an amino acid residue and/or a low aggregate content (% high molecular weight (% HMW)) in the antibody under light-exposed conditions, as compared with the histidine-free composition.

50. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition has a low aggregate content (% HMW) after being preserved at 40±2° C. for 2 weeks; and/or a low aggregate content (% HMW) after 5 cycles of freeze-thaw at −70±10° C./25° C., as compared with a composition comprising polysorbate 80 instead of polysorbate 20.

51. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition has a low acidic variant content (% Acidic) after 5 cycles of freeze-thaw at −70±10° C./25° C. or a low % Acidic difference (Δ % Acidic) before and after 5 cycles of freeze-thaw at −70±10° C./25° C., as compared with a composition comprising polysorbate 80 instead of polysorbate 20.

52. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition, when preserved at a temperature of 30±2° C. for 28 days to 35 days after being stored at a temperature of 5±3° C. for 32 months to 52 months, has one or more of the following characteristics: turbidity of 20 nephelometric turbidity units (NTU) or less, TNFα binding activity of about 90% or more, TNFα neutralizing activity of about 80% or more, total purity of 93% or more, an aggregate content (% HMW) of 2% or less, an acidic variant content (% Acidic) of 33% or less, a main protein content (% Main) of 55% or more, a basic variant content (% basic) of 14% or less, in 0.8 mL of the pharmaceutical composition, the number of particles having an average particle diameter of 10 μm or more is 1500 or less, in 0.8 mL of the pharmaceutical composition, the number of particles having an average particle diameter of 25 μm or more is 40 or less, and an endotoxin content of 8 EU/mL or less.

53. The pharmaceutical composition of claim 47, wherein the anti-TNFα antibody is adalimumab.

54. The pharmaceutical composition of claim 47, wherein the anti-TNFα antibody is comprised at a concentration of about 50 mg/mL or about 100 mg/mL.

55. The pharmaceutical composition of claim 47, wherein the histidine is comprised at a concentration of 50 mM to 89 mM.

56. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition is free of amino acids other than histidine.

57. The pharmaceutical composition of claim 47, wherein the polysorbate 20 is comprised at a concentration of more than 0.04% (w/v) and 0.2% (w/v) or less.

58. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition is free of polysorbate 80.

59. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition further comprises one or more selected from the group consisting of sorbitol, mannitol, meglumine, trehalose, sucrose, maltose, lactose, glucose, xylitol, arabitol, erythritol, lactitol, maltitol, and inositol.

60. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition further comprises citrate.

61. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition further comprises one or more selected from the group consisting of phosphate, succinate, and acetate.

62. The pharmaceutical composition of claim 61, wherein the pharmaceutical composition is free of citrate.

63. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition is free of one or more selected from the group consisting of ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium nitrate, sodium chloride, sodium sulfate, potassium chloride, sodium hydroxide, potassium hydroxide, and ethylenediaminetetraacetic acid (EDTA).

64. A container comprising: the pharmaceutical composition of claim 47, wherein the pharmaceutical composition has stability against temperature changes, after 3 cycles each consisting of exposure to a high temperature of 30±2° C. for 48 hours and exposure to a low temperature of −5±3° C. for 48 hours.

65. The container of claim 64, wherein the stability against temperature changes is determined by one or more selected from the group consisting of TNFα-binding activity, TNFα-neutralizing activity, the number of subvisible particles, oxidation of amino acid residue, clarity, pH, protein concentration, protein aggregation rate, purity, charge variants, and endotoxin.

66. The container of claim 64, wherein the pharmaceutical composition, after 3 cycles consisting of exposure to a high temperature of 30±2° C. for 48 hours and exposure to a low temperature of −5±3° C. for 48 hours, has one or more selected from the following characteristics: TNFα binding activity of about 90% or more, TNFα neutralizing activity of about 90% or more, an oxidation rate of methionine at position 34 of a heavy chain of the antibody of about 1.1% or less, an oxidation rate of methionine at position 83 of a heavy chain of the antibody of about 0.9% or less, an oxidation rate of methionine at position 256 of a heavy chain of the antibody of about 6.0% or less, an oxidation rate of methionine at position 432 of a heavy chain of the antibody of about 0.5% or less, an oxidation rate of methionine at position 4 of a light chain of the antibody of about 0.9% or less, turbidity of 20 nephelometric turbidity units (NTU) or less, total purity of 94% (w/v) or more, in 0.8 mL of the pharmaceutical composition, the number of particles having an average particle diameter of 10 μm or more is 2500 or less, in 0.8 mL of the pharmaceutical composition, the number of particles having an average particle diameter of 25 μm or more is 100 or less, and an endotoxin content of 8 EU/mL or less.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0164] FIG. 1 is a graph showing oxidation rates (average value: n=3) of an amino acid (Met.sub.256) in a protein (adalimumab: 100 mg/mL) formulation according to one embodiment under photostress conditions, according to the type and concentration of amino acid;

[0165] FIG. 2A is a graph showing % HMW of a protein in an antibody formulation according to one embodiment under photostress conditions, according to the type and concentration of amino acid;

[0166] FIG. 2B is a graph showing % Monomer of a protein in an antibody formulation according to one embodiment under photostress conditions, according to the type and concentration of amino acid;

[0167] FIG. 2C is a graph showing % LMW of a protein in an antibody formulation according to one embodiment under photostress conditions, according to the type and concentration of amino acid;

[0168] FIG. 3 is a graph showing oxidation rates (average value: n=2) of an amino acid (Met256) in a protein (adalimumab: 50 mg/mL) formulation according to one embodiment under photostress conditions, according to the presence or absence of histidine;

[0169] FIG. 4 is a graph showing high molecular weight % (% HMW) (average value: n=3) of a protein in a protein (adalimumab: 50 mg/mL) formulation according to one embodiment during preservation at 40±2° C. for 2 weeks, according to the type and concentration of surfactant, as measured by SE-HPLC analysis;

[0170] FIG. 5 is a graph showing % HMW (average value: n=3) of a protein in a protein (adalimumab: 50 mg/mL) formulation according to one embodiment under freeze-thaw conditions (5 cycles), according to the type and concentration of surfactant, as measured by SE-HPLC analysis;

[0171] FIG. 6 is a graph showing % HMW (average value: n=3) of a protein in a protein (adalimumab: 50 mg/mL) formulation according to one embodiment during preservation at 40±2° C. for 4 weeks, according to the presence or absence of histidine and the type of surfactant, as measured by SE-HPLC analysis;

[0172] FIG. 7 is a graph showing % HMW (average value: n=3) of a protein in a protein (adalimumab: 100 mg/mL) formulation according to one embodiment during preservation at 40±2° C. for 4 weeks, according to the presence or absence of histidine and the type of surfactant, as measured by SE-HPLC analysis;

