WO2018122053A1 - Anti-angiopoietin-2 antibody formulation - Google Patents

Abstract

This invention relates to a stable pharmaceutical formulation of an antibody against Angiopoietin-2, in particular a bispecific antibody binding Angiopoietin-2 and vascular endothelial growth factor, and a process for the preparation and uses of the formulation.

Description

Anti-Angiopoietin-2 antibody formulation

Field of the Invention

This invention relates to a stable pharmaceutical liquid formulation of an antibody molecule against Angiopoietin-2, in particular a bispecific antibody, and a process for the preparation and uses of the formulation.

Background

Antibodies against Angiopoietin-2 (ANG-2), including bispecific antibodies against Ang-2 and human vascular endothelial growth factor (VEGF, VEGF-A), are of therapeutic interest, in particular as medicaments for the treatment and prophylaxis of treatment of vascular diseases, including cancer. Antibodies against Ang-2/VEGF are for example described in WO2010040508 or WO2011/117329. These antibodies inhibit ANG-2 binding to Tie2 with an IC50 of 20 nM or less.

Antibody molecules, as part of the group of protein pharmaceuticals, are very susceptible to physical and chemical degradation. Chemical degradation includes any process that involves modification of the protein via bond formation or cleavage, yielding a new chemical entity. A variety of chemical reactions is known to affect proteins. These reactions can involve hydrolysis including cleavage of peptide bonds as well as deamidation, isomerization, oxidation and decomposition. Physical degradation refers to changes in the higher order structure and includes denaturation, adsorption to surfaces, aggregation and precipitation. Protein stability is influenced by the characteristics of the protein itself, e.g. the amino acid sequence, the glycosylation pattern, and by external influences, such as temperature, solvent pH, excipients, interfaces, or shear rates. So, it is important to define the optimal formulation conditions to protect the protein against degradation reactions during manufacturing, storage and administration. (Manning, M. C, et al. (1989), "Stability of protein pharmaceuticals", Pharm Res 6(11), 903-918; Zheng, J. Y., Janis, L. J. (2005), "Influence of pH, buffer species, and storage temperature on physicochemical stability of a humanized monoclonal antibody LA298", Int. J. Pharmaceutics 308, 46-51). Stable liquid formulations of therapeutic antibodies are particularly difficult to obtain when the formulation should include antibodies in a high concentration. It is therefore an object of the present invention to provide a stable, in particular highly concentrated, formulation of an anti-Ang-2 antibody with as few as necessary excipients, which enables the desired dosing and allows convenient administration of the antibody to a patient.

The formulation of the present invention shows good stability upon storage for 24 months at the intended storage temperature of 2 to 8 °C without formation of visible particles that will allow i.v. administration. Shaking and multiple freezing-thawing steps were applied to the liquid formulation to simulate physical stress conditions that potentially occur during manufacturing or transportation of the drug product. The formulation of the present invention shows good stability after applying shaking and freeze-thaw stress. Summary

The present invention relates to a pharmaceutical formulation of an antibody against ANG- 2, a process for the preparation and uses of the formulation. In particular, the pharmaceutical formulations of the present invention are hypertonic or isotonic.

In one aspect, the invention refers to a pharmaceutical formulation, in particular stable liquid pharmaceutical formulation, comprising:

- 15 to 200 mg/ml of an antibody against ANG-2

- 2 to 200 mM of a buffer

- 0.01-0.07% of surfactant

- a) 1 to 20mM of at least one stabilizer and 200-lOOOmM or more of a tonicity agent, or b) 200-lOOOmM or more of a tonicity agent,

at a pH in the range of 4.5 to 7.0

In one embodiment, the antibody against ANG-2 is a human or humanized antibody. In another embodiment, the antibody a monoclonal antibody.

In a further embodiment, the antibody against ANG-2 is a bispecific antibody, in particular a bispecific antibody against ANG-2 and VEGF. In particular, the antibody against ANG-2 and VEGF is the bispecific bivalent ANG-2/VEGF antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, characterized in that said antibody comprises: i) the heavy chain and the light chain of a first full length antibody that specifically binds VEGF with said first antigen-binding site comprising as heavy chain variable domain (VH) the SEQ ID NO: 13, and as light chain variable domain (VL) the SEQ ID NO: 14; and ii) the modified heavy chain and modified light chain of a second full length antibody that specifically binds ANG-2, wherein the constant domains CL and CHI are replaced by each other, with said second antigen-binding site comprising as heavy chain variable domain (VH) the SEQ ID NO: 15, and a light chain variable domain (VL) the SEQ ID NO: 16. Even more particular, the antibody against Ang-2 and VEGF is a bispecific, bivalent antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, characterized in comprising the amino acid sequences of SEQ ID NO: 17, of SEQ ID NO: 18, of SEQ ID NO: 19, and of SEQ ID NO: 20.

In another embodiment, the concentration of the antibody against ANG-2 is in the range of about 15 to 60mg/ml. In particular, the concentration of the antibody against Ang-2 is in the range of about 20 to 55mg/ml, or in the range of about 25 to 50mg/ml, more particularly the concentration is about 25mg/ml or about 50 mg/ml. In one embodiment, the buffer is selected from a histidine buffer (histidine/histidine hydrochloride monohydrate buffer), sodium acetate buffer, and arginine sucrose buffer, in particular the buffer is a histidine buffer.

In a further embodiment, the concentration of the buffer is about 10 mM to about 50mM or about 10 mM to about 30mM, in particular about 20mM. In another embodiment, the pH of the formulation is in the range 5.0 to 6.5. Particularly, the pH is in the range of 5.0 to 6.0, more particularly about pH6.

In one embodiment, the surfactant is Polysorbate 20, Polysorbate 80, or Poloxamer 188, in particular Polysorbate 20. In another embodiment, the concentration of the surfactant is in the range of about 0.02% to about 0.06% (w/v) or about 0.03% to about 0.05 % (w/v), in particular 0.04% (w/v).

In one embodiment, the at least one stabilizer is methionine. In a further embodiment, the concentration of the stabilizer is in the range of 5 to 15mM. Particularly, the concentration of the stabilizer is in the range of 9 to 11 mM, in particular about 10 mM.

In one embodiment, the tonicity agent is selected from sucrose, trehalose, sorbitol and ar- ginine hydrochloride, in particular the tonicity agent is sucrose. In a further embodiment, the concentration of the tonicity agent is more than about 200mM, in particular about 200mM to about lOOOmM. In a preferred embodiment, the concentration of the tonicity agent is in the range of about 200mM to about 800mM or 200mM to 600mM. Particularly, the concentration of the tonicity agent is in the range of about 200mM to about 550mM, about 200mM to about 300mM or about 400mM to about 550mM, more particularly, the concentration of the tonicity agent is about 240mM or about 500mM. In one embodiment, the pharmaceutical formulation comprises

- 15 to 200 mg/ml of a bispecific antibody against ANG-2 and VEGF;

- 2 to 200 mM of a histidine buffer;

- 0.01-0.07% of Polysorbate 20;

- a) 1 to 20mM of methionine, and 200mM to 600mM sucrose; or

- b) 200mM to 600mM sucrose

at pH 6.

In particular, the pharmaceutical formulation comprises

- about 25 mg/ml of a bispecific antibody against ANG-2 and VEGF;

- about 20mM of a histidine buffer;

- about 0.04% of Polysorbate 20;

- a) about lOmM of methionine, and about 240mM of sucrose; or

- b) about 240mM of a sucrose

at pH 6; or

- about 50 mg/ml of a bispecific antibody against ANG-2 and VEGF;

- about 20mM of a histidine buffer;

- about 0.04% of Polysorbate 20;

- a) about lOmM of methionine, and about 240mM of a sucrose; or

- b) about 240mM of a sucrose

at pH 6; or

- about 25 mg/ml of a bispecific antibody against ANG-2 and VEGF;

- about 20mM of a histidine buffer;

- about 0.04% of Polysorbate 20;

- a) about lOmM of methionine, and about 500mM of a sucrose; or

- b) about 500mM of a sucrose

at pH 6; or

- about 50 mg/ml of a bispecific antibody against ANG-2 and VEGF;

- about 20mM of a histidine buffer;

- about 0.04% of Polysorbate 20;

- a) about lOmM of methionine, and about 500mM of a sucrose; or - b) about 500mM of a sucrose

at pH 6.

In another aspect, the pharmaceutical formulation of the invention is used in the treatment of a vascular disease, in particular in the treatment of cancer. Description of the Figures

Figure 1 Turbidity of active formulations at 5°C. The data is shown in groups of four columns, the first column A: t=0, the second column B: t=2weeks, the third column C: t=4weeks, and the fourth column D: t=8weeks. The tested formulations are 1: GSM0002.01, 2: GSM0002.02, 3: GSM0002.03, 4: GSM0002.04, 5: GMS0002.05, 6: GSM0002.06, 7: GSM0002.07, 8: GSM0002.08, 9: GSM0002.09, 10: GSM0002.10. The composition of the formulations is given in Table 3, Example 2

Figure 2 Turbidity of active formulations at 25°C. The data is shown in groups of four columns, the first column A: t=0, the second column B: t=2weeks, the third column C: t=4weeks, and the fourth column D: t=8weeks. The tested formulations are 1: GSM0002.01, 2: GSM0002.02, 3: GSM0002.03, 4: GSM0002.04, 5: GMS0002.05, 6: GSM0002.06, 7: GSM0002.07, 8: GSM0002.08, 9: GSM0002.09, 10: GSM0002.10. The composition of the formulations is given in Table 3, Example 2.

Figures 3A, 3B Size exclusion chromatography (SEC) for all three buffer systems (His/His-HCl, NaAce and ArgSuc) stored at 5°C for 0- to 8 weeks. Figure 3A: SEC main peak; Figure 3B: SEC HMW's. The data is shown in groups of four columns, the first column A: t=0, the second column B: t=2weeks, the third column C: t=4weeks, and the fourth column D: t=8weeks. The tested formulations are 1: GSM0002.01, 2: GSM0002.02, 3: GSM0002.03, 4: GSM0002.04, 5: GMS0002.05, 6: GSM0002.06, 7: GSM0002.07, 8: GSM0002.08, 9: GSM0002.09, 10: GSM0002.10. The composition of the formulations is given in Table 3, Ex- ample 2.

Figures 4A, 4B Size exclusion chromatography (SEC) for all three buffer systems (His/His-HCl, NaAce and ArgSuc) stored at 25°C for 0 to 8 weeks. Figure 4A: SEC main peak; Figure 4B: SEC HMW's. The data is shown in groups of four columns, the first column A: t=0, the second column B: t=2weeks, the third column C: t=4weeks, and the fourth column D: t=8weeks. The tested formulations are 1: GSM0002.01, 2: GSM0002.02, 3: GSM0002.03, 4: GSM0002.04, 5: GMS0002.05, 6: GSM0002.06, 7: GSM0002.07, 8: GSM0002.08, 9: GSM0002.09, 10: GSM0002.10. The composition of the formulations is given in Table 3, Example 2. Figures 5A, 5B Size exclusion chromatography (SEC) for all three buffer systems (His/His-HCl, NaAce and ArgSuc) stored at 40°C for 0 to 8 weeks. Figure 5A: SEC main peak; Figure 5B: SEC HMW's. The data is shown in groups of four columns, the first column A: t=0, the second column B: t=2weeks, the third column C: t=4weeks, and the fourth column D: t=8weeks. The tested formulations are 1: GSM0002.01, 2: GSM0002.02, 3: GSM0002.03, 4: GSM0002.04, 5: GMS0002.05, 6: GSM0002.06, 7: GSM0002.07, 8: GSM0002.08, 9: GSM0002.09, 10: GSM0002.10. The composition of the formulations is given in Table 3, Example 2.

Figure 6 SEC overlay after 4 weeks storage at 40°C. 1: 20mM HisHisHCl pH 6.0 (GSM0002.02); 2: 20mM NaAce pH 5.0 (GSM0002.05); 3: 200mM ArgSuc pH 5.5 (GSM0002.08); 4: 200mM ArgSuc pH 6.0 (GSM0002.09). Measured at 280nm. The composition of the formulations is given in Table 3, Example 2.

Figures 7A, 7B IEC after 0 to 8 weeks storage at 5°C. Figure 7A: Acidic Variants; Figure 7B: Basic Variants. The data is shown in groups of four columns, the first column A: t=0, the second column B: t=2weeks, the third column C: t=4weeks, and the fourth column D: t=8weeks. The tested formulations are 1: GSM0002.01, 2: GSM0002.02, 3: GSM0002.03, 4: GSM0002.04, 5: GMS0002.05, 6: GSM0002.06, 7: GSM0002.07, 8: GSM0002.08, 9: GSM0002.09, 10: GSM0002.10. The composition of the formulations is given in Table 3, Example 2. Figures 8 A, 8B Acidic and basic variants by IEC after 0 to 8 weeks storage at 25 °C.

Figure 8A: Acidic Variants; Figure 8B: Basic Variants. The data is shown in groups of four columns, the first column A: t=0, the second column B: t=2weeks, the third column C: t=4 weeks, and the fourth column D: t=8weeks. The tested formulations are 1: GSM0002.01, 2: GSM0002.02, 3: GSM0002.03, 4: GSM0002.04, 5: GMS0002.05, 6: GSM0002.06, 7: GSM0002.07, 8: GSM0002.08, 9: GSM0002.09, 10: GSM0002.10. The composition of the formulations is given in Table 3, Example 2.

Figures 9A, 9B Acidic and Basic Variants by IEC after 0 to 8 weeks Storage at 40°C. Figure 9A: Acidic Variants; Figure 9B: Basic Variants. The data is shown in groups of four columns, the first column A: t=0, the second column B: t=2weeks, the third column C: t=4 weeks, and the fourth column D: t=8weeks. The tested formulations are 1: GSM0002.01, 2: GSM0002.02, 3: GSM0002.03, 4: GSM0002.04, 5: GMS0002.05, 6: GSM0002.06, 7: GSM0002.07, 8: GSM0002.08, 9: GSM0002.09, 10: GSM0002.10. The composition of the formulations is given in Table 3, Example 2. Figure 10 Turbidity data for 20mM His/HisHCl formulations (pH6). The data is shown in groups of four columns, the first column A: t=0; the second column B: 5°C, 1 week shaking; the third column C: 25°C, 1 week shaking, and the fourth column D: F/T (Freeze/Thaw cycles). The tested formulations are 1: GSM0005.01, 2: GSM0005.02, 3: GSM0005.03, 4: GSM0005.04, 5: GSM0005.05, 6: GSM0005.06, 7: GSM0005.07, 8: GSM0005.08, 9: GSM0005.09, 10: GSM0005.10, 11: GSM0005.i l, 12: GSM0005.12, 13: GSM0005.13. The composition of the formulations is given in Table 14, Example 3.

