Stable antibody formulation for intravenous use comprising nivolumab, histidine, sucrose, polysorbate and methionine

Abstract

The present invention relates to a stable antibody formulation for intravenous use comprising: a. about 10-40 mg / mL nivolumab, b. about 20 mM L-histidine buffer, c. about 270 mM sucrose, d. about 0.02 % w / v polysorbate 80, and e. about 10 mM L-methionine. The antibody formulation is stable at about 2 °C to about 8 °C for about 12 months. The stable antibody formulation is for use in the treatment of cancer.

Description

[0001] STABLE ANTIBODY FORMULATION FOR INTRAVENOUS USE COMPRISING NIVOLUMAB, HISTIDINE, SUCROSE, POLYSORBATE AND METHIONINE

[0002] TECHNICAL FIELD

[0003] The present invention relates to a stable liquid antibody formulation for intravenous use comprising nivolumab, histidine buffer, sucrose, methionine, and polysorbate 80. The present invention also relates to said formulations for use in the treatment of cancer.

[0004] BACKGROUND OF INVENTION

[0005] Nivolumab which is sold under the trademark Opdivo® is an antibody indicated for the treatment of melanoma, non-small cell lung cancer, malignant pleural mesothelioma, renal cell carcinoma, classical Hodgkin lymphoma, squamous cell carcinoma of the head and neck, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, esophageal or gastroesophageal junction cancer and / or gastric cancer [1], Opdivo® is administered as an intravenous infusion after a dilution based upon recommended infusion rate for each indication.

[0006] Opdivo® is a sterile, preservative-free, non-pyrogenic, clear to opalescent, colorless to paleyellow liquid that may contain few particles. The commercially sold dosage forms and strengths for injections are 40 mg / 4 mL, 100 mg / 10 mL, 120 mg / 12 mL, and 240 mg / 24 mL solutions in a single-dose vial, i.e. the concentration of nivolumab in a single-dose vial is 10 mg / ml for all formulations [1],

[0007] Each mL of an Opdivo® formulation contains nivolumab 10 mg, mannitol (30 mg), pentetic acid (0.008 mg), polysorbate 80 (0.2 mg), sodium chloride (2.92 mg), sodium citrate dihydrate (5.88 mg), and water for injection [1],

[0008] Nivolumab is a programmed death receptor-1 (PD-1) blocking antibody and is an IgG4 kappa immunoglobulin that has a calculated molecular mass of 146 kDa. The nivolumab antibody comprised in Opdivo® is expressed in a recombinant Chinese Hamster Ovary cell line as described in EP2161336 B2.

[0009] Biosimilars of nivolumab may be expressed as disclosed in SE543998 C2. A biosimilar is highly similar to a biologic medication already approved by a medical agency such as FDA and has no clinically meaningful differences from the reference product, i.e. a patient can expect the same safety and effectiveness from the biosimilar over the course of treatment as one would with the original product.

[0010] Pentetic acid which is used as an excipient in Opdivo® is a chelating agent which may potentially have toxic and hazardous properties at high doses. Hence, there is a need to formulate nivolumab in a composition which is free of pentetic acid.

[0011] Interestingly, the inventors of the present invention have discovered that nivolumab containing formulations comprising the same amounts of excipients as in Opdivo® result in relatively high amounts of sub-visible particles above 10 pm and 25 pm after being stored at -20 °C, -80 °C, 5 °C, 25 °C and 40 °C. Hence, there is a need to formulate nivolumab in a composition which results in lower amounts of sub-visible particles above 10 pm and 25 pm after being stored at the above-mentioned temperatures.

[0012] The amount of antibody that can be administered via the intravenous injection is limited by the physico-chemical properties of the antibody, in particularly by its solubility and stability in a suitable liquid formulation and by the volume of the infusion fluid. Hence, there is a need to formulate nivolumab in a composition which results in higher solubility and stability when compared to a formulation according to the chemical composition of Opdivo®.

[0013] OBJECT OF THE INVENTION

[0014] The object of the invention is to provide a stable antibody formulation comprising nivolumab which is free of pentetic acid.

[0015] The object of the invention is to provide a stable antibody formulation comprising nivolumab which retains its physical or chemical stability upon storage.

[0016] The object of the invention is to provide a stable antibody formulation comprising nivolumab which retains its biological activity upon storage.

[0017] A further object of the invention is to provide an antibody formulation comprising nivolumab which is more thermostable when compared to Opdivo® or a biosimilar formulation according to the chemical composition of Opdivo®. A further object of the invention is to provide an antibody formulation comprising nivolumab which has higher colloidal stability when compared to Opdivo® or a biosimilar formulation according to the chemical composition of Opdivo®.

[0018] A further object of the invention is to provide an antibody formulation comprising nivolumab which has less particles above 10 pm when compared to Opdivo® or a biosimilar formulation according to the chemical composition of Opdivo®.

[0019] A further object of the invention is to provide an antibody formulation comprising nivolumab which has less particles above 25 pm when compared to Opdivo® or a biosimilar formulation according to the chemical composition of Opdivo®.

[0020] A further object of the invention is to provide an antibody formulation comprising nivolumab which has higher solubility when compared to Opdivo® or a biosimilar formulation according to the chemical composition of Opdivo®.

[0021] A further object of the invention is to provide an antibody formulation comprising nivolumab which has higher stability when compared to Opdivo® or a biosimilar formulation according to the chemical composition of Opdivo®.

[0022] A further object of the invention is to provide an antibody formulation comprising nivolumab which has higher thermostability when compared to Opdivo® or a biosimilar formulation according to the chemical composition of Opdivo®.

[0023] A further object of the invention is to provide an antibody formulation comprising nivolumab which has less aggregation when compared to a formulation according to Opdivo® or a biosimilar formulation according to the chemical composition of Opdivo®.

[0024] A further object of the invention is to provide an antibody formulation comprising nivolumab which is stable at about 2 °C to about 8 °C for about 12 months.

[0025] The object of the invention is to provide a stable antibody formulation comprising nivolumab which retains its post translational modifications upon storage.

[0026] The object of the invention is to provide a stable antibody formulation comprising nivolumab which retains its charge variants and upon storage.

[0027] The object of the invention is to provide a stable antibody formulation comprising nivolumab which retains its acidic and basic distribution of species upon storage. SUMMARY OF INVENTION

[0028] The objects of the invention are attained by the subject-matter disclosed in the claims as well as the subject-matter disclosed in the below aspects and embodiments of the invention.

[0029] The first aspect of the invention relates to a stable antibody formulation comprising nivolumab, histidine buffer, sucrose or trehalose, and methionine. The antibody formulation is free of pentetic acid, enzymes and / or arginine.

[0030] In a preferred of embodiment, the antibody formulation comprises: a) about 10-40 mg / mL nivolumab, b) about 10-50 mM L-histidine buffer, c) about 70-270 mM sucrose or trehalose, d) about 10 mM L-methionine.

[0031] In a preferred of embodiment, the antibody formulation further comprises about 0.005-0.08 % weight / volume (w / v) polysorbate 80, preferably about 0.01-0.02 % w / v polysorbate 80, more preferably about 0.02 % w / v polysorbate 80.

[0032] In a preferred of embodiment, the antibody formulation comprises: a) about 10-40 mg / mL nivolumab, b) about 20 mM histidine buffer, c) about 140-270 mM sucrose or trehalose, d) about 0.02 % w / v polysorbate 80, and e) about 10 mM L-methionine.

[0033] In a preferred of embodiment, the antibody formulation consist of: a) about 10-40 mg / mL nivolumab, b) about 20 mM histidine buffer, c) about 140-270 mM sucrose or trehalose, d) about 0.02 % w / v polysorbate 80, e) about 10 mM L-methionine, and f) water, preferably water for injection.

[0034] In a preferred of embodiment, the antibody formulation comprises: a) about 10-40 mg / mL nivolumab, b) about 20 mM histidine buffer, c) about 270 mM sucrose, d) about 0.02 % w / v polysorbate 80, and e) about 10 mM L-methionine.

[0035] In a preferred of embodiment, the antibody formulation comprises: a) about 10 mg / mL nivolumab, b) about 20 mM histidine buffer, c) about 270 mM sucrose, d) about 0.02 % w / v polysorbate 80, and e) about 10 mM L-methionine.

[0036] In a preferred of embodiment, the antibody formulation consist of: a) about 10 mg / mL nivolumab, b) about 20 mM histidine buffer, c) about 270 mM sucrose, d) about 0.02 % w / v polysorbate 80, e) about 10 mM L-methionine, and f) water, preferably water for injection.

[0037] In a preferred of embodiment, the antibody formulation comprises: a) about 40 mg / mL nivolumab, b) about 20 mM histidine buffer, c) about 270 mM sucrose, d) about 0.02 % w / v polysorbate 80, and e) about 10 mM L-methionine.

[0038] In a preferred of embodiment, the antibody formulation consist of: a) about 40 mg / mL nivolumab, b) about 20 mM histidine buffer, c) about 270 mM sucrose, d) about 0.02 % w / v polysorbate 80, e) about 10 mM L-methionine, and f) water, preferably water for injection.

[0039] In a preferred of embodiment, the antibody formulation comprises: a) about 10-40 mg / mL nivolumab, b) about 20 mM histidine buffer, c) about 270 mM trehalose, d) about 0.02 % w / v polysorbate 80, and e) about 10 mM L-methionine.

[0040] In a preferred of embodiment, the antibody formulation comprises: a) about 10 mg / mL nivolumab, b) about 20 mM histidine buffer, c) about 270 mM trehalose, d) about 0.02 % w / v polysorbate 80, and e) about 10 mM L-methionine.

[0041] In a preferred of embodiment, the antibody formulation comprises: a) about 40 mg / mL nivolumab, b) about 20 mM histidine buffer, c) about 270 mM trehalose, d) about 0.02 % w / v polysorbate 80, and e) about 10 mM L-methionine.

[0042] In a preferred of embodiment, the antibody formulation comprises: a) about 10 mg / mL nivolumab, b) about 20 mM histidine buffer, c) about 140 mM sucrose, d) about 0.02 % w / v polysorbate 80, and e) about 10 mM L-methionine, wherein the antibody formulation optionally comprises NaCl, preferably 1-100 mM NaCl, more preferably 30-60 mM NaCl, most preferably about 50 mM NaCl.

[0043] In a preferred of embodiment, the antibody formulation comprises: a) about 10 mg / mL nivolumab, b) about 20 mM histidine buffer, c) about 140 mM trehalose, d) about 0.02 % w / v polysorbate 80, and e) about 10 mM L-methionine, wherein the antibody formulation optionally comprises NaCl, preferably 1-100 mM NaCl, more preferably 30-60 mM NaCl, most preferably about 50 mM NaCl. In a preferred of embodiment, antibody formulation has a Tmvalue which is at least about 69 °C when subjected to subjected to a thermal ramp from 25°C to 95°C at a rate of l°C / min.

[0044] In a preferred of embodiment, antibody formulation has a Turbidity value which is at least 71 °C when subjected to subjected to a thermal ramp from 25°C to 95°C at a rate of l°C / min.

[0045] In a preferred of embodiment, antibody formulation has a TON which is at least 65 °C when subjected to subjected to a thermal ramp from 25°C to 95°C at a rate of l°C / min.

[0046] In a preferred of embodiment, the pH of the antibody formulation is at least pH 6.0, more preferably pH 6.0-6.3, most preferably about pH 6.3.

[0047] In a preferred of embodiment, the antibody formulation is a liquid antibody formulation, preferably an aqueous antibody formulation.

[0048] In a preferred of embodiment, the antibody formulation is a reconstituted solution from a lyophilized formulation.

[0049] In a preferred of embodiment, the antibody formulation is a solid formulation, preferably lyophilized formulation.

[0050] In a preferred of embodiment, the antibody formulation comprises less than 6000 counts sub- visible particles above 10 pm.

[0051] In a preferred of embodiment, the antibody formulation comprises less than 600 counts sub- visible particles above 25 pm.

[0052] In a preferred of embodiment, the antibody formulation is for use in the treatment of cancer, preferably in the treatment of cancer, preferably treatment of cancer of a human patient.

[0053] In a preferred of embodiment, the antibody formulation is for use in the treatment of melanoma, non-small cell lung cancer, malignant pleural mesothelioma, renal cell carcinoma, classical Hodgkin lymphoma, squamous cell carcinoma of the head and neck, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, esophageal or gastroesophageal junction cancer, and / or gastric cancer.

[0054] In a preferred of embodiment, the antibody formulation is for use in intravenous administration, preferably for use in intravenous infusion.

[0055] In a preferred embodiment, the antibody formulation is stable at about 2 °C to about 8°C for at least 12 months, preferably stable at about 5 °C for at least 12 months. In a preferred embodiment, the formulation has been determined to be stable after exposure to about 2 °C to about 8 °C for about 12 months, preferably stable at about 5 °C for at least 12 months.

[0056] In a preferred embodiment, the antibody formulation is stable at about 2 °C to about 8 °C for about 12 months (preferably stable at about 5 °C for at least 12 months), wherein stability of the stable antibody formulation is defined based on one or more of: a. a relative main peak area of nivolumab > about 98 % as measured by SE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably about > 99 %, b. a relative peak area of HMW variants < about 0.5 % as measured by SE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably < about 0. 4%, c. a relative peak area of LMW variants < about 0.5 % as measured by SE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably < about 0.4 %, d. a relative main peak area of nivolumab > about 74 % as measured by CE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably about > 75 %, e. a relative acidic species of nivolumab < about 21 % as measured by CE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably < about 20 %, f. a relative basic species of nivolumab < about 5 % as measured by CE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, g. a relative main peak area of nivolumab > about 97 % as measured by non-reducing CE-SDS after exposure to about 2 °C to about 8 °C for about 12 months, preferably > about 98 %, h. a relative heavy chain (HC) peak of about 65 % to about 68 %, as measured by reducing CE-SDS after exposure to about 2 °C to about 8 °C for about 12 months, preferably about 67 % to about 68 %, and, i. a relative light chain (LC) peak of about 31 % to about 33 % as measured by reducing CE-SDS after exposure to about 2 °C to about 8 °C for about 12 months, preferably about 31 % to about 32 %, wherein said after exposure to about 2 °C to about 8 °C for about 12 months disclosed in steps a-i is preferably an exposure to about 5 °C for about 12 months.