[0173] FIG. 8 is a graph showing % Acidic (a content ratio of acidic variants) (average value: n=3) of a protein in a protein (adalimumab: 50 mg/mL) formulation according to one embodiment during preservation at 40±2° C. for 4 weeks, according to the presence or absence of histidine and the type of surfactant;

[0174] FIG. 9 is a graph showing % Acidic (average value: n=3) of a protein in a protein (adalimumab: 100 mg/mL) formulation according to one embodiment during preservation at 40±2° C. for 4 weeks, according to the presence or absence of histidine and the type of surfactant;

[0175] FIG. 10 is a graph showing % Acidic (average value: n=3) of a protein in a protein (adalimumab: 50 mg/mL) formulation according to one embodiment under freeze-thaw conditions (5 cycles), according to the type and concentration of surfactant, as measured by icIEF analysis;

[0176] FIGS. 11A, 11B, 11C and 11D show quality test results (11A: % HMW, 11B: % Total purity, 11C: TNFα binding activity, and 11D: TNFα neutralizing activity) of a prefilled syringe pre-filled with a protein (adalimumab; 50 mg/mL) formulation according to one embodiment, as tested by temperature excursions; and

[0177] FIGS. 12A, 12B, 12C, 12D and 12E show quality test results (12A: % HMW, 12B: % Total purity, 12C: % Main, 12D: TNFα binding activity, and 12E: TNFα neutralizing activity) of a prefilled syringe pre-filled with a protein (adalimumab; 50 mg/mL) formulation according to one embodiment, as tested by storage stability at room temperature.

BEST MODE

[0178] Hereinafter, the present disclosure will be described in more detail with reference to Examples and Experimental Examples. However, these Examples and Experimental Examples are only for illustrating the present disclosure, and they are not to be construed as limiting the present disclosure.

REFERENCE EXAMPLES

Reference Example 1. SE-HPLC Analysis

[0179] Size exclusion-high performance liquid chromatography (SE-HPLC) analysis was performed using the HPLC system of Waters Corporation according to the manufacturer's manual. A total of three peaks were separated depending on retention times (molecular weights of proteins). These three peaks accounted for a HMW peak (protein aggregation), a monomer peak, and an LMW peak (protein degradation), in order of shorter retention times (larger molecular weights of proteins).

% HMW (High molecular weight %)={area of HMW/area of (HMW+monomer+LMW)}*100)

% Monomer (Monomer weight %)={area of monomer/area of (HMW+monomer+LMW)}*100)

% LMW(Low molecular weight %)={area of LMW/area of (HMW+monomer+LMW)}*100)

Reference Example 2. Photostress

[0180] Formulations were subjected to a sample stability test under photostress conditions of Table 1 below.

TABLE-US-00001 TABLE 1 Light-exposed group Base group (Base, initial) Dark control * (Light-exposured) N/A (Not applicable) Cool White Fluorescent Cool White Fluorescent Lamp: ≥1.2M lux hours Lamp: ≥1.2M lux hours N/A (Not applicable) Near Ultraviolet Lamp: ≥200 Near Ultraviolet Lamp: ≥200 Watt hours/square meter Watt hours/square meter Chamber Chamber Environmental Chamber Environmental Environmental Conditions: 25 ± 2° C./60 ± 5% Conditions: 25 ± 2° C./60 ± 5% Conditions: 5 ± 3° C. RH (relative humidity) RH (relative humidity) * For a dark control, the sample was wrapped with foil, and then the experiment was performed under the above conditions. [0181] The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guideline, ‘Q1B Stability Testing: Photostability Testing of New Drug Substances and Products’

Reference Example 3. Measurement of % Oxidation Rate (% Oxidation Level) of Amino Acid Residue

[0182] With regard to % oxidation rate of amino acid residues in the protein included in the formulation, the mass of the 256th methionine amino acid residue of adalimumab heavy chain was measured using liquid-chromatography-mass spectrometry (LC-MS) of Waters Corporation. After performing the LC-MS experiment, a relative % oxidation rate was analyzed using MassLynx™ program.

Relative % Oxidation=(Control intensity of oxidized peptide*100)/{(Control intensity of non-oxidized peptide)+(Control intensity of oxidized peptide)

Reference Example 4. CEX-HPLC Analysis

[0183] Cation Exchange-High Performance Liquid Chromatography (CEX-HPLC) analysis was performed using the HPLC system of Waters Corporation according to the manufacturer's manual. A total of three peaks were separated depending on retention times (charges of proteins). These three peaks accounted for an acidic peak, a main peak, and a basic peak, in order of shorter retention times. [0184] Acidic % (content ratio of Acidic variants, % Acidic)={area of acidic/area of (acidic+main+basic)}*100) [0185] Main % (content ratio of Main protein, % Main)={area of main/area of (acidic+main+basic)}*100) [0186] Basic % (content ratio of Basic variants, % Basic)={area of basic/area of (acidic+main+basic)}*100)

Reference Example 5. icIEF Analysis

[0187] Imaged capillary isoelectric focusing (icIEF) analysis method identifies charge variants of a protein according to their isoelectric point (pI) in a capillary containing ampholytes, and the analysis was performed by analyzing an acidic isoform, a main isoform, and a basic isoform using the icIEF instrument (ProteinSimple, iCE3) according to the manufacturer's manual.

Reference Example 6. Quality (Stability) Test of Antibody-Containing Product (Antibody-Prefilled Syringe)

[0188] Stability of antibody-containing product samples was evaluated by methods summarized in Table 2 below:

TABLE-US-00002 TABLE 2 Formulation stability Category evaluation items Method Description Visible features Color Visual Color evaluation of sample inspection using Brown standards B1-B8 Clarity Turbidity Clarity evaluation of sample measurement (HACH Model 2100AN) Visible particles Visual inspection Measurement of number of visible particles in sample pH pH pH measurement pH evaluation of sample (Thermo Scientific Orion star A211) Chemical Protein A.sub.280 measurement Concentration evaluation of analysis concentration (UV-VIS protein (antibody) in sample spectrophotometer) Physicochemical HMW impurity SE-HPLC Measurement of content analysis ratio of high-molecular- weight (HMW) impurity in sample Total purity CE-SDS Measurement of total impurity in sample LMW impurity CE-SDS Measurement of content ratio of low-molecular- weight (LMW) impurity (e.g., NGHC) in sample Charge CEX-HPLC, Measurement of charge isoforms icIEF variants including acidic, main, basic variants Subvisible Subvisible HIAC 9703 + Measurement of number of particles particles HRLD-400 particles having an average particle diameter of 25 μm or more, 10 μm or more, 8 μm or more, 5 μm or more, 2 μm or more Biological activity TNF-alpha FRET Measurement of relative binding activity binding affinity to Reference standard TNF-alpha Luciferase reporter Measurement of relative neutralizing gene system assay potency to Reference activity standard

[0189] 6.1. Presence of Visible Particles, Color, and Clarity

[0190] Through visual inspection, the presence of visible particles in the formulation and color (Brown standard: B1-B8) were evaluated. Clarity of the formulation was measured using a turbidimeter (HACH Model 2100AN).

