STABLE ANTI-PD1 ANTIBODY PHARMACEUTICAL FORMULATIONS

Abstract

The present disclosure relates to a stable anti-PD-1 antibody pharmaceutical formulation and a method for preparing the same, the pharmaceutical formulation comprising: an anti-PD-1 antibody or an antigen binding fragment thereof; and a stabilizer, and not comprising a buffer.

Claims

1. A stable anti-PD-1 antibody pharmaceutical formulation, comprising: (a) an anti-PD-1 antibody or an antigen binding fragment thereof; and (b) a stabilizer, wherein the formulation does not comprise a buffer and has a pH of about 4.5 to about 6.5.

2. The pharmaceutical formulation of claim 1, wherein the formulation has a pH of about 5.0 to about 5.5.

3. The pharmaceutical formulation of claim 1, wherein the anti-PD-1 antibody includes a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3; and a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6.

4. The pharmaceutical formulation of claim 1, wherein the anti-PD-1 antibody is a pembrolizumab.

5. The pharmaceutical formulation of claim 1, wherein the concentration of the anti-PD-1 antibody or the antigen binding fragment thereof is about 5 to about 200 mg/ml.

6. The pharmaceutical formulation of claim 1, wherein the stabilizer is a polyol, an amino acid or a pharmaceutically acceptable salt, or a mixture thereof.

7. The pharmaceutical formulation of claim 6, wherein the polyol is sorbitol, sucrose, trehalose, mannose, maltose, mannitol, or a mixture thereof.

8. The pharmaceutical formulation of claim 6, wherein the amino acid is glycine, proline, phenylalanine, tyrosine, tryptophan, lysine, arginine, a pharmaceutically acceptable salt thereof, or a mixture thereof.

9. The pharmaceutical formulation of claim 1, wherein the stabilizer is about 1.0 to about 15.0% (w/v) of a sugar, about 1.0 to about 20.0% (w/v) of a sugar alcohol, about 0.1 to about 300.0 mM of an amino acid, or about 1.0 to about 300.0 mM of a metal salt.

10. The pharmaceutical formulation of claim 1, wherein the stabilizer is about 1.0 to about 15.0% (w/v) of a sugar and about 0.1 to about 300.0 mM of an amino acid.

11. The pharmaceutical formulation of claim 1, wherein the buffer is histidine, phosphoric acid, maleic acid, tartaric acid, succinic acid, citric acid, acetic acid, carbonic acid, a pharmaceutically acceptable salt thereof, or a mixture thereof.

12. The pharmaceutical formulation of claim 1, wherein the pharmaceutical formulation does not comprise a surfactant or further comprises a surfactant.

13. The pharmaceutical formulation of claim 12, wherein the surfactant is polysorbate, poloxamer, sorbitan ester of another fatty acid, or a mixture thereof.

14. The pharmaceutical formulation of claim 1, further comprising an antioxidant.

15. The pharmaceutical formulation of claim 14, wherein the antioxidant is methionine.

16. The pharmaceutical formulation of claim 1, wherein the pharmaceutical formulation is for administration by intravenous or subcutaneous injection.

17. The pharmaceutical formulation of claim 1, wherein the concentration of the anti-PD-1 antibody or the antigen binding fragment thereof is about 5 to about 200 mg/mL, and the stabilizer is: about 1.0 to about 15.0% (w/v) of sucrose, trehalose, a hydrate thereof, or a mixture thereof; about 1.0 to about 20.0% (w/v) of sorbitol, mannitol, a hydrate thereof, or a mixture thereof; about 0.1 to about 300.0 mM of arginine, lysine, proline, glycine, phenylalanine, tyrosine, tryptophan, a pharmaceutically acceptable salt thereof; or a mixture thereof; or a mixture of two or more thereof.

18. A method for treating a cancer in a subject, comprising administering a therapeutically effective amount of the pharmaceutical formulation according to claim 1 to the subject.

19. A method for preparing a stable pharmaceutical formulation, comprising: preparing a mixed solution by adding a stabilizer to a solvent; and adding a pembrolizumab or an antigen binding fragment thereof to the mixed solution, or comprising: preparing a solution of a pembrolizumab or an antigen binding fragment thereof by adding the pembrolizumab or the antigen binding fragment thereof to a solvent; and adding a stabilizer to the solution, wherein the method is performed without adding a buffer.