[0057] In a preferred embodiment, the antibody formulation is stable at about 2 °C to about 8 °C for about 12 months, as characterized by one or more of: a. a relative main peak area of nivolumab > about 98 % as measured by SE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably about > 99 %, b. a relative peak area of HMW variants < about 0.5 % as measured by SE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably < about 0.4 %, c. a relative peak area of LMW variants < about 0.5 % as measured by SE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably < about 0.4 %, d. a relative main peak area of nivolumab > about 74 % as measured by CE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably about > 75 %, e. a relative acidic species of nivolumab < about 21 % as measured by CE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably < about 20 %, f. a relative basic species of nivolumab < about 5 % as measured by CE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, g. a relative main peak area of nivolumab > about 97 % as measured by non-reducing CE-SDS after exposure to about 2 °C to about 8 °C for about 12 months, preferably > about 98 %, h. a relative heavy chain (HC) peak of about 65 % to about 68 %, as measured by reducing CE-SDS after exposure to about 2 °C to about 8 °C for about 12 months, preferably about 67 % to about 68 %, and i. a relative light chain (LC) peak of about 31 % to about 33 % as measured by reducing CE-SDS after exposure to about 2 °C to about 8 °C for about 12 months, preferably about 31 % to about 32 %, wherein said after exposure to about 2 °C to about 8 °C for about 12 months disclosed in steps a-i is preferably an exposure to about 5 °C for about 12 months.

[0058] In a preferred embodiment, the antibody formulation has one or more of the following characteristics: a. a relative main peak area of nivolumab > about 98 % as measured by SE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably about > 99 %, b. a relative peak area of HMW variants < about 0.5 % as measured by SE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably < about 0.4 %, c. a relative peak area of LMW variants < about 0.5 % as measured by SE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably < about 0.4 %, d. a relative main peak area of nivolumab > about 74 % as measured by CE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably about > 75 %, e. a relative acidic species of nivolumab < about 21 % as measured by CE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably < about 20 %, f. a relative basic species of nivolumab < about 5 % as measured by CE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, g. a relative main peak area of nivolumab > about 97 % as measured by non-reducing CE-SDS after exposure to about 2 °C to about 8 °C for about 12 months, preferably > about 98 %, h. a relative heavy chain (HC) peak of about 65 % to about 68 %, as measured by reducing CE-SDS after exposure to about 2 °C to about 8 °C for about 12 months, preferably about 67 % to about 68 %, and i. a relative light chain (LC) peak of about 31 % to about 33 % as measured by reducing CE-SDS after exposure to about 2 °C to about 8 °C for about 12 months, preferably about 31 % to about 32 %, wherein said after exposure to about 2 °C to about 8 °C for about 12 months disclosed in steps a-i is preferably an exposure to about 5 °C for about 12 months.

[0059] In a preferred embodiment, the antibody formulation is stable through 5 to 10 freeze-thaw cycles.

[0060] In a preferred embodiment, the antibody formulation is stable through 5 to 10 freeze-thaw cycles, as characterized by: a. a relative main peak area of nivolumab > about 99 % as measured by SE-HPLC, and / or b. a relative peak area of HMW variants < about 0.5 % as measured by SEC-HPLC.

[0061] In a preferred embodiment, the formulation has a volume of 4 ml, 10 ml, 12 ml or 24 ml.

[0062] In a preferred embodiment, nivolumab is a nivolumab biosimilar.

[0063] The second aspect of the invention relates to a stable antibody formulation comprising antibody, histidine buffer, sucrose or trehalose, and methionine. The antibody formulation is free of pentetic acid, enzymes and / or arginine.

[0064] A second aspect of the invention relates to a stable liquid antibody formulation for use in intravenous administration, comprising: a. about 10-40 mg / mL antibody, b. about 10-50 mM L-histidine buffer, c. about 70-270 mM sucrose or about 70-270 mM trehalose, d. about 0.005-0.08 % w / v polysorbate, and e. about 10-20 mM L-methionine.

[0065] In a preferred of embodiment, the antibody formulation comprises: a. about 10-40 mg / mL antibody, b. about 10-50 mM L-histidine buffer, c. about 140-270 mM sucrose or about 70-270 mM trehalose, d. about 0.005-0.08 % w / v polysorbate 80, and e. about 10-20 mM L-methionine.

[0066] In a preferred of embodiment, the antibody formulation comprises: a. about 10-40 mg / mL antibody, b. about 20 mM histidine buffer, c. about 270 mM sucrose, d. about 0.005-0.08 % w / v polysorbate 80, and e. about 10 mM L-methionine.

[0067] In a preferred of embodiment, the antibody formulation comprises: a. about 10-40 mg / mL antibody, b. about 20 mM histidine buffer, c. about 270 mM trehalose, d. about 0.005-0.08 % w / v polysorbate 80, and e. about 10 mM L-methionine.

[0068] In a preferred of embodiment, the antibody formulation comprises about 0.01-0.02 % w / v polysorbate 80, preferably about 0.02 % w / v polysorbate 80.

[0069] In a preferred of embodiment, the pH of the antibody formulation is at least pH 6.0, more preferably pH 6.0-6.3, most preferably about pH 6.3.

[0070] In a preferred of embodiment, the antibody is nivolumab.

[0071] In a preferred of embodiment, the antibody formulation comprises less than 6000 counts sub- visible particles above 10 pm. In a preferred of embodiment, the antibody formulation comprises less than 600 counts sub- visible particles above 25 pm.

[0072] In a preferred of embodiment, antibody formulation has a Tmvalue which is at least about 69 °C, preferably when subjected to subjected to a thermal ramp from 25°C to 95°C at a rate of l°C / min.

[0073] In a preferred of embodiment, antibody formulation has a Turbidity value which is at least 71 °C, preferably when subjected to subjected to a thermal ramp from 25°C to 95°C at a rate of l°C / min.

[0074] In a preferred of embodiment, antibody formulation has a TON which is at least 65 °C, preferably when subjected to subjected to a thermal ramp from 25°C to 95°C at a rate of l°C / min.

[0075] In a preferred of embodiment, the antibody formulation is for use in the treatment of cancer, preferably in the treatment of cancer, preferably treatment of cancer of a human patient.

[0076] In a preferred of embodiment, the antibody formulation is for use in the treatment of melanoma, non-small cell lung cancer, malignant pleural mesothelioma, renal cell carcinoma, classical Hodgkin lymphoma, squamous cell carcinoma of the head and neck, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, esophageal or gastroesophageal junction cancer, an / or gastric cancer.

[0077] A third aspect of the invention relates to a container comprising the antibody formulation according to the first or second aspects of the invention. The container may be a vial, bag, bottle and / or an injection device.

[0078] In a preferred of embodiment, the container is a glass vial comprising a pierceable stopper, preferably a glass vial comprising a bromobutyl or chlorobutyl stopper, more preferably a glass vial comprising a latex-free bromobutyl or chlorobutyl stopper.

[0079] A fourth aspect of the invention relates to a delivery device comprising the antibody formulation according to the first or second aspects of the invention. A fifth aspect of the invention relates to a kit of parts comprising the antibody formulation according to the first or second aspects of the invention or a container according to the third aspect of the invention.

[0080] A sixth aspect of the invention is a method of formulating a stable antibody formulation comprising or consisting of a) about 10 mg / mL nivolumab, b) about 20 mM histidine buffer, c) about 270 mM sucrose, d) about 0.02 % w / v polysorbate 80, e) about 10 mM L-methionine, and f) water, comprising the step of diluting a stable antibody formulation comprising or consisting of a) about 40 mg / mL nivolumab, b) about 20 mM histidine buffer; c) about 270 mM sucrose, d) about 0.02 % w / v polysorbate 80, e) about 10 mM L-methionine, and f) water, with an aqueous formulation buffer comprising or consisting of 20 mM L-histidine buffer, 10 mM L-methionine, 270 mM sucrose and 0.02 % w / v polysorbate 80. Preferably, the pH of the formulation buffer is at least 6.0, more preferably about 6.0-6.3, most preferably about 6.3.

[0081] In a preferred embodiment, the method of formulating a stable antibody formulation comprising or consisting of a) about 10 mg / mL nivolumab, b) about 20 mM histidine buffer; c) about 270 mM sucrose, d) about 0.02 % w / v polysorbate 80, e) about 10 mM L-methionine, and f) water, comprising the step of diluting 1 part of a stable antibody formulation comprising or consisting of a) about 40 mg / mL nivolumab, b) about 20 mM histidine buffer; c) about 270 mM sucrose, d) about 0.02 % w / v polysorbate 80, e) about 10 mM L-methionine, and f) water, with 3 parts of an aqueous formulation buffer comprising or consisting of 20 mM L-histidine buffer, 10 mM L-methionine, 270 mM sucrose and 0.02 % w / v polysorbate 80. Preferably, the pH of the formulation buffer is at least 6.0, more preferably about 6.0-6.3, most preferably about 6.3.

[0082] In a preferred embodiment of the method of formulating the stable antibody formulation, the resulting formulation is stored at about 2°C to about 8°C, preferably the resulting formulation is stored at about 5 °C.

[0083] In a preferred embodiment of the method of formulating the stable antibody formulation, nivolumab has been prepared by the steps comprising: a. fed batch cell culturing, b. harvesting the cell culture, c. clarifying the harvested cell culture suspension, d. affinity chromatography, e. ion exchange chromatography, and f ultrafiltrati on / diafiltration, wherein the step a-f are preferably in sequential order.

[0084] A seventh aspect of the invention is a stable antibody formulation obtainable by the method according to the sixth aspect of the invention. DESCRIPTION OF FIGURES

[0085] Figure 1 - Table for Excipient Screening - Thermal Unfolding.

[0086] Figure 2 - Table for Excipient Screening - Isothermal Incubation.

[0087] Figure 3 - 10 mg / ml nivolumab formulations F38, F46, FY and FX - Sub-visible particle counts in 2R vials for particles sizes above 10 pm.

[0088] Figure 4 - 10 mg / ml nivolumab formulations F38, F46, FY and FX - Sub-visible particle counts in 2R vials for particles sizes above 25 pm.

[0089] Figure 5 - 40 mg / ml nivolumab formulations F38-DS, F46-DS, FY-DS and FX-DS - Sub- visible particle counts in 2R vials for particle sizes above 10 pm.

[0090] Figure 6 - 40 mg / ml nivolumab formulations F38-DS, F46-DS, FY-DS and FX-DS - Sub- visible particle counts in 2R vials for particle sizes between 5-10 pm.

[0091] Figure 7 - 40 mg / ml nivolumab formulations F38-DS, F46-DS, FY-DS and FX-DS - Sub- visible particle counts at zero weeks in ambient temperature as well as at 2 week-time point at 5 °C, 25 °C, 40 °C, -20 °C and -80 °C.

[0092] Figures 8a and 8b - 10 mg / ml nivolumab formulations F38, F46, FY and FX - Sub-visible particle counts of particles sizes above 10 pm in 2R vials having bromobutyl stoppers. The formulations were stored inverted at 5 °C for 0 weeks, 1 week, 6 weeks, 7 weeks and 3 months.

[0093] Figures 9a and 9b - 10 mg / ml nivolumab formulations F38, F46, FY and FX - Sub-visible particle counts of particles sizes above 25 pm in 2R vials having bromobutyl stoppers. The formulations were stored inverted at 5 °C for 0 weeks, 1 week, 6 weeks, 7 weeks and 3 months.

[0094] Figure 10 - 40 mg / ml nivolumab formulations F38-DS, F46-DS, FY-DS and FX-DS - nrCE- SDS analysis of aggregates, i.e. high molecular weight (HMW) variants.

[0095] Figure 11 - 40 mg / ml nivolumab formulations F38-DS, F46-DS, FY-DS and FX-DS - nrCE- SDS analysis of nivolumab.

[0096] Figure 12 - 40 mg / ml nivolumab formulations F38-DS, F46-DS, FY-DS and FX-DS - SE- HPLC analysis of degraded products, i.e. low molecular weight (LMW) variants. Figure 13 - 40 mg / ml nivolumab formulations F38-DS, F46-DS, FY-DS and FX-DS - SE- HPLC analysis of aggregates, i.e. HMW variants.

[0097] Figure 14 - 40 mg / ml nivolumab formulations F38-DS, F46-DS, FY-DS and FX-DS - SE- HPLC analysis of nivolumab.

[0098] DETAILED DESCRIPTION

[0099] The present invention relates to stable formulations comprising nivolumab as active pharmaceutical ingredient and wherein said formulations further comprise pharmaceutical acceptable excipients such as buffer, stabilizer, surfactant and antioxidant. The preferred excipients are L-histidine as buffer, trehalose or sucrose as stabilizer, and methionine as antioxidant. The formulations will be used for the treatment of cancer by intravenous administration.

[0100] The formulations according to the present invention are more thermostable than Opdivo® as well as a nivolumab biosimilar formulated as Opdivo®. Additionally, the counts of sub- visible particles bigger than 10 pm and 25 pm are lower in the formulations according to the present invention when compared to a nivolumab biosimilar formulated as Opdivo®. Hence, the problems related to stability issues, as well as particle formation, are solved by the present invention.