Figure 11 Turbidity data for 200mM ArgSuc formulations (pH 5.5). The data is shown in groups of four columns, the first column A: t=0; the second column B: 5°C, 1 week shaking; the third column C: 25°C, 1 week shaking, and the fourth column D: F/T (Freeze/Thaw cycles). The tested formulations are 1: GSM0005.14, 2: GSM0005.15, 3: GSM0005.16, 4: GSM0005.17, 5: GSM0005.18, 6: GSM0005.19, 7: GSM0005.20, 8: GSM0005.21, 9: GSM0005.22; 10: GSM0005.23, 11: GSM0005.24, 12: GSM0005.25, 13: GSM0005.26. The composition of the formulations is given in Table 15, Example 3. Figure 12 Main Peak by SEC for 20mM His/HisHCl formulations (pH6). The data is shown in groups of four columns, the first column A: t=0; the second column B: 5°C, 1 week shaking; the third column C: 25°C, 1 week shaking, and the fourth column D: F/T (Freeze/Thaw cycles). The tested formulations are 1: GSM0005.01, 2: GSM0005.02, 3: GSM0005.03, 4: GSM0005.04, 5: GSM0005.05, 6: GSM0005.06, 7: GSM0005.07, 8: GSM0005.08, 9: GSM0005.09, 10: GSM0005.10, 11: GSM0005.i l, 12: GSM0005.12, 13: GSM0005.13. The composition of the formulations is given in Table 14, Example 3.

Figure 13 Main peak by SEC for 200mM ArgSuc formulations (pH 5.5). The data is shown in groups of four columns, the first column A: t=0; the second column B: 5°C, 1 week shaking; the third column C: 25°C, 1 week shaking, and the fourth column D: F/T (Freeze/Thaw cycles). The tested formulations are 1: GSM0005.14, 2: GSM0005.15, 3: GSM0005.16, 4: GSM0005.17, 5: GSM0005.18, 6: GSM0005.19, 7: GSM0005.20, 8: GSM0005.21, 9: GSM0005.22; 10: GSM0005.23, 11: GSM0005.24, 12: GSM0005.25, 13: GSM0005.26. The composition of the formulations is given in Table 15, Example 3.

Figure 14 High molecular weight (HMW)'s by SEC for 20mM His/HisHCl formula- tions (pH6). The data is shown in groups of four columns, the first column A: t=0; the second column B: 5°C, 1 week shaking; the third column C: 25°C, 1 week shaking, and the fourth column D: F/T (Freeze/Thaw cycles). The tested formulations are 1: GSM0005.01, 2: GSM0005.02, 3: GSM0005.03, 4: GSM0005.04, 5: GSM0005.05, 6: GSM0005.06, 7: GSM0005.07, 8: GSM0005.08, 9: GSM0005.09, 10: GSM0005.10, 11: GSM0005. i l, 12: GSM0005.12, 13: GSM0005.13. The composition of the formulations is given in Table 14, Example 3. Figure 15 HMW's by SEC for 200mM ArgSuc formulations (pH 5.5). The data is shown in groups of four columns, the first column A: t=0; the second column B: 5°C, 1 week shaking; the third column C: 25°C, 1 week shaking, and the fourth column D: F/T (Freeze/Thaw cycles). The tested formulations are 1: GSM0005.14, 2: GSM0005.15, 3: GSM0005.16, 4: GSM0005.17, 5: GSM0005.18, 6: GSM0005.19, 7: GSM0005.20, 8: GSM0005.21, 9: GSM0005.22; 10: GSM0005.23, 11: GSM0005.24, 12: GSM0005.25, 13: GSM0005.26. The composition of the formulations is given in Table 15, Example 3.

Figure 16 Turbidity of all formulations containing PS 20 (0.4%) stored at 5°C, 25°C or 40°C. The data is shown in groups of 12 columns, the 1^st column is A: t=0; the 2^nd column is B: t= 4 weeks, 5°C, C: t=12 weeks, 5°C; the 4^th column is D: t= 24 weeks, 5°C; the 5^th column is E: t= 4 weeks, 25°C; the 6^th column is F: t=12 weeks, 25°C; the 7^th column is G: t=24weeks, 25°C; the 8^th column is H: t=2 weeks, 40°C; the 9^th column is I: t=4weeks, 40°C; 10^th column is J: t=12 weeks, 40°C; the 12^th column is K: sh (shaking), 5°C, the 12^th column is L: sh, 25°C. The tested formulations are 1: GSM0007.01, 2: GSM0007.02; 3: GSM0007.03; 4: GSM0007.04; 5: GSM0007.05; 6: GSM0007. i l; 7: GSM0007.12; 8: GSM0007.16. The composition of the formulations is given in Table 19, Example 4.

Figure 17 Turbidity of all Formulations containing PS 80 (0.04%) stored at 5°C,_„25°C, or 40°C. The data is shown in groups of 12 columns, the 1^st column is A: t=0; the 2^nd column is B: t= 4 weeks, 5°C, C: t=12 weeks, 5°C; the 4^th column is D: t= 24 weeks, 5°C; the 5^th column is E: t= 4 weeks, 25°C; the 6^th column is F: t=12 weeks, 25°C; the 7^th column is G: t=24weeks, 25°C; the 8^th column is H: t=2 weeks, 40°C; the 9^th column is I: t=4weeks, 40°C; 10^th column is J: t=12 weeks, 40°C; the 12^th column is K: sh (shaking), 5°C, the 12^th column is L: sh, 25°C. The tested formulations are 1: GSM0007.06, 2: GSM0007.07; 3: GSM0007.08; 4: GSM0007.09; 5: GSM0007.10 6: GSM0007.13; 7: GSM0007.14. The composition of the formulations is given in Table 19, Example 4.

Figures 18A, 18B AHMW's [area %] by SEC. Figure 18A: OmM Met, or lOmM Met (hyperosmolar, or isoosmolar), at 5°C, 25°C or 40°C. The data is shown in groups of three lines. The continuous line with filled diamonds is A: GSM0007.03, ; the dashed line with filled squares is B: GSM0007.04; and the dotted line with filled circles is C: GSM0007.05. Group 1: 5°C, 2: 25°C, 3: 40°C Fig. 18B: GSM0007.16 at 5°C (A), 25°C (B) and 40°C (C). The composition of the formulations is given in Table 19, Example 4.

Figure 19 HMW's [area %] by SEC after shaking. The data is shown in groups of three columns, the 1^st column is A: t=0, the 2^nd column is B: shaking, at 5°C, the 3^rd column is C: shaking at 25°C. The tested formulations are 1: GSM0007.01; 2: GSM0007.02; 3: GSM0007.03, 4: GSM0007.04; 5: GSM0007.05; 6: GSM0007.06, 7: GSM0007.07; 8: GSM0007.08; 9: GSM0007.09; 10: GSM0007.10; 11: GSM0007.i l; 12: GMS0007.12; 13: GMS0007.13; 14: GMS0007.14; 15: GMS0007.15, 16: GMS0007.16. The composition of the formulations is given in Table 19, Example 4.

Figure 20 Basic Variants [ ] by IEC. The date is shown in groups of 12 columns. The 1^st column is A: t=0, the 2^nd column is B: t=4weeks, 5°C; the 3^rd column is C: t=12weeks, 5°C; the 4^th column is D: t=24weeks, 5°C; the 5^th column is E: t=4weeks, 25°C; the 6^th column is F: t=12weeks, 25°C; the 7^th column is G: t=24weeks, 25°C; the 8^th column is H: t=2weeks, 40°C; the 9^th column is I: t=4weeks, 40°C; the 10^th column is J: t=12weeks, 40°C; the 11^th column is K: =24weeks, -20°C; the 12^th column is L: t=24weeks, -40°C. The tested formulations are 1: GSM0007.03; 2: GSM0007.04; 3: GSM0007.05; 4: GSM0007.i l; 5: GSM0007.12; 6: GSM0007.13; 7: GSM0007.14; and 8: GSM0007.16. The composition of the formulations is given in Table 19, Example 4.

Figure 21 HMW's [area ] by SEC for Scratch Test I. The data is shown in groups of 15 columns. The 1^st column is A: t=0, the 2^nd column is B: untreated, -20°C, t=0, the 3^rd column is C: untreated, -20°C, t= 4weeks, the 4^th column is D: untreated, -20°C, t=12weeks, the 5^th column is E: untreated, -20°C, t= 26weeks, the 6^th column is F: scratched and sprinkled (SS), -20°C, t=0; the 7^th column is G: SS, -20°C, t=4weeks; the 8^th column is H: SS, -20°C, t=12weeks; the 9^th column is I: SS, -20°C, t=26 weeks; the 10^th column is J: SS -40°C, t=4weeks; the 11^th column is K: SS, -40°C, t=12weeks; the 12^th column is L: SS, -40°C, t=26weeks, the 13^th column is M: SS, -80°C, t=4weeks; the 14^th column is N: SS, -80°C, t=12weeks; and the 15^th column is O: SS, - 80°C, t=26weeks. The tested formulations are 1: GMS0011.01; 2: GMS0011.02; 3: GMS0011.03; and 4: GMS0011.04. The composition of the formulations is given in Table 26, Example 5.

Figure 22 LMW's [area ] by SEC for Scratch Test I. The data is shown in groups of 15 columns. The 1^st column is A: t=0, the 2^nd column is B: untreated, -20°C, t=0, the 3^rd column is C: untreated, -20°C, t= 4weeks, the 4^th column is D: untreated, -20°C, t=12weeks, the 5^th column is E: untreated, -20°C, t= 26weeks, the 6^th column is F: scratched and sprinkled (SS), -20°C, t=0; the 7^th column is G: SS, -20°C, t=4weeks; the 8^th column is H: SS, -20°C, t=12weeks; the 9^th column is I: SS, -20°C, t=26 weeks; the 10^th column is J: SS -40°C, t=4weeks; the 11^th column is K: SS, -40°C, t=12weeks; the 12^th column is L: SS, -40°C, t=26weeks, the 13^th column is M: SS, -80°C, t=4weeks; the 14^th column is N: SS, -80°C, t=12weeks; and the 15^th column is O: SS, - 80°C, t=26weeks. The tested formulations are 1: GMS0011.01; 2: GMS0011.02; 3: GMS0011.03; and 4: GMS0011.04. The composition of the formulations is given in Table 26, Example 5. Figure 23 HMW's [area %] by SEC for Scratch Test II. The data is shown in groups of 15 columns. The 1^st column is A: t=0, the 2^nd column is B: untreated, -20°C, t=0, the 3^rd column is C: untreated, -20°C, t= 4weeks, the 4^th column is D: untreated, -20°C, t=12weeks, the 5^th column is E: untreated, -20°C, t= 27weeks, the 6^th column is F: scratched and sprinkled (SS), -20°C, t=0; the 7^th column is G: SS, -20°C, t=4weeks; the 8^th column is H: SS, -20°C, t=12weeks; the 9^th column is I: SS, -20°C, t=27 weeks; the 10^th column is J: SS, -40°C, t=4weeks; the 11^th column is K: SS, -40°C, t=12weeks; the 12^th column is L: SS, -40°C, t=27weeks, the 13^th column is M: SS, -80°C, t=4weeks; the 14^th column is N: SS, -80°C, t=12weeks; and the 15^th column is O: SS, -80°C, t=27weeks. The tested formulations are 1: GSM00012.01; 2: GSM00012.02; 3: GSM00012.03; 4: GSM00012.04; 5: GSM00012.05; 6: GSM00012.06; 7: GSM00012.07; 8: GSM00012.08; 9: GSM00012.09; 10: GSM00012.10; and 11: GSM00012.i l. The composition of the formulations is given in Table 30, Example 5.

Figures 24A, 24B SEC overlays between sample GSM0012.10 at different time points. Figure 24A: untreated, Figure 24B: scratched and sprinkled. The data is shown as four lines, line 1: control (t=0); line 2: -20°C, t=4weeks; line 3: t=12weeks; -20°C; line 4: t=27weeks, -20°C. The composition of the formulation is given in Table 30, Example 5.

Figure 25 LMW's [area ] by SEC for Scratch Test II. The data is shown in groups of 15 columns. The 1^st column is A: t=0, the 2^nd column is B: untreated, -20°C, t=0, the 3^rd column is C: untreated, -20°C, t= 4weeks, the 4^th column is D: untreated, -20°C, t=12weeks, the 5^th col- umn is E: scratched and sprinkled (SS), -20°C, t=0; the 6^th column is F: SS, -20°C, t=4weeks; the 7^th column is G: SS, -20°C, t=12weeks; the 8^th column is H: SS, -40°C, t= 4weeks; the 9^th column is I: SS, -40°C, t=12 weeks; the 10^th column is J: SS, -80°C, t=4weeks; the 11^th column is K: SS, -80°C, t=12weeks; the 12^th column is L: untreated, -20°C, t=27weeks, the 13^th column is M: SS, -20°C, t=27 weeks; the 14^th column is N: SS, -40°C, t=27weeks; and the 15^th column is O: SS, -80°C, t=27weeks. The tested formulations are 1: GSM00012.01; 2: GSM00012.02; 3: GSM00012.03; 4: GSM00012.04; 5: GSM00012.05; 6: GSM00012.06; 7: GSM00012.07; 8: GSM00012.08; 9: GSM00012.09; 10: GSM00012.10; and 11: GSM00012.i l. The composition of the formulations is given in Table 30, Example 5.

