LU504834B1

LU504834B1 - Stable anti-vegf antibody formulation

Info

Publication number: LU504834B1
Application number: LU504834A
Authority: LU
Inventors: Zhimin Ma; Zhihao Wu; Yuanqiang Li; Cuihua Liu; Yong Wu; Di Cao
Original assignee: Bio Thera Solutions Ltd
Priority date: 2023-03-06
Filing date: 2023-07-31
Publication date: 2024-03-04
Also published as: WO2024183733A1; CN118593699A

Abstract

The present invention belongs to the field of bio-pharmaceuticals, and relates to a stable antibody formulation comprising an antibody, a buffer, a stabilizer and a surfactant, and a preparation method thereof. The antibody in the formulation of the present invention has better stability.

Description

BL-5733

STABLE ANTI-VEGF ANTIBODY FORMULATION 7006808

TECHNICAL FIELD

The present invention belongs to the field of bio-pharmaceuticals, and relates to a stable anti-

VEGF antibody formulation, and preparation and application thereof.

BACKGROUND

Vascular endothelial growth factor (VEGF), in particular VEGF-A, has been identified as a key factor in the induction of tumor angiogenesis. By specifically targeting and binding to human vascular endothelial growth factor (VEGF), and inhibiting the binding of VEGF to its receptor, the tumor angiogenesis is inhibited, and the supply of blood, oxygen and other nutrients to the tumor is reduced, thereby inhibiting tumor cell growth. Anti-VEGF antibodies such as bevacizumab have been currently applied for the treatment of various malignancies.

Antibodies, as macromolecular proteins, are more complex than traditional inorganic and organic drugs. Protein drug formulations are susceptible to degradation during storage due to chemical instability (e.g., deamidation, racemization, hydrolysis, oxidation, B-elimination or disulfide bond exchange, etc.) or physical instability (e.g., denaturation, aggregation, precipitation or adsorption). Instability of the antibody formulation can not only reduce or eliminate the efficacy, but also the aggregation of the antibody can improve the immunogenicity of the antibody formulation, thereby having adverse effects on the health of the patient. There remains a need in the art for more stable pharmaceutical formulations.

SUMMARY

In one aspect, the present invention provides a stable anti-VEGF antibody formulation comprising: about 20-100 mg/mL anti-VEGF antibody, a histidine buffer, a stabilizer and a surfactant, wherein the formulation has a pH value of 4.0-6.5.

In some embodiments, the anti-VEGF antibody comprises a heavy chain variable region set forth in SEQ ID NO: 1 and a light chain variable region set forth in SEQ ID NO: 2. In some embodiments, the anti-VEGF antibody comprises a heavy chain set forth in SEQ ID NO: 3 and

BL-5733 a light chain set forth in SEQ ID NO: 4. In some embodiments, the anti-VEGF antibody is HUS04834 bevacizumab (such as Avastin or a biosimilar thereof, e.g., Pobevcy (BAT1706), Pusintin,

Beianting, Hanbeitai, Airuituo, Boyounuo, Byvasda, Ankeda, Alymsys, Zirabev, Mvasi,

Vegzelma, Onbevzi, Oyavas, Aybintio, and Abevmy).

In some embodiments, the anti-VEGF antibody is at a concentration of about 20-100 mg/mL.

In some embodiments, the anti-VEGF antibody is at a concentration of about 25-80 mg/mL. In some embodiments, the anti-VEGF antibody is at a concentration of about 50-80 mg/mL. In some embodiments, the anti-VEGF antibody is at a concentration of about 25-50 mg/mL. In some embodiments, the anti-VEGF antibody is at a concentration of about 20 mg/mL, about mg/mL, about 30 mg/mL, about 35 mg/mL, about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about 55 mg/mL, about 60 mg/mL, about 65 mg/mL, about 70 mg/mL, about 75 mg/mL, or about 80 mg/mL, or a range between any two concentration values, including end values.

In some embodiments, the histidine buffer is at a concentration of about 1-30 mM. In some embodiments, the histidine buffer is at a concentration of about 8-15 mM. In some embodiments, the histidine buffer is at a concentration of about 8-12 mM. In some embodiments, the histidine buffer is at a concentration of about 10 mM. In some embodiments, the histidine buffer comprises histidine and histidine hydrochloride, e.g., a combination of L- histidine and Z-histidine hydrochloride.

In some embodiments, the stabilizer is a saccharide. In some embodiments, the saccharide is trehalose or sucrose. In some embodiments, the saccharide is at a concentration of about 5-200 mg/mL. In some embodiments, the saccharide is at a concentration of 10-100 mg/mL. In some embodiments, the saccharide is at a concentration of 30-80 mg/mL. In some embodiments, the saccharide is at a concentration of 50-300 mM. In some embodiments, the trehalose is at a concentration of about 158.6 mM, 1.e., corresponding to 54.3 mg/mL trehalose anhydrous, or 60 mg/mL trehalose dihydrate.

In some embodiments, the surfactant is polysorbate or poloxamer. In some embodiments, the surfactant is polysorbate 20, polysorbate 80, or poloxamer 188. In some embodiments, the surfactant is at a concentration of 0.001-10 mg/mL. In some embodiments, the surfactant is at a concentration of 0.01-2 mg/mL. In some embodiments, the surfactant is at a concentration of 2

BL-5733 0.1-2 mg/mL. In some embodiments, the surfactant is at a concentration of 0.2-1 mg/mL. In 17006886 some embodiments, the surfactant 1s 0.4 mg/mL polysorbate 20 or polysorbate 80.

In some embodiments, the formulation comprises: about 20-100 mg/mL anti-VEGF antibody, about 8-15 mM histidine buffer, about 10-100 mg/mL trehalose, and about 0.1-2 mg/mL polysorbate. In some embodiments, the anti-VEGF antibody is at a concentration of about 25- 80 mg/mL. In some embodiments, the anti-VEGF antibody is at a concentration of about 25 mg/mL.

In some embodiments, the formulation comprises: about 20-100 mg/mL anti-VEGF antibody, about 8-12 mM histidine buffer, about 30-80 mg/mL trehalose, and about 0.2-1 mg/mL polysorbate. In some embodiments, the anti-VEGF antibody is at a concentration of about 25- 80 mg/mL. In some embodiments, the anti-VEGF antibody is at a concentration of about 25 mg/mL.

In some embodiments, the formulation comprises: about 20-100 mg/mL anti-VEGF antibody, about 10 mM histidine buffer, about 60 mg/mL trehalose dihydrate, and about 0.4 mg/mL polysorbate.

In some embodiments, the formulation comprises: about 25-80 mg/mL anti-VEGF antibody, about 10 mM histidine buffer, about 60 mg/mL trehalose dihydrate, and about 0.4 mg/mL polysorbate.

In some embodiments, the formulation comprises: about 25-50 mg/mL anti-VEGF antibody, about 10 mM histidine buffer, about 60 mg/mL trehalose dihydrate, and about 0.4 mg/mL polysorbate.

In some embodiments, the formulation comprises: about 50-80 mg/mL anti-VEGF antibody, about 10 mM histidine buffer, about 60 mg/mL trehalose dihydrate, and about 0.4 mg/mL polysorbate.

In some embodiments, the formulation comprises: about 25 mg/mL anti-VEGF antibody, about mM histidine buffer, about 60 mg/mL trehalose dihydrate, and about 0.4 mg/mL polysorbate or polysorbate 80.

In some embodiments, the formulation comprises: about 50 mg/mL anti-VEGF antibody, about 10 mM histidine buffer, about 60 mg/mL trehalose dihydrate, and about 0.4 mg/mL polysorbate 80. 3

BL-5733

In some embodiments, the formulation comprises: about 80 mg/mL anti-VEGF antibody, about 7006808 mM histidine buffer, about 60 mg/mL trehalose dihydrate, and about 0.4 mg/mL polysorbate 80.

In some embodiments, the anti-VEGF antibody is bevacizumab.

In some embodiments, the formulation comprises: about 25 mg/mL bevacizumab, about 10 mM histidine buffer, about 60 mg/mL trehalose dihydrate, and about 0.4 mg/mL polysorbate 80.

In some embodiments, the formulation comprises: about 50 mg/mL bevacizumab, about 10 mM histidine buffer, about 60 mg/mL trehalose dihydrate, and about 0.4 mg/mL polysorbate 80.

In some embodiments, the formulation comprises: about 80 mg/mL bevacizumab, about 10 mM histidine buffer, about 60 mg/mL trehalose dihydrate, and about 0.4 mg/mL polysorbate 80.