20. The pharmaceutical formulation of claim 12, wherein the surfactant is a non-ionic surfactant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The above and other aspects, features, and advantages of certain embodiments of the disclosure will be more apparent from the following description taken in conjunction with the accompanying drawings, in which:

[0059] FIG. 1 shows Δ % HMW values of formulations including various buffers after applying temperature stress thereto for 4 weeks;

[0060] FIG. 2 shows the T.sub.agg values of buffer-free formulations or formulations including different amounts of histidine buffers and surfactants;

[0061] FIG. 3 shows the ratios (348/332) depending on the change in denaturant concentrations measured in a buffer-free formulation and a formulation including 20 mM of histidine; and

[0062] FIG. 4 shows Δ % HMW values of formulations after applying temperature stress to the formulations including various amounts of buffers and PS80 surfactants for 4 weeks.

DETAILED DESCRIPTION

[0063] Hereinafter, the present disclosure will be described in greater detail through the following examples. However, the following examples are provided only for illustration of the present disclosure, but the scope of the present disclosure is not limited by these examples.

[0064] Materials and Methods

[0065] 1. Size Exclusion Chromatography (SEC)

[0066] The purity of each sample was identified by size-exclusion chromatography. Percentages of antibody monomer, high molecular weight species (HMW), and low molecular weight species (LMW) are determined by the size-exclusion chromatography. In the size-exclusion chromatography, LMW is eluted later than HMW. The presence of HMW indicates formation of protein aggregates, and the presence of LMW indicates formation of protein fragments.

[0067] 2. Dynamic Light Scattering (DLS) Assay

[0068] Aggregates in each sample were measured by dynamic light scattering assay. In detail, each sample was diluted using each buffer and loaded onto wells of a 96-well plate. The plate was loaded onto DynoPro® Plate Reader™ II instrument (Wyatt Technology). The temperature of the sample was increased at a rate of 0.15° C./min in a temperature range of 25 to 70° C., and sizes of aggregates in the pharmaceutical formulation were measured by dynamic light scattering (DLS) assay. Evaluation of stability is performed to measure temperatures at a time point at which the aggregate size varies, and a higher T.sub.agg value means a higher level of stability.

[0069] 3. HUNKY (ΔG, AggPath) Assay

[0070] Aggregates in each sample were measured by HUNKY assay. HUNKY device (HUNKY, Unchained Labs.) is a device for identifying the stability of a protein through chemical denaturation, and enables C.sub.1/2, AG, AggPath, and ΔGtrend values to be identified using denaturants and samples. C.sub.1/2 ranks stability. AG quantifies stability. AggPath predicts aggregation. ΔG.sub.trend zooms in on aggregation.

[0071] 4. Imaging Capillary Isoelectric Focusing (icIEF)

[0072] % acidic values of samples were measured using icIEF. Specifically, each sample was put into a 1.5 mL polypropylene microtube, and then microtube was exposed to a 40° C. temperature stress condition in a stability thermostat for 4 weeks. The sample was injected into an iCE3 system (Protein Simple, USA), and charge variant values of proteins were measured using iCE CFR software. The charge variant values of proteins are indicated by % acidic values. Measurement of these values is for identifying charge variants of proteins. When a protein is denatured due to stress applied thereto, aggregation or a charge change thereof may occur. Therefore, the charge change of protein may serve as a factor for identifying the stability thereof.

[0073] 5. Preparation of Pharmaceutical Formulations

[0074] Various pharmaceutical formulations listed in Table 1 were prepared in the following manners and used in the following examples.

[0075] First, in the composition of each of the respective pharmaceutical formulations, a buffer and a stabilizer, except for pembrolizumab and a surfactant, were added to sterilized distilled water to prepare a buffer solution. The pembrolizumab was introduced into a dialysis cassette (Slide-A-Lyzer cassette, Thermo Fisher Scientific) and then placed in a beaker containing a solution of each of the compositions listed in Table 1 to perform dialysis, thereby exchanging the existing pembrolizumab solution with the buffer solution. Finally, PS80 as a surfactant was added to the pembrolizumab solution such that the concentration of the surfactant in the prepared buffer solution reached a 1× concentration. Thereafter, the concentration was adjusted using each solution such that the pembrolizumab finally reached a concentration of 25 mg/m L.