Figures 26 A, 26B Comparison of HMW's by SEC between samples at -20°C. Figure 26A: untreated, Figure 26B: scratched and sprinkled. The tested formulations are GSM00012.01, GSM00012.02, GSM00012.03, GSM00012.04, GSM00012.05, GSM00012.06, GSM00012.07, GSM00012.08, GSM00012.09, GSM00012.10 and GSM00012.i l. The composition of the formulations is given in Table 30, Example 5.

Figures 27 A, 27B Comparison of LMW's by SEC between samples at -20°C. Figure 27A: untreated; Figure 27B: scratched and sprinkled. The tested formulations are GSM00012.01, GSM00012.02, GSM00012.03, GSM00012.04, GSM00012.05, GSM00012.06, GSM00012.07, GSM00012.08, GSM00012.09, GSM00012.10 and GSM00012.i l. The composition of the formulations is given in Table 30, Example 5.

Detailed Description of the Invention The present invention relates to a stable liquid pharmaceutical formulation comprising an antibody against ANG-2.

Definitions

The term "pharmaceutical formulation" refers to preparations which are in such form as to permit the biological activity of the active ingredients to be unequivocally effective, and which contain no additional components which are toxic to the subjects to which the formulation is administered.

The term "liquid" as used herein in connection with the formulation according to the invention denotes a formulation which is liquid at a temperature of at least about 2 °C to about 8 °C under atmospheric pressure. A "stable" formulation is one in which the protein therein, e.g. the antibody, essentially retains its physical and chemical stability and thus its biological activity upon storage.

A "stable liquid pharmaceutical antibody formulation" is a liquid antibody formulation with no significant changes observed at a refrigerated temperature (2-8 °C) for at least 12 months, particularly 2 years, and more particularly 3 years. The criteria for stability are the following: no more than 10%, particularly 5%, of antibody monomer is degraded as measured by size exclusion chromatography (SEC-HPLC). Furthermore, the solution is colorless or clear to slightly opalescent by visual analysis. The protein concentration of the formulation has no more than +/- 10% change. No more than 10%, particularly 5% of aggregation is formed. The stability is measured by methods known in the art such UV spectroscopy, size exclusion chromatography (SEC-HPLC), Ion-Exchange Chromatography (IE-HPLC), turbidimetry and visual inspection.

"Bispecific antibodies" according to the invention are antibodies which have two different antigen-binding specificities. Bispecific antibodies of the present invention are in particular specific for two different antigens, VEGF as first antigen and ANG-2 as second antigen.

The term "Ang-2" or "Angiopoietin 2" as used herein refers to human angiopoietin-2 (ANG-2) (alternatively abbreviated with ANGPT2 or ANG2) (SEQ ID NO: 21) which is described e.g. in Maisonpierre, P. C, et al, Science 277 (1997) 55-60 and Cheung, A. H., et al, Genomics 48 (1998) 389-91. The angiopoietins-1 (ANG-1) and -2 were discovered as ligands for the Ties, a family of tyrosine kinases that is selectively expressed within the vascular endothelium. Yancopoulos, G. D., et al, Nature 407 (2000) 242-48. There are now four definitive members of the angiopoietin family. Angiopoietin-3 and -4 (Ang-3 and Ang-4) may represent widely diverged counterparts of the same gene locus in mouse and man. Kim, I., et al., FEBS Let, 443 (1999) 353-56; Kim, I., et al, J Biol Chem 274 (1999) 26523-28. ANG-1 and ANG-2 were originally identified in tissue culture experiments as agonist and antagonist, respectively (see for ANG-1 : Davis, S., et al, Cell 87 (1996) 1161-69; and for ANG-2: Maisonpierre, P. C, et al, Science 277 (1997) 55-60) All of the known angiopoietins bind primarily to Tie2, and both Ang-1 and -2 bind to Tie2 with an affinity of 3 nM (Kd). Maisonpierre, P. C, et al, Science 277 (1997) 55-60.

The term "VEGF" as used herein refers to human vascular endothelial growth factor (VEGF/VEGF-A) (SEQ ID NO: 22) which is described e.g. in Leung, D. W., et al, Science 246 (1989) 1306-9; Keck, P. J., et al., Science 246 (1989) 1309-12 and Connolly, D. T., et al, J. Biol. Chem. 264 (1989) 20017-24. VEGF is involved in the regulation of normal and abnormal angio- genesis and neovascularization associated with tumors and intraocular disorders (Ferrara, N., et al, Endocr. Rev. 18 (1997) 4-25; Berkman, R. A.,et al, J. Clin. Invest. 91 (1993) 153-159; Brown, L. F., et al, Human Pathol. 26 (1995) 86-91; Brown, L. F., et al, Cancer Res. 53 (1993) 4727- 4735; Mattern, J., et al, Brit. J. Cancer. 73 (1996) 931-934; and Dvorak, H. F., et al, Am. J. Pathol. 146 (1995) 1029-1039). VEGF is a homodimeric glycoprotein that has been isolated from several sources. VEGF shows highly specific mitogenic activity for endothelial cells.

The terms "antibody against human angiopoietin-2 (ANG-2)" and "anti-Ang-2 antibody" refer an antibody comprising a antigen-binding site that specifically binds to human ANG-2.

The terms "antibody against human vascular endothelial growth factor (VEGF/VEGF-A) and against human angiopoietin-2 (ANG-2)" and "anti-Ang-2/VEGF antibody" refer a bispecific, bivalent antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen binding site that specifically binds to human ANG-2.

As used herein, the term "binding" or "specifically binding" refers to the binding of the antibody to an epitope of the antigen (either human VEGF or human ANG-2) in an in vitro assay, preferably in an plasmon resonance assay (BIAcore, GE- Healthcare Uppsala, Sweden) (see e.g. Example 3 of WO2011/117329) with purified wild-type antigen. The affinity of the binding is defined by the terms ka (rate constant for the association of the antibody from the antibody/antigen complex), k[D] (dissociation constant), and K[D] (ko ka). In one embodiment binding or specifically binding means a binding affinity (K[D]) of 10("8) mo VI or less, preferably 10("9) M to 10("13) mol/l. The term "antibody" encompasses the various forms of antibody structures including but not being limited to whole antibodies and antibody fragments. The antibody comprised in the formulation of the present invention is in particular a human antibody, a humanized antibody, chimeric antibody, antibody fragment, or further genetically engineered antibody as long as the characteristic properties according to the invention are retained. More particularly, the antibody is a human or humanized monoclonal antibody, especially a recombinant human antibody.

"Antibody fragments" comprise a portion of a full length antibody, preferably the variable domain thereof, or at least the antigen binding site thereof. Examples of antibody fragments include diabodies, single-chain antibody molecules, and multispecific antibodies formed from an- tibody fragments. scFv antibodies are, e.g. described in Houston, J.S., Methods in Enzymol. 203 (1991) 46-96).

The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of a single amino acid composition.

The term "chimeric antibody" refers to an antibody comprising a variable region, i.e., bind- ing region, from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a murine variable region and a human constant region are of particular interest. Other forms of "chimeric antibodies" encompassed by the present invention are those in which the constant region has been modified or changed from that of the original antibody to generate the desired properties according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding. Such chimeric antibodies are also referred to as "class- switched antibodies". Chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies involve conventional re- combinant DNA and gene transfection techniques are well known in the art. See e.g. Morrison, S.L., et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; US Patent Nos. 5,202,238 and 5,204,244.

The term "humanized antibody" refers to antibodies in which the framework or "complementarity determining regions" (CDR) have been modified to comprise the CDR of an immuno- globulin of different specificity as compared to that of the parent immunoglobulin. In a preferred embodiment, a murine CDR is grafted into the framework region of a human antibody to prepare the "humanized antibody." See e.g. Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M.S., et al., Nature 314 (1985) 268-270. Particularly preferred CDRs correspond to those representing sequences recognizing the antigens noted above for chimeric antibodies. Oth- er forms of "humanized antibodies" encompassed by the present invention are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding.

The term "human antibody", as used herein, is intended to include antibodies having varia- ble and constant regions derived from human germ line immunoglobulin sequences. Human antibodies are well-known in the state of the art (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ- line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human antibodies can also be produced in phage display libraries (Hoogenboom, H.R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J.D., et al., J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et al., J. Immunol. 147 (1991) 86-95). As already mentioned for chimeric and humanized antibodies according to the invention the term "human antibody" as used herein also comprises such antibodies which are modified in the constant region to generate the properties according to the invention, especially in regard to Clq binding and/or FcR binding, e.g. by "class switching" i.e. change or mutation of Fc parts (e.g. from IgGl to IgG4 and/or IgGl/IgG4 mutation.).

The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as anti- bodies isolated from a host cell such as a NSO or CHO cell or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and constant regions in a rearranged form. The recombinant human antibodies according to the invention have been subjected to in vivo somatic hypermutation. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo.

The "variable region" (variable region of a light chain (V_L), variable region of a heavy chain (V_H)) or "variable domain" as used herein denotes each of the pair of light and heavy chain domains which are involved directly in binding the antibody to the antigen. The variable light and heavy chain domains have the same general structure and each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three "hypervari- able regions" (or complementary determining regions, CDRs). The framework regions adopt a β- sheet conformation and the CDRs may form loops connecting the β-sheet structure. The CDRs in each chain are held in their three-dimensional structure by the framework regions and form to- gether with the CDRs from the other chain the antigen binding site. The antibody's heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the invention. The term "antigen-binding portion of an antibody" when used herein refer to the amino acid residues of an antibody which are responsible for antigen-binding. The antigen-binding portion of an antibody comprises amino acid residues from the "complementary determining regions" or "CDRs". "Framework" or "FR" regions are those variable domain regions other than the hypervariable region residues as herein defined. Therefore, the light and heavy chain variable domains of an antibody comprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Especially, CDR3 of the heavy chain is the region which contributes most to antigen binding and defines the antibody's properties. CDR and FR regions are determined according to the standard definition of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) and/or those residues from a "hypervariable loop".

The term "epitope" includes any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, epitope determinant include chemically active surface group- ings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody.

The term "full length antibody" denotes an antibody consisting of two "full length antibody heavy chains" and two "full length antibody light chains". A "full length antibody heavy chain" is a polypeptide consisting in N-terminal to C- terminal direction of an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CHI), an antibody hinge region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3), abbreviated as VH-CH1-HR-CH2-CH3; and optionally an antibody heavy chain constant domain 4 (CH4) in case of an antibody of the subclass IgE. Preferably the "full length antibody heavy chain" is a polypeptide consisting in N-terminal to C-terminal direction of VH, CHI, HR, CH2 and CH3. A "full length antibody light chain" is a polypeptide consisting in N-terminal to C- terminal direction of an antibody light chain variable domain (VL), and an antibody light chain constant domain (CL), abbreviated as VL-CL. The antibody light chain constant domain (CL) can be k (kappa) or .lambda, (lambda). The two full length antibody chains are linked together via inter-polypeptide disulfide bonds between the CL domain and the CHI domain and between the hinge regions of the full length antibody heavy chains. Examples of typi- cal full length antibodies are natural antibodies like IgG (e.g. IgG 1 and IgG2), IgM, IgA, IgD, and IgE. The full length antibodies according to the invention can be from a single species e.g. human, or they can be chimerized or humanized antibodies. The full length antibodies according to the invention comprise two antigen binding sites each formed by a pair of VH and VL, which both specifically bind to the same antigen. The C- terminus of the heavy or light chain of said full length antibody denotes the last amino acid at the C-terminus of said heavy or light chain. The N-terminus of the heavy or light chain of said full length antibody denotes the last amino acid at the N- terminus of said heavy or light chain.

The term "peptide linker" as used within the invention denotes a peptide with amino acid sequences, which is preferably of synthetic origin. These peptides according to invention are used to connect the C-terminus of the light chain to the N-terminus of heavy chain of the second full length antibody (that specifically binds to a second antigen) via a peptide linker. The peptide linker within the second full length antibody heavy and light chain is a peptide with an amino acid sequence with a length of at least 30 amino acids, preferably with a length of 32 to 50 amino acids. In one the peptide linker is a peptide with an amino acid sequence with a length of 32 to 40 amino acids. In one embodiment said linker is (GxS)n with G = glycine, S = serine, (x =3, n= 8, 9 or 10 and m= 0, 1, 2 or 3) or (x = 4 and n= 6, 7 or 8 and m= 0, 1, 2 or 3), preferably with x = 4, n= 6 or 7 and m= 0, 1, 2 or 3, more preferably with x = 4, n= 7 and m= 2. In one embodiment said linker is (G₄S)6G2. The term "constant region" or "constant domains" as used within the current applications denotes the sum of the domains of an antibody other than the variable region. The constant region is not involved directly in binding of an antigen, but exhibits various effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies are divided in the classes: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses, such as IgGl, IgG2, IgG3, and IgG4, IgAl and IgA2. The heavy chain constant regions that correspond to the different classes of antibodies are called .alpha., .delta., .epsilon., .gamma., and .micro., respectively. The light chain constant regions which can be found in all five antibody classes are called k (kappa) and .lambda, (lambda).

The term "constant region derived from human origin" as used in the current application denotes a constant heavy chain region of a human antibody of the subclass IgGl, IgG2, IgG3, or IgG4 and/or a constant light chain kappa or lambda region. Such constant regions are well known in the state of the art and e.g. described by Kabat, E. A., (see e.g. Johnson, G., and Wu, T. T., Nucleic Acids Res. 28 (2000) 214-218; Kabat, E. A., et al, Proc. Natl. Acad. Sci. USA 72 (1975) 2785- 2788). anti-Ang-2 antibodies

The anti-Ang-2 antibodies may be monospecific or bispecific antibodies. In some embodiments the Ang-2 antibodies are bivalent antibodies. In a preferred embodiment, the Ang-2 antibodies are bivalent, bispecific antibodies. Preferred Ang-2 antibodies are bispecific, bivalent an- tibodies against ANG-2 and VEGF.

In some embodiment, the anti-Ang-2 antibodies are monoclonal antibodies. In some embodiment, the antibody against ANG-2 is a human, humanized or chimeric antibody.