In some embodiments, the formulation of the present invention does not comprise an antioxidant and/or a chelating agent. In some embodiments, the formulation of the present invention does not comprise an antioxidant. In some embodiments, the formulation of the present invention does not comprise a chelating agent. In some embodiments, the formulation of the present invention does not comprise an antioxidant and a chelating agent. In some embodiments, the formulation of the present invention does not comprise hyaluronidase,

EDTA (or edetate), methionine, or a combination thereof. In some embodiments, the formulation of the present invention does not comprise hyaluronidase. In some embodiments, the formulation of the present invention does not comprise methionine. In some embodiments, the formulation of the present invention does not comprise EDTA. In some embodiments, the formulation of the present invention does not comprise hyaluronidase and EDTA. In some embodiments, the formulation of the present invention does not comprise hyaluronidase and methionine. In some embodiments, the formulation of the present invention does not comprise

EDTA and methionine. In some embodiments, the formulation of the present invention does not comprise hyaluronidase, EDTA, and methionine. In some embodiments, the formulation can remain stable in the absence of hyaluronidase, EDTA, methionine, or a combination thereof. 4

BL-5733

In some embodiments, the formulation further comprises an antioxidant or a chelating agent. 17006886

In some embodiments, the antioxidant or the chelating agent is methionine or EDTA (or edetate).

In some embodiments, the formulation comprises 3-15 mM methionine. In some embodiments, the formulation comprises 5-10 mM methionine. In some embodiments, the formulation comprises 0.75-1.5 mg/mL methionine.

In some embodiments, the formulation comprises EDTA (or edetate). In some embodiments, the EDTA (or edetate) is at a concentration of less than 0.134 mM or 0.05 mg/mL. In some embodiments, the EDTA (or edetate) is at a concentration of less than 0.067 mM or 0.025 mg/mL. In some embodiments, the formulation comprises 0.05 mM EDTA. In some embodiments, the formulation comprises 0.019 mg/mL EDTA-2Na:2H20.

In some embodiments, the formulation comprises: about 20-100 mg/mL anti-VEGF antibody, about 8-15 mM histidine buffer, about 10-100 mg/mL trehalose, about 0.1-2 mg/mL polysorbate, and about 3-15 mM methionine or about 0.05 mM EDTA.

In some embodiments, the formulation comprises: about 20-100 mg/mL anti-VEGF antibody, about 8-15 mM histidine buffer, about 30-292 mM trehalose, about 0.1-2 mg/mL polysorbate, and about 3-15 mM methionine or 0.05 mM EDTA. In some embodiments, the anti-VEGF antibody is at a concentration of about 25-80 mg/mL. In some embodiments, the anti-VEGF antibody is at a concentration of about 25 mg/mL.

In some embodiments, the formulation comprises: about 20-100 mg/mL anti-VEGF antibody, about 8-15 mM histidine buffer, about 50-300 mM trehalose, about 0.1-2 mg/mL polysorbate, and about 3-15 mM methionine or 0.05 mM EDTA. In some embodiments, the anti-VEGF antibody is at a concentration of about 25-80 mg/mL. In some embodiments, the anti-VEGF antibody is at a concentration of about 25 mg/mL.

In some embodiments, the formulation comprises: about 20-100 mg/mL anti-VEGF antibody, about 8-12 mM histidine buffer, about 30-80 mg/mL trehalose, about 0.2-1 mg/mL polysorbate, and about 3-15 mM methionine or 0.05 mM EDTA. In some embodiments, the anti-VEGF antibody is at a concentration of about 25-80 mg/mL. In some embodiments, the anti-VEGF antibody is at a concentration of about 25 mg/mL.

In some embodiments, the formulation comprises: about 25 mg/mL anti-VEGF antibody, about

BL-5733 mM histidine buffer, about 60 mg/mL trehalose dihydrate, about 0.4 mg/mL polysorbate 7006808 80, and about 5 mM methionine.

In some embodiments, the formulation comprises: about 25 mg/mL anti-VEGF antibody, about 10 mM histidine buffer, about 60 mg/mL trehalose dihydrate, about 0.4 mg/mL polysorbate 80, and about 10 mM methionine.

In some embodiments, the formulation comprises: about 25 mg/mL anti-VEGF antibody, about 10 mM histidine buffer, about 60 mg/mL trehalose dihydrate, about 0.4 mg/mL polysorbate 80, and about 0.05 mM EDTA.

In some embodiments, the formulation has a pH value of 4.0-6.5. In some embodiments, the liquid formulation has a pH value of 4.9-5.7. In some embodiments, the liquid formulation has a pH value of 4.9-5.5.

In some embodiments, the formulation comprises: about 25 mg/mL bevacizumab, about 10 mM histidine buffer, about 60 mg/mL trehalose dihydrate, about 0.4 mg/mL polysorbate 80, and about 5 mM methionine, wherein the formulation has a pH value of 4.9-5.5.

In some embodiments, the formulation comprises: about 25 mg/mL bevacizumab, about 10 mM histidine buffer, about 60 mg/mL trehalose dihydrate, about 0.4 mg/mL polysorbate 80, and about 10 mM methionine, wherein the formulation has a pH value of 4.9-5.5.

In some embodiments, the formulation comprises: about 25 mg/mL bevacizumab, about 10 mM histidine buffer, about 60 mg/mL trehalose dihydrate, about 0.4 mg/mL polysorbate 80, and about 0.05 mM EDTA, wherein the formulation has a pH value of 4.9-5.5.

In some embodiments, the formulation of the present invention is a liquid formulation. In some embodiments, the solvent of the liquid formulation is water, such as water for injection.

In some embodiments, the formulation of the present invention is a solid formulation obtained by lyophilizing the liquid formulation described herein.

In some embodiments, the formulation of the present invention is a reconstituted formulation obtained by reconstituting the solid formulation described herein with a pharmaceutically acceptable solvent (such as water for injection or purified water).

In another aspect, the present invention provides a method for preparing the liquid formulation of the anti-VEGF antibody, comprising the following steps: (1) preparing a buffer solution; 6

BL-5733 (2) performing UF/DF ultrafiltration, and performing buffer exchange by ultrafiltration on an 17006886 antibody by adopting the buffer solution prepared in the step (1) to obtain an antibody solution; and (3) preparing an excipients stock solution containing a stabilizer and/or a surfactant, and adding the excipients stock solution into the antibody solution prepared in the step (2), and performing sterilizing and filtering to obtain an antibody formulation.

In another aspect, the present invention provides a method for treating a VEGF-associated disease, comprising administering to a patient an effective dose of the liquid formulation of the anti-VEGF antibody.

In some embodiments, the disease is cancer. In some embodiments, the cancer is selected from colorectal cancer, lung cancer, glioblastoma, hepatocellular carcinoma, epithelial ovarian cancer, fallopian tube cancer, primary peritoneal cancer, cervical cancer, breast cancer, renal cell carcinoma, glioblastoma, glioma, neuroglioma, tumor metastasis, neuromyelitis optica, B- cell chronic lymphocytic leukemia, sarcoma, liver cancer, multiple myeloma, and brain cancer.

In another aspect, the present invention provides an antibody pharmaceutical product for treating a VEGF-associated disease, comprising the liquid formulation described above and a container for storing the formulation. The pharmaceutical product may also comprise instructions for use. The container may be any container conventionally used in the art for storing drugs, such as vials (e.g., penicillin bottles), pre-filled syringes, injection pens, ampoules, sachets and the like. The antibody formulation of the present invention has good stability at high temperature and room temperature, can be kept in a liquid form, and is convenient to use.

The formulation of the present invention exhibits better stability than the control under the conditions of high temperature of 40 °C, illumination (4500 + 500 Ix, 25 °C), acceleration (25 °C) and the like, and is more suitable for clinical use and storage.

The liquid formulation of the present invention remain stable when left at 25 °C for at least 3 months, at high temperature of 40 °C for at least 4 weeks, or under illumination for at least 6 days. 7

BL-5733

BRIEF DESCRIPTION OF THE DRAWINGS 7006808

FIG. 1 is an aggregation curve of different formulation samples.

FIG. 2 shows the trend of the SEC-HPLC monomer contents with time under illumination conditions (left-upright sample; right-inverted sample).

FIG. 3 shows the trend of the SEC-HPLC monomer contents with time under high temperature conditions (left-upright sample; right-inverted sample).

FIG. 4 shows the trend of the IEC-HPLC main peak with time under high temperature conditions (left-upright sample; right-inverted sample).

FIG. 5 shows the trend of the SEC-HPLC aggregates contents with time under accelerated conditions (left-upright sample; right-inverted sample).

FIG. 6 shows the trend of the IEC-HPLC main peak under accelerated conditions (left-upright sample; right-inverted sample).

FIG. 7 shows the change of the SEC-HPLC monomer (left) and aggregates (right) contents with time under illumination conditions for samples with different pH values.

FIG. 8 shows the trend of the CE-SDS (NR) main peak (left) and fragment (right) with time under illumination conditions for samples with different pH values.