TABLE-US-00001 TABLE 1 Name Group Formulation composition 1A 1 (Buffer Buffer free, 7% sucrose, pH 5.5 1B free) Buffer free, 7% sucrose, 0.02% PS80, pH 5.5 1C Buffer free, 7% sucrose, 0.10% PS80, pH 5.5 1D Buffer free, 7% sucrose, 0.20% PS80, pH 5.5 2A 2 (histidine 20 mM histidine, 7% sucrose, pH 5.5 2B buffer) 20 mM histidine, 7% sucrose, 0.02% PS80, pH 5.5 2C 20 mM histidine, 7% sucrose, 0.10% PS80, pH 5.5 2D 20 mM histidine, 7% sucrose, 0.20% PS80, pH 5.5 2E 40 mM histidine, 7% sucrose, pH 5.5 2F 40 mM histidine, 7% sucrose, 0.02% PS80, pH 5.5 2G 40 mM histidine, 7% sucrose, 0.10% PS80, pH 5.5 2H 40 mM histidine, 7% sucrose, 0.20% PS80, pH 5.5 2I 10 mM histidine, 7% sucrose, 0.02% PS80, pH 5.5 (Keytruda formulation) 3A 3 (acetic acid 10 mM acetic acid, 7% sucrose, 0.20% PS80, pH buffer) 5.5 4A 4 (succinic 10 mM succinic acid, 7% sucrose, 0.20% PS80, acid buffer) pH 5.5 5A 5 (citric acid 10 mM citric acid, 7% sucrose, 0.20% PS80, pH buffer) 5.5

Example 1: Effect of Buffer on Stability of Pembrolizumab-Containing Pharmaceutical Formulation

[0076] In this example, the effect of a buffer on the stability of each of pembrolizumab-containing pharmaceutical formulations was identified. In detail, after applying temperature stress to formulations 1A, 2I, 3A, 4A, and 5A, purities of the formulation samples were measured.

[0077] The temperature stress was applied such that each 0.3 mL of the formulations was put into a type I, 2 cc glass vial and the vial was exposed to a 40° C. temperature stress condition in a stability thermostat (JEIO TECH Co., Ltd.) for 4 weeks. Specifically, the vial was placed into the stability thermostat and stored under the conditions of 40±2° C. in temperature and 75±5% in relative humidity for 4 weeks. SEC analysis was performed on the formulations stored for 4 weeks in the above-described manner.

[0078] The results are shown in Table 2 and FIG. 1. Table 2 shows purities of formulations including various buffers and purities of the formulations after applying temperature stress thereto for 4 weeks, as indicated by % HMW. FIG. 1 shows Δ % HMW values of formulations including various buffers after applying temperature stress thereto for 4 weeks.

TABLE-US-00002 TABLE 2 Name 0 week 4 weeks Name % HMW % HMW Δ% HMW 1B 0.20 0.59 0.39 2I 0.21 0.63 0.42 3A 0.22 0.73 0.51 4A 0.24 0.98 0.74 5A 0.25 1.13 0.88

[0079] As shown in Table 2 and FIG. 1, at week 4, the average Δ % HMW value of the formulations each including a buffer and the Δ % HMW value of the buffer-free formulation were 0.64% and 0.39%, respectively, suggesting that the buffer-free formulation had higher stability than the formulations each including a buffer.

Example 2: Effects of Amounts of Buffers and Surfactants on Stability of Pembrolizumab-Containing Pharmaceutical Formulation

[0080] In this example, the effects of the amounts of buffers and surfactants on the stability of each of pembrolizumab-containing pharmaceutical formulations were identified. In detail, T.sub.agg values of formulations 1A, 1B, 1C, 1D, 2A, 2B, 2C, 2D, 2E, 2F, 2G, and 2H were measured by DLS assay.

[0081] The results are shown in Table 3 and FIG. 2. Table 3 shows compositions of buffer-free formulations or formulations including histidine buffers and different amounts of PS80 surfactants, and T.sub.agg values thereof. FIG. 2 shows the T.sub.agg values of buffer-free formulations or formulations including histidine buffers and different amounts of surfactants.

TABLE-US-00003 TABLE 3 Name T.sub.agg (° C.) 1A 61.01 1B 61.71 1C 56.06 1D 50.80 2A 62.26 2B 61.97 2C 57.41 2D 53.28 2E 60.99 2F 62.51 2G 59.36 2H 59.35

[0082] As shown in Table 3 and FIG. 2, T.sub.agg values of the formulations not including PS80 or including 0.02% PS80 surfactants were higher than those of the formulations including 0.10% or 0.20% PS80 surfactants. This suggests that the formulations including less than 0.10% PS80 surfactants, that is, the formulations 1A, 1B, 2A, 2B, 2E, and 2F, had higher aggregation temperatures than the formulations including not less than 0.10% PS80 surfactants, that is, the formulations 1C, 1D, 2C, 2D, 2G, and 2H. That is, it is confirmed that the formulation including a low content, that is, less than 0.10%, of PS80, has higher stability than the formulation including a high content, that is, not less than 0.10%, of PS80.