[0191] 6.2. pH

[0192] pH of the sample was measured using a Thermo Scientific Orion star A211 (a combination of pH electrode and temperature probe; Thermo Scientific) adjusted to pH 4, 7, and 10 before use.

[0193] 6.3. Protein Concentration

[0194] Protein concentrations were measured by absorbance at a wavelength of 280 nm. In detail, all tested samples were diluted to a concentration of 1 mg/mL, based on the expected concentration of each sample in a 0.9% (w/v) NaCl solution, and absorbance at 280 nm (A280) was determined by a UV-VIS spectrophotometer (Agilent), and the obtained results were normalized using a 0.9% (w/v) NaCl solution as a blank solution.

[0195] 6.4. High-Molecular-Weight (HMW) Impurity

[0196] High-molecular-weight impurity in the sample was measured using size exclusion-high performance liquid chromatography (SE-HPLC) (Reference Example 1).

[0197] 6.5. Total Purity

[0198] Total purity of the sample was evaluated by measuring the prevalence of non-glycosylated heavy chains (NGHC) using capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) under non-reducing conditions.

[0199] 6.6. Charge Variants

[0200] Levels of charge variants in the sample were measured using CEX-HPLC (Reference Example 4) and icIEF (Reference Example 5).

[0201] 6.7. TNF-α Binding Activity (TNF-α Binding Affinity)

[0202] TNF-α binding activity of the antibody (adalimumab) included in the sample was measured by a competitive inhibition binding assay (CIBA) using fluorescence resonance energy transfer (FRET). TNF-α binding was measured by time-resolved fluorescence resonance energy transfer (TR-FRET). Adalimumab was labeled with a fluorescent europium (Eu) chelate (PerkinElmer), and human TNF-α was labeled with a Cy5 fluorophore, which were used in analysis. Eu-labeled adalimumab bound to Cy5-labeled TNF-α generates a fluorescent signal. When an antibody (adalimumab) in a fluorescent unlabeled sample is added thereto, the unlabeled antibody competes with Eu-labeled adalimumab for binding to Cy5-labeled TNF-α. Therefore, as the antibody in the sample binds to Cy5-labeled TNF-α, fluorescence generation is inhibited, and the measured fluorescence signal level is inversely proportional to the binding level of the unlabeled antibody in the sample to TNF-α. Eu-labeled adalimumab and Cy5-labeled TNF-α at a predetermined concentration and volume were sequentially added to an assay plate containing a reference standard and samples, followed by incubation at room temperature with gentle agitation, and the levels of generated fluorescence signal were measured by time-resolved fluorimetry (EnVision™, PerkinElmer). The TNF-α binding activity of adalimumab in the sample was calculated as a relative value (% relative binding activity) to the reference standard using a Parallel Line Analysis (PLA) software (Stegmann Systems). The reference standard means a reference substance having a reference value to which 100% is assigned, and the dose curves of the sample and the reference standard may be compared and analyzed through the PLA program to obtain % relative binding activity.

[0203] 6.8. TNF-α Neutralizing Activity (TNF-α Neutralization)

[0204] The TNF-α neutralizing activity of the antibody (adalimumab) included in the sample was measured using a luciferase reporter gene system. The sample was pre-incubated with TNF-α, and then incubated together with a cell line engineered to include a luciferase reporter gene (293-NF-κB-luc cell line (CVCL_KS34) engineered to include a human NF-κB response element upstream of the luciferase reporter gene, in which TNF-α binds to TNFR on the cell surface, thereby activating NF-κB signaling and promoting luciferase reporter gene expression). The sample was mixed with human TNF-α at a ratio of 1:1 (v/v) in a 96-well tissue culture plate, and incubated at room temperature for 30 minutes to 90 minutes. Thereafter, the prepared cells were put into the 96-well plate and incubated for 24 hours, and then the luminescence signal was measured using a Steady-Glo® Luciferase Assay System (Promega) on a microplate reader (EnVision™, PerkinElmer), thereby measuring TNF-α neutralization potency of adalimumab. The TNF-α neutralizing activity of adalimumab was calculated as a relative value (% relative potency) to a reference standard using a Parallel Line Analysis (PLA) software (Stegmann Systems). The reference standard means a reference substance having a reference value to which 100% is assigned.

[0205] 6.9. Number of Particles Per Size

[0206] Particles with an average particle diameter of 2 μm or more, 5 μm or more, 8 μm or more, 10 μm or more, and 25 μm or more were counted using HIAC 9703+ and HRLD 400 instrument (Beckman Coulter, Inc.).

[0207] 6.10. Endotoxin

[0208] Based on the reaction in which endotoxin activates limulus amebocyte lysate (LAL) to cause gelation, endotoxin was detected and quantified using a Bio Tek Elx808 IU Microplate Reader System (Bio Tek Instruments).

Example 1. Amino Acid Screening

[0209] 1-1. Photostability of Formulation Containing High Concentration of Protein Under Photostress Conditions

[0210] By evaluating stability of a protein formulation according to the type and concentration of amino acid, the type and concentration range of amino acid showing advantageous effects on the stability of the protein formulation were identified. In detail, photostability of the formulation containing a high concentration of protein under photostress conditions was examined in terms of antioxidant and anti-aggregation effects.

[0211] 1-1-1. Preparation of Formulation Containing High Concentration of Protein

[0212] Aqueous liquid formulations including 100 mg/mL of an anti-TNFα antibody (Adalimumab; anti-TNFα monoclonal antibody; CAS Registry Number: 331731-18-1) as a protein and having the compositions of the following Table 3 were prepared:

TABLE-US-00003 TABLE 3 Concentration Sample of protein No. (mg/mL) pH Buffer Excipient 1 Excipient 2 Surfactant 1 100 5.2 2.6 mM Na- 59 mM His 3.0% mannitol 0.08% PS 20 phosphate 2 2.6 mM Na- 3 phosphate + N/A 5.0% mannitol 4 5 mM Na- 59 mM Arg-HCl 3.0% mannitol 5 succinate 10 mM His 4.5% mannitol 6 80 mM His 2.0% mannitol  7* N/A N/A 4.2% mannitol 0.1% PS80 (*a sample including the same formulation ingredients as Humira ® including 100 mg/mL of adalimumab; PS 20: polysorbate 20, PS 80: polysorbate 80; N/A: not applicable; %: %(w/v))

[0213] 1-1-2. Anti-Oxidation Effect

[0214] With respect to each of the formulations prepared according to Table 3, an oxidation rate (% Met256) of Met256 (methionine residue at position 256) in the protein under photostress conditions was measured. The photostress conditions were prepared with reference to Reference Example 2, and the oxidation rate (% Met256) of Met256 in the protein was measured with reference to Reference Example 3.