FIG. 9 shows the change of the SEC-HPLC monomer (left) and aggregates (right) contents with time under high temperature conditions for samples with different pH values.

FIG. 10 shows the trend of the IEC-HPLC main peak (left) and acidic variant (right) with time under high temperature conditions for samples with different pH values.

FIG. 11 shows the trend of CE-SDS (NR) main peak (left) and fragment (right) with time under high temperature conditions for samples with different pH values.

FIG. 12 shows the trend of the IEC-HPLC main peak (left) and acidic variant (right) with time under accelerated conditions for samples with different pH values.

FIG. 13 shows the trend of the SEC-HPLC monomer (left) and aggregates (right) contents with time under illumination conditions for samples.

FIG. 14 shows the trend of the CE-SDS (NR) main peak (left) and fragment contents (right) with time under illumination conditions for samples.

FIG. 15 shows the trend of the SEC-HPLC monomer (left) and aggregates (right) contents with 8

BL-5733 time under high temperature conditions for samples. 7006808

FIG. 16 shows the trend of the IEC-HPLC main peak (left) and acidic variant contents (right) with time under high temperature conditions for samples.

FIG. 17 shows the trend of the CE-SDS (NR) main peak (left) and fragment contents (right) with time under high temperature conditions for samples.

DETAILED DESCRIPTION

The technical solution of the present invention will be further illustrated below through specific examples, which are not intended to limit the protection scope of the present invention. Some nonessential modifications and adjustments made by other people according to the concept of the present invention still fall within the protection scope of the present invention.

Terms

It should be noted that “%” related to components in the present invention refers to a weight/volume (w/v) percentage, wherein the weight unit may be g, and the volume unit may be mL. For example, 1% stabilizer in a solution means 1 g stabilizer in 100 mL solution or the stabilizer has a content of 0.01 g/mL. For example, 6% trehalose dihydrate means 60 mg/mL trehalose dihydrate, and 0.04% polysorbate means 0.4 mg/mL polysorbate. “About” or “approximately” refers to a general error range for corresponding values as readily understood by those skilled in the relevant art. In some embodiments, “about” or “approximately” mentioned herein refers to the numerical values described as well as its ranges of + 10%, + 5%, + 1% or + 0.1%. “Comprise” or “include” means that the composition, method and the like include the listed elements (e.g., components of the compositions, steps of the methods, and the like), but do not exclude the others. When used to define the compositions and methods, “consisting essentially of...” means excluding other elements that have a fundamental impact on the combination for its intended use, but not excluding elements that do not substantially affect the characteristics of the compositions or methods. “Consisting of...” means excluding elements not specifically listed. Embodiments defined by each of these transition terms are all within the scope of the 9

BL-5733 present invention. For example, when a composition is described as including components A, 7006808

B, and C, a composition consisting essentially of A, B, and C, and a composition consisting of

A, B, and C are independently within the scope of the present invention.

The “treatment” of a patient’s disease means: (1) preventing a disease from occurring in a patient who is prone to the disease or has not shown symptoms of the disease; (2) inhibiting the disease or preventing its development; or (3) relieving the disease or making it regress.

The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary cancer, liver tumor, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma (including low-grade/follicular non-Hodgkin’s lymphoma (NHL), small lymphocytic (SL)

NHL, intermediate-grade/follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphomas, and Waldenstrom’s macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia and post-transplant lymphoproliferative disorder (PTLD), and abnormal vascular proliferation associated with phakomatoses, edema (such as those associated with brain tumors) and Meigs’ syndrome, brain tumor and brain cancer, head and neck cancer as well as related metastases. In certain embodiments, cancers suitable for treatment by the antibody of the present invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-

BL-5733

Hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic 17006886 cancer, soft tissue sarcoma, kaposi’s sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some embodiments, the cancer 1s selected from: small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. In some embodiments, the cancer is selected from: non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer, including metastatic forms of those cancers.

The term “buffer” also refers to a buffer system, and a histidine buffer includes histidine and histidine salts such as hydrochloride. The amount of the buffer in the present invention means the total amount of the buffer pair in the buffer system constituting the buffer. In some embodiments, molar concentration is taken as a unit of the amount of the buffer, the value of which refers to the molar concentration of the buffer pair in the buffer system of the buffer. For example, where histidine buffer consisting of histidine and histidine hydrochloride is used as the buffer, a given concentration of histidine buffer (e.g., 10 mM) is the combined concentration of histidine and histidine hydrochloride.

The formulation of the present invention can be prepared with the excipients or the hydrate thereof. For example, histidine hydrochloride, also known as histidine hydrochloride, may be anhydrous histidine hydrochloride, or histidine hydrochloride hydrate, such as histidine hydrochloride monohydrate. As another example, trehalose is usually present as trehalose dihydrate in a solid state. Trehalose herein may be a trehalose hydrate containing an equivalent amount of trehalose such as trehalose dihydrate. For example, trehalose at a concentration of 54.3 mg/mL may be 60 mg/mL trehalose dihydrate. The formulation may be prepared by adopting trehalose dihydrate, and may also be prepared by adopting trehalose in other forms (such as anhydrous trehalose).

In some embodiments, the chelating agent is selected from salts of EDTA (i.e., edetate), including dipotassium edetate, disodium edetate, calcium disodium edetate, sodium edetate, trisodium edetate, and potassium edetate. EDTA-2Na may be anhydrous EDTA-2Na, and may also be EDTA-2Na hydrate, such as EDTA-2Na-2H;0. For example, “0.05 mM EDTA-2Na” or 0.0019% w/v EDTA-2Na:2H,0, may be 1 mL solution formed by dissolving 0.019 mg

EDTA-2Na:2H20 in a solvent. 11

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Hyaluronidases are generally neutral or acid active enzymes found throughout the animalia. 7006808

Hyaluronidases differ with respect to their substrate specificity and mechanism of action (WO2004/078140). Neutral-active soluble hyaluronidase glycoproteins (SHASEGP), for example, may be human soluble PH20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENE (registered trademark), Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. patent publication NOs. 2005/0260186 and 2006/0104968.

Tween is also known as polysorbate, e.g., Tween 20 is known as polysorbate 20 (i.e., PS20), and Tween 80 is known as polysorbate 80 (i.e., PS80). “Stability” and “stable” herein mean that the antibody (including the antibody fragment thereof) does not, or only minimally, undergo aggregation, degradation, or fragmentation under given conditions of manufacture, preparation, transportation and/or storage in a liquid formulation comprising the antibody. A “stable” formulation maintains biological activity under given conditions of manufacture, preparation, transportation and/or storage. The stability of the antibody can be evaluated by the extent of aggregation, degradation or fragmentation of the formulation and the like as measured by technologies such as SEC-HPLC, IEC-HPLC, CE-

SDS (NR), visual inspection and turbidity, insoluble particles, and detection of particle size by

DLS. In some embodiments, “stable” refers to a decrease in monomer purity of no more than 10%, 5%, or 2% over a period of time (e.g., two weeks, one month, or 3 months) under certain storage conditions. “FRA” herein refers to a fragment; “HHL” refers to an impurity with two heavy chains and one light chain; “HWM” refers to a high weight molecule, referred to as an aggregation; and “MAIN” refers to a main peak.

In the following examples, reagents and instruments used are those conventional in the art and commercially available unless otherwise specified; the methods used are conventional in the art, and those skilled in the art can undoubtedly implement the methods and obtain the corresponding results according to the content of the examples.

In some embodiments, the anti-VEGF antibody is bevacizumab BAT1706, and may be expressed in CHO cells by recombinant DNA techniques and obtained after purification by a series of standard chromatographic steps. The sequence structure of BAT 1706 is shown in the 12

BL-5733 . LU504834 following table:

Sequence | Amino acid sequence Description

ID NOs 1 EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVG | VH

WINTYTGEPTYAADFKRRFTFSLDTSKSTAYLOQMNSLRAEDTAVYYCAKYP

HYYGSSHWYFDVWGQGTLVTVSS

2 DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQOQKPGKAPKVLIYFTSS | VL

LHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEI

K

3 EVQOLVESGGGLVQPGGSLRLSCAASGYTFINY GMNWVRQAPGKGLEWVGW | heavy

INTYTGEPTYAADFKRRFTFSLDTSKSTAYLOMNSLRAEDTAVYYCAKYPHY | chain

YGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD

YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV

NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR

TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV

LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE

MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL

TVDKSRWQQGNVFSCSVMHEALHNHYTQOKSLSLSPGK

4 DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSS | light chain

LHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS

QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR

GEC

Preparation method for 10 mM histidine buffer with a pH value of 5.5 (hereinafter referred to as His buffer): 1.55 g/L histidine solution and 2.09 g/L histidine hydrochloride solution were prepared, and the two solutions were adjusted to pH 5.5 with each other.