[0101] The invention may be carried out at various concentrations of nivolumab and excipients such as about 10-40 mg / mL nivolumab, about 10-50 mM L-histidine buffer, about 70-270 mM sucrose or trehalose, and about 10 mM L-methionine. The formulation may further comprise a polysorbate such as polysorbate 80 as a surfactant.

[0102] An antibody formulation according to the present invention may comprise either sucrose or trehalose, i.e. preferred formulations do not comprise a mixture of sucrose and trehalose. The amount of nivolumab may be varied 10-40 mg / ml in a formulation. The preferred concentration in the drug product (DP) which is to be administered to the patient is about 10 mg / ml nivolumab while the drug substance (DS) may comprise a higher concentration such as about 40 mg / ml.

[0103] The concentration of sucrose and trehalose may be varied between 70-270 mM. In formulations which comprise NaCl, the most optimal concentration of sucrose and trehalose is about 140 mM. In formulations which are free from NaCl, the most optimal concentration of sucrose and trehalose is about 270 mM. The preferred compositions comprising 140 nm or 270 nm carbohydrate all comprise 20 mM L-histidine buffer, 10 mM L-methionine and 0.02 w / v % polysorbate 80.

[0104] Although any type of nivolumab may be used in the present invention, the preferred nivolumab is the nivolumab biosimilar prepared by using the DNA construct, expression vector, expression cassette, host cell and / or method of expressing disclosed in the claims of granted patents SE543998,C2, SE544006 C2, AU2021239744 B2 and KR102520468 Bl. This nivolumab biosimilar is hereinafter referred to as Xdivane.

[0105] In an embodiment of the invention, the cell culture process of expressing Xdivane is a fed batch process involving a single use bioreactor. The harvested cell suspension is clarified before being subjected to chromatography such as affinity chromatography. The material may further be purified by ion exchange chromatography as well as ultrafiltration / diafiltration (UF / DF). The resulting bulk drug substance is preferably filled into sterile single use bags that provide thermal performance down to -80 °C.

[0106] In the present specification, results from extensive comparative experiments have been presented on the formulations which have been researched and developed (see examples 1- 13). In example 1, the results of Thermal Unfolding Analysis, Isothermal Incubation Analysis and kDAnalysis of screened formulations have been discussed. Thermal Unfolding Analysis, Isothermal Incubation Analysis and kn Analysis have been used to assess the thermal stability, colloidal stability and aggregation propensity, respectively.

[0107] According to said comparative experiments discussed in example 1, the two most stable formulations are formulation 38 (F38) and formulation 46 (F46) having the following compositions:

[0108] F38: 10 mg / ml nivolumab, 20 mM L-histidine buffer, 270 mM sucrose, 10 mM L- methionine and 0.02 w / v % polysorbate 80, pH 6.3, and

[0109] F46: 10 mg / ml nivolumab, 20 mM L-histidine buffer, 270 mM trehalose, 10 mM L- methionine and 0.02 w / v % polysorbate 80, pH 6.3

[0110] The above-mentioned formulations F38 and F46, which both comprise the nivolumab biosimilar Xdivane, had higher thermostability and colloidal stability when compared to all other screened and tested formulation. Moreover, said two formulations were also more stable than commercially sold Opdivo® as well as Xdivane formulated according to Opdivo® formulation.

[0111] Furthermore, as discussed in examples 2 and 5, sub-visible particle formation in the abovediscussed two formulations F38 and F46 was compared with sub-visible particle formation in the following two formulations referred to as FX and FY:

[0112] - FX: Xdivane formulated according to Opdivo®, i.e. an Opdivo® biosimilar, and

[0113] - FY: Xdivane formulated according to Opdivo® but lacking pentetic acid.

[0114] The results indicate that formulations F38 and F46 have lower amounts of sub-visible particles above 10 pm when compared to formulations FX and FY. Moreover, the results also indicate that formulations F38 and F46 have lower amounts of sub-visible particles above 25 pm when compared to formulations FX and FY. Thus, formulations F38 and F46 have less formation of sub-particles above 10 pm and 25 pm when compared to formulation FX which is an Opdivo® biosimilar.

[0115] Hence, in summary, formulations F38 and F46 have higher thermostability and colloidal stability, as well as lower amounts of particle formation, when compared to Opdivo® and a biosimilar of Opdivo®.

[0116] Furthermore, as indicated in examples 2-7 formulations F38 and F46, as well as their versions comprising 40 mg / ml nivolumab referred to as F38-DS and F46-DS, have further technical advantages when compared with a biosimilar of Opdivo®. These technical advantages are lower amounts of (a) sub-visible particles, (b) high molecular weight variants, and (c) low molecular weight variants. Consequently, formulations F38 and F46, as well as their version comprising 40 mg / ml nivolumab, are more stable when compared to a biosimilar of Opdivo®.

[0117] The antibody formulations according to the present invention are preferably administered as liquid formulations which are in the art also referred to as parenteral solutions, injectable solution or injectables. The liquid formulations may be packaged as Small Volume Injectable (SVI) solutions, preferably as SVI solutions having a volume less than 100 ml packaged in containers such as ampoules, vials, small bags and pre-filled syringes. The preferred containers are single-dose vials which have the inner volume to allow containment of 4 ml, 10 ml, 12 ml or 24 ml of an antibody formulation according to the present invention. The preferred strengths of the antibody formulations are 40 mg / 4 ml nivolumab, 100 mg / 10 ml nivolumab, 120 mg / 12 ml nivolumab, and 240 mg / 24 ml nivolumab in each single-dose vial. The SVI solutions are to be stored under refrigeration at 2 °C to 8 °C, or alternatively at lower temperatures such as -80 °C, and then protected from light until the time of use.

[0118] A liquid antibody formulation can be made by taking a composition comprising nivolumab (e.g. 8 mg / ml) and buffer exchanging it into a histidine buffer at one of the last steps of the downstream purification process and then concentrating it to about 40 mg / ml. Sucrose or trehalose, as well as L-methionine and polysorbate 80, are then added to the composition and the resulting antibody formulation may be filtered into containers such as bulk drug substance (BDS) bags and thereafter stored under refrigeration at 2 °C to 8 °C or alternatively at lower temperatures such as -80 °C.

[0119] A first preferred embodiment of the target levels of the compounds in a container such as a BDS bag is about 40 mg / ml nivolumab, about 270 mM sucrose, about 10 mM L-methionine and about 0.02 % w / v polysorbate 80. In a second preferred embodiment, 270 mM sucrose is exchanged with about 270 mM trehalose. These two embodiments, referred to as F38-DS and F46-DS in examples 3 and 4, showed lower sub-visible particle formation when compared to formulation FX-DS which comprises 40 mg / ml nivolumab Xdivane biosimilar formulated as Opdivo®.

[0120] For the production of the drug product, the contents of the BDS bags are diluted using a histidine buffer to final concentration of about 10 mg / ml nivolumab and the excipients are spiked into the resulting formulation to attain target levels of each excipient. An example of the target levels of the compounds in the drug product is about 10 mg / ml nivolumab, about 270 mM sucrose, about 10 mM L-methionine and about 0.02 % w / v polysorbate 80 at a pH of about 6.3. In an alternative example, 270 mM sucrose is exchanged with 270 mM trehalose. These two embodiments, referred to as F38 and F46 in examples 2 and 5, showed lower sub-visible particle formation when compared to Xdivane biosimilar formulated as Opdivo®.

[0121] The drug product is preferably stored under refrigeration at 2 °C to 8 °C until the drug product is to be filtered into containers such as ampoules, vials, small bags and pre-filled syringes. The preferred containers are vials that are suitable for pharmaceutical packaging, preferably Type 1 borosilicate glass vials made from materials such as Schott’s Fiolax® clear tubings which are clear, neutral and chemically highly resistant. Although a liquid antibody formulation is the preferred type of formulation, a formulation according to the present invention may also be provided as a lyophilized formulation or as a liquid antibody formulations that is reconstituted from a lyophilized formulation.

[0122] An antibody formulation according to the present invention is used for the treatment of disease, preferably treatment of cancer, more preferably treatment of melanoma, non-small cell lung cancer, malignant pleural mesothelioma, renal cell carcinoma, classical Hodgkin lymphoma, squamous cell carcinoma of the head and neck, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, esophageal or gastroesophageal junction cancer and / or gastric cancer.

[0123] The following compositions referred to as F03 and comprising NaCl may also be used as an antibody formulation although it appears to be less thermostable than NaCl-free formulations F38 and F46:

[0124] F03: 10 mg / ml nivolumab, 50 mM NaCl, 20 mM L-histidine buffer, 140 mM sucrose, 10 mM L-methionine, 0.02 w / v % polysorbate 80 and pH 6.3

[0125] NaCl-comprising formulations such as F03 may have uses when a lower sucrose concentration is more favorable in an antibody formulation.

[0126] It is to be noted that, in the present invention, the singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise. The present invention also contemplates other embodiments "comprising," "consisting of and "consisting essentially of," the embodiments or elements presented herein, whether explicitly set forth or not.

[0127] The term "about", when used for indicating the quantity or concentration (e.g. mM, mg / ml or w / v %) of a formulation component, or the pH value of a formulation, or relative peak area, refers to variations in the numerical quantity that can occur. Due to said variations "about" can mean a variation of plus or minus (±) 10 percent (%).

[0128] The term "buffer" indicates compounds that maintain the solution pH of the antibody formulations of the present invention in an acceptable range, or, for lyophilized formulations of the invention, provide an acceptable solution pH before lyophilization. A "stable" antibody formulation is one in which nivolumab substantially retains its physical, chemical, thermal, colloidal and / or biological stability.

[0129] The term "cancer" indicates the physiological condition in a human which is characterized by unregulated cell growth.

[0130] The publications referenced in the present specification are incorporated for the purpose of describing and disclosing methods and products that might be employed in connection with the present invention.

[0131] The following examples are offered by way of illustration and not by way of limitation.

[0132] EXAMPLES

[0133] Antibody formulations comprising nivolumab according to the present invention were screened as explained in example 1. Furthermore, the analytical methods outlined in examples 2-13 were used for assessing the stability of the formulations.

[0134] Example 1 relates to the high-throughput screening methodology which was used for identifying stable formulations comprising nivolumab. Although the high-throughput screening was conducted by employing screening methods described in the art [2, 3], the specific high-throughput screening described in Example 1 was conducted with similar instrument, methodology and excipients employed in US2023355757 in which 36 formulations were subjected to excipient screening (paragraphs 162-175; Table 1) to arrive at the most stable formulations which comprised either 205 mM trehalose (claim 16) or 205 mM mannitol (claim 17) in combination with 10 mM L-histidine / histidine hydrochloride, 0.1 mg / ml to 0.4 mg / ml polysorbate 20 or polysorbate 80, 25 mg / ml pembrolizumab and water for injection, and furthermore having a pH of 5.5 to 5.9 (claims 16 and 17). However, US2023355757 explicitly discloses that the claimed invention performs better without methionine (paragraph 0214) and US2023355757 consequently relates to formulations that do not contain methionine (paragraph 0080 and 0082) and the claimed formulations of US2023355757 therefore do not disclose methionine (claims 1-21). In other words, US2023355757 teaches away from the present invention which instead shows that methionine-containing formulations are more thermostable and have higher colloidal stability. Hence, although pembrolizumab and nivolumab are used for the same diseases (and both are anti-PDl inhibitors), they are thermostable in different types of chemical environments. In other words, every antibody is unique and must therefore be formulated with a unique “blend” of excipients having unique concentrations of said excipients.

[0135] Examples 2-5 relate to analysis of sub-visible particles in formulations comprising nivolumab. Particulate matter consists of mobile solids, randomly sourced, extraneous substances, other than gas bubbles, that cannot be quantified because of their heterogenous composition. These solids, which are unintentionally present, may contain glass, rubber, metal or plastic fragments derived from sources such as manufacturing environment, manufacturing personnel and packaging components [4],

[0136] Examples 6 and 7 relates to analyzing aggregates and degraded products in formulations comprising nivolumab. Aggregates of monoclonal antibodies can be induced during the manufacturing process due to parameters such as pH, temperature, reactive oxygen species or shear forces and contribute to the heterogeneity profile of the drug substance and drug product [5], These types of aggregates are regarded as product-related impurities since they are known to affect drug activity and safety [5], Formation of aggregates and degraded products have been analyzed by using nr-CE-SDS and SE-HPLC as described in examples 6 and 7, respectively.

[0137] Example 8 relates to freeze-thaw (F / T) characterization of formulations comprising nivolumab. Proteins are commonly exposed to the freeze-thawing events during bulk drug substance handling, manufacturing, and storage, as well as potential excursions during shipping [6], Changes to the protein conformation such as partial unfolding of protein molecules during F / T may result in protein aggregation [6], Formation of aggregates and degraded products have been analyzed by using SE-HPLC.

[0138] Example 9 relates to evaluating the effect of freezing on aggregate formation in a formulation comprising 10 mg / ml nivolumab. Minimizing aggregation can reduce immunogenic responses and thereby render the nivolumab formulation safer for patients to use.

[0139] Example 10 relates to assessment of stability of the of formulation F38 by using SE-HPLC, nr-CE-SDS and r-CE-SDS analytical methods. Stability was characterized by analyzing monomers of nivolumab (i.e. main peak) as well as aggregates and degraded products in the F38 formulation stored upright in vials for 1, 2 and 12 months at 5 °C. The effects of stressed degradation (25 °C) and accelerated degradation (40 °C) on formulation F38 was also analyzed.