[0215] The oxidation rate measurement was repeated three times (n=3), and the average values of the obtained results are shown in the following Table 4, and FIG. 1:

TABLE-US-00004 TABLE 4 Oxidation rate (% Met256) of amino acid (Met256) and Change (Δ% Met256) in amino acid oxidation rate under photostress conditions Δ% Met.sub.256 % Met.sub.256 [Light- [Light- Sample Dark- Light- exposured % Met.sub.256] − exposured % Met.sub.256] − No. Base control exposured [Base % Met.sub.256] [Dark-control % Met.sub.256] 1 4.23 4.43 55.80 51.57 51.37 SD 0.06 0.31 1.04 1.08 1.34 2 4.67 4.57 57.07 52.40 52.50 SD 0.42 0.32 0.49 0.10 0.66 3 4.37 4.57 77.77 73.40 73.20 SD 0.12 0.40 2.99 3.04 3.35 4 4.23 4.57 80.83 76.60 76.27 SD 0.21 0.32 4.03 3.82 3.72 5 4.07 4.57 70.23 66.17 65.67 SD 0.21 0.25 0.35 0.38 0.31 6 4.20 4.80 57.63 53.43 52.83 SD 0.26 0.46 3.51 3.62 3.88 7 4.13 4.43 73.27 69.13 68.83 SD 0.21 0.35 2.11 1.92 1.76

[0216] As confirmed from the above results, the Met.sub.256 oxidation rate and a change thereof according to the type of amino acid under the light-exposed conditions were observed in this order of Arg-HCl>Amino acid-free>His, indicating that the histidine-containing formulations are more suitable than the amino acid-free formulations or the Arg-HCl-containing formulations, in terms of photostability of formulation. In addition, the histidine-containing formulations exhibited remarkably low amino acid (Met256) oxidation rates and changes thereof to have excellent antioxidant effect, as compared with the amino acid-free formulation (Sample No. 7) having the same composition as the commercially available Humira® 100 mg/mL (pH 5.2, 4.2% mannitol, 0.1% PS 80).

[0217] In addition, as a result of analysis according to the histidine concentration, the change in the amino acid oxidation rate was observed in the order of 10 mM His>59 mM His≈80 mM His, indicating that formulations containing 59 mM or more of His had higher antioxidant effects.

[0218] As above, it was confirmed that histidine has activity as an anti-oxidant agent. In particular, when the histidine content in the antibody formulation is 10 mM or more, or more than 10 mM, and 80 mM or less (e.g., about 59 mM), the highest antioxidant effect was observed.

[0219] 1-1-3. Anti-Aggregation Effect

[0220] With respect to the formulations prepared according to Table 3, high molecular weight % (% HMW), monomer weight % (% monomer), and low molecular weight % (% LMW) of the protein under photostress conditions of Reference Example 2 were measured by SE-HPLC analysis according to Reference Example 1. The test was repeated three times (n=3), and the average values of the obtained results are shown in the following Tables 5 to 7, and FIGS. 2A to 2C:

TABLE-US-00005 TABLE 5 % HMW under photostress conditions Δ% HMW % HMW [Light- [Light- Sample Dark- Light- exposured % HMW] − exposured % HMW] − No. Base control exposured [Base % HMW] [Dark-control % HMW] 1 0.35 0.46 8.92 8.57 8.46 SD 0.01 0.02 0.15 0.14 0.13 2 0.33 0.43 8.19 7.86 7.76 SD 0.00 0.01 0.12 0.12 0.12 3 0.65 0.79 18.13 17.49 17.34 SD 0.02 0.03 0.61 0.60 0.58 4 0.40 0.50 20.79 20.39 20.29 SD 0.00 0.01 0.85 0.85 0.86 5 0.47 0.58 14.53 14.07 13.96 SD 0.01 0.01 0.34 0.34 0.34 6 0.31 0.40 7.45 7.14 7.05 SD 0.00 0.01 0.20 0.20 0.20 7 0.77 0.95 19.14 18.38 18.20 SD 0.01 0.01 0.26 0.26 0.26

TABLE-US-00006 TABLE 6 % Monomer under photostress conditions Δ% Monomer % Monomer [Light- [Light- Sample Dark- Light- exposured % Monomer] − exposured % Monomer] − No. Base control exposured [Base % Monomer] [Dark-control % Monomer] 1 99.40 99.29 89.62 −9.77 −9.66 SD 0.01 0.02 0.17 0.16 0.15 2 99.42 99.31 90.37 −9.05 −8.94 SD 0.00 0.01 0.11 0.11 0.12 3 99.10 98.95 79.97 −19.13 −18.98 SD 0.01 0.03 0.62 0.61 0.60 4 99.35 99.25 77.40 −21.95 −21.85 SD 0.00 0.01 0.83 0.83 0.84 5 99.28 99.17 83.73 −15.56 −15.44 SD 0.01 0.02 0.35 0.34 0.35 6 99.44 99.40 91.15 −8.29 −8.25 SD 0.02 0.11 0.23 0.25 0.30 7 98.98 98.80 78.93 −20.05 −19.88 SD 0.01 0.01 0.24 0.24 0.24

TABLE-US-00007 TABLE 7 % LMW under photostress conditions Δ% LMW % LMW [Light- [Light- Sample Dark- Light- exposured % LMW] − exposured % LMW] − No. Base control exposured [Base % LMW] [Dark-control % LMW] 1 0.25 0.25 1.46 1.21 1.21 SD 0.00 0.00 0.02 0.02 0.02 2 0.25 0.25 1.44 1.19 1.19 SD 0.00 0.01 0.02 0.02 0.02 3 0.25 0.26 1.89 1.64 1.64 SD 0.01 0.01 0.02 0.03 0.03 4 0.25 0.26 1.81 1.56 1.55 SD 0.00 0.01 0.02 0.02 0.02 5 0.25 0.26 1.74 1.49 1.48 SD 0.00 0.01 0.02 0.02 0.02 6 0.25 0.20 1.40 1.15 1.20 SD 0.01 0.10 0.03 0.04 0.11 7 0.25 0.25 1.93 1.68 1.68 SD 0.01 0.00 0.03 0.03 0.03

[0221] As confirmed from the above results, % HMW and change of % HMW under the light-exposed conditions were observed in this order of Arg-HCl>Amino acid-free>His, indicating that the histidine-containing formulations exhibited more improved anti-aggregation effect than the amino acid-free formulations or the Arg-HCl-containing formulations, and this effect shows a similar trend when compared to Sample 7 having the same composition as Humira® 100 mg/mL (pH 5.2, 4.2% mannitol, 0.1% PS 80), which is an amino acid-free formulation.