Preparation method for 50 mM phosphate buffer with a pH value of 6.1 (hereinafter referred to as PB buffer): 1.20 g of anhydrous disodium hydrogen phosphate and 5.8 g of sodium dihydrogen phosphate monohydrate were added to 1 L of solution.

Example 1: Buffer Optimization and Screening

A stock solution of a BAT1706 antibody (hereinafter referred to as an antibody) was exchanged into the corresponding prepared buffer by ultrafiltration to obtain an antibody stock solution.

An excipients stock solution containing trehalose and Tween with 6 times of target concentration was prepared and added to the antibody stock solution. The excipients 13

BL-5733 . . . ; LU504834 concentration was adjusted to prepare a formulation shown in Table 1, wherein the antibody was at a concentration of about 25 mg/mL. The sample was filtered by using a filter flask with a filter membrane with an aperture of 0.22 um, filled into a 6 mL vial (4 mL/bottle, Schott vial), and covered with a rubber plug. The stability test was carried out on the above samples under the conditions of illumination (4500 + 500 Ix, 25 °C), high temperature (40 °C), acceleration (25 °C) and 2-8 °C, respectively.

Table 1. Formulation composition

Note: Tre is a,a-trehalose dihydrate, and PS is polysorbate.

All formulations had Tm values above 70 °C and Tonset values above 62 °C, and good structural stability (see Table 2). It can also be seen from the sample aggregation curve (FIG. 1) that PB buffer system samples showed significant aggregation at about 68 °C, and no significant aggregation was detected in the His buffer system samples throughout the entire warming process, indicating that the His buffer system had better colloidal stability compared to the PB buffer system.

B»2 and kD of the His buffer system samples were both positive values, which indicated that weak mutual repulsive force exists between molecules, and the formulation was more favorable for the protein-solvent interactions, predicting a tendency for long-term placement to be stable.

B»z and kD of the PB buffer system samples were negative values, indicating a weak mutual attraction between protein molecules and a tendency to aggregate.

Table 2. Detection results of antibody stability for different formulation samples

Formulation Tm (°C) Tonset (°C) Tagg473 (°C) B»» (mol-mL/g?) kD (mL/g) 1-PB 73.5 66.9 68.8 -7.21E-05 -9.12 3-His-PS20 71.2 62.5 70.9 5.29E-04 15.4 4-His-PS20 70.6 62.1 71.4 9.09E-04 19.92 5-His-PS80 70.5 62.4 70.7 6.39E-04 23.69 14

BL-5733 1. Illumination test (4500 + 500 Ix, 25 °C) 17006886

The sample to be detected was placed under an illumination condition (GZ) for 14 days (upright (ZF) or inverted (DF) sample), sampled and detected on days 0, 7 and 14 (OD, 7D and 14D).

The results were shown in Table 3.

As can be seen from the SEC-HPLC detection data (FIG. 2), with the extension of illumination time, regardless of the upright (ZF) or inverted (DF) samples, the formulation of the His buffer system (hereinafter referred to as His formulation) consistently had higher SEC monomer content than that of the formulation of the PB buffer system (hereinafter referred to as PB formulation), and consistently had lower SEC polymer content than that of the PB buffer system. The inverted PS80 formulation had a slightly lower SEC aggregation content than that of the PS20 formulation.

According to IEC-HPLC detection results, after 14 days under illumination condition, the IEC main peak content of the His formulation was higher than that of the PB formulation, and the acidic variant content and the basic variant content of the His formulation were lower than that of the PB formulation regardless of the upright or the inverted samples; the difference between different formulations of PS20 and PS80 was not significant in the upright samples, and formulation 5-His-PS80 had a slightly lower IEC acidic variant compared to PS20 formulation (His formulations 3 and 4) in the inverted samples.

According to CE-SDS (NR) detection results, the main peak content of formulation CE (NR) of the His buffer system was higher than that of the PB formulation, and the fragments (FAR) and polymer (HWM) contents were lower than that of the PB formulation regardless of the upright or the inverted samples. The difference between different formulations of PS20 and

PS80 was not significant.

Table 3. SEC-HPLC, IEC-HPLC and CE-SDS (NR) detection results of different formulation samples under the illumination condition

SEC-HPLC (%) IEC-HPLC (%) CE-SDS (NR) (%) ne Formu fatio Aggregatio Monome Fragmen Acidic Main Basic FR HH MAI HW n r t , Peak + A L ON M 0D 1-PB 1.59 98.37 0.04 17.29 Pi 7.00 188 1.00 97.87 0.25

BL-5733 76.4 LU504834 3-His-PS20 0.65 99.30 0.04 17.12 1 6.48 1.72 0.92 98.28 0.00 . 76.3 4-His-PS20 0.65 99.31 0.04 17.09 2 6.59 183 0.99 98.17 0.00 5-His-PS80 0.66 99.30 0.04 17.19 763 6.50 1.76 0.96 98.24 0.00 42.6 1-PB 6.97 92.77 0.27 33.25 4 24.10 437 2.43 93.12 2.51 3-His-PS20 3.07 96.78 0.15 31.82 47.2 20.98 3.47 1.94 9595 0.58

GZ-7D- 0

ZF . 48.5 4-His-PS20 2.75 97.12 0.14 31.19 8 20.23 3.30 1.91 96.14 0.56 5-His-PS80 2.95 96.91 0.14 31.22 3 20.46 3.35 1.90 95.99 0.66 40.6 1-PB 7.56 92.13 0.31 34.27 1 25.12 487 2.72 9245 2.68 3-His-PS20 4.17 95.62 0.21 34.75 419 23.28 3.95 2.25 95.09 0.96

GZ-7D- 7

DF . 42.6 4-His-PS20 3.99 95.80 0.21 34.48 1 2291 4.02 2.21 95.19 0.79 5-His-PS80 3.54 96.27 0.19 32.89 i 21.69 3.81 2.18 9545 0.74 25.3 1-PB 11.16 88.26 0.57 43.56 9 31.05 7.24 372 89.10 3.66

GZ 3-His-PS20 5.36 94.35 0.29 41.11 07 28.17 5.02 2,78 93.89 1.09 14D-ZF . 29.4 4-His-PS20 5.67 94.02 0.30 41.99 6 2855 5.07 2.74 93.81 1.12 5-His-PS80 5.36 94.35 0.29 41.63 300 2830 4.98 2.83 93.98 1.04 23.0 1-PB 12.53 86.81 0.66 45.46 2 31.52 7.57 3.70 88.08 4.35

GZ 3-His-PS20 6.97 92.64 0.39 44.33 20 30.02 5.75 3.06 92.74 1.51 14D-DF . 24.0 4-His-PS20 7.43 92.14 0.43 45.50 5 3045 6.33 3.38 91.78 1.89 5-His-PS80 6.58 93.06 0.37 43.04 1 29.61 5.66 3.03 92.63 1.71

Under the illumination condition, the activity decrease trend and amplitude of each formulation were basically consistent, and no obvious difference was detected.

As can be seen from the detection result of the polysorbate content under the illumination condition (Table 4), PS20 was more stable in the His buffer system than in the PB buffer system. PS80 was more stable than PS20.

Table 4. Detection results of the polysorbate content of different formulation samples under the illumination condition 16

BL-5733

Formulation Polysorbate content (ng/mL) LUS04834 0D GZ-7D-ZF GZ-7D-DF GZ-14D-ZF GZ-14D-DF 1-PB 392 374 359 373 351 3-His-PS20 380 372 362 370 352 4-His-PS20 373 366 357 350 343 5-His-PS80 379 373 375 368 367

Taking into account all the above illumination investigation results, the formulation of His buffer system had better stability than the formulation of PB buffer system, and PS80 degraded more slowly than PS20. 2. High temperature test (40 + 2 °C)

The samples were placed under the high temperature (GW) condition of 40 °C for 4 weeks, and sampled at week 0, week 2, and week 4 for detection (0D-2W-4W). The results were shown in Table 5.

As can be seen from the SEC-HPLC detection data (FIG. 3), after 4 weeks of high temperature investigation, the PB formulation had a greater decrease in the SEC monomer content compared to the His formulation regardless of the upright or the inverted samples.

As can be seen from the IEC-HPLC detection results (FIG. 4), after 4 weeks of high temperature investigation, the PB formulation had a greater decrease in the IEC main peak content and a greater increase in the acidic and basic variants compared to the His formulation regardless of the upright or the inverted samples.

As can be seen from the CE-SDS (NR) detection results (FIG. 8), after 4 weeks of high temperature investigation, the His formulation showed a slightly higher CE (NR) main peak content and slightly lower fragment and polymer contents compared to the PB formulation regardless of the upright or the inverted samples.

The activity decline trend and amplitude of each formulation were basically consistent, and no obvious difference was detected when the samples were placed under the high temperature condition for 4 weeks.