[0140] Example 11 relates to assessment of charge variants of nivolumab in formulation F38 by applying CE-HPLC on F38 formulations stored upright in vials for 1, 2 and 12 months at 5 °C. The effects of stressed degradation (25 °C) and accelerated degradation (40 °C) on formulation F38 was also analyzed.

[0141] Example 12 relates to assessment of stability of the of formulation F38 by using SE-HPLC, nr-CE-SDS and r-CE-SDS analytical methods. Stability was characterized by analyzing monomers of nivolumab (i.e. main peak) as well as aggregates and degraded products in the F38 formulation stored inverted in vials for 1, 2 and 12 months at 5 °C.

[0142] Example 13 relates to assessment of charge variants of nivolumab in formulation F38 by applying CE-HPLC on F38 formulations stored inverted in vials for 1, 2 and 12 months at 5 °C.

[0143] EXAMPLE 1 - High-throughput screening

[0144] The high-throughput screening methodology for testing liquid antibody formulations comprised the two main stages of solubility screening and excipient screening as summarized in table 1.

[0145] Table 1 - Screening parameters and ranges.

[0146] Solubility screening

[0147] The methods of Thermal Unfolding Analysis, Isothermal Incubation Analysis and kn Analysis described in the previous section were used for assessing the optimal stability under thermal stress of various formulation comprising 10 mg / mL nivolumab across different pH conditions and NaCl concentrations ranging from pH 4-8 and 10-135 mM NaCl.

[0148] Based on the results, which involved 36 screened formulations (data not shown), a highly stable formulation comprising 20 mM histidine buffer and 50 mM NaCl having a pH of 6.3 was selected as the most promising steppingstone for the next step in the screening process, i.e. excipient screening.

[0149] Excipient screening

[0150] The aim of the excipient screening was to identify formulations having improved colloidal stability. The excipient screening of formulations comprising 10 mg / mL nivolumab was analyzed across different concentrations of histidine, NaCl, arginine, methionine, sucrose, sorbitol and trehalose. The results from the Thermal Unfolding Analysis are shown in the table disclosed in figure 1. As explained earlier, the more thermostable a given formulation, the higher the T m, Tturbidity and TON.

[0151] Surprisingly, 10 mg / mL nivolumab was more stable in NaCl-free formulations comprising methionine as well as a high concentration of either sucrose or trehalose. This is indicated by the Tm, Tturbidity and TON values that are higher for formulations 35-46 which are NaCl-free when compared to formulations 1-34 which all comprise NaCl and have lower T m, Tturbidity and TON values.

[0152] Importantly, formulations 38 and 46 have the highest Tm, Tturbidity and / or TON values of the screened formulations which renders them the most thermostable formulations of all that were tested:

[0153] - Formulation 38 has Tm, Tturbidity and TON of 69.50, 71.22 and 65.36, and

[0154] - Formulation 46 has Tm, Tturbidity and TON of 69.65, 71.30 and 65.44.

[0155] More importantly, formulations 38 and 46 have higher Tm, Tturbidity and / or TON values when compared to the originator formulation of Opdivo® as well the nivolumab biosimilar Xdivane formulated with the same concentrations of excipients as in Opdivo® (see the last two formulations in the table in figure 1):

[0156] Xdivane formulated as Opdivo® has Tm, Tturbidity and TON of 67.73, 68.03 and 63.52, and

[0157] Opdivo® has Tm, Tturbidity and TON of 67.66, 67.96 and 63.50.

[0158] Consequently, formulations 38 and 46 are more thermostable than commercially sold Opdivo® as well as Xdivane formulated as Opdivo®.

[0159] The results from the Isothermal Incubation Analysis are shown in the table in figure 2. As explained earlier, the more stable the formulation, the lower the percentage change observed for Tm, Tturbidity and TON.

[0160] Based on the resulting excipient screen isothermal incubation data, optimal isothermal stability was observed in NaCl-free formulations comprising methionine and high concentration of carbohydrates. As indicated in the table in figure 2, formulations 38 and 46 displayed the lowest % changes of 72.2 % and 67.9 % in their respective TON values indicating the lowest levels of aggregation.

[0161] Hence, just like Thermal Unfolding Analysis, also Isothermal Incubation Analysis indicated formulations 38 and 46 as having the best technical effects:

[0162] - Formulation 38 having % change of Tm, Turbidity and TON of 0.1 %, 0 % and 72.2 %, and

[0163] - Formulation 46 having % change of Tm, Turbidity and TON of 0.3 %, 0.1 % and 67.9 %

[0164] More importantly, formulations 38 and 46 had the lowest % changes of Tm, Turbidity and / or TON values when compared to the originator formulation of Opdivo® as well the nivolumab biosimilar Xdivane formulated with the same concentrations of excipients as in Opdivo® (see the last two formulations the table in figure 2):

[0165] Xdivane formulated as Opdivo® having % change of Tm, Turbidity and TON of 4.7 %, 57.5 % and 1315.8 %, and

[0166] Opdivo® having % change of Tm, Turbidity and TON of 4.5 %, 76.6 % and 3566.2 %

[0167] Consequently, formulations 38 (F38) and 46 (F46) form less aggregates than commercially sold Opdivo® as well as Xdivane formulated as Opdivo®.

[0168] The results from the kDAnalysis are shown in the below table 2. As explained earlier, a large negative value is indicative of increased propensity to aggregate while a positive value is indicative of a lower propensity for aggregation. Interestingly, formulations F38 and F46 emerged as the top performers also in the kDAnalysis.

[0169] Table 2 - kn Analysis

[0170] In summary, Thermal Unfolding Analysis, Isothermal Incubation Analysis and kDAnalysis all showed the unexpected and surprising technical effects of formulation 38 and 46 having higher stability than commercially sold Opdivo®.

[0171] In other words, the two most stable formulations are formulation 38 (F38) and formulation 46 (F46) having the following compositions:

[0172] F38: 10 mg / ml nivolumab biosimilar Xdivane, 20 mM L-histidine buffer, 270 mM sucrose and 10 mM L-methionine, pH 6.3, and

[0173] F46: 10 mg / ml nivolumab biosimilar Xdivane, 20 mM L-histidine buffer, 270 mM trehalose and 10 mM L-methionine, pH 6.3.

[0174] The results relating to formulation 38 comprising 270 mM sucrose was especially surprising since state of the art documents relating to high-throughput screening do not disclose methionine-comprising pharmaceuticals being co-formulated with sucrose having concentrations as high as 270 mM sucrose which corresponds to 92.4 mg / ml sucrose [2, 3], Moreover, the use of high sucrose concentrations is typically not utilized for relatively low antibody concentrations of 10-40 mg / ml [2, 3, 11], Hence, this was a further surprising effect of formulation 38. EXAMPLE 2 - Sub-visible Particulate counts in 2R vials - 10 mg / ml nivolumab - F38, F46, FX and FY

[0175] Sub-visible particles can be inherent (e.g. protein aggregation), intrinsic (e.g. silicone particles from filling process) or extrinsic (e.g. human skin or dust) particles that are too large for detection by SE-HPLC and too small for the naked eye. This is important for antibody pharmaceuticals, as sub-visible particles contained in formulations may cause undesired immune reactions in patients.

[0176] The following formulations which had been prepared at ambient temperature were subjected to SvP analysis directly after being applied to 2R vials (i.e. time = 0):

[0177] F38: 10 mg / ml nivolumab biosimilar Xdivane, 20 mM L-histidine buffer, 270 mM sucrose, 10 mM L-methionine and 0.02 % w / v polysorbate 80, pH 6.3,

[0178] F46: 10 mg / ml nivolumab biosimilar Xdivane, 20 mM L-histidine buffer, 270 mM trehalose, 10 mM L-methionine and 0.02 % w / v polysorbate 80, pH 6.3,

[0179] FX: 10 mg / ml nivolumab biosimilar Xdivane, 20 mM citrate, 50 mM NaCl, 30 g / L mannitol, 0.008 g / L pentetic acid, 0.02 % w / v polysorbate 80, pH 6.0, and

[0180] FY: 10 mg / ml nivolumab biosimilar Xdivane, 20 mM citrate, 50 mM NaCl, 30 g / L mannitol, 0.02 % w / v polysorbate 80, pH 6.0.

[0181] As indicated in chapter 11 of the HIGHLIGHTS OF PRESCRIBING INFORMATION, Opdivo® may contain few visible particles [1], Hence, it is a common knowledge that Opdivo® has issues with formation of visible particles. However, the HIGHLIGHTS OF PRESCRIBING INFORMATION is altogether silent about sub-visible particles, especially particulates larger than 10 pm and 25 pm. As already indicated, US Pharmacopoeia, Japanese Pharmacopoeia and Japanese Pharmacopoeia all require that sub-visible particles in small volume injectables must conform to the limits for USP <787> and USP <788> which are:

[0182] < 6000 counts of particles sizes above 10 pm, and

[0183] < 600 counts of particles sizes above 20 pm.

[0184] Example 1 indicated Opdivo® and nivolumab biosimilar Xdivane formulated as Opdivo® had similar thermal unfolding and k characteristics (see figure 1 and table 2). Hence, rather than comparing the formulations according to the present invention with Opdivo®, instead formulations F38 and F46 were compared with nivolumab biosimilar Xdivane formulated as Opdivo® (i.e. formulation FX). Moreover, the effect of pentetic acid on particle formation was investigated by also including Xdivane formulated as Opdivo® but lacking pentetic acid (i.e. formulation FY) in the comparative experiments.

[0185] The results of the experiments indicate that formulations F38 and F46 have lower amounts of sub-visible particles above 10 pm (figure 3) as well as lower amounts of sub-visible particles above 25 pm (figure 4) when compared to nivolumab biosimilar Xdivane formulated as Opdivo® (i.e. formulation FX).

[0186] Interestingly, removing pentetic acid appears to have advantageous effects since formulation FY had lower counts of sub-visible particles when compared to formulation FX. However, formulation FY had higher counts of sub-visible particles when compared to formulation F38 and F46.

[0187] As a final remark it is important to note that more than twice as many sub-visible particles per milliliter was formed in formulation FX when compared with formulations F38 and F46. Consequently, formulations F38 and F46 are more stable than nivolumab biosimilar Xdivane formulated as Opdivo®.

[0188] EXAMPLE 3 - SvP counts - 40 mg / ml nivolumab - F38-DS, F46-DS, FX-DS and FY-DS

[0189] SvP count analysis was also conducted on versions of F38, F46, FX and FY which comprised 40 mg / ml nivolumab biosimilar Xdivane instead of 10 mg / ml of the same biosimilar; however, the concentrations of the other compounds (i.e. excipients) were the same as in F38, F46, FX and FY. The resulting formulations which were named F38-DS, F46-DS, FX-DS and FY-DS had the following compositions:

[0190] F38-DS: 40 mg / ml nivolumab biosimilar Xdivane, 20 mM L-histidine buffer, 270 mM sucrose, 10 mM L-methionine and 0.02 % w / v polysorbate 80, pH 6.3, F46-DS: 40 mg / ml nivolumab biosimilar Xdivane, 20 mM L-histidine buffer, 270 mM trehalose, 10 mM L-methionine and 0.02 % w / v polysorbate 80, pH 6.3, FX-DS: 40 mg / ml nivolumab biosimilar Xdivane, 20 mM citrate, 50 mM NaCl, 30 g / L mannitol, 0.008g / L pentetic acid, 0.02 % w / v polysorbate 80, pH 6.0, and FY-DS: 40 mg / ml nivolumab biosimilar Xdivane, 20 mM citrate, 50 mM NaCl, 30 g / L mannitol, 0.02 % w / v polysorbate 80, pH 6.0. The samples were prepared at ambient temperature and subjected to SvP analysis directly after being applied to 2R vials (i.e. time = 0).

[0191] The results indicate that formulations F38-DS and F46-DS have lower amounts of sub-visible particles above 10 pm (figure 5) when compared to formulations FX-DS and FY-DS. Consequently, either one of the formulations F38-DS and F46-DS are more stable than FX- DS and FY-DS.

[0192] Moreover, although not required by USP <787> and USP <788>, the counts of sub-particles between 5 pm and 10 pm were also investigated and the results show that formulations F38- DS and F46-DS have lower amounts of sub-visible particles between 5 pm and 10 pm (figure 6) when compared to formulations FX-DS and FY-DS.

[0193] Hence, in summary, formulations F38-DS and F46-DS according to the present invention are more stable than formulations FX-DS and FY-DS.

[0194] EXAMPLE 4 - SvP counts after storage at different temperatures - 40 mg / ml nivolumab - F38-DS, F46-DS, FX-DS and FY-DS

[0195] SvP count analysis was conducted on formulations F38-DS, F46-DS, FX-DS and FY-DS after two weeks of storage at 5 °C, 25 °C, 40 °C, - 20 °C and - 80 °C. Moreover, said formulations were also subjected to SvP count analysis at ambient temperature directly after being placed in the 2R vials, i.e. at time = 0 weeks (further referred to as “t = Ow” in figure 7).

[0196] The results illustrated in figure 7 indicate that formulations F38-DS and F46-DS have lower amounts of sub-visible particles above 10 pm when compared to formulations FX-DS and FY-DS.

[0197] In summary, SvP analysis indicated that formulations F38-DS and F46-DS are more stable than FX-DS and FY-DS at 5 °C, 25 °C, 40 °C, - 20 °C and -80 °C after two weeks of storage as well more stable as t = 0 weeks analyzed at ambient temperature.

[0198] EXAMPLE 5 - SvP counts with bromobutyl stopper - 10 mg / ml nivolumab - F38, F46, FX and FY SvP count analyses for particles above 10 pm (figure 8) and 25 pm (figure 9) were conducted on formulations F38, F46, FX and FY which had been enclosed in 2R vials having bromobutyl stoppers. The formulations were stored inverted at 5 °C for 0 weeks, 1 week, 6 weeks, 7 weeks and 3 months. The bromobutyl stoppers which are commercially sold by West Pharmaceutical Services are latex-free and have product code INJW13RTS (West Art. 1104).