[0083] Meanwhile, in the formulations 2A, 2B, 2E and 2F, each including less than 0.10% PS80, changes in the T.sub.agg are not significantly affected by the amount of histidine.

[0084] In addition, among the formulations including 20 mM histidine buffers, the formulation not including PS80, that is, 2A, has a higher T.sub.agg value than the formulations 2B, 2C, and 2D including PS80. By contrast, among the formulations including 40 mM histidine buffers, the formulation 2F including 0.02% PS80 has a higher T.sub.agg value than the formulations 2E, 2G, and 2H.

Example 3: Effects of Buffers on Stability of Pembrolizumab-Containing Pharmaceutical Formulations

[0085] In this example, the effects of absence of buffer and 20 mM histidine buffer on the stability of each of the pembrolizumab-containing pharmaceutical formulations were identified. In detail, ΔG values of the formulations 1B and 2B were measured by HUNKY assay.

[0086] Unlike in the DLS assay for measuring protein aggregation depending on the temperature increase, in the HUNKY assay, the stability against chemical stress is measured using guanidine-HCl (Gdn-HCl). Specifically, in the HUNKY assay, an absorbance intensity ratio, that is, a ratio (348/332) is measured in a Gdn-HCl concentration gradient of 0 to 5.5 M at 348 nm and 332 nm. As a protein is denatured, the ratio increases.

[0087] The results are shown in Table 4 and FIG. 3. Table 4 shows compositions of a buffer-free formulation and a formulation including 20 mM histidine, and AG values thereof. FIG. 3 shows the ratios (348/332) depending on the change in denaturant concentrations measured in a buffer-free formulation and a formulation including 20 mM histidine

TABLE-US-00004 TABLE 4 Name ΔG1 (kcal/mol) 1B 4.26 2B 4.50

[0088] As shown in Table 4 and FIG. 3, the AG values of the formulations 1B and 2B were equal levels. This suggests that the stability of the pembrolizumab-containing pharmaceutical formulation is not significantly affected by the presence of histidine buffer.

Example 4: Effects of Amounts of Buffers and Surfactants on Stability of Pembrolizumab-Containing Pharmaceutical Formulations

[0089] In this example, the effects of amounts of buffers and surfactants on the stability of each of the pembrolizumab-containing pharmaceutical formulations were identified. In detail, purities of formulation samples were measured by SEC after applying temperature stress to the formulations 1A, 1B, 1C, 1D, 2A, 2B, 2C, 2D, 2E, 2F, 2G, and 2H.

[0090] The temperature stress was applied such that each 0.3 mL of the formulations was put into a type I, 2 cc glass vial, and the vial was exposed to the 40° C. temperature stress condition in a stability thermostat for 4 weeks. Specifically, the vial was placed into the stability thermostat and stored under the conditions of 40±2° C. in temperature and 75±5% in relative humidity for 4 weeks. SEC analysis was performed on the formulations stored for 4 weeks in the above-described manner.

[0091] The results are shown in Table 5 and FIG. 4. Table 5 shows compositions of formulations including various amounts of buffers and PS80 surfactants and purities of the formulations after applying temperature stress thereto for 4 weeks, as indicated by % HMW. FIG. 4 shows Δ % HMW values of formulations including various amounts of buffers and PS80 surfactants after applying temperature stress thereto for 4 weeks.

TABLE-US-00005 TABLE 5 0 week 4 weeks Name % HMW % HMW Δ% HMW 1A 0.19 0.50 0.31 1B 0.20 0.59 0.39 1C 0.28 0.75 0.47 1D 0.41 0.90 0.49 2A 0.18 0.48 0.30 2B 0.18 0.62 0.44 2C 0.25 0.87 0.62 2D 0.39 1.05 0.66 2E 0.18 0.60 0.42 2F 0.19 0.84 0.65 2G 0.27 1.20 0.93 2H 0.41 1.59 1.18

[0092] As shown in Table 5 and FIG. 4, at week 4, the average Δ % HMW value of the formulations each including 20 mM histidine buffer and the Δ % HMW value of the buffer-free formulation were 0.51% and 0.42%, respectively, suggesting that the buffer-free formulation had higher stability than the formulations each including 20 mM histidine buffer. By contrast, the average Δ % HMW value of the formulations each including 40 mM histidine buffer was 0.80%, meaning that the stability was lowest.