[0222] In addition, % HMW and change of % HMW under the light-exposed conditions according to His concentrations were observed in the order of 10 mM His>59 mM His 80 mM His, indicating that formulations containing 59 mM to 80 mM of His had higher anti-aggregation effects than the formulation containing 10 mM of His.

[0223] This indicates that when the His concentration is 59 mM or more, more excellent anti-aggregation effects are obtained. In addition, % Monomer and % LMW results also showed that histidine-containing formulations (Sample Nos. 1, 2, 5, and 6) had higher anti-aggregation effects than amino acid-free formulations or formulations containing amino acids (e.g., arginine) other than histidine.

[0224] 1-2. Photostability of Formulation Containing Low Concentration of Protein Under Photostress Conditions

[0225] Photostability of the formulations containing histidine, which was confirmed in Example 1-1 to have excellent photostability, and low concentration of protein were evaluated under photostress conditions.

[0226] Aqueous liquid formulations including 50 mg/mL of an anti-TNFα antibody (Adalimumab; anti-TNFα monoclonal antibody; CAS Registry Number: 331731-18-1) as a protein and having the compositions of the following Table 8 were prepared:

TABLE-US-00008 TABLE 8 Concen- tration Sample of protein Excip- Excip- Surfac- No. (mg/mL) pH Buffer ient 1 ient 2 tant 1 50 5.2 10 mM Na- 59 mM His 2.5% 0.08% 2 citrate N/A sorbitol PS 20 (N/A: not applicable; %: %(w/v))

[0227] With respect to the prepared histidine (His)-containing formulations and histidine-free formulations, an oxidation rate (% Met.sub.256) of Met.sub.256 (methionine residue at position 256), which is known as a main oxidized site in the protein, under photostress conditions was measured. The photostress conditions were prepared with reference to Reference Example 2, and the oxidation rate (% Met.sub.256) of Met.sub.256 in the protein was measured with reference to Reference Example 3.

[0228] The oxidation rate measurement was repeated twice (n=2), and the average values of the obtained results are shown in the following Table 9, and FIG. 3:

TABLE-US-00009 TABLE 9 Oxidation rate (% Met.sub.256) of amino acid (Met.sub.256) and Change (Δ% Met.sub.256) in amino acid oxidation rate under photostress conditions Δ% Met.sub.256 [Light- [Light- exposured % Met.sub.256 exposured % Met.sub.256] − Dark- Light- % Met.sub.256] − [Dark- con- expo- [Base control Sample No. Base trol sured % Met.sub.256] % Met.sub.256] 1 Average 16.50 17.10 40.40 23.90 23.30 SD (N = 2) 0.14 0.42 0.57 0.42 0.99 2 Average 15.10 16.35 64.90 49.80 48.55 SD (N = 2) 0.28 0.07 4.95 4.67 4.88

[0229] As shown in Table 9, it was confirmed that % Met.sub.256 of the histidine-containing formulation even with a low protein concentration under light-exposed conditions was 24.50% lower than that of the histidine-free formulation. In addition, as a result of analyzing the change (Δ % Met.sub.256) in the amino acid oxidation rate of the light-exposed group, as compared with a base group (Base) and a dark control (Dark control), according to the presence or absence of histidine, Δ % Met.sub.256 of the histidine-containing formulation were 25.90% (Δ % Met.sub.256 relative to the base group) and 25.25% (Δ % Met.sub.256 relative to dark control) lower than that of the histidine-free formulation. These results showed a similar tendency to the experimental results of the formulations containing a high concentration of protein of Example 1-1. Consequently, it was confirmed that the histidine-containing formulations had the excellent antioxidant effect on amino acid residues in the protein, as compared with the histidine-free formulation, demonstrating the function of histidine as an anti-oxidant agent in the protein formulations.

Example 2. Surfactant Screening

[0230] 2-1. Stability of Protein Formulation According to Type of Surfactant

[0231] To evaluate stability of the protein formulation according to the type of surfactant, aqueous liquid formulations including 50 mg/mL of an anti-TNFα antibody (Adalimumab; anti-TNFα monoclonal antibody; CAS Registry Number: 331731-18-1) as a protein and having the compositions of the following Table 10 were prepared:

TABLE-US-00010 TABLE 10 Concen- tration Sample of protein Excip- Excip- Surfac- No. (mg/mL) pH Buffer ient 1 ient 2 tant 1 50 5.2 10 mM Na- 59 mM 2.5% 0.04% PS 20 2 citrate His sorbitol 0.08% PS 20 3  0.1% PS 20 4  0.2% PS 20 5 0.04% PS 80 6 0.08% PS 80 7  0.1% PS 80 8  0.2% PS 80 (PS 20: polysorbate 20, PS 80: polysorbate 80; %: %(w/v))

[0232] 2-1-1. Stability of Formulation Under Heat Stress Conditions

[0233] The formulations prepared in Example 2-1 were stored at 40±2° C. for 2 weeks, and then high molecular weight % (% HMW) of the protein in the formulation at 0th week (initial), 1.sup.st week, and 2.sup.nd weeks was measured by SE-HPLC analysis according to Reference Example 1. The measurement was repeated three times (n=3), and the average values of the obtained results are shown in the following Table 11, and FIG. 4:

TABLE-US-00011 TABLE 11 % HMW upon Storage over Two Weeks at 40° C. % HMW Sample No. Initial 1 Wk 2 Wk 1 Average 0.48 0.62 0.67 SD (N = 3) 0.00 0.01 0.00 2 Average 0.49 0.62 0.67 SD (N = 3) 0.00 0.00 0.00 3 Average 0.49 0.63 0.68 SD (N = 3) 0.00 0.01 0.00 4 Average 0.51 0.64 0.69 SD (N = 3) 0.01 0.01 0.00 5 Average 0.49 0.62 0.67 SD (N = 3) 0.00 0.00 0.01 6 Average 0.51 0.63 0.68 SD (N = 3) 0.01 0.01 0.01 7 Average 0.57 0.67 0.70 SD (N = 3) 0.01 0.01 0.01 8 Average 0.68 0.79 0.78 SD (N = 3) 0.01 0.01 0.02

[0234] As shown in Table 11 and FIG. 4, initial % HMW of the formulations including polysorbate 20 (Samples 1˜4) was maintained at 0.48% to 0.51% with increasing concentrations, whereas initial % HMW of the formulations including polysorbate 80 (Samples 5˜8) increased from 0.49% to 0.68% with increasing concentrations. In particular, initial % HMW of the formulation including 0.2% polysorbate 20 (Sample 4) was 0.17% lower than that of the formulation including 0.2% polysorbate 80 (Sample 8), and % HMW at 2nd weeks of the formulation including 0.2% polysorbate 20 was 0.09% lower than that of the formulation including 0.2% polysorbate 80.