[0199] The results indicate that formulation F38 has lower amounts of sub-visible particles above 10 pm at all storage times when compared to all of the other formulations; see figures 8a and 8b, wherein figure 8b is a zoomed-in version of figure 8a.

[0200] Similarly, the results indicate that formulations F38 has lower amounts of sub-visible particles above 25 pm at all storage times when compared to all of the other formulations; see figures 9a and 9b, wherein figure 9b is a zoomed-in version of figure 9a.

[0201] Interestingly, the bromobutyl stopper resulted in the formation of high levels of sub-visible particles in formulation F46 and appears therefore not to be compatible with trehalose.

[0202] In summary, SvP analysis indicated that formulation F38 contained in 2R vials enclosed with bromobutyl stoppers is more stable than all other formulations when stored at 5 °C and incubated for 0 weeks, 1 week, 6 weeks, 7 weeks and 3 months.

[0203] EXAMPLE 6 - nrCE-SDS - 40 mg / ml nivolumab F38-DS, F46-DS, FX-DS and FY-DS

[0204] The stability of formulations F38-DS, F46-DS, FX-DS and FY-DS stored in 2R vials and which were thermally stressed at 40 °C for 6 weeks was analyzed by non-reduced CE-SDS (i.e. nrCE-SDS). The monomeric peak of nivolumab as well as aggregates and degraded fragments were separated and quantified. The aggregates and degraded fragments are in the art also referred to as high-molecular weight (HMW) variants and low-molecular weight (LMW) variants, respectively.

[0205] Figure 10 illustrates a bar graph which shows the percentage of HMW variants on the y-axis and degradation time in weeks in the x-axis. As clearly illustrated by figure 10, after 2, 4 and 6 weeks of thermal stress at 40 °C, formulations F38-DS and F46-DS comprised less HMWs when compared to FX-DS and FY-DS. Consequently, formulations F38-DS and F46-DS were more stable than FX-DS and FY-DS since less aggregates had been formed in formulations F38-DS and F46-DS. Figure 11 illustrates a line graph which shows the percentage of nivolumab on the y-axis and degradation time in weeks on the x-axis. The slope equations of the tested formulations have been disclosed in parenthesis wherein a less negative slope is indicative of less amounts of nivolumab being degraded over time: F38-DS (y=-0.705x + 97.94), F46-DS (y=-0.72x + 98.01), FX-DS (y = -0.74x + 97.82) and FY-DS (y = -0.78x + 97.89).

[0206] As indicated by figure 11 and above-mentioned slope equations, after 2, 4 and 6 weeks of thermal stress at 40 °C, formulations F38-DS and F46-DS exhibited less degradation of nivolumab over time (i.e. less negative slope) when compared to the percentage of nivolumab being degraded over time in formulations FX-DS and FY-DS. Consequently, formulations F38-DS and F46-DS were more stable than FX-DS and FY-DS since more intact protein was retained in formulations F38-DS and F46-DS.

[0207] In summary, CE-SDS analysis indicated that formulations F38-DS and F46-DS are more stable than formulations FX-DS and FY-DS.

[0208] EXAMPLE 7 - SE-HPLC - 40 mg / ml nivolumab - F38-DS, F46-DS, FX-DS and FY-DS

[0209] The monomeric peak of nivolumab as well as HMW and LMW variants were separated and quantified by using SE-HPLC, i.e. size-exclusion chromatography (SEC). The stability of each of the formulations F38-DS, F46-DS, FX-DS and FY-DS stored in 2R vials was analyzed at the below listed three storage conditions:

[0210] - time = 0 weeks (t=0w) at ambient temperature,

[0211] 6 weeks storage at 5 °C, and

[0212] 6 weeks storage at -80 °C.

[0213] Figure 12 illustrates a graph which shows the percentage of LMW variants on the y-axis and the tested formulations in the x-axis wherein each tested formulation is represented by three bar graphs illustrating the above listed three storage conditions. All of the tested formulations show similar results, i.e. low percentage of degradation at t=0w but more than twice the percentage after 6 weeks storage at 5 °C as well as -80 °C albeit at very low percentages of about 0.12-0.14.

[0214] Figure 13 illustrates a graph which shows the percentage of HMW variants. At all tested conditions, formulations F38-DS and F46-DS comprised less percentage of HMW variants when compared to FX-DS and FY-DS. Figure 14 illustrates a graph which shows the percentage of nivolumab (i.e. monomer). At all tested conditions, formulations F38-DS and F46-DS comprised higher percentage of nivolumab when compared to FX-DS and FY-DS.

[0215] In summary, SE-HPLC analysis indicated that formulations F38-DS and F46-DS are more stable than formulations FX-DS and FY-DS.

[0216] EXAMPLE 8 - SE-HPLC - Freeze-thaw (F / T) characterization - 40 mg / ml nivolumab - F38-DS, F46-DS, FX-DS and FY-DS

[0217] Formulations F38-DS, F46-DS and FX were subjected to 5 and 10 freeze-thaw (F / T) cycles in 2 ml 2R glass vials and aggregate formation was compared with samples which had not been subjected to any F / T cycles (i.e. t=0). Freeze-thaw (F / T) cycles were conducted by thawing a sample at ambient temperature for 1 hour before being refrozen. A maximum of 1 F / T cycle was performed each day.

[0218] The vials were sealed with bromobutyl stoppers and stored in upright position. One F / T cycle involved freezing at -80 °C overnight, i.e. not less than 16 hours, and thawing for not less than 6 hours at 5±3 °C. Samples were kept at -80 °C for the last F / T cycle before being thawed and analyzed.

[0219] As indicated in below table 3, none of the tested formulations were susceptible to aggregation by F / T stress although there was a very minor tendency for formation of HMW variants after 5 and 10 F / T cycles. In other words, the percentages of nivolumab was very high in all formulation, especially F38-DS and F46-DS which both comprised > 99.5 % nivolumab. LMW variants were not detectable in any of the samples and have therefore been omitted from the table.

[0220] Table 3 - SE-HPLC - 5 and 10 F / T cycles in vials Formulation F38-DS was also subjected to 3 F / T cycles in 6 liter Celsius® FFT bags. One F / T cycle involved freezing at -80 °C for at least 48 hours and thawing for not less than 24 hours at ambient temperature. Samples were kept at 5±3 °C after the final F / T cycle until they were analyzed.

[0221] The results shown in table 4 indicate that the percentages of nivolumab remained very high, 99.57 %, after 3 F / T cycles. LMW variants were not detectable.

[0222] Table 4 - SE-HPLC - 3 F / T cycles in 6 liter bags

[0223] EXAMPLE 9 - SE-HPLC - Freezing - 10 mg / ml nivolumab - Formulation F38

[0224] Formulation F38 was subjected to the temperature conditions of 5 °C, -20 °C and -80 °C for 1 month in order to evaluate the impact of freezing on nivolumab stability. The SE-HPLC studies were conducted on duplicate samples taken from 5 ml Flexboy® bags in which formulation F38 was stored.

[0225] The results shown in table 5 indicate that freezing did not generate significant levels of HMW variants, i.e. aggregates. More importantly, the level of nivolumab was > 99.5 % at all tested temperatures. Hence, formulation F38 having the below disclosed composition has high a very stability at 5 °C, -20 °C and -80 °C, and therefore, formulation 38 is optimal as a commercial pharmaceutical candidate:

[0226] F38: 10 mg / ml nivolumab biosimilar Xdivane, 20 mM L-histidine buffer, 270 mM sucrose, 10 mM L-methionine and 0.02 % w / v polysorbate 80, pH 6.3. Table 5 - SE-HPLC - Freezing in 5 ml bags

[0227] EXAMPLES 10-13 - F38 formulation - Background information In examples 10-13, the F38 formulation which had been stored upright (examples 10 and 11) and inverted (examples 12 and 13) in 2R vials was subjected to the analytical methods summarized in table 6 in order to assess stability and charge variants (i.e. post translational modifications) at various timepoints and temperatures.

[0228] Table 6: Analytical methods and their quality attributes

[0229] As indicated earlier the F38 formulation is an aqueous formulation consisting essentially of: a. about 10 mg / mL nivolumab, b. about 20 mM L-histidine buffer, c. about 270 mM sucrose, d. about 0.02 % w / v polysorbate 80, and e. about 10 mM L-methionine. EXAMPLE 10 - F38 formulation - Stored upright - Stability

[0230] The drug product according to the F38 formulation which is to be sold commercially will be stored under refrigeration at 2 °C to 8 °C until it is time to administer it to the patient via an intravenous infusion. Hence, short and long term stability, as well as charge variants (see example 11), were analyzed by carrying out the analytical methods at timepoints 0 months (TO), 1 month (Tl), 2 months (T2) and 12 months (T12) of samples stored at 5 °C. The vials which were stored upright were each sealed with a West® bromobutyl stopper.

[0231] SE-HPLC (SEC) data in table 7 shows that the percentage of nivolumab (i.e. “SEC Main”) in formulation F38 stored at 5 °C had only decreases by 0.1 % when timepoint TO (99.7 %) is compared with T2 (99.6 %). There was no decrease when timepoints TO and Tl are compared. Moreover, there was no increase of LMW variants when TO was compared with T2. Furthermore, only an increase of 0.1 % was observed for HMW variants when TO was compared with T2.

[0232] More importantly, SEC data at timepoint T12 showed that the percentage of nivolumab had only decreased by 0.5 % over the 12 month storage period. The high stability of the F38 formulation was further corroborated by the fact that during the 12 month storage period, HMW and LMW variants had only increased by 0.1 % and 0.4 %, respectively.

[0233] Hence, in summary, according to SEC data summarized in table 7, the F38 formulation had very high short and long term stability.

[0234] Table 7: SE-HPLC (SEC) data As illustrated by table 8, data derived from reduced capillary gel electrophoresis SDS (r-CE- SDS), hereinafter also referred to as r-CGE, further corroborated that the F38 formulation stored at 5 °C had very high short and long term stability when TO, Tl, T2 and T12 values were compared for light chain (LC) and heavy chain (HC) which had very small changes in LC and HC levels during the 1, 2 and 12 months storage periods. Moreover, the changes of levels of impurities, i.e. LMW, HMW and non-glycosylated heavy chain (ng-HC) variants, remained at very low levels.

[0235] Table 8: CE-SDS (CGE) data As illustrated by table 8, data derived from non-reduced capillary gel electrophoresis SDS (nr-CE-SDS), herein after also referred to as nr-CGE, also corroborated that the F38 formulation stored at 5 °C had very high short and long term stability when TO, T1,T2 and T12 values were compared for nivolumab (“nr-CGE Main ”) which had very small changes during the 1, 2 and 12 months storage periods. The changes of levels of impurities, i.e. LMW and non-glycosylated nivolumab (ng-Main) variants, remained at very low levels.

[0236] Consequently, the SEC (SE-HPLC), r-CGE (r-CE-SDS) and nr-CGE (nr-CE-SDS) analytical methods all showed that the F38 formulation had short and long term stability at 5 °C. As already implied, 5 °C was chosen as since it which is within the storage temperature interval of 2°C to 8°C which will be used for storing the drug product to be commercialized. Hence, in view of the results indicated by tables 7 and 8, a preferred stable antibody formulation according to present invention (i.e. the F38 formulation), is stable at about 2 °C to about 8 °C (e.g. 5 °C) for about 12 months, as characterized by (or as determined by, or as assessed by) one or more of : a. a relative main peak area of nivolumab > about 98 % as measured by SE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably about > 99 %, b. a relative peak area of HMW variants < about 0.5 % as measured by SE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably < about 0.4 %, c. a relative peak area of LMW variants < about 0.5 % as measured by SE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably < about 0.4 %, d. a relative main peak area of nivolumab > about 97% as measured by non-reducing CE-SDS after exposure to about 2 °C to about 8 °C for about 12 months, preferably > about 98 %, e. a relative heavy chain (HC) peak of about 65 % to about 68 %, as measured by reducing CE-SDS after exposure to about 2 °C to about 8 °C for about 12 months, preferably about 67 % to about 68 %, and / f. a relative light chain (LC) peak of about 31 % to about 33 % as measured by reducing CE-SDS after exposure to about 2 °C to about 8 °C for about 12 months, preferably about 31 % to about 32 %.

[0237] Although the drug product will be stored at 2 °C to 8 °C until it is time to administer it to the patient, the stability at 1 month (Tl) and 2 month (T2) time points was nevertheless analyzed at 25 °C and 40 °C in order to assess the effects of stressed degradation (25 °C) and accelerated degradation (40 °C). The data collected by the SEC, r-GCE and nr-GCE analytical methods shown in tables 7 and 8 indicated that the F38 formulation had high stability despite being subjected to stressed and accelerated degradation for 1 and 2 months at both at 25°C and 40°C. EXAMPLE 11 - F38 formulation - Stored upright - Charge variants

[0238] The F38 formulation which had been stored upright in 2R vials was subjected to CE-HPLC (CEX) in order to assess the charge variants at various timepoints and temperatures. The vials were each sealed with a West® bromobutyl stopper.

[0239] Since the drug product according to the F38 formulation will be stored at 2 °C to 8 °C until it is time to administer it to the patient, basic and acidic species (i.e. charge variants) as well as main species (i.e. nivolumab) were analyzed by carrying out CEX at timepoints 0 months (TO), 1 month (Tl), 2 months (T2) and 12 months (T12) of samples stored at 5 °C.