Bispecific antibodies particularly useful in the invention comprise a) the heavy chain and the light chain of a first full length antibody that specifically binds to VEGF; b) the heavy chain and the light chain of a second full length antibody that specifically binds to ANG-2, wherein the N-terminus of the heavy chain is connected to the C- terminus of the light chain via a peptide linker; and wherein the constant domains CL and CHI are replaced by each other (e.g. an anti-Ang-2/VEGF antibody as described in WO2010/040508, in one preferred embodiment the bispecific anti-Ang-2/VEGF antibody is XMabl as described in WO2011/117329). Preferably, the CH3 domains of the bispecific, bivalent antibody according to the invention is altered by the "knob-into-holes" technology which is described in detail with several examples in e.g. WO 96/027011, Ridgway J. B., et al, Protein Eng 9 (1996) 617-621; and Merchant, A. M., et al, Nat Biotechnol 16 (1998) 677- 681. In this method the interaction surfaces of the two CH3 domains are altered to increase the heterodimerisation of both heavy chains containing these two CH3 domains. Each of the two CH3 domains (of the two heavy chains) can be the "knob", while the other is the "hole". The introduction of a disulfide bridge stabilizes the heterodimers (Merchant, A. M, et al, Nature Biotech 16 (1998) 677- 681; Atwell, S., et al. J. Mol. Biol. 270 (1997) 26-35) and increases the yield.

In a preferred aspect of the invention all bispecific antibodies comprised in formulation of the invention are characterized in that the CH3 domain of one heavy chain and the CH3 domain of the other heavy chain each meet at an interface which comprises an original interface between the antibody CH3 domains; wherein said interface is altered to promote the formation of the bispecific antibody, wherein the alteration is characterized in that: a) the CH3 domain of one heavy chain is altered, so that within the original interface the CH3 domain of one heavy chain that meets the original interface of the CH3 domain of the other heavy chain within the bispecific antibody, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the interface of the CH3 domain of one heavy chain which is positionable in a cavity within the interface of the CH3 domain of the other heavy chain and b) the CH3 domain of the other heavy chain is altered, so that within the original inter- face of the second CH3 domain that meets the original interface of the first CH3 domain within the bispecific antibody an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the interface of the second CH3 domain within which a protuberance within the interface of the first CH3 domain is positionable. Thus, the antibody useful in the invention is preferably characterized in that the CH3 domain of the heavy chain of the full length antibody of a) and the CH3 domain of the heavy chain of the full length antibody of b) each meet at an interface which comprises an alteration in the original interface between the antibody CH3 domains; wherein i) in the CH3 domain of one heavy chain an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the interface of the CH3 domain of one heavy chain which is positionable in a cavity within the interface of the CH3 domain of the other heavy chain and wherein ii) in the CH3 domain of the other heavy chain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the interface of the second CH3 domain within which a protuberance within the interface of the first CH3 domain is positionable. Preferably said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W). Preferably said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), valine (V). In one aspect of the invention both CH3 domains are further altered by the introduction of cyste- ine (C) as amino acid in the corresponding positions of each CH3 domain such that a disulfide bridge between both CH3 domains can be formed. In one embodiment, the bispecific antibody comprises a T366W mutation in the CH3 domain of the "knobs chain" and T366S, L368A, Y407V mutations in the CH3 domain of the "hole chain". An additional interchain disulfide bridge between the CH3 domains can also be used (Merchant, A. M, et al., Nature Biotech 16 (1998) 677-681) e.g. by introducing a Y349C mutation into the CH3 domain of the "knobs chain" and a E356C mutation or a S354C mutation into the CH3 domain of the "hole chain".

In another embodiment, the bispecific antibody useful in the invention comprises Y349C, T366W mutations in one of the two CH3 domains and E356C, T366S, L368A, Y407V mutations in the other of the two CH3 domains. In a another preferred embodiment the bispecific antibody comprises Y349C, T366W mutations in one of the two CH3 domains and S354C, T366S, L368A, Y407V mutations in the other of the two CH3 domains (the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain forming a interchain disulfide bridge) (numbering always according to EU index of Kabat; (Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991))). But also other knobs-in-holes technologies as described by EP 1 870 459 Al, can be used alternatively or additionally. Thus another example for the bispecific antibody are R409D; K370E mutations in the CH3 domain of the "knobs chain" and D399K; E357K mutations in the CH3 domain of the "hole chain" (numbering always according to EU index of Kabat; (Kabat, E. A., et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991))). In another embodiment the bispecific antibody comprises a T366W mutation in the CH3 domain of the "knobs chain" and T366S, L368A, Y407V mutations in the CH3 domain of the "hole chain" and additionally R409D; K370E mutations in the CH3 domain of the "knobs chain" and D399K; E357K mutations in the CH3 domain of the "hole chain". In another embodiment the bispecific antibody comprises Y349C, T366W mutations in one of the two CH3 domains and S354C, T366S, L368A, Y407V mutations in the other of the two CH3 domains or said trivalent, bispecific antibody comprises Y349C, T366W mutations in one of the two CH3 domains and S354C, T366S, L368A, Y407V mutations in the other of the two CH3 domains and additionally R409D; K370E mutations in the CH3 domain of the "knobs chain" and D399K; E357K mutations in the CH3 domain of the "hole chain". In one embodiment, the bispecific Ang-2/VEGF antibody is an antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, wherein: i) said antigen-binding sites each comprise an antibody heavy chain variable domain and an antibody light chain variable domain; ii) said first antigen-binding site comprises in the heavy chain variable domain: a CDR3 region having an amino acid sequence of SEQ ID NO: 1; a CDR2 region having an amino acid sequence selected of SEQ ID NO: 2; and a CDR1 region having an amino acid sequence of: SEQ ID NO: 3, and in the light chain variable domain: a CDR3 region having an amino acid sequence of SEQ ID NO: 4, a CDR2 region having an amino acid sequence of SEQ ID NO: 5; and a CDR1 region having an amino acid sequence of SEQ ID NO: 6; and iii) said second antigen-binding site comprises in the heavy chain variable domain: a CDR3 region having an amino acid sequence of SEQ ID NO: 7; a CDR2 region having an amino acid sequence of SEQ ID NO: 8; and a CDR1 region having an amino acid sequence of SEQ ID NO: 9; and in the light chain variable domain: a CDR3 region having an amino acid sequence of SEQ ID NO: 10; a CDR2 region having an amino acid sequence of SEQ ID NO: 11 ; and a CDR1 region having an amino acid sequence of SEQ ID NO: 12.

In a more preferred embodiment, the bispecific bivalent anti-Ang-2/VEGF antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen- binding site that specifically binds to human ANG-2, characterized in that said antibody comprises: i) the heavy chain and the light chain of a first full length antibody that specifically binds VEGF with said first antigen-binding site comprising as heavy chain variable domain (VH) the SEQ ID NO: 13, and as light chain variable domain (VL) the SEQ ID NO: 14; and ii) the modified heavy chain and modified light chain of a second full length antibody that specifically binds ANG-2, wherein the constant domains CL and CHI are replaced by each other, with said second antigen-binding site comprising as heavy chain variable domain (VH) the SEQ ID NO: 15, and a light chain variable domain (VL) the SEQ ID NO: 16. In one embodiment of the invention the bispecific, bivalent antibody comprises a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, characterized in comprising the amino acid sequences of SEQ ID NO: 17, of SEQ ID NO: 18, of SEQ ID NO: 19, and of SEQ ID NO: 20. In one embodiment, the C-terminal lysine of the sequences of the heavy chains (SEQ ID NOs: 19 and 20) is truncated.

The concentration of the anti-Ang-2 antibody comprised in the pharmaceutical formulation is in the range of 15 mg/ml to 200 mg/ml, particularly in the range of 15 mg/ml to 100 mg/ml, more particularly in the range of 15 mg/ml to 60 mg/ml or 20 mg/ml to 55 mg/ml and most particularly of about 25mg/ml or about 50 mg/ml. Surfactants

The pharmaceutical formulation of the present invention comprises a surfactant to reduce aggregation of the antibodies and particle formation. The term "surfactant" as used herein denotes a pharmaceutically acceptable excipient which is used to protect protein formulations against mechanical stresses like agitation and shearing. Examples of pharmaceutically acceptable surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (for example those sold under the trademark Brij™) and polyoxyethylene- polyoxypropylene copolymer (Poloxamer, Pluronic).

Preferably, the surfactant is a polyoxyethylenesorbitan-fatty acid ester or a polyxamer. Examples of polyoxyethylenesorbitan-fatty acid esters are polysorbate 20 (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). The preferred polyoxyethylenesorbitan-fatty acid is polysorbate 20. The above mentioned surfactants are generally used in an amount of 0.01 (w/v) or higher, e.g. 0.01 to about 0.09% (w/v). The surfactant in a pharmaceutical composition of the present invention are in particular used in the range of about 0.02% to about 0.06% (w/v), more particular in the range of about 0.03% to about 0.05% (w/v), even more particularly in an amount of about 0.04% (w/v).

The term "poloxamer" as used herein includes a polyoxyethylene-polyoxypropylene triblock copolymer composed of a central hydrophobic chain of polyoxypropylene flanked by two hydrophilic chains of polyoxyethylene known as poloxamer 188, sold under the trade name PLURONIC® F68 by BASF (Parsippany, N.J.). Other poloxamers which may be utilized in the formulations of the present invention include poloxamer 403 (sold as PLURONIC® P123), poloxamer 407 (sold as PLURONIC® P127), poloxamer 402 (sold as PLURONIC® P122), poloxamer 181 (sold as PLURONIC® L61), poloxamer 401 (sold as PLURONIC® L121), poloxamer 185 (sold as PLURONIC® P65), and poloxamer 338 (sold as PLURONIC® F108).

Buffers The term "buffer" as used herein denotes a pharmaceutically acceptable excipient, which stabilizes the pH of a pharmaceutical preparation. Suitable buffers are well known in the art and can be found in the literature. Preferred pharmaceutically acceptable buffers comprise but are not limited to histidine -buffers, citrate-buffers, succinate -buffers, acetate -buffers, arginine -buffers, phosphate-buffers or mixtures thereof. Buffers of particular interest comprise L-histidine or mix- tures of L-histidine and L-histidine hydrochloride with pH adjustment with an acid or a base known in the art. The abovementioned buffers are generally used in an amount of about 2 mM to about 200 mM or about 5 mM to about 100 mM, particularly in an amount of about 10 mM to about 50 mM or about 10 mM to about 30 mM and more particularly of about 20 mM. Independently from the buffer used, the pH can be adjusted to a value in the range from 4.5 to 7.0 and particularly to a value in the range from 5.0 to 6.5 and most particularly to pH 6.0 + 0.03 with an acid or a base known in the art, e.g. hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid and citric acid, sodium hydroxide and potassium hydroxide.

Stabilizers

The term "stabilizer" denotes a pharmaceutical acceptable excipient, which protects the ac- tive pharmaceutical ingredient and/or the formulation from chemical and/or physical degradation during manufacturing, storage and application. Chemical and physical degradation pathways of protein pharmaceuticals are reviewed by Cleland et al. (1993), Crit Rev Ther Drug Carrier Syst 10(4):307-77, Wang (1999) Int J Pharm 185(2): 129-88, Wang (2000) Int J Pharm 203(1-2): 1-60 and Chi et al. (2003) Pharm Res 20(9): 1325-36. Stabilizers include but are not limited to sugars, amino acids, polyols, cyclodextrines, e.g. hydroxypropyl- -cyclodextrine, sulfobutylethyl-β- cyclodextrin, β-cyclodextrin, polyethylenglycols, e.g. PEG 3000, PEG 3350, PEG 4000, PEG 6000, albumin, human serum albumin (HSA), bovine serum albumin (BSA), salts, e.g. sodium chloride, magnesium chloride, calcium chloride, chelators, e.g. EDTA as hereafter defined. Sta- bilizers that are particularly used in the present invention, are selected from the group consisting of sugars, polyols and amino acids. More particularly, the stabilizers are selected from the group consisting of sucrose, trehalose, sorbitol and methionine. More preferably, the stabilizer is methionine. Stabilizers can be present in the formulation in an amount of about ImM to about 600mM or about 2 mM to about 600 mM, particularly in an amount of about ImM to about 20mM or about 2mM to about 20mM or 5 to 15mM; more particularly in an amount of about of 9 to 1 ImM or about lOmM.

In some embodiments, the stable liquid pharmaceutical formulation of the present invention comprises an antioxidant as a second stabilizer. An "antioxidant" is a pharmaceutically acceptable excipient, which prevents oxidation of the active pharmaceutical ingredient. Antioxi- dants include but are not limited to chelating agents such as EDTA, citric acid, ascorbic acid, bu- tylated hydroxytoluene (BHT), butylated hydroxy anisole (BHA), sodium sulfite, p-amino benzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol, benzyl alcohol and n-acetyl cysteine. Antioxidants can be used in an amount of about 0.01 to about 100 mM, particularly in an amount of about 5 to about 50 mM and more particularly in an amount of about 5 to about 25 mM. In particular, methionine is chosen as a second stabilizer, particularly in a concentration of about 5 to about 25 mM, more particularly in a concentration of about 10 mM.

The term "sugar" as used herein denotes a monosaccharide or an oligosaccharide. A monosaccharide is a monomeric carbohydrate which is not hydrolysable by acids, including simple sugars and their derivatives, e.g. aminosugars. Examples of monosaccharides include glucose, fructose, galactose, mannose, sorbose, ribose, deoxyribose, neuraminic acid. An oligosaccharide is a carbohydrate consisting of more than one monomeric saccharide unit connected via glyco- sidic bond(s) either branched or in a chain. The monomeric saccharide units within an oligosaccharide can be identical or different. Depending on the number of monomeric saccharide units the oligosaccharide is a di-, tri-, tetra- penta- and so forth saccharide. In contrast to polysaccha- rides, the monosaccharides and oligosaccharides are water soluble. Examples of oligosaccharides include sucrose, trehalose, lactose, maltose and raffinose. In particular, sugars are selected from sucrose and trehalose.