Table 5. SEC-HPLC, IEC-HPLC and CE-SDS (NR) detection results of different formulation samples under the high temperature condition 17

BL-5733

LU504834

SEC-HPLC (%) IEC-HPLC (%) CE-SDS (NR) (%)

Samplin Formulatio idi i ; gtime n aggregatio Monome Fragmen Acidic Main D3 FR HH Main HW varian varian n r t { peak | A L peak M 75.7 97.8 1-PB 1.59 98.37 0.04 17.29 1 7,00 1.88 1.00 7 0.25 . 76.4 98.2 3-His-PS20 0.65 99.30 0.04 17.12 1 648 1.72 0.92 8 0.00 0D ; 76.3 98.1 4-His-PS20 0.65 99.31 0.04 17.09 5 6.59 1.83 0.99 7 0.00 ; 76.3 98.2 5-His-PS80 0.66 99.30 0.04 17.19 1 76.50 1.76 0.96 4 0.00 56.1 95.9 1-PB 2.87 96.82 0.31 34.46 9 9.34 3.59 1.32 0 0.51 ; 63.8 96.9 3-His-PS20 0.80 98.92 0.28 27.96 3 8.22 2.93 1.08 0.14

GW-2W- 3

ZF 63.8 96.7 4-His-PS20 0.78 98.95 0.27 27.84 3 8.33 3.10 1.11 8 0.12 . 64.3 96.7 5-His-PS80 0.82 98.92 0.26 27.46 0 8.24 3.09 1.14 4 0.17 552 95.7 1-PB 3.34 96.35 0.31 35.27 3 9.44 3.72 1.37 1 0.57 . 63.4 96.6 3-His-PS20 0.80 98.93 0.27 28.21 7 8.32 321 1.16 0.14

GW-2W- 5

DF ; 63.6 96.8 4-His-PS20 0.81 98.92 0.27 28.00 7 8.33 3.05 1.14 3 0.12 ; 63.9 96.8 5-His-PS80 0.83 98.90 0.27 27.76 7 8.27 3.06 1.16 0 0.14 42 4 94.0 1-PB 4.56 92.82 2.62 48.42 2 9.16 5.26 1.63 8 0.66 ; 52.9 95.5 3-His-PS20 1.02 96.44 2.54 38.80 7 8.23 433 1.28 0.12

GW-4W- 5

ZF ; 53.2 95.6 4-His-PS20 0.98 97.07 1.95 38.60 8 8,12 421 1.25 6 0.13 ; 53.2 95.3 5-His-PS80 1.01 96.70 2.29 38.58 0 822 4.52 1.41 3 0.15 40.8 93.3 1-PB 5.26 92.24 2.50 49.17 4 “9.99 574 1.85 > 0.94

GW-4W- i 52.5 95.2

DF 3-His-PS20 1.04 96.49 2.47 39.17 3 8,25 4.59 142 ) 0.19 4-His-PS20 1.01 96.63 2.36 38.99 52.7 822 465 1.51 95.1 0.16 18

BL-5733 - LU5 04834 9 9 . 52.7 95.2 5-His-PS80 1.13 96.61 2.26 38.84 9 © 8.38 4.59 1.52 6 0.15

After 4 weeks examination under high temperature condition, the polysorbate content was shown in Table 6, and PB formulation showed a greater degradation in PS20 than the His formulation (formulation 1-PB and formulation 3-His-PS20). PS80 degraded more slowly than

PS20.

Taking into account all the high temperature investigation results, under the same condition, the PB formulation showed a greater degradation in PS20 than the His formulation, and the His buffer system had better stability than the PB buffer system by combining physicochemical detection results. In addition, PS80 was more stable than PS20, and PS80 degraded much lower than PS20 under high temperature for 4 weeks.

Table 6. Detection results of the polysorbate content of different formulation samples under the high temperature condition

Polysorbate content (ug/mL)

Formwlaton +—{ÆŒ—Æ 0D GW-2W-ZF GW-2W-DF GW-4W-ZF GW-4W-DF 1-PB 392 291 241 262 198 2-PB-Coating 394 307 311 281 279 3-His-PS20 380 340 313 325 279 4-His-PS20 373 328 288 295 249 5-His-PS80 379 369 355 359 339 3. Accelerated test (25 + 2 °C)

The formulation sample was placed at 25 + 2 °C for 3 months, and sampled at day 0, month 1, month 2 and month 3 (0D-1M-2M-3M) for accelerated stability study. The results were shown in Table 7 and Table 8.

According to SEC-HPLC detection results, the upright and inverted samples had no obvious difference, and all the formulations were basically unchanged after acceleration for 1 month; after acceleration for 3 months, all formulations had a slight decrease in the SEC monomer 19

BL-5733 . . . . . . . . LU504834 content and a slight increase in the aggregation content, with PB formulations having a slightly greater increase in the aggregation content compared to His formulations (FIG. 5).

According to IEC-HPLC detection results, under acceleration for 3 months, the upright and inverted samples had no obvious difference, and the PB formulation had a greater decrease in the IEC main peak content (FIG. 6) and a greater increase in the acidic and basic variants compared to the His formulation.

According to CE-SDS (NR) detection results, under acceleration for 3 months, the upright and inverted samples had a generally consistent trend, and all formulations had a slight decrease in the CE (NR) main peak and an increase in fragments, with a slightly greater decrease in the CE (NR) main peak for the PB formulation compared to the His formulation.

The activity of different formulations was basically consistent in the descending trend and the amplitude after accelerated investigation for 3 months.

Table 7. SEC-HPLC and IEC-HPLC detection results of different formulation samples under the accelerated condition . SEC-HPLC (%) IEC-HPLC (%)

Sampling ; css time Formulation aggregation Monomer Fragment Acidic Main Basic 88165 8 variant peak variant 1-PB 1.59 98.37 0.04 17.29 75.71 7.00 oD 3-His-PS20 0.65 99.30 0.04 17.12 76.41 6.48 4-His-PS20 0.65 99.31 0.04 17.09 76.32 6.59 5-His-PS80 0.66 99.30 0.04 17.19 76.31 6.50 1-PB 1.58 98.27 0.15 23.38 69.15 7.47 3-His-PS20 0.67 99.20 0.14 20.79 72.34 6.88

JS-1M-ZF . 4-His-PS20 0.67 99.19 0.14 20.70 72.51 6.79 5-His-PS80 0.69 99.17 0.14 20.81 72.64 6.55 1-PB 1.61 98.24 0.16 23.56 68.97 7.47 3-His-PS20 0.68 99.19 0.14 20.82 72.31 6.87

JS-1M-DF . 4-His-PS20 0.67 99.20 0.13 20.87 72.32 6.81 5-His-PS80 0.69 99.18 0.14 20.86 72.49 6.64 1-PB 2.20 97.59 0.21 26.47 65.00 8.53 3-His-PS20 0.79 99.03 0.18 22.38 70.27 7.34

JS-2M-ZF . 4-His-PS20 0.77 99.04 0.18 22.51 70.00 7.49 5-His-PS80 0.79 99.03 0.18 22.45 70.18 7.37 1-PB 2.16 97.63 0.21 27.01 64.50 8.49 3-His-PS20 0.77 99.04 0.19 22.83 69.81 7.36

JS-2M-DF . 4-His-PS20 0.77 99.05 0.18 22.91 69.58 7.52 5-His-PS80 0.82 99.00 0.19 22.93 69.58 7.49 1-PB 1.98 97.73 0.29 29.98 61.64 8.38

JS-3M-ZF . 3-His-PS20 0.79 98.95 0.26 24.68 68.16 7.16

BL-5733 4-His-PS20 0.78 98.97 0.26 24,74 6808 7.18 LU504834 5-His-PS80 0.84 98.91 0.26 24.96 67.76 7.28 1-PB 2.29 97.41 0.30 30.70 60.46 8.84 3-His-PS20 0.77 98.97 0.26 25.00 67.63 7.37

JS-3M-DF . 4-His-PS20 0.79 98.95 0.26 24.94 67.94 7.12 5-His-PS80 0.87 98.88 0.26 25.19 67.46 7.35

Table 8. CE-SDS (NR) Detection results of different formulation samples under the accelerated condition

CE-SDS (NR) (%)

Sampling time Formulation ———

FRA HHL MAIN HWM

1-PB 1.88 1.00 97.87 0.25 3-His-PS20 1.72 0.92 98.28 0.00 0D 4-His-PS20 1.83 0.99 98.17 0.00 5-His-PS80 1.76 0.96 98.24 0.00 1-PB 2.47 1.01 97.16 0.37 3-His-PS20 2.21 0.89 97.69 0.10