[0240] CEX data in table 9 shows that there were only very small changes in percentage of acidic, main (i.e. nivolumab) and basic species in formulation F38 which had been stored at 5 °C for 1 month (Tl) and 2 months (T2). There was no change in levels of basic species after 12 months (T12) of storage. A decrease of 1.8 % of acidic species and 1.7 % increase of nivolumab was observed after 12 months (T12) of storage.

[0241] Hence, in summary the levels of nivolumab in F38 formulation showed very small changes when stored upright at 5 °C for 1 month (Tl), 2 months (T2) and 12 months (T12).

[0242] Consequently, there was only very small or no effect on post translational modifications when F38 formulation was stored upright at 5 °C for 1 month, 2 months and 12 months timepoints.

[0243] Table 9: CE-HPLC (CEX) data Hence, in view of the results indicated by table 9, a preferred stable antibody formulation according to present invention (i.e. the F38 formulation), is stable at about 2 °C to about 8 °C (e.g. 5 °C) for about 12 months, as characterized by (or as determined by, or as assessed by) one or more of : a. a relative main peak area of nivolumab > about 74 % as measured by CE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably about > 75 %, b. a relative acidic species of nivolumab < about 21% as measured by CE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably < about 20 %, and c. a relative basic species of nivolumab < about 5 % as measured by CE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months.

[0244] Interestingly, CEX data indicated that at 1 month (Tl) and 2 month (T2) timepoints the F38 formulation stored at 25 °C and 40 °C showed a relatively high increase in acidic species and a relatively high decrease of nivolumab. The basic species was neither affected by stressed degradation (25 °C) nor accelerated degradation (40 °C) at timepoints Tl and T2. This shows that it is important to store the sample at 2 °C to 8 °C, preferably at 5 °C.

[0245] EXAMPLE 12 - F38 formulation - Stored inverted at 5 °C - Stability

[0246] The stability of the F38 formulation was also tested when it was stored inverted in 2R vials which had been sealed with Daikyo® bromobutyl stopper, West® bromobutyl stopper and West® chlorobutyl stopper.

[0247] For all of the tested stoppers, SE-HPLC (SEC) data in table 10 shows that the percentage of nivolumab (i.e. “SEC Main”) in formulation F38 stored at 5 °C had only decreases by 0.1 % when timepoint Tl (99.7 %) is compared with T2 (99.6 %). There was no decrease when timepoints TO and Tl are compared. Moreover, there was no increase of LMW variants when TO was compared with T2. Furthermore, only an increase of 0.1 % was observed for HMW variants when TO was compared with T2.

[0248] More importantly, for all of the tested stoppers, SEC data at timepoint T12 showed that the percentage of nivolumab had only decreased by 0.5 % over the 12 month storage period. The high stability of the F38 formulation was further corroborated by the fact that during the 12 month storage period, HMW and LMW variants had only increased by 0.4 % and 0.4 %, respectively.

[0249] Table 10: SE-HPLC (SEC) data Hence, in summary, according to SEC data summarized in table 10, the F38 formulation had very high short and long term stability when stored inverted at 5 °C for 1, 2 and 12 months in 2R vials which had been sealed with Daikyo® bromobutyl stopper, West® bromobutyl stopper and West® chlorobutyl stopper. As illustrated by table 11, data derived from reduced capillary gel electrophoresis SDS (r-CE- SDS), hereinafter also referred to as r-CGE, further corroborated that the F38 formulation stored inverted at 5 °C had very high stability when TO, Tl, T2 and T12 values were compared for light chain (LC) and heavy chain (HC) which showed relatively minor changes during the 1, 2 and 12 months storage periods. Moreover, the changes of levels of impurities, i.e. LMW, HMW and non-glycosylated heavy chain (ng-HC) variants, remained at relatively low levels. Table 11 : CE-SDS (CGE) data

[0250] As illustrated by table 11, data derived from non-reduced capillary gel electrophoresis SDS (nr-CE-SDS), herein after also referred to as nr-CGE, also corroborated that the F38 formulation stored at 5 °C had very high short and long term stability when TO, T1,T2 and T12 values were compared for nivolumab (“nr-CGE Main ”) which had very small changes during the 1, 2 and 12 months storage periods. The changes of levels of impurities, i.e. LMW and non-glycosylated nivolumab (ng-Main) variants, remained at relatively low levels.

[0251] Consequently, the SEC (SE-HPLC), r-CGE (r-CE-SDS) and nr-CGE (nr-CE-SDS) analytical methods all showed that the F38 formulation had short and long term stability at 5 °C. As already implied, 5 °C was chosen as since it which is within the storage temperature interval of 2°C to 8°C which will be used for storing the drug product to be commercialized.

[0252] It is important to note that the vials that are to be sold commercially will be stored upright. EXAMPLE 13 - F38 formulation - Stored inverted at 5 °C - Charge variants

[0253] The stability of the F38 formulation was also tested when it was stored inverted in 2R vials which had been sealed with Daikyo® bromobutyl stopper, West® bromobutyl stopper and West® chlorobutyl stopper.

[0254] The F38 formulation which had been stored inverted in 2R vials was subjected to CE-HPLC (CEX) in order to assess the charge variants at various timepoints and temperatures. The vials were sealed with Daikyo® bromobutyl stopper, West® bromobutyl stopper and West® chlorobutyl stopper.

[0255] Since the drug product according to the F38 formulation will be stored at 2 °C to 8 °C until it is time to administer it to the patient, basic and acidic species (i.e. charge variants) as well as main species (i.e. nivolumab) were analyzed by carrying out CEX at timepoints 0 months (TO), 1 month (Tl), 2 months (T2) and 12 months (T12) of samples stored at 5 °C.

[0256] CEX data in table 12 shows that there were only very small changes in percentage of acidic, main (i.e. nivolumab) and basic species in formulation F38 which had been stored inverted at 5 °C for 1 month (Tl) and 2 months (T2).

[0257] F38 inverted in vials comprising a Daikyo® bromobutyl stopper showed an increase of 1.8 % of acidic species and 0.4 % decrease of nivolumab after 12 months (T12) of storage. There was no change in levels of basic species after 12 months (T12) of storage.

[0258] F38 inverted in vials comprising a West® bromobutyl stopper showed a decrease of 0.1 % of acidic species and no change for nivolumab after 12 months (T12) of storage. There was no change in levels of basic species after 12 months (T12) of storage.

[0259] F38 inverted in vials comprising a West® chlorobutyl stopper showed an increase of 0.9 % of acidic species and 1.1 % decrease of nivolumab after 12 months (T12) of storage. There was no change in levels of basic species after 12 months (T12) of storage.

[0260] Hence, in summary, irrespective of the type of stopper was used, the levels of nivolumab in F38 formulation showed very small changes when stored inverted in vials at 5 °C for 1 month (Tl), 2 months (T2) and 12 months (T12).

[0261] Consequently, there was only very small or no effect on post translational modifications when F38 formulation was stored inverted with various stoppers at 5 °C for 1 month, 2 months and 12 months timepoints. Table 12: CE-HPLC (CEX) data

[0262] MATERIALS AND METHODS

[0263] 1. High-throughput screening - Example 1

[0264] A Prometheus Panta® instrument (NanoTemper Technologies) was used for high-throughput screening [7, 8, 9], Prometheus Panta® comprises nanoDSF (nano differential scanning fluorimetry), b ackreflection, DLS (dynamic light scattering), and SLS (Static Light Scattering) technologies for the characterization of biologies such as antibodies. The nanoDSF, b ackreflection, DLS, and SLS technologies rely on the intrinsic fluorescence and light scattering properties of analyzed molecules so that one doesn’t need dyes or additives. Prometheus Panta® was used for the below described methods of Thermal Unfolding Analysis, Isothermal Incubation Analysis and kDAnalysis.

[0265] Thermal Unfolding Analysis

[0266] For Thermal Unfolding (TU), samples were loaded onto capillaries and subjected a thermal ramp from 25°C to 95°C at a rate of 1 °C / min. Thermal and colloidal stability was monitored using nano-DSF, turbidity by back-reflection, and DLS to determine the fluorescence ratio Melting Temperature (Tm), Turbidity Melting Temperature inflection point (Turbidity ), and the cumulant radius onset temperature (TON), respectively. These points represent the temperature of the mid-point in the main 350nm / 330nm fluorescence slope, the inflection point in the turbidity slope, and the point at which cumulant radius change becomes detectable, respectively. The more stable a given formulation, the higher the T m, Tturbidity, and / or TON at which it is measured to change.

[0267] Isothermal Incubation Analysis

[0268] Formulations and references were subjected to thermal stress at a consistent temperature several degrees below the average TON and Tmvalues observed during the above-described Thermal Unfolding Analysis . This is used to stratify the thermal degradation rates of the different formulations and produce plots that make identifying the more stable formulations. A time-point along the plot where heavy stratification between the formulations can be observed is chosen as the final timepoint for generating percent change values for each formulation. These percentage change values are then compared to identify stable formulation candidates, the more stable the formulation the lower the percent change observed. For both the solubility and excipient screens, Isothermal Incubation analysis is used as the primary benchmark for ranking stability among the formulations being tested. All three reportable (fluorescence, turbidity, and cumulant radius) trended together, though cumulant radius was chosen as a leading indicator due to wider stratification of formulation performance.

[0269] In the solubility screening, the isothermal incubation temperature was 53 °C while the isothermal incubation temperature of 60 °C was chosen for excipient screening to produce more rapid degradation and stronger stratification at an earlier timepoint. A time-point where heavy stratification between the formulations can be observed is chosen as the final timepoint for generating percent change values for each formulation. In the solubility screening, a stratification point of 755 minutes at 53 °C was chosen. In the excipient screening, a stratification point of 225 minutes at 60 °C was chosen. These differences between solubility and excipient screenings are also indicated in table 13. kn Analysis

[0270] The k analysis was used to stratify the three leading formulations following isothermal incubation. It can finely parse stability between formulations that were indicated as highly stable in the isothermal analysis and as a result can often determine the most promising candidate to carry on as the base for the next screening stage during the formulation development process. The k analysis compares several measurements of diffusion rate across a dilution series of a formulation to approximate a ko value for each formulation. The approximated k value was interpreted as a relative measurement of the intermolecular attraction between the different proteins in the formulation. A large negative value would indicate a strong level of attraction, which is correlated to increased propensity to aggregate in solution. In comparison, a value close to zero would indicate neither attraction nor repulsion between proteins in solution. A positive value indicates a level of repulsion and a lower propensity for aggregation. The k results are generally evaluated and used in conjunction with other more consistent analytical methods such as Isothermal Incubation to make definitive decisions on ranking formulation stability, taking into account the low R2values for ko observed during this study. Prometheus Panta® configuration

[0271] Prometheus Panta® was configured as summarized in below table 13 for Thermal Unfolding

[0272] Analysis, Isothermal Incubation Analysis and kDAnalysis.

[0273] Table 13. Configuration of Prometheus Panta® and measured parameters. 2. Chemical compositions of formulations

[0274] The formulations discussed in the examples 2-13 were prepared by using the amounts of compounds described below.

[0275] Each mL of formulation F38 comprises nivolumab 10 mg, L-histidine (1.965 mg), L-histidine HC1 monohydrate (1.537 mg), sucrose (92.42 mg), L-methionine (1.492 mg), polysorbate 80 (0.2 mg) and water for injection. The final pH is 6.3.

[0276] Each mL of formulation F38-DS comprises nivolumab 40 mg, L-histidine (1.965 mg), L- histidine HC1 monohydrate (1.537 mg), sucrose (92.42 mg), L-methionine (1.492 mg), polysorbate 80 (0.2 mg) and water for injection. The final pH is 6.3.

[0277] Each mL of formulation F46 comprises nivolumab 10 mg, L-histidine (1.965 mg), L-histidine HC1 monohydrate (1.537 mg), a, a-trehalose dihydrate (102.15 mg), L-methionine (1.492 mg), polysorbate 80 (0.2 mg) and water for injection. The final pH is 6.3.

[0278] Each mL of formulation F46-DS comprises nivolumab 40 mg, L-histidine (1.965 mg), L- histidine HC1 monohydrate (1.537 mg), a, a-trehalose dihydrate (102.15 mg), L-methionine (1.492 mg), polysorbate 80 (0.2 mg) and water for injection. The final pH is 6.3.

[0279] Each mL of formulation FX comprises nivolumab 10 mg, tri-sodium citrate dihydrate (5.254 mg), citric acid monohydrate (0.445 mg), sodium chloride (2.922 mg), D-mannitol (30 mg), polysorbate 80 (0.2 mg), pentetic acid (0.008 mg) and water for injection. The final pH is 6.0.

[0280] Each mL of formulation FX-DS comprises nivolumab 40 mg, tri-sodium citrate dihydrate (5.254 mg), citric acid monohydrate (0.445 mg), sodium chloride (2.922 mg), D-mannitol (30 mg), polysorbate 80 (0.2 mg), pentetic acid (0.008 mg) and water for injection. The final pH is 6.0.

[0281] Formulations FY and FY-DS differed from formulations FX and FX-DS only in that pentetic acid was not present in formulations FY and FY-DS.

[0282] Although pH adjustments were not necessary in the preparation of the above described formulations, such adjustments can be made if necessary by using hydrochloric acid and / or sodium hydroxide.

[0283] The above described formulations are preferably prepared by using substantially pure nivolumab which has been subjected to midstream and downstream purification methods such as affinity chromatography, ion exchange chromatography, and / or ultrafiltration / diafiltration (UF / DF). The source of the substantially pure nivolumab discussed and used in examples 8 and 9, as well as examples 10-13, were prepared by using all of the methods listed in the previous sentence as described in sections 9-11 in MATERIALS AND METHODS. A detailed description of the method of preparing formulations F38, F38-DS, F46, F46-DS, FX and FX-DS having alternative compositions than the above mentioned are also described in sections 9-11 in MATERIALS AND METHODS.