The term "amino acid" as used herein denotes a pharmaceutically acceptable organic molecule possessing an amino moiety located at a-position to a carboxylic group. Examples of amino acids include arginine, glycine, ornithine, lysine, histidine, glutamic acid, asparagic acid, isoleu- cine, leucine, alanine, phenylalanine, tyrosine, tryptophane, methionine, serine, proline. The term "polyols" as used herein denotes pharmaceutically acceptable alcohols with more than one hydroxy group. Suitable polyols comprise to but are not limited to mannitol, sorbitol, glycerine, dextran, glycerol, arabitol, propylene glycol, polyethylene glycol, and combinations thereof. Polyols can be used in an amount of about 10 mM to about 500 mM, particularly in an amount of about 10 to about 250 mM and more particularly in an amount of about 200 to about 250 mM.

The term "stabilizers" also includes lyoprotectants. The term "lyoprotectant" denotes a pharmaceutical acceptable excipient, which protects the labile active ingredient (e.g. a protein) against destabilizing conditions during the lyophilisation process, subsequent storage and recon- stitution. Lyoprotectants comprise but are not limited to the group consisting of sugars, polyols (such as e.g. sugar alcohols) and amino acids. In particular, lyoprotectants can be selected from the group consisting of sugars such as sucrose, trehalose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, neuraminic acid, amino sugars such as glucosamine, ga- lactosamine, N-methylglucosamine ("Meglumine"), polyols such as mannitol and sorbitol, and amino acids such as arginine and glycine or mixtures thereof. Lyoprotectants are generally used in an amount of about 10 to about 600 mM, particularly in an amount of about 10 to about 250 mM and more particularly in an amount of about 100 to about 250 mM.

Tonicity Agents

The pharmaceutical formulation may also contain tonicity agents. The term "tonicity agents" as used herein denotes pharmaceutically acceptable tonicity agents which are used to modulate the tonicity of the formulation. The formulation can be hypotonic, isotonic or hypertonic. Isotonicity in general relates to the osmotic pressure relative of a solution usually relative to that of human blood serum. The formulation according to the invention can be hypotonic, isotonic or hypertonic, preferably the pharmaceutical formulation is isotonic or hypertonic. An hypertonic or hypoosmolar formulation is liquid or liquid reconstituted from a solid form, e.g. from a lyophilised form and denotes a solution having a higher tonicity as some other solution with which it is compared, such as physiologic salt solution and the blood serum. Suitable tonicity agents comprise but are not limited to sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars, in particular sucrose. In one embodiment of the present invention, the tonicity agent is sucrose. Tonicity agents are generally used in an amount of about 5mM to about lOOOmM, in particular about 200mM to about 800mM or about 200mM to about 600mM, more particular about 240mM to about 500mM. Tonicity agents for hypertonic formulations of the present invention are generally used in an amount of about 300 mM to about lOOOmM, in particular about 400mM to about 800mM. More particularly, tonicity agents for hypertonic formulations are used in an amount of 400mM to 600mM, or 450mM to 550mM, and even more particularly in an amount of about 500mM. Tonicity agents for isotonic formulations of the present invention are generally used in an amount of about 100 mM to about 300mM, in particular about 200mM to about 300mM. More particularly, tonicity agents for isotonic formulations are used in an amount of about 240mM.

Within the stabilizers and tonicity agents there is a group of compounds which can function in both ways, i.e. they can at the same time be a stabilizer and a tonicity agent. Examples thereof can be found in the group of sugars, amino acids, polyols, cyclodextrines, polyeth- yleneglycols and salts. An example for a sugar which can at the same time be a stabilizer and a tonicity agent is sucrose and trehalose, in particular sucrose.

Adjuvants

The pharmaceutical formulation may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. Preservatives are generally used in an amount of about 0.001 to about 2 %(w/v). Preservatives comprise but are not limited to ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or pro- pyl parabens, benzalkonium chloride.

Use

The stable liquid pharmaceutical formulation of the antibody against ANG-2 according to the invention can be used in the prevention or treatment of vascular diseases. The term "vascular diseases" includes Cancer, Inflammatory diseases, Atherosclerosis, Ischemia, Trauma, Sepsis, COPD, Asthma, Diabetes, AMD, Retinopathy, Stroke, Adipositas, Acute lung injury, Hemorrhage, Vascular leak e.g. Cytokine induced, Allergy, Graves' Disease, Hashimoto's Autoimmune Thyroiditis, Idiopathic Thrombocytopenic Purpura, Giant Cell Arteritis, Rheumatoid Arthritis, Systemic Lupus Erythematosus (SLE), Lupus Nephritis, Crohn's Disease, Multiple Sclerosis, Ulcerative Colitis, intraocular neovascular syndromes such as proliferative retinopathies or age- related macular degeneration (AMD), rheumatoid arthritis, and psoriasis (Folkman, J., et al., J. Biol. Chem. 267 (1992) 10931-10934; Klagsbrun, M., et al., Annu. Rev. Physiol. 53 (1991) 217- 239; and Garner, A., Vascular diseases, In: Pathobiology of ocular disease, A dynamic approach, Garner, A., and Klintworth, G.K., (eds.), 2nd edition, Marcel Dekker, New York (1994), pp 1625-1710). More particularly, the stable liquid pharmaceutical formulation of the antibody against ANG-2 can be used in the prevention or treatment of cancer, especially solid tumors.

The term cancer as used herein refers to proliferative diseases, such as lymphomas, lymphocytic leukemias, lung cancer, non small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma and Ewings sarcoma, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers. Cancer includes especially solid tumors. In particular, cancer includes rectal cancer, especially colorectal cancer (CRC), ovarian cancer, especially platin-resistant ovarian cancer (PROC), and hepatocellular cancer.

Administration The stable liquid pharmaceutical formulation according to the invention can be administered by intravenous (i.v.), subcutaneous (s.c.) or any other parental administration means such as those known in the pharmaceutical art. In a particular embodiment the formulation is administered i.v.

To administer a composition of the invention by certain routes of administration, it may be necessary to dilute the composition in a diluent. Pharmaceutically acceptable diluents include saline, glucose, Ringer and aqueous buffer solutions. In one particular embodiment the diluent is saline.

In view of their high stability the pharmaceutical formulation according to the invention can be administered i.v. without the need of an in-line filter and is thus much more convenient to handle than conventional formulations that need to be administered with an in-line filter. In-line filters such as Sterifix® have to be installed in the infusion line of i.v. medications to prevent the administration of any particles, air, or microorganisms that may be in the i.v. solution or line. Particles of 5 to 20 microns size and larger have the capability of obstructing blood flow through pulmonary capillaries, which could lead to complications such as pulmonary embolism. Foreign particles can also cause phlebitis at the injection site and filters may help to reduce the incidence of phlebitis.

The stable formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. The stable liquid pharmaceutical formulation according to the invention can be prepared by methods known in the art, e.g. ultrafiltration-diafiltration, dialysis, addition and mixing, lyophi- lisation, reconstitution, and combinations thereof. Examples of preparations of formulations according to the invention can be found herein after. The stable liquid pharmaceutical formulations according to the invention can also be in a lyophilized form or in a liquid form reconstituted from the lyophilized form. The "lyophilized form" is manufactured by freeze-drying methods known in the art. The lyophilizate usually has a residual moisture content of about 0.1 to 5% (w/w) and is present as a powder or a physically stable cake. The "reconstituted form" can be obtained from the lyophilizate by a fast dissolution after addition of reconstitution medium. Suitable reconstitution media comprise but are not limited to water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g. 0.9% (w/v) NaCl), and glucose solutions (e.g. 5% (w/v) glucose).

Production of the antibodies

Anti-Ang-2 antibodies that are particularly useful for the invention are produced by re- combinant means. Methods for recombinant production are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody and usually purification to a pharmaceutically acceptable purity. For the expression of the antibodies as aforementioned in a host cell, nucleic acids encoding the respective modified light and heavy chains are inserted into expression vectors by standard methods. Expression is performed in appropriate prokaryotic or eukaryotic host cells like CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, or E.coli cells, and the antibody is recovered from the cells (supernatant or cells after lysis). General methods for recombinant production of antibodies are well-known in the state of the art and described, for example, in the review articles of Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al, Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R.J., Mol. Biotechnol 16 (2000) 151-160; Werner, R.G., Drug Res. 48 (1998) 870-880. A method for the preparation of an antibody useful in the invention, comprises the steps of a) transforming a host cell with vectors comprising nucleic acid molecules encoding said antibody; b) culturing the host cell under conditions that allow synthesis of said antibody molecule; and c) recovering said antibody molecule from said culture. The antibodies are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. DNA and RNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures. The hybridoma cells can serve as a source of such DNA and RNA. Once isolated, the DNA may be inserted into expression vectors, which are then transfected into host cells such as HEK 293 cells, CHO cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of recombinant monoclonal antibodies in the host cells.

Amino acid sequence variants (or mutants) of the bispecific antibody are prepared by in- traducing appropriate nucleotide changes into the antibody DNA, or by nucleotide synthesis. Such modifications can be performed, however, only in a very limited range. For example, the modifications do not alter the above mentioned antibody characteristics such as the IgG isotype and antigen binding, but may improve the yield of the recombinant production, protein stability or facilitate the purification. The term "host cell" as used in the current application denotes any kind of cellular system which can be engineered to generate the antibodies comprised in the formulation of the current invention. In one embodiment HEK293 cells and CHO cells are used as host cells.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

Expression in NSO cells is described by, e.g., Barnes, L.M., et al, Cytotechnology 32 (2000) 109-123; Barnes, L.M., et al, Biotech. Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g., Durocher, Y., et al, Nucl. Acids. Res. 30 (2002) E9. Cloning of variable domains is described by Orlandi, R., et al, Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P., et al, Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Norderhaug, L., et al, J. Immunol. Methods 204 (1997) 77-87. A preferred transient expression system (HEK 293) is described by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996) 191-199.

The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, enhancers and polyadenylation signals. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre -protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oli- gonucleotide adaptors or linkers are used in accordance with conventional practice.

Purification of antibodies is performed in order to eliminate cellular components or other contaminants, e.g. other cellular nucleic acids or proteins, by standard techniques, including al- kaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See Ausubel, F., et al, ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987). Different methods are well established and widespread used for protein purification, such as affinity chromatography with microbial proteins (e.g. protein A or protein G affinity chromatography), ion exchange chromatography (e.g. cation exchange (carboxymethyl resins), anion exchange (amino ethyl resins) and mixed-mode exchange), thiophilic adsorption (e.g. with beta-mercaptoethanol and other SH lig- ands), hydrophobic interaction or aromatic adsorption chromatography (e.g. with phenyl- sepharose, aza-arenophilic resins, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g. with Ni(II)- and Cu(II)-affinity material), size exclusion chromatography, and electrophoretical methods (such as gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, M.A., Appl. Biochem. Biotech. 75 (1998) 93-102). Amino acid sequences disclosed in the application:

SEQ ID NO: Description

1 heavy chain variable domain CDR3 of <VEGF>bevacizumab

2 heavy chain variable domain CDR2 of <VEGF> bevacizumab

3 heavy chain variable domain CDR1 of <VEGF> bevacizumab

4 light chain variable domain CDR3 of <VEGF> bevacizumab

5 light chain variable domain CDR2 of <VEGF> bevacizumab

6 light chain variable domain CDR1 of <VEGF> bevacizumab

7 heavy chain variable domain CDR3 of <ANG-2> Ang2i_LC06

8 heavy chain variable domain CDR2 of <ANG-2> Ang2i_LC06

9 heavy chain variable domain CDR1 of <ANG-2> Ang2i_LC06

10 light chain variable domain CDR3 of <ANG-2> Ang2i_LC06 SEQ ID NO: Description

11 light chain variable domain CDR2 of <ANG-2> Ang2i_LC06

12 light chain variable domain CDR1 of <ANG-2> Ang2i_LC06

13 heavy chain variable domain (VH) of <VEGF> bevacizumab

14 light chain variable domain (VL) of <VEGF> bevacizumab

15 heavy chain variable domain (VH) of <ANG-2> E6Q

16 light chain variable domain (VL) of <ANG-2> E6Q

17 XMab 1 -<VEGF> light chain

18 XMab 1 -<ANG2> light chain

19 XMab 1 -<VEGF> heavy chain

20 XMab 1 -<ANG2> heavy chain

21 Human angiopoietin-2 (ANG-2) with leader and His-tag

22 human vascular endothelial growth factor (VEGF)

Examples

Liquid drug product formulations for intravenous (i.v.) administration according to the invention were developed as follows. Example 1: Material and Study Overview

The bispecific Ang-2/VEGF antibody Ang2vEGF (Vanucizumab, XMabl) prepared and purified as described in WO2011/117329 was provided at a concentration of approximately 50mg/mL.

A summary of materials (including supplier) used during the preparation of the formula- tions and their primary packaging is given in Table 1 and Table 2

Table 1 Chemicals used for formulations

Table 2 Primary Packaging

Container Closure System

Colorless 50-mL glass vial (type 1 glass) closed by means of a rubber stopper (D 777-1, 20mm) and an aluminium overseal with flip-off cap. Size Exclusion Chromatography (SE-HPLC)

Size Exclusion Chromatography (SEC) was used to detect soluble high molecular weight species (aggregates) and low molecular weight hydrolysis products (LMW) in the formulations. The method was performed with a TSK-Gel G3000SWXL, 7.8 x 300 mm, 5 μιη (Tosoh Bioscience^, no. 08541) or BioSuite 250, 7.8 x 300 mm, or 5 μιη (Waters,Cat. no. 186002165). In- tact monomer, aggregates and hydrolysis products were separated by an isocratic elution profile, using 0.2 M Potassium phosphate, 0.25 M KC1, pH 7.0 as mobile phase, and were detected at a wavelength of 280 nm.

Ion Exchange Chromatography (IE-HPLC)

Ion Exchange Chromatography (IEC) was performed to detect chemical degradation prod- ucts altering the net charge of the test antibody in the formulations. The method used a Waters Alliance 2695 HPLC instrument and detection wavelength of 280nm). 20 mM His/His-HCl, pH 6.0 was used as eluent A and 20 mM His/L-His, 1 M NaCl, pH 6.0 as eluent B, respectively, at a flow rate of 1.0 ml/min. The formulation buffer contained 20 mM His/ His-HCl, 240 mM Sucrose, 0.04 % Tween 20, pH 6.0, and the Conditioning buffer A 20 mM NaOH; Conditioning buffer B: 20 mM MES, pH 6.5

Gradient program:

CE-SDS (Capillary Electrophoresis).