JS-1M-ZF 4-His-PS20 2.32 0.98 97.56 0.12 5-His-PS80 2.99 0.93 96.93 0.08 1-PB 2.51 1.01 97.13 0.36 3-His-PS20 2.34 0.94 97.55 0.11

JS-1M-DF 4-His-PS20 2.24 0.91 97.64 0.12 5-His-PS80 2.21 0.89 97.65 0.14 1-PB 3.12 1.16 96.19 0.69 3-His-PS20 2.86 1.07 96.78 0.36

JS-2M-ZF 4-His-PS20 2.84 1.10 96.88 0.28 5-His-PS80 2.91 1.05 96.64 0.45 1-PB 3.19 1.30 96.11 0.70 3-His-PS20 3.06 1.20 96.70 0.24

JS-2M-DF 4-His-PS20 2.68 1.00 97.12 0.20 5-His-PS80 2.69 1.02 96.98 0.33 1-PB 3.47 1.25 95.91 0.62 3-His-PS20 2.97 1.09 96.83 0.20

JS-3M-ZF 4-His-PS20 2.95 1.05 96.79 0.26 5-His-PS80 3.01 1.11 96.77 0.22 1-PB 3.62 1.32 95.66 0.72

JS-3M-DF 3-His-PS20 3.00 1.08 96.82 0.18 21

BL-5733 4-His-PS20 3.02 1.09 96.80 0.18 5-His-PS80 3.07 1.09 96.74 0.19

The detection result of the polysorbate content was shown in Table 9 after accelerated investigation for 3 months, and the PB formulation showed a greater degradation in PS20 than the His formulation. PS80 degraded more slowly than PS20.

Taking into account all the accelerated investigation results, the PB formulation showed a greater degradation in PS20 than the His formulation. PS80 was more stable than PS20, and

PS80 degraded much lower than PS20 under acceleration for 3 months.

Table 9. Detection results of the polysorbate content of different formulation samples under the acceleration condition

Polysorbate content (ng/mL)

Formulation —m08 7 X—X——™M8M8M8M8M8 —M8¥ MM —————————— 0D JS-1M-ZF JS-1M-DF JS-2M-ZF JS-2M-DF JS-3M-ZF JS-3M-DF 1-PB 392 316 284 262 229 220 173 3-His-PS20 2380 355 335 323 302 290 270 4-His-PS20 373 339 312 307 284 268 246 5-His-PS80 379 370 366 361 355 352 351

As can be seen from the physicochemical detection results, the His formulation had better quality and stability than the PB formulation under various compulsory investigation conditions.

As can be seen from the detection results of the polysorbate content, PS20 was more stable in the His formulation than in the PB formulation. The PB formulation showed a greater degradation in PS20 than the His formulation in high temperature and 25 °C accelerated investigations, and the stability of PS80 was superior to that of PS20.

Example 2: pH Optimization and Screening Experiments

BAT1706 antibodies were exchanged into the buffer by ultrafiltration respectively, and the samples were prepared into formulations as shown in Table 10 with an antibody concentration of about 25 mg/mL. The samples were filtered using a 0.22 um filter and dispensed into 0.6 mL sterile EP tubes (200 pL/tube), and the SEC-HPLC, IEC-HPLC, CE-SDS (NR) stability 22

BL-5733 parameters of the formulation samples were detected under illumination (4500 + 500 Ix, 25 °C), 7006808 high temperature (40 °C), and accelerated (25 °C) experimental conditions, respectively.

Table 10. Formulation composition concentration mM His buffer 6% Tre 0.04% PS20 25 mg/mL = —

Note: Tre is a,a-trehalose dihydrate, and PS20 is polysorbate 20. 1. Illumination test (4500 + 500 Ix, 25 + 2 °C)

The samples were placed under the illumination condition for 10 days, and sampled for detection on days 0, 5 and 10 (0D-5D-10D). The results were shown in Table 11 and Table 12.

As can be seen from the SEC-HPLC detection data, His formulations with a pH value of 4.9- 5.7 were all obviously superior to PB formulation, and the formulation 06-PB6.1 sample had the highest contents of the aggregation and the fragment under the illumination condition (FIG. 7).

From IEC-HPLC detection results, the His formulation with a pH value of 4.9-5.5 was superior to the PB formulation; and the lower the pH, the better the stability in the His formulation.

As can be seen from the CE-SDS (NR) detection results, the His formulation was superior to the PB formulation under illumination for 10 days; there was no significant difference between formulations with different pH values in the His formulation, (FIG. 8). Formulation 06-PB6.1 sample had a significant increase in FAR and HWM, and a larger increase amplitude than the

His formulation.

Taking into account all the above illumination investigation results, among the His formulations, the formulations with a pH value of 4.9-5.5 had better stability; formulation 05-

His5.7 had slightly poor stability, and formulation 06-PB6.1 had the worst stability.

Table 11. SEC-HPLC and IEC-HPLC detection results of different pH samples under the 23

BL-5733 ; a _ LU504834 illumination condition

SEC-HPLC (%) IEC-HPLC (%)

Sampling time Formulation i Acidic Main Basic aggregation Monomer Fragment . . variant peak variant 01-His4.9 0.49 99.45 0.05 16.96 75.61 7.44 02-His5.1 0.54 99.39 0.07 17.03 75.59 7.38 oD 03-His5.3 0.62 99.32 0.06 17.10 75.72 7.17 04-His5.5 0.76 99.18 0.06 17.17 75.73 7.10 05-His5.7 1.08 98.86 0.06 17.22 75.60 7.18 06-PB6.1 1.61 98.32 0.08 18.08 74.30 7.62 01-His4.9 3.23 96.42 0.36 30.76 31.48 37.76 02-His5.1 4.17 95.49 0.34 33.49 31.75 34.76

GZ-5D 03-His5.3 5.01 94.65 0.34 36.73 31.26 32.01 04-His5.5 5.63 94.06 0.31 38.69 28.80 32.51 05-His5.7 7.63 92.05 0.32 43.63 26.11 30.27 06-PB6.1 12.98 84.42 2.60 40.92 24.15 34.93 01-His4.9 7.70 88.81 3.49 38.04 15.99 45.96 02-His5.1 8.40 88.24 3.36 45.75 12.26 41.98

GZ-10D 03-His5.3 9.88 86.81 3.31 50.54 11.72 37.74 04-His5.5 13.00 84.50 2.49 54.48 11.61 33.91 05-His5.7 11.20 85.63 3.17 58.44 12.01 29.54 06-PB6.1 23.19 72.60 4.20 52.65 9.86 37.49

Table 12. CE-SDS (NR) detection results of different pH samples under the illumination condition

CE-SDS (NR) (%)

Sampling time Formulation -_— ——

FRA HHL MAIN HWM

01-His4.9 1.94 0.95 97.92 0.14 02-His5.1 1.89 0.91 97.93 0.18 03-His5.3 2.02 0.99 97.84 0.14 0D 04-His5.5 1.91 0.91 97.89 0.20 05-His5.7 1.98 0.96 97.80 0.22 06-PB6.1 1.93 0.95 97.67 0.40 01-His4.9 6.45 3.65 92.33 1.22 02-His5.1 6.16 3.47 92.18 1.66 03-His5.3 6.06 3.37 92.20 1.74

GZ-5D 04-His5.5 5.75 3.17 92.54 1.71 05-His5.7 5.93 3.29 92.07 2.00 06-PB6.1 7.45 3.89 87.78 4.77 01-His4.9 10.66 5.52 86.43 2.91

GZ-10D 02-His5.1 10.61 5.45 86.23 3.16 24

BL-5733 03-His5.3 10.58 5.56 86.04 3.38 04-His5.5 10.29 5.45 86.06 3.65 05-His5.7 9.61 5.06 86.76 3.63 06-PB6.1 12.15 5.68 79.94 7.91 2. High temperature test (40 + 2 °C)

The samples were placed under the high temperature condition for 4 weeks, and sampled at week 0, week 2, and week 4 for detection (0D-2W-4W). The results were shown in Table 13 and Table 14.

As can be seen from the SEC-HPLC detection data (FIG. 13), among the His formulations, the difference between the formulations with a pH value of 4.9-5.5 was not large, and the PB formulation had poor stability compared to the His formulation at high temperature for 4 weeks.

As can be seen from the IEC-HPLC detection results, among the His formulations, the main peak difference between the formulations with a pH value of 4.9-5.7 was not large, and the PB formulation had poor stability compared to the His formulation at high temperature for 4 weeks.

As can be seen from the CE-SDS (NR) detection results (FIG. 14), among the His formulations, the formulations with a pH value of 4.9-5.7 had no obvious difference, and the PB formulation had poor stability compared to the His formulation.

Taking into account all the high temperature investigation results, the His formulations with a pH value of 4.9-5.5 all had good stability.