[0235] 2-1-2. Stability of Formulation Under Freeze-Thaw Conditions

[0236] High molecular weight % (% HMW) of proteins in the formulations prepared in Example 2-1 were measured under freeze-thaw conditions (Freeze/thaw, 5 cycles; FT5; each cycle: ‘frozen at −70° C.±10° C. for 18 hours or longer’+‘thawed at room temperature (25° C.) for 1 hour or longer’) by SE-HPLC analysis according to Reference Example 1. The experiment was repeated three times (n=3) and the average values of the results are shown in the following Table 12 and FIG. 5.

TABLE-US-00012 TABLE 12 % HMW under FT5 (Freeze/thaw, 5 cycle) conditions % HMW Sample No. Initial FT5 1 Average 0.48 0.48 SD (N = 3) 0.00 0.00 2 Average 0.49 0.48 SD (N = 3) 0.00 0.00 3 Average 0.49 0.48 SD (N = 3) 0.00 0.01 4 Average 0.51 0.49 SD (N = 3) 0.01 0.00 5 Average 0.49 0.49 SD (N = 3) 0.00 0.01 6 Average 0.51 0.49 SD (N = 3) 0.01 0.00 7 Average 0.57 0.50 SD (N = 3) 0.01 0.01 8 Average 0.68 0.59 SD (N = 3) 0.01 0.02

[0237] As shown in Table 12 and FIG. 5, initial % HMW of the formulations including polysorbate 20 (Samples 1˜4) was maintained at 0.48% to 0.51% with increasing concentrations, whereas initial % HMW of the formulations including polysorbate 80 (Samples 5˜8) increased from 0.49% to 0.68% with increasing concentrations. In particular, initial % HMW and % HMW after F5 of the formulation including 0.2% polysorbate 20 (Sample 4) was 0.17% and 0.1% lower than that of the formulation including 0.2% polysorbate 80 (Sample 8), respectively, indicating excellent stability.

[0238] 2-2. Evaluation of Stability of Formulations Containing Histidine and Polysorbate 20

[0239] 2-2-1. Preparation of Formulation

[0240] With respect to the protein formulations including histidine and polysorbate 20, which were confirmed in Examples 1 and 2-1 to have excellent formulation stability, various exemplary formulations were prepared, and thermal stability thereof was evaluated.

[0241] To this end, experimental formulations and control formulations were prepared according to the following compositions: [0242] Experimental Formulation 1: 50 mg/ml of adalimumab, pH 5.2, 10 mM Na-citrate, 59 mM histidine, 2.5% (w/v) sorbitol, 0.08% (w/v) polysorbate 20; [0243] Experimental Formulation 2: 100 mg/ml of adalimumab, pH 5.2, 2.5 mM Na-citrate, 59 mM histidine, 2.5% (w/v) sorbitol, 0.08% (w/v) polysorbate 20; [0244] Control Formulation 1 (a formulation having the same composition as Cyltezo®): 50 mg/ml of adalimumab, pH 5.2, 24.7 mM Na-acetate, 8.1% trehalose, 0.1% polysorbate 80; [0245] Control Formulation 2 (a formulation having the same composition as Amgevita®): 50 mg/ml of adalimumab, pH 5.2, 10 mM acetate, 9.0% sucrose, 0.1% polysorbate 80 [0246] Control Formulation 3 (a formulation having the same composition as Cyltezo®, except that 100 mg/ml of adalimumab was included instead of 50 mg/ml of adalimumab): 100 mg/ml of adalimumab, pH 5.2, 24.7 mM Na-acetate, 8.1% trehalose, 0.1% polysorbate 80; [0247] Control Formulation 4 (a formulation having the same composition as Amgevita®, except that 100 mg/ml of adalimumab was included instead of 50 mg/ml of adalimumab): 100 mg/ml of adalimumab, pH 5.2, 10 mM acetate, 9.0% sucrose, 0.1% polysorbate 80; [0248] Control Formulation 5 (a formulation having the same composition as Humira® (100 mg/mL)): 100 mg/ml of adalimumab, pH 5.2, 4.2% (w/v) mannitol, 0.1% (w/v) polysorbate 80.

[0249] 2-2-2. Stability of Formulation Under Heat Stress Conditions

[0250] % HMW

[0251] The experimental formulations and control formulations prepared in Example 2-2-1 were stored at 40±2° C. for 4 weeks, and then high molecular weight (%) (% HMW) of the protein in each formulation at 0th week, 1St week, 2nd week, and 4th week was measured by SE-HPLC analysis according to Reference Example 1. Further, changes in % HMW (Δ % HMW=(% HMW at corresponding week)−(% HMW at 0th week)) for 1 week, 2 weeks, and 4 weeks were calculated using the measured % HMW values.

[0252] Average values (N=3) of the obtained results of % HMW and Δ % HMW are shown in Table 13 and FIG. 6 (a formulation having a protein concentration of 50 mg/mL) and Table 14 and FIG. 7 (a formulation having a protein concentration of 100 mg/mL):

TABLE-US-00013 TABLE 13 % HMW Δ% HMW 0 1 2 4 1 2 4 Formulation Wk Wk Wk Wk Wk Wk Wk Experi- Average 0.20 0.35 0.43 0.57 0.15 0.23 0.37 mental SD 0.00 0.01 0.01 0.01 formula- (N = 3) tion 1 Control Average 0.48 0.70 0.82 1.07 0.22 0.34 0.60 formula- SD 0.01 0.04 0.05 0.06 tion 1 (N = 3) Control Average 0.46 0.68 0.79 1.05 0.24 0.32 0.59 formula- SD 0.02 0.03 0.03 0.05 tion 2 (N = 3)

TABLE-US-00014 TABLE 14 % HMW Δ% HMW 0 1 2 4 1 2 4 Formulation Wk Wk Wk Wk Wk Wk Wk Experi- Average 0.27 0.55 0.69 0.95 0.27 0.41 0.68 mental SD 0.01 0.03 0.03 0.02 formula- (N = 3) tion 2 Control Average 0.59 1.01 1.19 1.56 0.43 0.60 0.98 formula- SD 0.05 0.05 0.03 0.06 tion 3 (N = 3) Control Average 0.58 1.10 1.28 1.68 0.52 0.72 1.10 formula- SD 0.02 0.01 0.01 0.04 tion 4 (N = 3) Control Average 0.53 0.90 1.33 1.60 0.37 0.80 1.07 formula- SD 0.01 0.01 0.02 0.05 tion 5 (N = 3)

[0253] The results of Tables 13 and 14 and FIGS. 6 and 7 confirmed that experimental formulations including histidine and polysorbate 20, wherein the protein concentration was 50 mg/mL or 100 mg/mL, showed remarkably low % HMW and Δ % HMW at each measurement point, as compared with control formulations free of histidine and polysorbate 20, indicating that experimental formulations had excellent stability.