[0093] In addition, among the formulations including 20 mM histidine buffers, the formulation not including PS80, that is, 2A, had a lower Δ % HMW value than the formulations each including PS80, that is, 2B, 2C and 2D. In addition, among the formulations including 40 mM histidine buffers, the formulation not including PS80, that is, 3A, has a lower Δ % HMW value than the formulations each including PS80, that is, 3B, 3C and 3D. This suggests that the formulations each free of buffer and PS80 have higher stability than the formulations each including buffer and PS80.

[0094] For the formulations stored under stress conditions for 4 weeks, pH values thereof were also measured. Table 6 shows pH measurement results of the samples left under 40° C. temperature stress conditions for 4 weeks.

TABLE-US-00006 TABLE 6 pH Name 40° C./0 week 40° C./4 weeks 1A 5.50 5.57 1B 5.47 5.60 1C 5.46 5.53 1D 5.49 5.55 2A 5.59 5.55 2B 5.57 5.53 2C 5.58 5.54 2D 5.64 5.52 2E 5.56 5.52 2F 5.54 5.51 2G 5.55 5.50 2H 5.53 5.51

[0095] As shown in Table 6, the formulations including 20 mM histidine buffers and 40 mM histidine buffers and the buffer-free formulations showed no significant difference in the pH variation under 40° C./4 week stress conditions, compared to initial pH values. It was confirmed that a pH buffering action was exerted in the formulation not including a buffer, like in the formulation including a buffer.

Example 5: Effects of Buffer and pH Change on Stability of Pembrolizumab-Containing Pharmaceutical Formulation

[0096] In this example, the effects of presence of buffer and pH change on the stability of each of the pembrolizumab-containing pharmaceutical formulations were identified.

[0097] First, the respective pharmaceutical formulations listed in Table 7 were prepared in the same manner as described above.

TABLE-US-00007 TABLE 7 Name Pembrolizumab Buffer Stabilizer Surfactant pH 2I 25 mg/ml 10 mM histi- 7% sucrose 0.02% PS80 5.5 dine 6A 25 mg/ml — 7% sucrose 0.02% PS80 5.5 6B 25 mg/ml 10 mM histi- 7% sucrose 0.02% PS80 5.0 dine 6C 25 mg/ml — 7% sucrose 0.02% PS80 5.0

[0098] Purities of the formulations were measured by performing SEC analysis after applying temperature stress to the respective formulations in the same manner as in Example 1.

[0099] The results are shown in Table 8. Table 8 shows purities of the buffer-free formulations and formulations including a buffer and having pH variations, as indicated by % HMW.

TABLE-US-00008 TABLE 8 0 week Name Composition % HMW 2I 10 mM histidine, 7% sucrose, 0.02% PS80, pH 5.5 0.34 6A buffer free, 7% sucrose, 0.02% PS80, pH 5.5 0.18 6B 10 mM histidine, 7% sucrose, 0.02% PS80, pH 5.0 0.16 6C buffer free, 7% sucrose, 0.02% PS80, pH 5.0 0.14

[0100] As shown in Table 8, the buffer-free formulations had lower % HMW values than the formulations each including a buffer. In addition, the % HMW value of the formulation having a pH 5.0 was lower than that of the formulation having a pH 5.5. Specifically, the % HMW value of the buffer-free formulation having pH 5.0 was lowest. Accordingly, it was confirmed that the buffer-free formulation had higher stability than the formulation including a buffer, the formulation having pH 5.0 had higher stability than the formulation having pH 5.5, and the stability of the buffer-free formulation having pH 5.0 was highest.

[0101] In addition, % acidic values of the formulations were measured in the same manner as described above, using icIEF, after applying temperature stress to the respective formulations listed in Table 7.

[0102] The results are shown in Table 9. Table 9 shows % acidic values of buffer-free formulations and formulations including a buffer and having various pH levels.