Table 13. SEC-HPLC and IEC-HPLC detection results of different pH samples under the high temperature condition

SEC-HPLC (%) IEC-HPLC (%)

Sampling time Formulation di M F 0 Acidic Main Basic aggregation onomer ragmen variant peak variant 01-His4.9 0.49 99.45 0.05 16.96 75.61 7.44 02-His5.1 0.54 99.39 0.07 17.03 75.59 7.38 oD 03-His5.3 0.62 99.32 0.06 17.10 75.72 7.17 04-His5.5 0.76 99.18 0.06 17.17 75.73 7.10 05-His5.7 1.08 98.86 0.06 17.22 75.60 7.18 06-PB6.1 1.61 98.32 0.08 18.08 74.30 7.62 01-His4.9 0.50 99.14 0.36 28.59 62.23 9.18 02-His5.1 0.56 99.11 0.33 28.51 62.75 8.74

GW-2W ; 03-His5.3 0.69 99.01 0.30 28.54 63.03 8.43 04-His5.5 0.88 98.84 0.28 28.43 63.57 8.01

BL-5733 05-His5.7 1.34 98.39 0.26 2864 6361 7.75 LUS04834 06-PB6.1 3.40 96.27 0.33 35.44 55.66 8.90 01-His4.9 0.63 96.70 2.67 38.83 49.93 11.24 02-His5.1 0.72 96.88 2.40 38.95 50.68 10.37 03-His5.3 0.89 96.90 2.20 38.80 51.71 9.49

GW-4W ; 04-His5.5 1.18 96.73 2.08 38.56 51.93 9.51 05-His5.7 1.94 95.50 2.56 40.75 51.44 7.81 06-PB6.1 5.63 92.04 2.34 49.47 39.76 10.77

Table 14. CE-SDS (NR) detection results of different pH samples under the high temperature condition

CE-SDS (NR) (%)

Sampling time Formulation. —- ————}>}>}>}>}>}>}_

FRA HHL MAIN HWM

01-His4.9 1.94 0.95 97.92 0.14 02-His5.1 1.89 0.91 97.93 0.18 03-His5.3 2.02 0.99 97.84 0.14 0D 04-His5.5 1.91 0.91 97.89 0.20 05-His5.7 1.98 0.96 97.80 0.22 06-PB6.1 1.93 0.95 97.67 0.40 01-His4.9 3.38 1.09 95.36 0.31 02-His5.1 2.99 0.96 96.38 0.11 03-His5.3 3.17 0.97 95.59 0.15

GW-2W 04-His5.5 2.87 0.92 95.40 0.47 05-His5.7 2.97 0.96 93.45 0.36 06-PB6.1 3.84 1.17 93.80 0.69 01-His4.9 4.67 1.27 95.14 0.19 02-His5.1 4.45 1.27 95.24 0.31 03-His5.3 4.61 1.26 95.16 0.23

GW-4W 04-His5.5 4.37 1.17 95.34 0.29 05-His5.7 5.28 1.33 94.56 0.16 06-PB6.1 6.06 1.95 92.22 1.72 3. Accelerated test (25 + 2 °C)

The samples were placed under the 25 °C accelerated test conditions for 3 months, and sampled at day 0, month 1, month 2 and month 3 (0D-1M-2M-3M) to evaluate the accelerated stability of the formulations, as shown in Table 15 and Table 16.

As can be seen from the SEC-HPLC detection data, His formulation samples with a pH value 26

BL-5733 a . . LU504834 of 4.9-5.5 all had a slight decrease in SEC monomers, and a slight increase in aggregations and fragments, and showed good stability.

As can be seen from the IEC-HPLC detection results (FIG. 12), His formulations with a pH value of 4.9-5.5 had no large difference, and all were stable.

As can be seen from the CE-SDS (NR) detection results, His formulations with a pH value of 4.9-5.7 had no obvious difference, and were relatively stable, and formulation 06-PB6.1 was relatively slightly poor.

Taking into account all the accelerated investigation results, the His formulations with a pH value of 4.9-5.5 all had good stability, and the His formulation with a pH value of 5.7 had slightly poor stability, and the PB formulation had the worst stability.

Table 15. SEC-HPLC and IEC-HPLC detection results of different pH samples under the accelerated condition . SEC-HPLC (%) IEC-HPLC (%)

Sampling ; css time Formulation aggregation Monomer Fragment Acidic Main Basic 881C8 8 variant peak variant 01-His4.9 0.49 99.45 0.05 16.96 75.61 7.44 02-His5.1 0.54 99.39 0.07 17.03 75.59 7.38 oD 03-His5.3 0.62 99.32 0.06 17.10 75.72 7.17 04-His5.5 0.76 99.18 0.06 17.17 75.73 7.10 05-His5.7 1.08 98.86 0.06 17.22 75.60 7.18 06-PB6.1 1.61 98.32 0.08 18.08 74.30 7.62 01-His4.9 0.50 99.35 0.15 20.67 71.90 7.44 02-His5.1 0.60 99.26 0.14 20.90 71.60 7.50

JS-IM 03-His5.3 0.69 99.15 0.16 22.18 70.65 7.17 04-His5.5 0.84 99.04 0.12 21.27 71.53 7.20 05-His5.7 0.37 99.44 0.19 22.89 68.11 9.00 06-PB6.1 2.12 97.73 0.15 24.62 67.21 8.17 01-His4.9 0.52 99.24 0.24 22.76 68.19 9.05 02-His5.1 0.64 99.14 0.23 22.90 68.07 9.04

JS-2M 03-His5.3 0.70 99.09 0.21 23.05 68.30 8.65 04-His5.5 0.89 98.91 0.20 23.14 68.36 8.50 05-His5.7 1.30 98.50 0.20 23.45 68.11 8.44 06-PB6.1 2.32 97.43 0.25 28.08 62.32 9.61 01-His4.9 0.64 99.02 0.34 25.82 64.54 9.64 02-His5.1 0.60 99.09 0.30 25.80 65.13 9.07

JS-3M 03-His5.3 0.91 98.80 0.29 26.12 65.00 8.89 04-His5.5 0.94 98.79 0.27 26.03 65.65 8.32 05-His5.7 2.95 96.79 0.26 26.71 63.80 9.50 06-PB6.1 2.69 96.99 0.33 32.18 58.51 9.31 27

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Table 16. CE-SDS (NR) detection results of different pH samples under the accelerated 17006886 condition

CE-SDS (NR) (%)

Sampling time Formulation _—

FRA HHL MAIN HWM

01-His4.9 1.94 0.95 97.92 0.14 02-His5.1 1.89 0.91 97.93 0.18 03-His5.3 2.02 0.99 97.84 0.14 op 04-His5.5 1.91 0.91 97.89 0.20 05-His5.7 1.98 0.96 97.80 0.22 06-PB6.1 1.93 0.95 97.67 0.40 01-His4.9 2.30 1.04 97.51 0.19 02-His5.1 2.34 1.07 97.47 0.19 03-His5.3 2.96 0.96 96.91 0.13

IM 04-His5.5 2.33 1.04 97.41 0.26 05-His5.7 2.52 1.11 97.30 0.18 06-PB6.1 2.59 1.15 96.92 0.49 01-His4.9 2.89 1.06 96.92 0.19 02-His5.1 3.08 1.20 96.73 0.19 03-His5.3 2.87 1.06 96.96 0.17

M 04-His5.5 2.91 1.11 96.92 0.17 05-His5.7 2.96 1.05 96.84 0.20 06-PB6.1 3.23 1.20 96.11 0.66 01-His4.9 2.79 1.16 97.04 0.17 02-His5.1 2.81 1.13 97.01 0.18 03-His5.3 2.95 1.02 96.81 0.24

M 04-His5.5 2.77 0.99 97.06 0.17 05-His5.7 2.87 1.02 96.93 0.20 06-PB6.1 3.70 1.32 95.49 0.81

In conclusion, the stability of the BAT 1706 antibody in the His buffer system with a pH value of 4.9-5.7 is superior to that of the PB buffer system, and the stability tends to be better as the pH value is lower.

Example 3: Stabilizer Screening Experiment

The stability and antioxidant stability of BAT1706 formulations of different protein concentrations were investigated. 28

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Table 17. Formulation composition 0504834

Formulation | Co | Antibody /mg/mL

Comoro tomers jw Js

Note: Tre is a,a-trehalose dihydrate, PS is polysorbate, and Met is methionine.

BAT1706 antibodies were exchanged into the corresponding prepared buffer by ultrafiltration, and prepared into formulations as shown in Table 17. The samples were filtered using a 0.22 um filter and dispensed into 0.6 mL sterile EP tubes (200 uL/tube), and the stability indicators such as SEC-HPLC, IEC-HPLC, CE-SDS (NR), the polysorbate content and activity of the antibody formulation were detected under illumination (4500 + 500 Ix, 25 °C) and high temperature (40 °C) conditions, respectively. 1. Illumination test (4500 + 500 Ix, 25 + 2 °C)

The samples were placed under the illumination condition (4500 + 500 Ix, 25 + 2 °C) for 6 days, sampled at days 0, 3 and 6 and detected for stability, as shown in Table 18.