The method used a Beckman PA800 Enhanced/Plus, with LIF detector at 488 nm excitation equipped with NAP™5 columns, Sephadex® G-25 Medium, DNA Grade (GE Healthcare). Example 2: pH/buffer Screen

2.1 Setup

The scope of the pH/buffer screen is to select the optimal pH and buffer for the commercial formulation of Ang2vEGF. A pH range of 5.5 to 6.5 was selected based on the pH selected for the Phl/II formulation (pH 6.0). Three different buffer systems were selected with 20mM (His/HisHCl and NaAce) and 200mM (ArgSuc). The setup of the active formulations (Table 3) is shown below.

Table 3 Formulation Codes GSM0002

2.2 Stability Program The formulations (active) were kept on stability at 2-8°C, 25 and 40°C for 8 weeks. Samples were drawn and analysed after 2, 4 and 8 weeks storage. 2.3 Materials and Methods

A summary of materials used during the preparation of the formulations and their primary packaging is given Table 1 and Table 2).

2.4 Analytical Program

The analytical program per stability timepoint is shown for the active formulations in Table 4. The analytical methods used are listed below

• Static storage conditions, sample handling chain, sample transfer chain

• Visible particles:

o Visual Inspection using Seidenader V90-T

o Visual Inspection by Optima lamp Simplex

• Color - Dr. Lange LICO 500

• Turbidity - Turbidimeter HACH 2100 AN

• Subvisible Particles -HIAC Royco PharmaSpec Model 9703

• Protein determination by UV - Perkin Elmer

• pH measurement - Metrohm pH Meter 744

• Osmolality- Osmomat Vogel OM 802

• Viscosity by rotation- Anton Paar MCR301

• Density- Paar DMA 38

• SEC

• IEC

• CE-SDS (Capillary Electrophoresis).

Table 4 Analytical Program for GSM0002

2.5 Results a) Initial data on pH, Osmolality, density, concentration by UV and Viscosity pH and protein concentration were matching the theoretical values. Osmolality, density and viscosity are only shown for information.

Table 5 Initial Data of the pH/ buffer Screen

b) Visual control by Seidenader (S A) and Optima ( Opt)

In the active formulations Table 6 only 0 to 2 visible particles were observed at t=0 in the Seidenader. Visual control by Optima showed only 1 particle in GSM0002.02. At 5°C there was a variability in visible particles over time observed by Seidenader, but no trend. The visual control by Optima did not show an increase in visible particles.

For 25 °C both Seidenader and Optima showed a slight increase in visible particles over time, but no trend. At 40°C more than 10 particles were observed in Seidenader, but not in Optima.

Only 0- 3 particles were observed by Seidenader and Optima for all placebo formulations (data not shown). Table 6 Visual control of active formulations (pH/buffer screen)

c) Color

No changes in color were observed. The 20mM His/HisHCl solutions started with B6 with some minor variations during the stability. Also all NaAce formulations were B6 with some variability over stability. All ArgSuc formulations started with B5 initially and showed slight variation between B5 and B6 (slightly brownish). No clear trend was observed for a specific buffer (Table 7).

All placebo formulations showed B9 (colorless) values (data not shown).

Table 7 Color of Active Formulations

B5,B6: slightly brownish; BG6: slightly brownish yellow d) Turbidity

The formulations in His/HisHCl 20mM showed slightly increasing turbidity with pH (from pH5.5 to 6.5). An more obvious increase in turbidity with increasing pH was determined in the 20mM NaAce formulations at 5°C. All 200mM ArgSuc formulations showed a turbidity >20FTU at 5°C (Figure 1).

At 25 °C storage temperature the in His/HisHCl 20mM formulations showed slightly increasing turbidity with pH as at 5°C. This increase in turbidity was more pronounced in the 20mM NaAce formulations. Again, All 200mM ArgSuc formulations showed a turbidity >20FTU (Figure 2). overall, the Data at 5°C was comparable to 25°C storage temperature. At 40°C all formulations showed an increase in turbidity over time (Data not shown).

No buffer dependent turbidity was seen for the placebo solution (Data not shown). e) Light obscuration by HIAC

The subvisible particle counts (per mL) for the active formulations (GSM0002.01-10) stored at 5°C were below 6000 for the fraction >10μιη and below 600 for the fraction >25μιη. Overall it seems that the 2 weeks timepoint showed slightly increased values of more than 1000 subvisible particles for the fraction >2μιη, but also slightly increased values for the fractions >5μιη, >10μιη, >25μιη.

The placebo solutions (GSM0003.01-10) showed lower subvisible particle counts for all formulations compared to the active formulations stored at 5°C.

All active formulations (GSM0002.01-10) stored at 25°C were below 6000 for the fraction >10μιη and below 600 for the fraction >25μιη. Again, a slight increase in subvisible particles of more than 1000 was observed for the fraction >2μιη, but also slightly increased values for the fractions >5μιη, >10μιη, >25μιη.

The placebo solutions stored at 25°C did not show any significant trend. For the active formulations (GSM0002.01-10) stored at 40°C the fraction >10μιη was below 6000 for the fraction >25μιη was below 600. Again, a slight increase in subvisible particles of more than 1000 was observed for the fraction >2μιη, but also slightly increased values for the fractions >5μιη, >10μιη, >25μιη. All placebo solutions stored at 40°C did not show any significant trend. Overall, all formulations fulfill the pharmacopoeia requirements (USP <787>) of below 6000 for the fraction >10μιη and below 600 for the fraction >25μιη. f) Size Exclusion Chromatography

All three buffer systems (His/His-HCl, NaAce and ArgSuc) showed a clear decrease in the SEC main peak with increasing pH over 8 weeks storage at 5°C (Figures 3A, 3B). This observation was confirmed by SEC HMW's. An increase in HMW's could be seen for all three buffer systems with increasing pH over the storage time. Comparing all formulations at pH 5.5 Arg Sue (200mM) showed the least increase in HMW's from 2.0 to 2.1% over 8 weeks at 5°C. When comparing the 3 buffer systems at pH 6.0 an increase for 20mM His/HisHCl from 3.0 to 3.1%, for 20mM NaAce from 3 .4 to 3.9% and for 200mM ArgSuc from 2.3 to 2.4% was observed.

The decrease in main peak and increase in HMW's by SEC was more prone in the formulations stored at 25°C for 8 weeks (Figures 4A, 4B). Comparing the 3 buffer systems at pH 5.5 the lowest increase of 0.3% HMW's was observed for 20mM His/HisHCl and 0.5% for 20mM NaAce and 200mM ArgSuc after 8 weeks storage at 25°C (Table 8). For the 3 buffer systems at pH 6.0 the lowest increase in HMW's of 0.7% for 20mM His/HisHCl and 0.6% for 200mM ArgSuc was determined within 8 weeks storage.

After 8 weeks storage at 40°C a decrease in main peak and concomitant increase in HMW's by SEC was seen with increasing pH (Figures 5A, 5B). When comparing all buffer systems at pH 6.0, 20mM His/HisHCl showed the least increase of 4.1% in HMW's compared to 5.2% for 20mM NaAce and 5.6% for 200mM ArgSuc. This confirms the results seen for 5 and 25°C storage temperature (Table 8). All three buffer systems at pH 5.5 showed a HMW's increase between 3.3 and 4.5% over 8 weeks storage at 40°C. Overall, the lowest increase in HMW's was seen in 20mM His/HisHCl buffer both at pH 5.5 and 6.0.

A different peak pattern was observed in the SEC chromatogram after 4w storage at 40°C in 200mM ArgSuc buffer atpH5.5 and pH6.0 (Figure 6).

Table 8 Increase in HMW's (Δ HMW's) in pH/buffer screen after 8 weeks storage

g) Ion exchange chromatography

For formulations GSM0002.01-.09 no trend was observed in the main peak by Ion exchange chromatography (IEC). During 8 weeks storage at 5°C the formulations GSM0002.01- .09 were stable (Table 9). It can be pointed out that formulation GSM0002.10 (200mM ArgSuc at pH 6.5) showed a different behavior with regards to the main peak by IEC during 8 weeks storage at 5°C. All formulations showed a decrease after 2 weeks in main peak by IEC when stored at 40°C.

Figures 7 A, 7B and Figures 8 A, 8B show the acidic and basic peak distribution in IEC. Formulation GSM0002.10 showed a completely different behavior in the acidic peak by IEC compared to formulation GSM0002.01-.09 during storage at 5°C. The values for the basic peak by IEC shows slightly increased values for the 200mM ArgSuc formulations compared to the 20mM His/His HC1 and 20mM NaAce formulations. For all 200mM ArgSuc formulations stored at 5°C a decrease in basic peak was observed with increasing pH (from 5.5 to 6.5). During the storage at 25°C an increase in acidic peak by IEC was observed for the 20mMHis/HisHCl formulation at pH 6.5 (Figures 8A, 8B). The formulation GSM0002.10, again showed a different pattern for the acidic peak by IEC. In the basic peak distribution by IEC all 20mM His/HisHCl and 20mM NaAce formulations showed a decrease over time with increasing pH. Also the 200mM ArgSuc formulations showed a decrease in basic peak by IEC with increasing pH.

During storage at 40°C , again, an increase in acidic peak by IEC is visible for increasing pH within all three buffer systems (20mM His/His HC1, 20mM NaAce and 200mM ArgSuc). A concurrent decrease in basic peak by IEC was visible for all three buffer systems.

The formulations 1-9 showed a stable IEC pattern over 8 weeks storage at 5 and 25°C. Only formulation GSM0002.10 (200mM ArgSuc at pH 6.5) showed a harsh increased values in acidic and decreased values in basic peak by IEC over 8 weeks at 5°C and 25°C.

Table 9 Main Peak by IEC

h) Capillary Electrophoresis

In the non-reduced CE- SDS slight differences have been observed between t=0 and t= 4 weeks, but no composition related trend within 4 weeks storage at 5°C and 40°C was determined (Table 10 and Table 11).

Table 10 Data by CED SDS (Non reduced)

Table 11 Data by CED SDS (Reduced)

) Protein A Chromatography and Peptide Mapping

Selected data points were analyzed by Protein A chromatography (Table 12) and Peptide Mapping (Volker, Schnaible, PTDE-A,).

The starting material (Flex bulk) already showed a high degree of oxidation, but no increase after 4 weeks at 25 °C. This was also confirmed by Peptide Map and is reported in the Memo from 24.06.2014/JW.

Table 12 Data by Protein A Chromatography after 25°C Storage

2.6 Conclusions

In summary, the data shown under 2.5 is collected in Table 13. The formulations in 20mM His/HisHCl buffer showed a lower turbidity compared to the 200mM ArgSuc formulations, the 20mM NaAce formulations showed a dependency on the pH level.

Visible and subvisible particle determination did not show a risk on particle formation within the stability program.

In SEC a trend to reduce the formation of HMW s over time by decreasing the pH was observed. Also a pH dependent acidic and basic peak formation could be observed in IEC for 20mM His/HisHCl at and 20mM NaAce formulations. But this pH dependence could not be confirmed for the 200mM ArgSuc formulations at pH 6.5.

With regards to Methionine oxidation, it seemed that Ang2vEGF in general is sensitive. Further investigations will be performed in the excipient screen.

Based on the data presented above, it was proposed to further continue the surfactant screen with 20mM His/His HC1 buffer pH 6.0 and 20mM NaAce buffer pH 5.5.

Table 13 PH buffer screen summary

Example 3: Surfactant screen 3.1 Setup

The scope of the surfactant screen is to select the optimal surfactant type and the surfactant concentration for the commercial formulation of Ang2vEGF. The two buffer systems 20mM His/His HC1 at pH 6.0 as lead buffer and 20mM NaAce at pH 5.5 as backup buffer were selected to be used for the surfactant screen. Three different surfactants (Polysorbate 20 and 80 and Poloxamer 188) at 4 different concentrations were investigated. The composition of the active formulations is shown below (Tables 14 and 15).

Table 14 Formulation Codes for the Surfactant Screen (Lead buffers), Buffer system: His/ HisHCl, pH 6)

Table 15 Formulation Codes for the Surfactant Screen (back-up buffers); Buffer s stem: ArgSuc; pH: 5.5)

3.2 Stability Program

During the surfactant screen mechanical stress test conditions of 1 week horizontal shaking at 2-8°C (200rpm), 1 week horizontal shaking at 25°C (200 rpm) and 5 cycles Freeze/Thaw (- 20°C/ 2-8°C) were applied.

3.3 Materials and Methods

A summary of materials (including supplier, material and Lot number) used during the preparation of the formulations and their primary packaging is given in (Table 1 and Table 2).

3.4 Analytical Program

Active samples are analyzed for visible (Seidenader and Optima) and sub-visible (HIAC) particles, turbidity, osmolality (t=0), pH (t=0), SEC, UV (t=0) and surfactant concentration (t=0). The analytical program for the surfactant screen is shown in Table 16. The used analytical methods are listed in below:

• Static storage conditions, sample handling chain, sample transfer chain

• Visual Inspection by Optima lamp Simplex

• Color - Dr. Lange LICO 500

• Turbidity - Turbidimeter HACH 2100 AN

• Subvisible Particles - HIAC Royco PharmaSpec Model 9703 (new)

• pH measurement - Metrohm pH Meter 744

• Osmolality- Osmomat Vogel OM 802

• Protein determination by UV - Perkin Elmer

• Density- Paar DMA 38

• SEC • IEC

• CE-SDS

Table 16 Analytical Program for Mechanically Stressed Samples

3.5 Results for Surfactant Selection a) Initial Data on pH, osmolality, concentration by UV, density and surfactant concentration

Table 17 summarizes the data on osmolality, pH, concentration by UV, density and surfactant for the 26 screened formulations. All values were within the expected range.