TABLE-US-00009 TABLE 9 Temperature stress (40° C.): icIEF 0 week Name % acidic 2I 23.90 6A 20.51 6B 20.38 6C 20.41

[0103] As shown in Table 9, the buffer-free formulations had lower % acidic values than the formulations each including a buffer. In addition, the % acidic value of the formulation having pH 5.0 was lower than that of the formulation having pH 5.5. Specifically, the % acidic value of the buffer-free formulation having pH 5.0 was lowest. Accordingly, it was confirmed that the buffer-free formulation had higher stability than the formulation including a buffer, the formulation having pH 5.0 had higher stability than the formulation having pH 5.5, and the stability of the buffer-free formulation having pH 5.0 was highest.

Example 6: Extension of Stabilizers

[0104] In this example, the effects of changes in kinds of stabilizers and addition of an antioxidant on the stability of each of the pembrolizumab-containing pharmaceutical formulations were identified.

[0105] First, the respective pharmaceutical formulations listed in Table 10 were prepared in the same manner as described above.

TABLE-US-00010 TABLE 10 Pembro- Anti- Name lizumab Buffer Stabilizer Surfactant oxidant pH 7A 25 — 7% 3.3 mM 0.02% 5.0 mM 5.0 mg/ml sucrose Arg PS80 Met 7B 25 — 4% 3.3 mM 0.02% 5.0 mM 5.0 mg/ml sorbitol Arg PS80 Met 7C 25 — 7.6% 3.3 mM 0.02% 5.0 mM 5.0 mg/ml trehalose Arg PS80 Met 6A 25 10 mM 7% — 0.02% — 5.5 mg/ml histidine sucrose PS80

[0106] Purities of the respective formulations listed in Table 10 were measured by performing SEC analysis after applying temperature stress to the respective formulations in the same manner as in Example 1.

[0107] The results are shown in Table 11. Table 11 shows purities of the formulations including various kinds of stabilizers and having an antioxidant added thereto, as indicated by % HMW.

TABLE-US-00011 TABLE 11 0 week 1 week 2 weeks Name % HMW % HMW Δ% HMW % HMW Δ% HMW 7A 0.32 ND ND 0.64 0.32 7B 0.17 0.43 0.26 0.55 0.38 7C 0.19 0.40 0.21 0.54 0.35 6A 0.36 ND ND 0.76 0.40 * ND: Not determined

[0108] As shown in Table 11, the buffer-free formulations each having an antioxidant added thereto and having a pH 5.0, that is, formulations 7A, 7B, and 7C, had lower Δ % HMW values than the formulation including a buffer, not including an antioxidant, and having a pH 5.5, that is, the formulation 6A. That is, the buffer-free formulation having an antioxidant added thereto and having a pH 5.0 had higher stability than the formulation including a buffer, not including an antioxidant, and having a pH of 5.5.

[0109] In addition, the buffer-free formulations each having an antioxidant added thereto and having a pH of 5.0 showed excellent stability even when the kinds of stabilizers were variously changed to sucrose, sorbitol, trehalose, arginine, or a combination thereof.

[0110] In addition, % acidic values of the respective formulations indicated in Table 10 were measured in the same manner as described above, using icIEF, after applying temperature stress to the respective formulations.

[0111] The results are shown in Table 12. Table 12 shows % acidic values of the formulations including various kinds of stabilizers and having antioxidant added thereto.

TABLE-US-00012 TABLE 12 Temperature stress (40° C.): icIEF 0 week 1 week 2 weeks 4 weeks % % Δ % % Δ % % Δ % Name acidic acidic acidic acidic acidic acidic acidic 7A 22.48 ND ND 24.58 2.10 29.42 6.95 7B 20.57 21.95 1.38 24.08 3.51 ND ND 7C 20.97 21.95 0.98 24.05 3.08 ND ND 6A 22.34 ND ND 24.92 2.58 30.78 8.44 * ND: Not determined

[0112] As shown in Table 12, the buffer-free formulations each having an antioxidant added thereto and having a pH of 5.0, that is, formulations 7A, 7B, and 7C, had Δ % acidic values similar to or lower than the Δ % acidic value of the formulation including a buffer, not including an antioxidant, and having a pH of 5.5, that is, the formulation 6A. specifically, the Δ % acidic value of the formulation 7A was lowest, suggesting that the formulation 7A had highest stability.

Example 7: Effect of Kind of Buffer on Stability of Pembrolizumab-Containing Pharmaceutical Formulation

[0113] In this example, the effect of the kind of buffer on the stability of each of the pembrolizumab-containing pharmaceutical formulations was identified.

[0114] First, the respective pharmaceutical formulations listed in Table 13 were prepared in the same manner as described above.