[0284] However, the source of nivolumab for the formulations discussed in examples 2-7 was a composition comprising nivolumab, buffer, carbohydrate, salt and polysorbate. This composition was subjected to affinity chromatography (by using Cytiva MabSelect PrismA chromatography resin) and the resulting material was used for preparing formulations F38, F38-DS, F46, F46-DS, FX, FX-DS, FY and FY-DS.

[0285] The concentrations and amounts of compounds in each of the above listed formulation can deviate up to 10 %. In an embodiment of the invention, the concentration of nivolumab in formulations F38, F46 and FX is 10 ± 2 mg / ml, preferably 10 ± 1 mg / ml. In a further embodiment, the concentration of nivolumab in formulations F38-DS, F46-DS and FX-DS is 40 ± 4 mg / ml, preferably 40 ± 2 mg / ml.

[0286] 3. Sub-visible particle count analysis - Examples 2-5

[0287] Flow imaging microscopy (FIM) is an orthogonal method to light obscuration for determining sub-visible particulate content. In flow imaging microscopy, particles move through a flow cell as microscopic images are captured of particles in real-time by a highspeed camera and processed by image analysis software to measure particle size and morphology.

[0288] Sub-visible particle (SvP) counts were determined by using FlowCam® 8100 and FlowCam® LO (Yokogawa Fluid Imaging Technologies) which are instruments for conducting flow imaging microscopy. FlowCam® 8100 performs flow imaging and thereby provides particle sizing and counting according to USP < 1788> (Subvisible Particulate Matter). FlowCam® LO performs both flow imaging and light obscuration measurements sequentially in the same instrument and thereby provides compendial particle sizing and counting according to USP <787> (Subvisible Particulate Matter in Therapeutic Protein Injections) and <788> (Particulate Matter in Injections). The captured particles are categorized by size by using the statistical software “Visual Spreadsheet” (Yokogawa Fluid Imaging Technologies). The qualification is carried out in particle counts per milliliter (counts / ml).

[0289] In examples 3-7 , 2R vials were filled with 500 pl formulation and 100 pl were subjected SvP count analysis. In examples 3-5, the samples were placed in 2R-type vials and thereafter mixed by pipetting up and down at least 20 times before SvP count analysis. In example 6, the samples were mixed by inverting the vials at least 20 times before SvP count analysis.

[0290] US Pharmacopoeia, Japanese Pharmacopoeia and Japanese Pharmacopoeia all require that sub-visible particles in small volume injectables must conform to the limits for USP <787> and USP <788> which are [4]:

[0291] < 6000 counts of particles sizes above 10 pm, and

[0292] < 600 counts of particles sizes above 20 pm.

[0293] 4. CE-SDS - Example 6

[0294] All experiments were performed on the LabChip® GXII Touch Protein Characterization System (Perkin Elmer) and wherein Protein Express Assay Reagent Kit (Perkin Elmer - Mat. Nr 760499) and Protein Express Assay LabChip® HT (Perkin Elmer - Mat. Nr. CLS960008) were used for conducting the assays

[0010] , The LabChip® was prepared according to the manufacturer’s instructions and CE-SDS analysis was conducted according to “HT Antibody Analysis 200” assay.

[0295] For non-reduced CE-SDS (nrCE-SDS), samples were denatured at 70 C° for 10 minutes in the presence of 0.25 M iodoacetamide.

[0296] 5. SE-HPLC (SEC) - Examples 7-9

[0297] Aggregates were analyzed by SE-HPLC (i.e. size exclusion chromatography - SEC) with UV absorbance (280 nm). SE-HPLC measurements were made on a Thermo Scientific Vanquish HPLC device with an ACQUITY UPLC Protein BEH SEC 200A column (1.7 pm, 4.6 mm x 150 nm). 20 pg samples were injected into the system. An isocratic gradient comprising PBS (Phosphate Buffered Saline) buffer at pH 7.4 was used. The levels of high molecular weight (HMW) variants, monomer (i.e. nivolumab) and low molecular weight (LMW) variants were detected and their distribution in percentage of total peak were evaluated. The standard deviation for the measured values in examples 8 was 0.02 %. Furthermore, in example 8, the relative standard deviation (RSD) was 4.72 % per sample for the HMW peak while the RSD was 0.02 % per sample for the nivolumab (i.e. main) peak.

[0298] 6. SE-HPLC (SEC) - Examples 10 and 12

[0299] Aggregates were analyzed by SE-HPLC (i.e. SEC) with UV absorbance (280 nm). SEC measurements were made on an Acquity H-Class UPLC with UV Detector (Acquity TUV) device with an ACQUITY UPLC Protein BEH SEC 200A column (1.7 pm, 4.6 mm x 150 nm). 20 pg samples were injected into the system. An isocratic gradient comprising PBS (Phosphate Buffered Saline) buffer was used. The levels of high molecular weight (HMW) variants, monomer / main (i.e. nivolumab) and low molecular weight (LMW) variants were detected and their distribution in percentage of total peak were evaluated.

[0300] The main peak accuracy for the tested range (10 - 30 pg) was 100±0.05 %. The HMW peak accuracy for the tested range (10 - 30 pg) was 100±3.02 %.

[0301] The principle behind SEC is that different components of a sample are eluted in order of their decreasing molecular size and are detected using UV absorption at 280 nm. The peaks observed are classified as high molecular weight (HMW) peaks, main (Main) peak, low molecular weight (LMW) peaks, and buffer component peaks. The amount of different molecular weight species is determined by relating the peak area of the different forms to the total peak area of protein. In other words, SEC is applicable in the purity assessment of formulations with regard to size distribution of proteins.

[0302] 7. CE-SDS (CGE) - Examples 10 and 12

[0303] All experiments were performed on the Maurice system with Compass for iCE software (ProteinSimple) by using Maurice CE-SDS Plus Cartridge (ProteinSimple - Part No. PS- MC02-SP). Appropriate consumables and reagents provided by ProteinSimple were used to conduct CE-SDS (CGE) analysis.

[0304] For non-reduced CGE (nr-CGE), samples were denatured at 70 C° for 10 minutes in the presence of 0.25 M iodoacetamide. The heavy chain (HC) peak accuracy for the tested range (0.25 - 0.75 mg / ml nominal protein concentration) was 99.8-100.1 %. The light chain (LC) peak accuracy for the tested range (0.25 - 0.75 mg / ml nominal protein concentration) was 99.6-102.4%. The sum of LC and HC peaks accuracy for the tested range (0.25 - 0.75 mg / ml nominal protein concentration) was 99.9-100.7%. The ng-HC peak accuracy for the tested range (0.25 - 0.75 mg / ml nominal protein concentration) was 96.3-101.9 %.

[0305] The principle behind CGE is that separation is performed using capillary electrophoresis (CE) in a polyacrylamide gel. Loading of the sample is done in the presence of sodium dodecyl sulphate (SDS). SDS is distributed evenly over the analyte and denatures it. As a result, all analytes will have a fairly constant charge / mass ratio. The separation is therefore performed according to mass since larger molecules move slower through the porous gel. CGE is applicable for both analyzing intact molecules and reduced molecules. Detection is made using UV-absorbance at 280 nm as the analytes migrate through the capillary detection window. In other words, CGE was used in the purity assessment of formulations primarily with regard to size distribution.

[0306] 8. CEX-HPLC (CEX) - Examples 11 and 13

[0307] Charge variants were analyzed by CEX-HPLC (CEX) conducted with an Acquity H-Class UPLC with UV Detector (Acquity TUV) device equipped with Waters BioResolve SCX mAb 3 pm, 4.6*100 mm (part no. 186009059) column. 25 pg samples were injected into the CEX system. A gradient involving Mobile phase A (Themo CX-1 pH gradient buffer A pH 5.6) and Mobile phase B (Themo CX-1 pH gradient buffer A pH 10.2) was utilized. The UPLC software Empower3 was set-up to calculate relative areas of acidic peaks, main peak and basic peaks.

[0308] Accuracy at column loads of 20 - 38 pg is as follows:

[0309] Main peak: 99.5 - 101.0 %.

[0310] Acidic peaks: 98.7 - 100.2 %

[0311] - Basic peaks: 95.2 - 103.0 %.

[0312] The principle behind CEX is that separation is performed using a strong cation exchange column on a UPLC system. Protein isomers with a positive net charge are retained in the column and are eluted using a pH gradient. The retention time reflects the solvent exposed net-charge of the analyte; a higher positive net-charge gives a longer retention time. Consequently, deamidation products will be eluted prior to the main peak and incompletely processed N- and C-terminal variants will elute after the main peak. Detection occurs using UV-spectroscopy at 280 nm. In other words, CEX was used in the purity assessment of nivolumab primarily with regard to charge distribution typically caused by deamidation or partial processing of N- and C-terminal amino acids.

[0313] 9. Preparation of formulation F38-DS and F38

[0314] Nivolumab biosimilar Xdivane was cultivated in a 200L single-use bioreactor and processed according to the established manufacturing process including steps such as affinity chromatography, ion exchange chromatography and nano filtration.

[0315] A drug substance stock solutions comprising 49.2 g / L nivolumab was prepared by ultrafiltration / diafiltration of the purified protein over a 30 kDa cellulose membrane. During this step, the material was transferred to a diafiltration buffer (20 mM L-histidine buffer, 10 mM L-methionine, pH 6.3; see table 14).

[0316] Formulation F38-DS (i.e. the drug substance) was formulated by adding a spike buffer in a ratio of 0.2 L spike buffer per L protein stock solution. The spike buffer comprised the excipients polysorbate 80 and sucrose in a 6-fold higher concentration than the target concentration in the drug substance (i.e. 20 mM L-histidine buffer, 10 mM L-methionine, 1.62 M Sucrose, 1.2 g / L polysorbate 80, pH 6.3, see table 15).

[0317] After spiking, formulation F38-DS comprised 41.5 g / L (i.e. 41.5 mg / ml) nivolumab, 20 mM L-histidine buffer, 270 mM sucrose, 10 mM L-methionine, 0.2 g / L (i.e. 0.02 w / v %) polysorbate 80 and pH 6.3

[0318] The formulation was sterile filtered into sterile glass or plastic bottles and stored at 2-8 °C until it was filled into glass vials and 5 mL plastic bags. Table 14 - Diafiltration buffer

[0319] Table 15 - Spike buffer

[0320] Formulation F38 (i.e. the drug product) was obtained by diluting 75 mL of the above described formulation F38-DS with 225 mL formulation buffer (20 mM L-histidine buffer, 10 mM L-methionine, 270 mM sucrose, 0.2 g / L polysorbate 80, pH 6.3; see table 16) in a sterile environment. The resulting formulation F38 comprised 10.7 g / L (i.e. 10.7 mg / ml) nivolumab, 20 mM L-histidine buffer, 270 mM sucrose, 10 mM L-methionine, 0.2 g / L polysorbate 80 and pH 6.3. The formulation was then filtered into a sterile plastic bottle using autoclaved tubing and filters. The formulation was stored at 2-8 °C until it was filled into glass vials and 5 mL plastic bags.

[0321] Table 16 - Formulation buffer 10. Preparation of formulations F46-DS and F46

[0322] Nivolumab biosimilar Xdivane was cultivated in a 200 L single-use bioreactor and processed according to the established manufacturing process including steps such as affinity chromatography, ion exchange chromatography and nano filtration.

[0323] A drug substance stock solution having 49.2 g / L nivolumab concentration was prepared by ultrafiltration / diafiltration of the purified protein over a 30 kDa cellulose membrane. During this step, the material was transferred to a diafiltration buffer (20 mM L-histidine buffer, 10 mM L-methionine, pH 6.3; see table 14).

[0324] Formulation F46-DS (i.e. the drug substance) was formulated by adding a spike buffer in a ratio of 0.2 L spike buffer per L protein stock solution. The spike buffer comprised the excipients polysorbate 80 and trehalose in a 6-fold higher concentration than the target concentration in the drug substance (i.e. 20 mM L-histidine buffer, 10 mM L-methionine 1.62 M trehalose, 1.2 g / L polysorbate 80, pH 6.3; see table 17). The spike buffer was heated before it was used for spiking.

[0325] After spiking, the resulting formulation F46-DS comprised 42.0 g / L (42.0 mg / ml) nivolumab, 20 mM L-histidine buffer, 270 mM trehalose, 10 mM L-methionine, 0.2 g / L polysorbate 80 and pH 6.3.

[0326] The formulation was sterile filtered into sterile glass or plastic bottles and stored at 2-8 °C until it was filled into glass vials and 5 mL plastic bags.

[0327] Table 17 - Spike buffer

[0328] Formulation F46 (i.e. the drug product) was obtained by diluting 12.5 mL of the above described formulation F46-DS with 37.5 mL formulation buffer (20 mM L-histidine buffer, 10 mM methionine, 270 mM trehalose, 0.2 g / L polysorbate 80, pH 6.3; see table 18) in a sterile environment. The resulting formulation F46 comprised 10.5 g / L (i.e. 10.5 mg / ml) nivolumab, 20 mM L-histidine buffer, 270 mM trehalose, 10 mM L-methionine and 0.2 g / L polysorbate 80 and pH 6.3.

[0329] The formulation was then filtered into a sterile plastic bottle using autoclaved tubing and filters. The formulation was stored at 2-8 °C until it was filled into glass vials and 5 mL plastic bags.

[0330] Table 18 - Formulation buffer

[0331] 11. Preparation of nivolumab formulation FX-DS and FX

[0332] Nivolumab biosimilar Xdivane was cultivated in a 200 L single-use bioreactor and processed according to the established manufacturing process including steps such as affinity chromatography, ion exchange chromatography and nano filtration.