Table 17 Summary on Initial Data for the Surfactant Screen

b) Visual control by Optima (SA)

During the visual control by Optima only 0-1 visible particles were seen at the initial timepoint. For all mechanical stress conditions the formulations without surfactant (GSM0005.01 and GSM0005.14) showed particles or milky appearance. No visible particles were observed by Optima in all 20mM His/HisHCl formulations. For the 200mM ArgSuc formulations more than 0-1 visible particles have been observed in Optima for poloxamer 188 formulations at 0.01 and 0.03% (w/v) concentration. The concentrations 0.05% and 0.07% did not show appearance of visible particles in Optima. No Increase in visible particle counts for placebo formulations was seen (data not shown). c) Color

No changes in color were determined during the mechanical stress test program (data not shown). d) Light Obscuration by HIAC

An increase in subvisible particles by HIAC was observed for 20mM His/His HCl as well as for 200mM ArgSuc formulations without surfactant. The formulations containing Polysorbate 20 and Polysorbate 80 showed comparable subvisible particle counts for 20mM His/His HCl and 200mM ArgSuc buffer.

Increased subvisible particle (> 2 μιη) counts of more than 1000 were observed for Poloxamer 188 containing formulations (with at least 0.03%(w/v) ) after shaking at 25°C.

All placebo formulations showed lower subvisible particle values compared to the active formulations. e) Turbidity

An increased turbidity was observed after shaking at 25°C for 20mM His/His HCl (Figure 10) as well as 200mM ArgSuc (Figure 11) formulations without surfactant. For the formulation GSM0005.01 the value after 1 week shaking at 25°C could not be determined (n.d.). For GSM0005.14 the value after 1 week shaking at 25°C was 87.6 FTU (value exceeds scale in (Figure 11).

Overall the turbidity in surfactant containing 20mM His/HisHCl formulations was below 15 FTU (for all tested conditions) compared to 16.5- 21.8 FTU for the 200mM ArgSuc formulations. f) Size exclusion chromatography (SEC)

The main peak by SEC was decreased for 20mM His/HisHCl formulations at pH 6.0 containing none, 0.01% polysorbate 20 and polysorbate 80 after 1 week of shaking at 25°C (Figure 12).

HMW's by SEC increased for the respective formulations without and 0.01% polysorbate 20/80 accordingly after 1 week of shaking at 25°C (Figure 14). For a 0.03% polysorbate 80 containing formulation HMW's by SEC still showed a slight increase from initial 3.2 to 3.5% after 1 week shaking at 25°C. For the poloxamer containing formulations no changes in SEC were observed.

For the 200mM ArgSuc formulations at pH 5.5 a less pronounced decrease in main peak by SEC after 1 week of shaking at 25°C was also observed (without and with 0.01% polysorbate 20/80) (Figure 13). This observation was also in alignment with the increase in HMW's by SEC (Figure 15).

Overall all 20mM His/HisHCl formulations at pH 6.0 showed 1% more HMW's by SEC compared to the 200mM ArgSuc formulations at pH 5.5.

In general, all formulations showed F/T stability by SEC.

3.6 Conclusions

In summary (Table 18), during the surfactant screen no color changes were observed. Initially no increased numbers of visible and subvisible particles were determined. After mechanical stress an increase in visible particles was observed for formulations without surfacteant and selected poloxamer concentrations by Optima. Also subvisible particles were determined in formulations without surfactant after mechanical stress conditions. The 20mM His/His HCl formulations at pH 6.0 and 200mM ArgSuc at pH 5.5 showed similar stability in the mechanical stress conditions with polysorbate 20 compared to polysorbate 80. Some increased subvisible particle counts were found for poloxamer 188 formulations, both in 20mM His/His HCl formulations at pH 6.0 and 200mM ArgSuc at pH 5.5.

The turbidity results showed an increased turbidity without surfactant, but also an increase in 200mM ArgSuc at pH 5.5.

A less pronounced increase in HMW's was observed at a surfactant concentration of more than 0.03%(w/v). Based on the data presented above, it was proposed to further continue the excipient screen with 20mM His/His HC1 buffer pH 6.0, 20mM NaAce buffer pH 5.5 and polysorbate 20 and 80, both at a concentration of 0.04%(w/v).

Table 18 Surfactant Screen Summary

Example 4 Excipient screen 4.1 Setup

The scope of the excipient screen is to select the final composition for the commercial formulation of Ang2vEGF. Two buffer systems of 20mM His/His HCl at pH 6.0 and 20mM NaAce at pH 5.5 were selected. As surfactant components Polysorbate 20 and 80 at a concentration of 0.04% (w/v) were investigated. In addition, Sucrose and ArgHCl were investigated as tonicity adjusters. Finally, the formulations were tested with and without lOmM Methionine. The setup of the active formulations (GSM007.01-16) and the placebo formulations (GSM008.01-16) is shown in Table 19 below. The drug substance used for the excipient screen was Upscale 07 (Second VE batch or first

Lonza upscale), for which a decrease in productivity after 250 hours and an undesirable lactic acid profile were observed during upstream processing.

During drug substance stability this batch showed a different behavior in HMW's by SEC during 12 weeks of storage at 40°C (refer to meeting minutes TDT from November 3rd 2014). A similar behavior had already been observed for a Phi clinical batch

(D011.01E/G003.01E). For this clinical batch all release data were within specifications. However after 10 weeks of storage at 40°C a slight increase of HMW species, in comparison to other Phl/Ph2 batches, was observed which was confirmed and pronounced after 12 weeks of storage. An extended characterization didn't show any unexpected results and HCP levels were in a simi- lar range as for the other batches.

Table 19 Excipient Screen Formulations

4.2 Stability Program

The formulations were kept on stability at 2-8°C, 25 and 40°C for 24 months. Samples were drawn and analysed initially, after 4 and 10 weeks, 3, 6,9,12,18 and 24 months. In addition, samples were shaken for 1 week at 2-8°C and 25 °C. Another sample set was put on 5 consecu- tive Freeze/Thaw cycles (-20°C to 2-8°C) and analyzed.

In addition long term storage at -20°C, -40°C and -80°C in stainless steel containers was performed. Samples were drawn and analyzed after 6, 12 and 24 months.

4.3 Materials and Methods

A summary of materials used during the preparation of the formulations and their primary packaging is given in (Table 1 and Table 2).

4.4 Analytical Program

The samples are analyzed for visible (Seidenader and Optima) and sub-visible (HIAC) particles, color, turbidity, content by UV, pH, osmolality density, surfactant and methinone concentration at t= 0.). For all stability timepoints SEC, IEC and Protein A chromatography will be per- formed. CE-sds, Bioassay and Peptide Map will be performed at selected timepoints.

The analytical program for the excipient screen is shown below and in Table 20.

Static storage conditions, sample handling chain, sample transfer chain

Visual Inspection by Seidenader V90-T

Visual Inspection by Optima lamp Simplex

Color - Dr. Lange LICO 500

Turbidity - Turbidimeter HACH 2100 AN

Subvisible Particles - HIAC Royco PharmaSpec Model 9703 (new)

pH measurement - Metrohm pH Meter 744

Osmolality- Osmomat Vogel OM 802

Protein determination by UV - Perkin Elmer

Density- Paar DMA 38

SEC IEC

CE-SDS

Protein A Chromatography

PepMap

Bioassay Table 20 Analytical Program for the Excipient Screen

4.5 Results a) Initial Data on pH, Osmolality, concentration by UV, density, surfactant and methionine concentration Table 21 summarizes the data on osmolality, pH, concentration by UV, density, surfactant and methionine concentration for the 16 screened formulations. All values were within the expected range.

Table 21 Initial data of the excipient screen

b) Visual control by Optima and Seidenader (SA)

All values were within the expected range. No trend was observed. c) Color

All values were within the expected range. No trend was observed. d) Light obscuration by HIAC

All values were within the expected range. No trend was observed. e) Turbidity

An increase in turbidity with ArgHCl and ArgSuc was observed during storage for all three storage temperatures (5, 25 and 40°C). Also, all 50mg/mL formulations show comparable turbid- ity to the 25mg/mL (GSM0007.16, Figure 16). GSM0007.04 (hyperosmolar) showed less increase in turbidity than GSM0007.05 (isoosmolar). A lower increase in turbidity was also observed for GSM0007.09 (hyperosmolar, incl. PS80) compared to GSM0007.10 (isoosmolar, incl. PS80, Figure 17). f) Size exclusion chromatography (SEC) A decrease in the main peak [area ] was pronounced in 25mg/mL formulation

(GSM007.16) during 6months stability, except for storage at -20°C and -40°C (24w) (

An increase in AHMW's [area %] by SEC was more pronounced in the 25mg/mL formulation compared to 50mg/mL formulations (Figure 18A, 18B). In the formulations including Polysorbate 80 (GSM0007.06, .07 and .08) also an increase in HMW's [area %] by SEC was more pronounced than in the formulations including Polysorbate 20 (GSM0007.01, .02 and .03). The lowest increase in HMW's [area ] by SEC was seen in GSM0007.04 and .05, as well as in GSM0007. 09 and .10. These 4 formulations all contained lOmM Methionine. Low initial HMW values were determined in all ArgSuc Formulations (<His/HisHCl buffer for .04 and .05 and in .09 and .10). A slight increase in HMW's [area ] by SEC was seen during storage at -20°C and -40°C (24w) observed (in green) for ArgHCl formulations.

During shaking stress at 5 and 25°C for one week an increase in HMW's [area ] by SEC was more pronounced in 20mM His/HisHCl formulations compared to all 200mM ArgSuc containing formulations (Figure 19). The lowest initial values for the 20mM His/HisHCl formulations as well as the lowest increase in HMW's was observed for all sucrose compared to ArgHCl containing formulations (GSM0007.04,.05, .09 and .10). No significant changes in LMW's [area %] by SEC was determined during 6 months storage at 5 and 25°C. It seems that LMWs are slightly increased at 40°C storage in formulations containing Polysorbate 80.

Generally LMW's were low, but during long term storage at -20°C and -40°C, an increase was observed from initially 0.2/0.3% to 0.6/0.7% after 6 months storage. It was not clear if this increase in LMW's was related to the storage condition or due to the Drug substance quality (Example 4). Therefore the long term storage was further investigated in the Scratch test (excipi- ent crystallization test) I and II (Example 5.1 and Example 5.2) g) Ion exchange chromatography (IEC) In main peak by IEC no changes were visible for different formulations or storage conditions.

A slightly faster increase in basic variants by IEC was observed in 200mM ArgSuc formulations (Figure 20) during storage at 25°C and 40°C over time. This slight increase in basic variant was accompanied by a slower decrease in acidic variants (data not shown) compared to the 20mM His/HisHCl formulations.

An increase in basic variants was even more pronounced as in the early phase formulations (GSM007.16) compared to all screened commercial formulations (GSM007.01- GSM007.14) h) Protein A Chromatography, Bioassay and Pepmap

In Protein A Chromatography an increase in oxidized species was determined after 24w/25°C storage for formulations without Methionine (Table 22). Also a more pronounced increase in oxidized species was determined for formulation containing Polysorbate 80. Overall, the early phase formulation (GSM007.16) showed an increase of 19.1%, which was a higher increase than any of the screened commercial formulation compositions. GSM007.03 (hyperosmolar, no Methionine) showed a slight increase of oxidized species over time. Whereas no increase was observed in GSM007.04 (hyperosmolar) and GSM007.05 (isoosmolar), both containing lOmM Methionine.

When the oxidized species by Protein A Chromatography increased for GSM0007.03 during stability, the non-oxidized species decreased in value (Table 23).

The activity for GSM007.03-.05, as well as GSM007.16 was determined by Bioassay after 3months of storage at 5°C and 25°C. A slight decrease could be observed for the early phase formulation after storage of 3months at 25°C (Table 24), but this value is still within Method variation. No initial potency value was determined by bioassay. In GSM007.03-.05 there were no difference in Methionine oxidation observed in Peptide mapping (refer to Memo from Jan 19^th 2015) In contrast GSM007.16 showed a significant Methionine oxidation in peptide T19(HC-VEGF)/T23(HC-Ang2) a slightly increased (Met-) oxidation in the peptides T2(HC-VEGF) and T39(HCVEGF)=Met434/T43(HC-Ang2). No trypto- phane oxidation was determined for GSM007.03-.05, but for GSM007.16 in peptide T4(HC- VEGF).

Succinimide formation seemed to increase in general with increasing storage time.

Table 22 Oxidized species [%] by Protein A Chromatography

Table 23 Non-oxidized species [%] by Protein A Chromatography

Table 24 Activity [%] by Bioassay

4.6 Conclusions

In summary, 20mM His/HisHCl buffer at pH 6.0 is suggested for the commercial formulation. Compared to the 200mM ArgSuc buffer at pH 5.5 a lower turbidity, as well as a better HMW profile over time and a less pronounced increase in basic peak formation was observed (Table 25).

Polysorbate 20 at a concentration of 0.04%(w/v) is proposed as surfactant due to a slightly higher increase in HMW's by SEC in formulations containing Polysorbate 80. Also oxidized species in protein a Chromatography seemed to be increased in Polysorbate 80 formulations compared to Polysorbate 20. As stabilizer sucrose showed a beneficial effect compared to ArgHCl with regards to turbidity and increase in HMW's during long term storage at -20°C and -40°C. In addition, a concentration of 500mM sucrose compared to 240mM showed a beneficial effect in HMW's by SEC during storage time. Nevertheless, LMW's by SEC need to be further investigated (scratch test I and II) if they are due to the hyperosmolar formulation (500mM sucrose) or the drug sub- stance quality. With regard to turbidity the formulations with 50mg/mL protein and 500mM sucrose concentration showed comparable results to the early phase formulation with 25mg/mL protein concentration.

As additional stabilizer lOmM methionine is proposed, because of its beneficial effect in color, HMW's by SEC and oxidized species by protein A chromatography. The proposed commercial formulation is shown in Table 25 below.

Table 25 Summary Table for Excipient Screen

Example 5: Scratch Tests

Two scratch tests (excipient crystallization tests) were performed in order to define the long them storage conditions for the Ang2vEGF commercial drug substance. A frozen storage in 300L cryo-vessels is considered. The storage temperature of either -20°C or -40°C needs to be defined.

Scratch test I was performed for 4 different formulations with the same drug substance used in the excipient screen (Second VE batch or first Lonza upscale), where during upstream processing a decrease in productivity after 250 hours and an undesirable lactic acid profile were observed (see see Example 4.1). An increase in HMW's by SEC over time was observed in the early phase formulation (GSM007.16 ) as well as an increase in LMW's by SEC during long term frozen storage in hyperosmolar solutions (see excipient screen 0).