TABLE-US-00013 TABLE 13 Name Pembrolizumab Buffer Stabilizer Surfactant pH 8A 25 mg/ml 10 mM acetate 128 mM 0.02% PS80 5.5 Arg-HCl 8B 25 mg/ml 10 mM acetate 0.7% NaCl 0.02% PS80 5.5 8C 25 mg/ml 10 mM histi- 128 mM 0.02% PS80 5.5 dine Arg-HCl 8D 25 mg/ml 10 mM histi- 0.7% NaCl 0.02% PS80 5.5 dine 8E 25 mg/ml 10 mM citrate 128 mM 0.02% PS80 5.5 Arg-HCl 8F 25 mg/ml 10 mM citrate 0.7% NaCl 0.02% PS80 5.5 8G 25 mg/ml 10 mM succi- 128 mM 0.02% PS80 5.5 nate Arg-HCl 8H 25 mg/ml 10 mM succi- 0.7% NaCl 0.02% PS80 5.5 nate

[0115] Purities of the formulation samples of Table 13 were measured by performing SEC analysis after applying temperature stress to the respective formulations in the same manner as in Example 1.

[0116] The results are shown in Table 14. Table 14 shows purities of the formulations having different kinds of buffers, as indicated by % HMW.

TABLE-US-00014 TABLE 14 0 week 1 week 2 weeks 4 weeks % % Δ % % Δ % % Δ % Name HMW HMW HMW HMW HMW HMW HMW 8A 0.25 0.85 0.60 1.23 0.98 1.90 1.65 8B 0.27 0.90 0.63 1.27 1.00 1.93 1.66 8C 0.25 0.90 0.65 1.32 1.07 2.02 1.77 8D 0.26 0.83 0.57 1.21 0.95 1.86 1.60 8E 0.24 0.75 0.51 1.07 0.83 1.71 1.47 8F 0.28 0.82 0.54 1.11 0.83 1.65 1.37 8G 0.26 0.73 0.47 1.05 0.79 1.76 1.50 8H 0.28 0.77 0.49 1.10 0.82 1.71 1.43

[0117] As shown in Table 14, the formulations each including histidine, that is, 8C and 8D, had higher Δ % HMW values than the formulation including other kinds of buffers. Therefore, it was confirmed that the formulations each including histidine were lower stability than the formulation including other kinds of buffers.

Example 8: Stabilities of High-Content Pembrolizumab-Containing Pharmaceutical Formulations

[0118] In this example, the stability of each of high-content pembrolizumab-containing pharmaceutical formulations was identified.

[0119] First, the pharmaceutical formulations listed in Table 15 were prepared in the same manner as described above.

TABLE-US-00015 TABLE 15 Pembro- Sur- Anti- Name lizumab Buffer Stabilizer factant oxidant pH 9A 150 — 7% 10 50 0.02% 30 mM 5.5 mg/ml sucrose mM mM PS80 Met Arg Lys 9B 150 — 1% 50 50 0.02% 30 mM 5.5 mg/ml sucrose mM mM PS80 Met Arg Lys 90 150 — 7% 50 — 0.02% 30 mM 5.5 mg/ml sucrose mM PS80 Met Arg 9D 150 — 7% 20 — 0.02% 30 mM 5.0 mg/ml sucrose mM PS80 Met Arg 9E 150 — — 20 — 0.02% — 5.0 mg/ml mM PS80 Arg 9F 150 — 5% 60 — 0.02% 30 mM 5.0 mg/ml sucrose mM PS80 Met Arg 9G 150 — 7% 30 — 0.02% 30 mM 5.0 mg/ml sucrose mM PS80 Met Pro 9H 150 — 7% 20 — 0.02% 30 mM 5.0 mg/ml sucrose mM PS80 Met Lys 9I 150 — 7% 20 — 0.02% 30 mM 5.0 mg/ml sucrose mM PS80 Met Arg 9J 150 — 7% 20 — 0.02% 30 mM 5.0 mg/ml sucrose mM PS80 Met Lys 9K 150 10 mM 7% — — 0.02% — 5.5 mg/ml histidine sucrose PS80

[0120] Purities of formulations 9A to 9D and 9K, among the formulation samples of Table 15, were measured by performing SEC analysis after applying temperature stress to the formulations 9A to 9D and 9K in the same manner as in Example 1. The results are shown in Table 16.