[0254] % Acidic

[0255] The experimental formulations and control formulations prepared in Example 2-2-1 were stored at 40±2° C. for 4 weeks, and then % Acidic (a content ratio of acidic variants) of the protein in each formulation at 0.sup.th week, 2.sup.nd week, and 4.sup.th week was measured by CEX-HPLC analysis of Reference Example 4 (control formulations 1, 2, 3, and 4) or by icIEF analysis of Reference Example 5 (experimental formulations 1 and 2, and control formulation 5). Further, changes in % Acidic (Δ % Acidic=(% Acidic at corresponding week)−(% Acidic at 0.sup.th week)) were calculated using the measured % Acidic values.

[0256] The obtained results (average value: N=3) of % Acidic and Δ % Acidic are shown in Table 15 and FIG. 8 (a formulation having a protein concentration of 50 mg/mL) and Table 16 and FIG. 9 (a formulation having a protein concentration of 100 mg/mL):

TABLE-US-00015 TABLE 15 % Acidic Δ% Acidic 0 2 4 2 4 Formulation Wk Wk Wk Wk Wk Experimental Average 25.32 35.33 42.40 10.01 17.08 formulation 1 SD (N = 3) 0.62 1.64 0.66 Control Average 26.50 40.65 55.02 14.16 28.52 formulation 1 SD (N = 3) 0.89 0.75 1.34 Control Average 25.93 41.35 56.23 15.42 30.30 formulation 2 SD (N = 3) 0.30 0.61 1.00

TABLE-US-00016 TABLE 16 % Acidic Δ% Acidic 0 2 4 2 4 Formulation Wk Wk Wk Wk Wk Experimental Average 24.79 33.79 39.28 8.99 14.49 formulation 2 SD (N = 3) 0.41 0.41 0.80 Control Average 25.26 38.37 51.46 13.11 26.20 formulation 3 SD (N = 3) 1.39 0.78 1.23 Control Average 24.58 39.19 52.84 14.61 28.26 formulation 4 SD (N = 3) 0.44 0.30 0.62 Control Average 25.08 35.87 45.07 10.78 19.98 formulation 5 SD (N = 3) 1.99 0.78 0.92

[0257] The results of Tables 15 and 16 and FIGS. 8 and 9 confirmed that experimental formulations including histidine and polysorbate 20, wherein the protein concentration was 50 mg/mL or 100 mg/mL, showed remarkably low % Acidic and Δ % Acidic at each measurement point, as compared with control formulations free of histidine and polysorbate 20, indicating that experimental formulations had excellent stability.

Example 3. Optimization of Surfactant (Polysorbate 20) Concentration

[0258] % Acidic of the protein in each formulation prepared in Example 2-1 under freeze-thaw conditions (Freeze/thaw, 5 cycles; FT5; each cycle: ‘frozen at −70° C.±10° C. for 18 hours or longer’+‘thawed at room temperature (25° C.) for 1 hour or longer’) was measured by icIEF analysis according to Reference Example 5. Further, changes (Δ % Acidic) in % Acidic before and after FT5 were calculated using the measured % Acidic values [Δ % Acidic=(FT5% Acidic)−(Initial % Acidic)]. The experiment was repeated three times (n=3), and the average values of the obtained results are shown in the following Table 17, and FIG. 10:

TABLE-US-00017 TABLE 17 % Acidic under FT5 (Freeze/thaw, 5 cycles) conditions % Acidic Δ% Acidic [(FT5 % Acidic) − Formulation Initial FT5 (Initial % Acidic)] 0.04% PS 20 Average 23.92 26.91 2.99 SD (N = 3) 0.28 0.22 0.46 0.08% PS 20 Average 24.40 26.82 2.42 SD (N = 3) 0.46 0.43 0.88  0.1% PS 20 Average 24.79 25.62 0.83 SD (N = 3) 0.34 0.85 0.83  0.2% PS 20 Average 24.93 25.72 0.79 SD (N = 3) 0.39 0.79 1.09 0.04% PS 80 Average 24.22 26.92 2.70 SD (N = 3) 0.31 0.36 0.10 0.08% PS 80 Average 23.82 26.97 3.15 SD (N = 3) 0.32 0.05 0.28  0.1% PS 80 Average 23.64 26.94 3.29 SD (N = 3) 0.34 0.19 0.20  0.2% PS 80 Average 23.74 27.37 3.63 SD (N = 3) 0.58 0.31 0.43

[0259] As shown in Table 17 and FIG. 10, the formulations including polysorbate 20 as a surfactant showed overall low Δ % Acidic values, as compared with formulations including polysorbate 80. In particular, when the concentration was 0.08% or more, the formulations including polysorbate 20 showed remarkably low Δ % Acidic values of about 2.5% or less, as compared with the formulations including polysorbate 80. In detail, it was confirmed that when the concentration of the surfactant was in the range of 0.08% or more, the Δ % Acidic values of the formulations including polysorbate 20 tend to decrease with increasing concentration of polysorbate 20, whereas the Δ % Acidic values of the formulations including polysorbate 80 tend to increase with increasing concentration of polysorbate 80. These results indicate that the formulations including polysorbate 20 had excellent stability, as compared with the formulations including polysorbate 80, and in particular, when the concentration of polysorbate 20 was 0.08% or more, the stability was particularly excellent.

Example 4. Quality Evaluation of Antibody-Containing Product—Temperature Excursion Test

[0260] During commercialization, storage, distribution, and clinical trials of the formulations containing the antibody, temperature excursions outside the recommended temperature for the formulations may occur, which may impede proper prescription. In this exemplary embodiment, to examine storage stability of the antibody (adalimumab) formulations, which were confirmed in Examples 1 to 3 to have excellent stability, formulation stability of pre-filled syringe (PFS) products, which were exposed to high temperature and low temperature conditions for a short period of time, was evaluated.

[0261] Compositions of the antibody formulations (samples) used in this exemplary embodiment are as follows:

[0262] 50 mg/ml of adalimumab, pH 5.2, 10 mM Na-citrate, 59 mM histidine, 2.5% (w/v) sorbitol, 0.08% (w/v) polysorbate 20.

[0263] Pre-filled syringes with the antibody (adalimumab) formulation in a single dose (0.8 mL) were prepared (n=132), and 3 cycles of freeze/thaw were performed using the pre-filled syringes. Each cycle was performed by exposure to a high temperature (30±2° C. and relative humidity (RH) of 65±5%) for 48 hours, and then exposure to a low temperature (−5±3° C.) for 48 hours (3 cycles: high temperature exposure (30±2° C.) for a total of 144 hours and low temperature exposure (−5±3° C.) for a total of 144 hours). Thereafter, analysis was performed by methods of evaluating various items as follows: appearance, pH, protein concentration, container closure integrity, protein aggregation rate (% HMW), purity, charge variants (e.g., % Acidic, % Main, % Basic), oxidation of amino acid residues, endotoxin, particulates, and biological activity.