[0333] A drug substance stock solution with 50.1 g / L nivolumab concentration was prepared by ultrafiltration / diafiltration of the purified protein over a 30 kDa cellulose membrane. During this step, the material was transferred to a diafiltration buffer (20 mM sodium citrate, 50 mM sodium chloride, pH 6.0; see table 19).

[0334] Formulation FX-DS was formulated by adding a spike buffer in a ratio of 0.2 L spike buffer per L protein stock solution. The spike buffer comprised the excipients polysorbate 80, mannitol and pentetic acid in a 6-fold higher concentration than the target concentration in the drug substance (i.e. 20 mM sodium citrate, 50 mM sodium chloride, 988.1 mM mannitol, 1.2 g / L polysorbate 80, 0.12 mM pentetic acid, pH 6.0; see table 20).

[0335] After spiking, formulation FX-DS comprised 41.0 g / L (41.0 mg / ml) nivolumab, 20 mM citrate buffer, 50 mM NaCl, 30 g / L mannitol, 0.008 g / L pentetic acid, 0.2 g / L polysorbate 80 and pH 6.0.

[0336] The solution was sterile filtered into sterile glass or plastic bottles and the material was stored at 2-8 °C until it was filled into glass vials and 5 mL plastic bags.

[0337] Table 19 - Diafiltration buffer Table 20 - Spike buffer

[0338] Formulation FX was obtained by diluting 12.5 mL of the above described formulation FX-DS with 37.5 mL formulation buffer (20 mM sodium citrate, 50 mM sodium chloride, 165 mM mannitol, 0.2 g / L polysorbate 80, 0.02 mM pentetic acid, pH 6.0; see table 21) in a sterile environment. The resulting formulation FX comprised 10.4 g / L (10.4 mg / ml) nivolumab, 20 mM sodium citrate buffer, 50 mM sodium chloride, 165 mM mannitol, 0.2 g / L polysorbate 80, 0.02 mM pentetic acid and pH 6.0. The formulation was then filtered into a sterile plastic bottle using autoclaved tubing and filters. The formulation was stored at 2-8 °C until it was filled into glass vials and 5 mL plastic bags.

[0339] Table 21 - Formulation buffer

[0340] REFERENCES

[0341] 1. Highlights of prescribing information for Opdivo® - Revised version from February 2023 - https: / / www.accessdata.fda.gov / drugsatfda_docs / label / 2023 / 125554sll91bl.pdf

[0342] 2. Warne, Nicholas W. “Development of high concentration protein biopharmaceuticals: the use of platform approaches in formulation development.” European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V vol. 78,2 (2011): 208-12

[0343] 3. “Formulation and Process Development Strategies for Manufacturing Biopharmaceuticals”, Wiley & Sons, 2010. Chapter 6: “Formulation Development of Phase 1-2 Biopharmaceuticals: The used of Platform Approaches in Formulation Development”

[0344] 4. “Particle counting in Injectable Solutions”. Particle Measuring Systems - a Spectris company © 2018 (https: / / www.pmeasuring.com / PMS / files / fl / flba872d-077b-4dbb-b857- 526cd0027f46.pdf)

[0345] 5. Katterle, Bettina “How stable are new biologies?” Pharm. Ind. 80, Nr. 11, 1557-1563 (2018)

[0346] 6. Jain K, Salamat-Miller N, Taylor K. “Freeze-thaw characterization process to minimize aggregation and enable drug product manufacturing of protein based therapeutics” Sci Rep. 2021 May 31 ; 11(1): 11332

[0347] 7. NanoTemper Technologies. Prometheus Panta. BR-PA-092020 (downloaded from https: / / resources.nanotempertech.com / prometheus / prometheus-panta-biologics-brochure)

[0348] 8. https: / / nanotempertech.com / prometheus / biologics /

[0349] 9. US2023355757: paragraphs 0162-0170 PerkinElmer, Inc. Protein Express Assay User Guide for LabChip® GXII Touch and LabChip® GXII, PN CLS140156 Rev. F, Publication date March 20, 2019 Wang SS, Yan YS, Ho K. US FDA-approved therapeutic antibodies with high- concentration formulation: summaries and perspectives. Antib Then 2021 Nov 18;4(4):262-272

Claims

CLAIMS1. A stable antibody formulation for use in intravenous administration, comprising: a. about 10-40 mg / mL nivolumab, b. about 20 mM L-histidine buffer, c. about 270 mM sucrose, d. about 0.02 % w / v polysorbate 80, and e. about 10 mM L-methionine.

2. Antibody formulation according to claim 1, wherein the antibody formulation is free of pentetic acid, enzymes and arginine.

3. Antibody formulation according to claim 1, consisting of: a. about 10-40 mg / mL nivolumab, b. about 20 mM L-histidine buffer, c. about 270 mM sucrose, d. about 0.02 % w / v polysorbate 80, e. about 10 mM L-methionine, and f. water.

4. Antibody formulation according to claim 2, comprising: a. about 10 mg / mL nivolumab, b. about 20 mM histidine buffer, c. about 270 mM sucrose, d. about 0.02 % w / v polysorbate 80, and e. about 10 mM L-methionine.

5. Antibody formulation according to claim 3, consisting of: a. about 10 mg / mL nivolumab, b. about 20 mM histidine buffer, c. about 270 mM sucrose, d. about 0.02 % w / v polysorbate 80, e. about 10 mM L-methionine, and f. water.

6. Antibody formulation according to claim 2, comprising: a. about 40 mg / mL nivolumab, b. about 20 mM histidine buffer, c. about 270 mM sucrose, d. about 0.02 % w / v polysorbate 80, and e. about 10 mM L-methionine.

7. Antibody formulation according to claim 3, consisting of: a. about 40 mg / mL nivolumab, b. about 20 mM histidine buffer, c. about 270 mM sucrose, d. about 0.02 % w / v polysorbate 80, and e. about 10 mM L-methionine, and f. water.

8. Antibody formulation according to any one of the previous claims 1-7, wherein a pH of the antibody formulation is at least 6.0, more preferably about 6.0-6.3, most preferably about 6.3.

9. Antibody formulation according to any one of the previous claims 1, 2, 4, 6 or 8, wherein the antibody formulation is a liquid antibody formulation, preferably an aqueous antibody formulation.

10. Antibody formulation according to any one of the previous claims 1-9 for use in the treatment of cancer, preferably for use in the treatment of melanoma, non-small cell lung cancer, malignant pleural mesothelioma, renal cell carcinoma, classical Hodgkin lymphoma, squamous cell carcinoma of the head and neck, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, esophageal or gastroesophageal junction cancer, an / or gastric cancer.

11. Antibody formulation according to any one of the previous claims 1-10 for use in intravenous infusion.

12. Antibody formulation according to any one of the previous claims 1-11, wherein the antibody formulation is stable at about 2 °C to about 8 °C for about 12 months.

13. Antibody formulation according to any one of the previous claims 1-12, wherein the antibody formulation is stable at about 2 °C to about 8 °C for about 12 months, wherein stability of the stable antibody formulation is defined based on one or more of: a. a relative main peak area of nivolumab > about 98 % as measured by SE- HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably about > 99 %, b. a relative peak area of HMW variants < about 0.5 % as measured by SE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably < about 0.4 %, c. a relative peak area of LMW variants < about 0.5 % as measured by SE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably < about 0.4 %, d. a relative main peak area of nivolumab > about 74 % as measured by CE- HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably about > 75 %, e. a relative acidic species of nivolumab < about 21 % as measured by CE- HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably < about 20 %, f. a relative basic species of nivolumab < about 5 % as measured by CE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, g. a relative main peak area of nivolumab > about 97 % as measured by nonreducing CE-SDS after exposure to about 2 °C to about 8 °C for about 12 months, preferably > about 98 %, h. a relative heavy chain (HC) peak of about 65 % to about 68 %, as measured by reducing CE-SDS after exposure to about 2 °C to about 8 °C for about 12 months, preferably about 67 % to about 68 %, and i. a relative light chain (LC) peak of about 31 % to about 33 % as measured by reducing CE-SDS after exposure to about 2 °C to about 8 °C for about 12 months, preferably about 31 % to about 32 %.

14. Antibody formulation according to any one of the previous claims 1-12, wherein the antibody formulation is stable at about 2 °C to about 8 °C for about 12 months, as characterized by one or more of: a. a relative main peak area of nivolumab > about 98 % as measured by SE- HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably about > 99 %, b. a relative peak area of HMW variants < about 0.5 % as measured by SE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably < about 0.4 %, c. a relative peak area of LMW variants < about 0.5% as measured by SE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably < about 0.4 %, d. a relative main peak area of nivolumab > about 74 % as measured by CE- HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably about > 75 %, e. a relative acidic species of nivolumab < about 21 % as measured by CE- HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably < about 20 %, f. a relative basic species of nivolumab < about 5 % as measured by CE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, g. a relative main peak area of nivolumab > about 97 % as measured by nonreducing CE-SDS after exposure to about 2 °C to about 8 °C for about 12 months, preferably > about 98 %, h. a relative heavy chain (HC) peak of about 65 % to about 68 %, as measured by reducing CE-SDS after exposure to about 2 °C to about 8 °C for about 12 months, preferably about 67 % to about 68 %, and i. a relative light chain (LC) peak of about 31 % to about 33 % as measured by reducing CE-SDS after exposure to about 2 °C to about 8 °C for about 12 months, preferably about 31 % to about 32 %.

15. Antibody formulation according to any one of the previous claims 1-12, wherein the antibody formulation has one or more of the following characteristics:a. a relative main peak area of nivolumab > about 98 % as measured by SE- HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably about > 99 %, b. a relative peak area of HMW variants < about 0.5 % as measured by SE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably < about 0.4 %, c. a relative peak area of LMW variants < about 0.5 % as measured by SE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably < about 0.4 %, d. a relative main peak area of nivolumab > about 74 % as measured by CE- HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably about > 75 %, e. a relative acidic species of nivolumab < about 21 % as measured by CE- HPLC after exposure to about 2 °C to about 8 °C for about 12 months, preferably < about 20%, f. a relative basic species of nivolumab < about 5 % as measured by CE-HPLC after exposure to about 2 °C to about 8 °C for about 12 months, g. a relative main peak area of nivolumab > about 97 % as measured by nonreducing CE-SDS after exposure to about 2 °C to about 8 °C for about 12 months, preferably > about 98 %, h. a relative heavy chain (HC) peak of about 65 % to about 68 %, as measured by reducing CE-SDS after exposure to about 2 °C to about 8 °C for about 12 months, preferably about 67 % to about 68 %, and i. a relative light chain (LC) peak of about 31 % to about 33 % as measured by reducing CE-SDS after exposure to about 2 °C to about 8 °C for about 12 months, preferably about 31 % to about 32 %.

16. Antibody formulation according to any one of the previous claims 1-15, wherein the antibody formulation is stable at about 5 °C for about 12 months.

17. Antibody formulation according to any one of the previous claims 13-15, wherein said exposure to about 2 °C to about 8 °C for about 12 months disclosed in steps a-h is exposure to about 5 °C for about 12 months.

18. Antibody formulation according to any one of the previous claims 1-17, wherein the antibody formulation is stable through 5 to 10 freeze-thaw cycles.

19. Antibody formulation according to any one of the previous claims 1-18, wherein the antibody formulation is stable through 5 to 10 freeze-thaw cycles, as characterized by: a. a relative main peak area of nivolumab > about 99 % as measured by SE- HPLC, and / or b. a relative peak area of HMW variants < about 0.5 % as measured by SEC- HPLC.

20. Antibody formulation according to any one of the previous claims 1-19, comprising: a. less than 6000 counts sub-visible particles above 10 pm, and / or b. less than 600 counts sub-visible particles above 25 pm.

21. Antibody formulation according to any one of the previous claims 1-20, wherein the formulation has a volume of 4 ml, 10 ml, 12 ml or 24 ml.

22. Antibody formulation according to any one of the previous claims 1-21, wherein nivolumab is a nivolumab biosimilar.

23. A container comprising the antibody formulation according to any one of the previous claims 1-22, preferably the container is a vial, bag and / or an injection device, more preferably a glass vial, most preferably a glass vial comprising a stopper.

24. A container according to the previous claim, wherein the container is a glass vial comprising a stopper, preferably a glass vial comprising a pierceable stopper, more preferably a glass vial comprising a pierceable bromobutyl or chlorobutyl stopper, most preferably a glass vial comprising a pierceable bromobutyl stopper.

25. A kit of parts comprising the antibody formulation according to any one of the previous claims 1-22.

26. A kit of parts comprising the container according to claim 23 or 24.

27. A method of formulating a formulation of claim 4 or claim 5, comprising the step of diluting a formulation according to claim 6 or claim 7 with an aqueous formulation buffer comprising 20 mM L-histidine buffer, 10 mM L-methionine, 270 mM sucrose and 0.02 % w / v polysorbate 80.

28. Method according to claim 27 of formulating a formulation of claim 4 or claim 5, comprising the step of diluting 1 part of a formulation according to claim 6 or claim 7 with 3 parts of an aqueous formulation buffer comprising 20 mM L-histidine buffer, 10 mM L-methionine, 270 mM sucrose and 0.02 % w / v polysorbate 80.

29. Method according to the previous claims 27 or 28, wherein the resulting formulation is stored at about 2 °C to about 8 °C, preferably the resulting formulation is stored at about 5 °C.

30. Method according to any one of the previous claims 27-29, wherein nivolumab has been prepared by the steps comprising: a. fed batch cell culturing, b. harvesting the cell culture, c. clarifying the harvested cell culture suspension, d. affinity chromatography, e. ion exchange chromatography, and f. ultrafiltration / diafiltration.

31. A stable antibody formulation obtainable by any one of claims 27-30.

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