TABLE-US-00016 TABLE 16 0 week 1 week 2 weeks Name % HMW % HMW Δ% HMW % HMW Δ% HMW 9A 0.50 1.35 0.85 1.70 1.20 9B 0.44 1.58 1.14 2.03 1.59 9C 0.49 1.35 0.86 1.70 1.21 9D 0.43 1.63 1.20 2.13 1.70 9K 0.64 ND ND 2.35 1.71 * ND: Not determined

[0121] As shown in Table 16, the formulations 9A to 9D each including a high content, that is, 150 mg/ml, of pembrolizumab, and not including a buffer, showed higher stability than the formulation including a buffer, that is, the formulation 9K.

[0122] In addition, % acidic values of the respective formulations indicated in Table 15 were measured in the same manner as described above, using icIEF, after applying temperature stress to the respective formulations. The results are shown in Table 17.

TABLE-US-00017 TABLE 17 Temperature stress (40° C.): icIEF 0 week 1 week 2 weeks 4 weeks % % Δ % % Δ % % Δ % Name acidic acidic acidic acidic acidic acidic acidic 9A 20.74 21.93 1.19 23.84 3.10 ND ND 9B 20.72 21.87 1.15 23.68 2.96 ND ND 9C 20.42 21.76 1.34 23.61 3.19 ND ND 9D 20.96 22.05 1.09 24.29 3.33 ND ND 9E 22.86 23.34 0.48 24.89 2.03 28.86 6.00 9F 22.44 23.26 0.82 25.00 2.56 28.41 5.97 9G 22.80 23.86 1.06 25.64 2.84 29.99 7.19 9H 22.92 23.34 0.42 25.16 2.24 28.00 5.08 9I 20.02 21.52 1.50 24.19 4.17 28.86 8.84 9J 20.21 20.55 0.34 23.83 3.62 28.86 8.65 9K 24.6 ND ND 28.9 4.3 36.69 12.09 * ND: Not determined

[0123] As shown in Table 17, the formulations 9A to 9J each including a high content, that is, 150 mg/ml, of pembrolizumab, and not including a buffer, showed higher stability than the formulation including a buffer, that is, the formulation 9K.

[0124] In addition, the respective pharmaceutical formulations listed in Table 18 were prepared in the same manner as described above.

TABLE-US-00018 TABLE 18 Pembro- Sur- Anti- Name lizumab Buffer Stabilizer factant oxidant pH 10A 200 — 5% 80 — 0.02% 30 mM 5.0 mg/ml sucrose mM PS80 Met Arg 10B 200 — 7% 40 — 0.02% 30 mM 5.0 mg/ml sucrose mM PS80 Met Lys 10C 200 — 7% 26.4 — 0.02% 40 mM 5.0 mg/ml sucrose mM PS80 Met Arg 10D 200 — 7% 13.2 13.2 0.02% 40 mM 5.0 mg/ml sucrose mM mM PS80 Met Arg Lys 10E 200 — 4% 13.2 13.2 0.02% 40 mM 5.0 mg/ml sorbitol mM mM PS80 Met Arg Lys 10F 200 — 7% 33.3 — 0.02% 50 mM 5.0 mg/ml sucrose mM PS80 Met Arg 10G 200 — 7% 33.3 — 0.02% 50 mM 5.0 mg/ml sucrose mM PS80 Met Lys 10H 200 — 7% 16.7 16.7 0.02% 50 mM 5.0 mg/ml sucrose mM mM PS80 Met Arg Lys 10I 200 — 4% 16.7 16.7 0.02% 50 mM 5.0 mg/ml sorbitol mM mM PS80 Met Arg Lys

[0125] % acidic values of the respective formulations indicated in Table 18 were measured in the same manner as described above, using icIEF, after applying temperature stress to the respective formulations. The results are shown in Table 19.

TABLE-US-00019 TABLE 19 0 week 4 weeks Name % acidic % acidic Δ% acidic 10A 22.19 28.73 6.54 10B 22.66 28.86 6.20 10C 19.72 28.86 9.14 10D 19.41 28.86 9.45 10E 20.01 28.86 8.85 10F 19.76 28.86 9.10 10G 19.56 28.86 9.30 10H 19.76 28.86 9.10 10I 19.92 28.86 8.94

[0126] As shown in Table 19, the buffer-free formulations 10A to 101 each including a high content, that is, 200 mg/ml or 250 mg/ml, of pembrolizumab, and not including a buffer, showed higher stability than the formulation including a buffer, that is, the formulation 9K.