[0264] The effects of the freeze-thaw treatment (3 thermal cycles) on quality of the antibody formulation products (pre-filled syringes), as compared with those of a baseline (antibody formulation preserved at 5° C.), are shown in Table 18 below, and among them, % HMW, % Total purity, TNFα binding activity, and TNFα neutralizing activity are shown in FIGS. 11A to 11D (11A: % HMW, 11B: % Total purity, 11C: TNFα binding activity, and 11D: TNFα neutralizing activity), respectively.

TABLE-US-00018 TABLE 18 Quality of Antibody Formulation Product (3 cycled sample) Under Extreme Temperature Cycling Conditions Test Item Test method Baseline Temperature Cycle 3 Color Reference Colourless B8 ≤ Sample < B7 Example 6.1 Clarity Reference Example 18 NTU 17 NTU 6.1 (Nephelometric Turbidity Unit; NTU) Visible particles Reference Practically free Practically free from Example 6.1 from particles particles pH Reference 5.3 5.3 Example 6.2 Protein concentration Reference 51.6 49.7 (mg/mL) Example 6.3 Polymer aggregate Reference Example 0.2%  0.2%  (impurity) (% HMW) 6.4 and Reference Example 1 Total purity Reference Example 96.8%   96.6%   Single highest 6.5 (CE-SDS; 2.1 2.0 impurity Capillary Electrophoresis- Sodium Dodecyl Sulfate, Non-reducing) Main peak icIEF (Reference 8.6 8.6 % Acidic Example 6.6 and 21.8 25.0 % Main Reference 67.1 64.5 % Basic Example 5) 11.2 10.5 TNFα binding activity Competitive Binding 92% 98% (% relative binding Assay (FRET) activity) (Reference Example 6.7) TNFα neutralizing Cell-based, NF-kB 94% 105%  activity (% relative Reporter Gene assay potency) (Reference Example 6.8) Number of particles Reference 1521 1494 having 10 μm or Example 6.9 more (particles/ syringe) Number of particles 15 18 having 25 μm or more (particles/ syringe) Number of particles 12217 13111 having 2 μm or more (particles/ syringe) Number of particles 6257 6882 having 5 μm or more (particles/ syringe) Number of particles 2476 2579 having 8 μm or more (particles/ syringe) Endotoxin Endotoxin Units <5 EU/mL <5 EU/mL per ml (EU/mL) (Reference Example 6.10) % acidic CEX-HPLC 23.5 24.0 % main (Reference Example 67.2 65.2 % basic 6.6 and Reference 9.3 10.9 Example 4) % Oxidation of heavy Reference 0.6 0.6 chain Met34 Example 3 % Oxidation of heavy 0.3 0.4 chain Met83 % Oxidation of heavy 5.8 4.7 chain Met256 % Oxidation of heavy Not Not chain Met432 detected detected % Oxidation of light 0.2 0.5 chain Met4

[0265] As shown in Table 18 and FIGS. 11A to 11D, the pre-filled syringes which were filled with the antibody formulation and treated with 3 cycles (each cycle: high temperature (30±2° C.) exposure and low temperature (−5±3° C.) exposure), as tested in the present exemplary embodiment, showed no significant changes in four critical quality attributes (CQA) regarding % HMW by SE-HPLC, % Total purity by non-reducing CE-SDS, % relative binding activity by TNF-α binding assay, and % relative potency by a cell-based TNF-α neutralization assay, as compared with the baseline, and the results met stability acceptance criteria.

[0266] In addition, charge variants of antibody, oxidation degree of amino acid residue, endotoxin levels, appearance (clarity, visible particles, etc.), pH, protein concentration, and subvisible particles in the antibody formulation products (prefilled syringes) tested in the present exemplary embodiment also showed no significant changes, as compared with those of the reference, and the results met stability acceptance criteria.

[0267] Further, none of the tested pre-filled syringes showed closure breaches of the container.

[0268] As described above, the antibody formulations tested in the present exemplary embodiment showed excellent stability inside the products even after exposed to several times of freeze-thaw cycles (thermal cycles) immediately after commercialization. These results allow prediction of the effects of temperature excursions during shipment or preservation of the antibody formulation products, and thus are expected to contribute to prescribing the antibody.

Example 5. Evaluation of Storage Stability at Room Temperature—Patient Convenience Stability Study

[0269] With respect to the formulation samples of Example 4 preserved for 48 months under long-term storage conditions (5±3° C.), physical properties of Table 19 were measured at the time point of 0 day, 15 days, and 30 days while preserved for 30 days under room temperature conditions (30±2° C. and RH of 60±5%). Detailed measurement methods are as performed in Example 4.

TABLE-US-00019 TABLE 19 Stability results under room-temperature storage conditions (30 ± 2° C./RH of 65 ± 5%) Time Point Test Item 0 Day 15 Days 30 Days Appearance Color B8 < sample < N/A B9 < sample < B8 B7 Clarity 17 17 Visible Practically free Practically free particles from particles from particles pH 5.3 5.3 Protein content (A.sub.280) 50.1 50.7 Size exclusion chromatography 0.7 0.8 0.9 (SE-HPLC) CE-SDS 96.9 95.6 96.1 (non-reducing) 1.9 1.9 1.7 icIEF 29 31 33 62 59 56 9 10 11 Competitive inhibition binding 102 100 106 assay (CIBA) of TNF-α by FRE Analysis of TNF-α neutralization 93 100 86 by NF-κB reporter gene Particulate matter 6 N/S 12 1,231 923 N/A: not applicable

[0270] As shown in Table 19 and FIGS. 12A to 12E, the antibody formulation samples tested in the present exemplary embodiment were tested under room temperature conditions (30±2° C. and RH of 60±5%) for 30 days, after being preserved for 48 months under long-term storage conditions (5±3° C.), and as a result, they showed no significant changes in four critical quality attributes (CQA) regarding % HMW by SE-HPLC, % Total purity by non-reducing CE-SDS, % relative binding activity by TNF-α binding assay, and % relative potency by a cell based TNF-α neutralization assay, as compared with the baseline (0 day), and the results met stability acceptance criteria.

[0271] Further, they also showed no significant changes in % Main by icIEF, as compared with the baseline (0 day), and the results met stability acceptance criteria.

[0272] Accordingly, the pharmaceutical compositions of the present disclosure exhibit significantly improved patient convenience and a longer lifetime characteristic during commercialization, as compared with the original and biosimilars thereof.