Scratch test II was then initiated later with an extended setup (including a protein and sucrose concentration range) with drug substance, where no issues were observed during upstream processing (fourth VE-batch or third Lonza upscale, Upscale 09).

5.1 Scratch Test I

5.1.1 Study Design

The scratch test is performed in order to evaluate the risk of potential formation of HMW by SEC ("trailing edge dimers" (TED)) during freeze and thaw operations and long term storage of the drug substance (DS) at the recommended storage temperature of -20°C (backup- 40°C).

The setup of the study is shown in Table 26. The four different formulations are scratched on the surface and sprinkled with sucrose in order to initiate potential crystallization. As reference unscratched (+unsprinkled) samples are put on stability in parallel.

Table 26 Setu of Scratch Test I

5.1.2 Stability Program

The four formulations (scratched and unscratched) were kept at 2-8°C, -20°C, -40°C and - 80°C for 24 months. Samples were drawn initially, after initial freezing and at t=l, 3, 6, 12 and 24 months for analysis.

5.1.3 Analytical Program

Table 27 Analytical Program for Scratch Test I

5.1.4 Results a) Initial data on pH, Osmolality, concentration by UV, surfactant and methionine concentration

Table 8 summarizes the data on osmolality, pH, concentration by UV, surfactant and methionine concentration for the 4 formulations screened. All values were within the expected range.

Table 28 Initial Data for Scratch Test I

a) Visible particles by Seidenader

Only 0-1 visible particles were determined by Seidenader initially. No further determination of visible particles during long term storage was performed. Table 29 Initial Visible Particles by Seidenader

b) Size Exclusion Chromatography

In the scratch test I the same drug substance was used as in the excipient screen . Therefore the long term storage was further investigated in the Scratch test I and II with special interest in HMW's and LMW's by SEC in hyperosmolar solutions. Figure 21 shows slightly lower HMW's by SEC in the hyper-osmolar solutions (GSMOO 1.03 and .04) compared to the iso-osmolar solutions (GSM0011.01 and .02). With regards to LMW's by SEC 0.6-0.8% were observed for both hyper-osmolar solutions compared to the iso-osmolar solutions (Figure 22). Still, it is not clear if the change in LMW's by SEC in the hyperosmolar solutions is due to the formulation or the drug substance quality. Therefore Scratch test II was initiated with a different drug substance quality.

5.2 Scratch Test II

5.2.1 Study Design

The scratch test is performed in order to evaluate the risk of potential formation of HMW by SEC ("trailing edge dimers" (TED)) during freeze and thaw operations and long term storage of the drug substance (DS) at the recommended storage temperature of -20°C (backup- 40°C). For the scratch test II a DoE setup was chosen in order to evaluate the variation between sucrose and protein content and a potential interaction effect. Three centerpoints with the target formulation composition are included into the design (GSM0012.09-.i l). Table 30 Study Design of Scratch Test II

5.2.2 Stability Program

The stability program is according the stability program of scratch test I.

5.2.3 Analytical Program

The analytical program is according the stability program of scratch test I.

5.2.4 Results a) Initial data on pH, Osmolality, concentration by UV, surfactant and methionine concentration

Table 31 summarizes the data on osmolality, pH, concentration by UV, surfactant and methionine concentration for the 4 formulations screened. All values were within the expected range.

b) Visible particles by Seidenader

Only 0-1 visible particles were determined by Seidenader initially. No further determination of visible particles during long term storage was performed

Table 32 Initial Visible Particles by Seidenader

In the HMW's by SEC very little variation between 1.6-2.0% was seen for the centerpoints (GSM0012.09-11), but independent of storage temperature or time. Slightly increased HMW's (up to 2.2% within 6 months storage time) were determined for the formulations with 60mg/mL protein concentration (GSMOO 12.02, .04, .06, Figure 23). No significant changes in the HMw profile over storage time have been observed in the SEC chromatograms (Figures 24A, 24B) for untreated or scratched and sprinkled samples.

For the LMW's by SEC no increase was observed for the hyperosmolar solutions with time or temperature. Surprisingly, slightly lower LMW's were determined for the formulations with 60mg/mL protein concentration (GSMOO 12.02, .04, .06, Figure 25). A comparison (HMW's and LMW's by SEC) between the untreated and scratched& sprinkled samples was done at the storage temperature of -20°C (Figures 26A, 26B and Figures 27 A, 27B). But no significant difference was observed c) Glass transition temperature

The glass transition temperature Tg' was determined by Differential Scanning Calorimetry (DSC) at a temperature of -27.8°C. 5.3 Summary Scratch Tests and Intended Storage Temperature

Overall, no increase in HMW's by SEC was observed due to storage temperature or time for up to 6 months. Also, no increase in LMW's by SEC could be confirmed with Scratch test II for the different drug substance quality. Therefore, a storage temperature of-20°C for the com- mercial formulation (Example 4.6) is proposed.

Example 6: Summary

6.1 Study Overview

For the selection of the commercial formulation the following studies have been performed (Table 33).

The overview includes the starting material used for the respective study

Table 33 Formulation Development Studies

6.2 Performed Formulation Development Studies

The commercial formulation development was performed using a sequential approach. During a first pH/buffer screen a 20mM His/HisHCl buffer was selected due to a lower turbidity compared to the two other buffer systems (20mM Sodium acetate and 200mM Arginine succin- ate) tested Figure 1). A pH of 6.0 was finally defined, due to less HMW's at low pH, but also due to the basic peak (by IEC) decrease over time with increasing pH (Figures 8 A, 8B). A pH dependent acidic and basic peak formation could be observed in IEC for 20mM His/HisHCl at and 20mM NaAce formulations.

In a following surfactant screen a similar stability in HMW's was observed for polysorbate 20 and 80 during the mechanical stress conditions at a concentration of more than 0.03%(w/v). Some increased subvisible particle counts were found for poloxamer 188 formulations.

In the final excipient screen the 20mM His/HisHCl buffer at pH 6.0 was confirmed due to lower turbidity compared to the 200mM ArgSuc buffer at pH 5.5, as well as the HMW profile over time and a less pronounced increase in basic peak formation in 20mM His/HisHCl buffer (Table 25).

Polysorbate 20 at a concentration of 0.04%(w/v) was selected as surfactant due to a slightly higher increase in HMW's by SEC in formulations containing Polysorbate 80. In addition, a concentration of 500mM sucrose compared to 240mM showed a beneficial effect in HMW's by SEC during storage time. In turbidity the formulations with 50mg/mL protein and 500mM su- crose concentration showed comparable results to the early phase formulation with 25mg/mL protein concentration.

As additional stabilizer lOmM methionine is proposed, because of its beneficial effect in color, HMW's by SEC (Figure 18 A, 18B) and oxidized species by protein A chromatography.

The proposed commercial formulation is shown in Table 34.

Table 34 Drug Product Composition (Ro 552-0985/F03-01)

L-Histidine/ L-Histidine hydrochloride monohydrate buffer pH 6.0

Function: Buffer to maintain solution pH at 6.0

Concentration: 20 mM in Drug substance and Drug product

L-histidine / L- histidine hydrochloride monohydrate provides buffering capacity at pH 6.0. A L-histidine / L-histidine hydrochloride monohydrate concentration of 20 mM was shown to be sufficient to maintain the formulation pH through the manufacturing of the drug product as well as during storage of the drug substance and drug product.

Sucrose

Function: Tonicity agent

Concentration: 500 mM in drug substance and drug product

Sucrose is used as a tonicity agent. A concentration of 500 mM sucrose was found to be sufficient to ensure stability of vanucizumab drug substance and drug product.

L-Methionine

Function: Stabilizing agent

Concentration: 10 mM in drug substance and drug product

A concentration of 10 mM of L-methionine was found to avoid HMW increase by SEC for vanucizumab during formulation processes or storage.

Polysorbate 20

Function: Surfactant to prevent losses due to surface adsorption as well as to minimize the potential formation of soluble aggregates and/or insoluble proteinaceous particles.

Concentration: 0.04% (w/v) in drug substance and drug product A polysorbate 20 level of 0.04% (w/v) was shown to be sufficient to maintain the integrity of vanucizumab during stability and processing of drug substance and drug product.

Abbreviations

Claims

Claims

1. A stable liquid pharmaceutical formulation comprising:

- 15 to 200 mg/ml of an antibody against anti-Angiopoietin-2 (ANG-2)

- 2 to 200 mM of a buffer

- 0.01-0.07% of surfactant

- a) 1 to 20mM of at least one stabilizer and 200-1000mM or more of a tonicity agent, or b) 200-1000mM or more of a tonicity agent

at a pH in the range of 4.5 to 7.0

2. The pharmaceutical formulation according to claim 1, wherein the concentration of the anti- body against ANG-2 is in the range 15 to 60mg/ml.

3. The pharmaceutical formulation according to claim 1, wherein the concentration of the antibody against ANG-2 is in the range 20 to 55mg/ml, in particular of about 25mg/ml or about 50 mg/ml.

4. The pharmaceutical formulation according to any of the previous claims, wherein the buffer is selected from a histidine buffer (histidine/histidine hydrochloride monohydrate buffer), sodium acetate buffer, and arginine sucrose buffer, in particular the buffer is a histidine buffer.

5. The pharmaceutical formulation according to any one of the previous claims, wherein the concentration of the buffer is 10 to 50mM, in particular 20mM.

6. The pharmaceutical formulation according to any one of the previous claims, wherein the pH of the formulation is in the range 5.0 to 6.5.

7. The pharmaceutical formulation according to any of the previous claims, wherein the pH is in the range of 5.0 to 6.0, in particular about pH6.

8. The pharmaceutical formulation according to any of the previous claims, wherein the surfactant is Polysorbate 20, Polysorbate 80, or Poloxamer 188, in particular Polysorbate 20.

9. The pharmaceutical formulation according to any of the previous claims, wherein the concentration of the surfactant is in the range of about 0.02-0.06% (w/v), or about 0.03-0.05 % (w/v), in particular 0.04% (w/v).

10. The pharmaceutical formulation according to anyone of the previous claims, wherein the at least one stabilizer is methionine.

11. The pharmaceutical formulation according to any one of the previous claims, wherein the concentration of the stabilizer is in the range of 5 to 15mM.

12. The pharmaceutical formulation according to any one of the previous claims, wherein the concentration of the stabilizer is in the range of 9 to 11 mM, in particular about 10 mM.

13. The pharmaceutical formulation according to anyone of the previous claims, wherein the tonicity agent is selected from sucrose, trehalose, sorbitol and arginine hydrochloride, in particular sucrose.

14. The pharmaceutical formulation according to anyone of the previous claims, wherein the concentration of the tonicity agent is in the range of 200mM to 800mM, or 200mM to 600mM.

15. The pharmaceutical formulation according to anyone of the previous claims, wherein the concentration of the tonicity agent is in the range of 200mM to 300mM or 400mM to 550mM, in particular about 240mM or about 500mM.

16. The pharmaceutical formulation according to any one of claims 1 to 15, wherein the antibody against ANG-2 is

a) a bispecific antibody, in particular a bispecific antibody against ANG-2 and VEGF;

and/or

b) a human or humanized antibody; and/or

c) a monoclonal antibody.

17. The pharmaceutical formulation according to any one of the previous claims wherein it comprises

- 15 to 200 mg/ml of a bispecific antibody against ANG-2 and VEGF;

- 2 to 200 mM of a a histidine buffer;

- 0.01-0.07% of Polysorbate 20;

- a) 1 to 20mM of methionine, and 200mM -600mM sucrose; or - b) 200mM-600mM sucrose

at pH 6

18. The pharmaceutical formulation according to any one of the previous claims wherein it comprises

A) - about 25 mg/ml of a bispecific antibody against ANG-2 and VEGF;

- about 20mM of a histidine buffer;

- about 0.04% of Polysorbate 20;

- a) about lOmM of methionine, and about 240mM of sucrose; or

- b) about 240mM of a sucrose

at pH 6; or

B) - about 50 mg/ml of a bispecific antibody against anti-ANG-2 and VEGF;

- about 20mM of a histidine buffer;

- about 0.04% of Polysorbate 20;

- a) about lOmM of methionine, and about 240mM of a sucrose; or

- b) about 240mM of a sucrose

at pH 6; or

C) - about 25 mg/ml of a bispecific antibody against ANG-2 and VEGF;

- about 20mM of a histidine buffer;

- about 0.04% of Polysorbate 20;

- a) about lOmM of methionine, and about 500mM of a sucrose; or

- b) about 500mM of a sucrose

at pH 6; or

D) - about 50 mg/ml of a bispecific antibody against ANG-2 and VEGF;

- about 20mM of a histidine buffer;

- about 0.04% of Polysorbate 20;

- a) about lOmM of methionine, and about 500mM of a sucrose; or

- b) about 500mM of a sucrose

at pH 6

19. The pharmaceutical formulation according to any one of claims 1 to 18, wherein the antibody against ANG-2 is the bispecific bivalent Anti-Ang-2/VEGF antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, characterized in that said antibody comprises:the heavy chain and the light chain of a first full length antibody that specifically binds VEGF with said first antigen-binding site comprising as heavy chain variable domain (VH) the SEQ ID NO: 13, and as light chain variable domain (VL) the SEQ ID NO: 14; and ii) the modified heavy chain and modified light chain of a second full length antibody that specifically binds ANG-2, wherein the constant domains CL and CHI are replaced by each other, with said second antigen-binding site comprising as heavy chain variable domain (VH) the SEQ ID NO: 15, and a light chain variable domain (VL) the SEQ ID NO: 16.

20. The pharmaceutical formulation according to any one of claims 1 to 19, wherein the antibody against ANG-2 is a bispecific, bivalent antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, characterized in comprising the amino acid sequences of SEQ ID NO: 17, of SEQ ID NO: 18, of SEQ ID NO: 19, and of SEQ ID NO: 20.

21. The pharmaceutical formulation according to any one of claims 1 to 20 for use in the treatment of a vascular disease.

22. The pharmaceutical formulation for use according to claim 21, wherein the vascular diseases is cancer.

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