As can be seen from the SEC-HPLC detection data (FIG. 13), the SEC aggregation of formulation C3 with an antibody concentration of 80 mg/mL was 4% higher than that of His formulation C1 with a concentration of 25 mg/mL, and 2.6% lower than that of formulation 07-PB after 6 days of illumination, indicating that the high concentration formulation had decreased light stability, and was more prone to generate aggregations, but still better than 25 mg/mL PB formulation. The addition of Met or EDTA had no obvious effect on improving the stability of the formulation.

As can be seen from the IEC-HPLC detection results, the higher the protein concentration of the His formulation, the greater the rise in the IEC acidic variant and basic variant, and the acidic variant of the His formulation with an antibody concentration of 80 mg/mL was slightly 29

BL-5733 . . . . LU504834 lower than that of the PB formulation with an antibody concentration of 25 mg/mL after 6 days under illumination condition. The formulation with the addition of Met had the smallest decrease in the IEC main peak relative to the other formulations, indicating that the addition of

Met had a beneficial effect on improving the light stability of the antibody. The stability of the formulation added with EDTA was not obviously improved.

As can be seen from the CE-SDS (NR) detection results (FIG. 14), the formulation with high concentration of antibody protein had a larger decrease in the CE (NR) main peak relative to the formulation with low concentration, with the greatest decrease in the CE (NR) main peak for formulation 07-PB and the least decrease in the CE (NR) main peak for His formulations with the addition of Met or EDTA (formulations C4, CS and C6).

Taking into account all the above illumination investigation results, the formulation comprising high concentration of antibody protein had a decrease in the stability relative to the formulation with low concentration, but still had better quality than the PB formulation, and the addition of

Met or EDTA had a certain effect on enhancing the illumination stability of the His formulation.

Table 18. Detection results of different formulation samples under the illumination condition

F SEC-HPLC (%) IEC-HPLC (%) CE-SDS (NR) (%) rx ——— — …=H".-"" —— tation aggreg Mono Fragm Acidic Main Basic FRA HHL MAIN HWM ation mer ent variant peak variant

Cl 117 98.78 0.06 18.23 73.84 7.92 2.15 1.04 97.45 0.40

C2 132 98.62 0.06 18.30 73.92 7.79 1.90 1.02 97.73 0.37

C3 1.53 98.40 0.06 18.35 73.86 7.79 1.97 1.07 97.55 0.48 0D C4 1.16 98.79 0.06 18.32 73.71 7.97 1.96 1.05 97.65 0.39

C5 1.16 98.78 0.06 18.33 73.58 8.08 1.96 0.98 97.67 0.37

C6 115 98.79 0.06 18.35 73.62 8.03 1.96 0.97 97.73 0.31

C7 166 98.28 0.06 18.55 73.30 8.15 1.90 0.92 97.74 0.36

Cl 347 96.35 0.18 28.29 4930 22.40 4.50 2.62 94.63 0.87

C2 467 95.15 0.18 2934 47.06 23.60 4.09 2.36 94.15 1.76

C3 6.08 93.76 0.16 30.48 43.00 26.52 4.12 2.34 93.51 2.37 3D C4 3.56 96.27 0.17 27.65 49.78 22.57 4.62 2.59 94.57 0.81

C5 353 9631 0.16 27.00 50.73 22.28 4.31 2.54 94.76 0.93

C6 341 96.41 0.18 29.08 47.58 23.34 4.72 2.69 94.40 0.88

C7 9.05 90.64 0.30 33.45 3991 26.63 5.74 3.00 90.87 3.39

BL-5733

Cl 488 9486 0.25 33.57 38.25 28.18 5.74 3.28 92.61 1.65 LUS04834

C2 8.04 91.69 0.27 36.27 33.56 30.17 5.49 3.14 91.68 2.83

C3 888 90.88 0.24 36.20 31.28 32.52 4.92 2.76 91.60 3.48 6D C4 539 9436 0.25 32.47 39.18 28.34 5.41 3.13 93.20 1.39

C5 517 94.60 0.23 31.23 41.81 26.96 5.23 2.96 93.26 1.51

C6 452 9523 0.24 33.83 38.13 28.04 5.35 2.96 93.59 1.06

C7 11.49 88.06 0.44 38.64 30.35 31.01 6.89 3.63 88.64 4.47 2. High temperature test (40 + 2 °C)

The prepared sample aliquots were placed at 40 °C for 4 weeks, and sampled at day 0, week 1, week 2, week 3 and week 4 (0D-1W-2W-3W-4W) respectively for detection. The results were shown in Table 19.

As can be seen from the SEC-HPLC detection data (FIG. 15), after 4 weeks of high temperature investigation, the His formulation had an increase in the SEC aggregation with the increase of the antibody protein concentration, and the addition of Met or EDTA had no obvious effect on enhancing the stability of the antibody. Formulation 07-PB had the largest decrease in the SEC monomer and the largest increase in the aggregation.

As can be seen from the IEC-HPLC detection results (FIG. 16), after 4 weeks of high temperature investigation, the His formulations with different antibody protein concentrations had no obvious difference in the IEC main peak decrease, and the addition of Met or EDTA had no obvious effect on enhancing the antibody stability. Formulation 07-PB had the largest decrease in the IEC main peak and the largest increase in the acidic variant.

As can be seen from the CE-SDS (NR) detection results (FIG. 17), after 4 weeks of high temperature investigation, the His formulations with different antibody protein concentrations had no obvious difference in the CE (NR) main peak decrease, and the addition of Met or EDTA had no obvious effect on enhancing the antibody stability. Formulation 07-PB had the largest decrease in the CE (NR) main peak.

Taking into account all the high temperature investigation results, when the formulation buffer system was a His buffer, the formulation with high concentration of antibody protein had a decrease in the stability relative to the formulation with low concentration, but still had better quality than the PB formulation with low concentration. The addition of Met or EDTA had no 31

BL-5733

LU504834 significant effect on enhancing the formulation stability of the His formulation.

Table 19. SEC-HPLC, IEC-HPLC and CE-SDS (NR) detection results of different formulation samples under the high temperature condition © SECHPLC(%) ___ IECHPLC(%) CE-SDS(NR) (%) t Formulation aggregation Monomer Fragment Acidic ak Basic FRA HHL MAIN HWM

Cl 1.17 98.78 0.06 18.23 73.84 7.92 2.15 1.04 9745 0.40

C2 1.32 98.62 0.06 18.30 73.92 7.79 1.90 1.02 97.73 0.37

C5 1.16 98.78 0.06 18.33 73.58 8.08 1.96 0.98 97.67 0.37

C6 1.15 98.79 0.06 18.35 73.62 8.03 1.96 0.97 97.73 0.31

C7 1.66 98.28 0.06 18.55 73.30 8.15 1.90 0.92 97.74 0.36

Cl 1.02 98.76 0.23 25.02 66.42 8.56 2.66 0.90 96.92 0.42

C2 1.35 98.42 0.23 24.97 66.21 8.81 2.54 0.82 97.04 0.42

C3 1.77 98.00 0.22 24.87 65.52 9.61 2.64 0.84 9694 0.42 1W C4 0.95 98.82 0.22 25.08 66.38 8.55 2.18 0.74 9755 0.27

C5 0.95 98.83 0.22 24.94 66.66 8.40 2.38 0.81 97.19 0.43

C6 0.96 98.82 0.22 25.05 66.41 8.54 2.29 0.77 9739 0.32

C7 2.61 97.15 0.23 29.35 61.84 8.81 2.66 0.90 96.92 0.42

Cl 1.19 96.42 2.39 34,57 56.62 8.81 3.63 1.11 9595 042

C2 1.73 95.83 2.43 34,50 55.56 9.94 3.68 1.15 95.83 0.49

C3 2.29 95.19 2.53 34.18 55.93 9.89 3.69 1.21 95.71 0.60 3W C4 1.03 96.57 2.40 34,52 56.38 9.10 3.55 0.96 96.17 0.28

C5 1.03 96.58 2.39 34,57 56.92 8.51 3.48 1.05 96.09 043

C6 1.10 96.46 2.44 3470 56.67 8.63 3.51 1.03 96.21 0.28

C7 4.04 93.52 2.44 42.74 47.97 9.29 427 1.20 9489 0.84

Cl 1.30 95.99 2.71 40.32 51.65 8.02 432 1.07 9538 0.30

C2 2.01 95.08 2.91 42.10 48.93 8.96 492 1.17 9465 0.43

C3 2.63 94.66 2.71 39.85 51.12 9.02 463 1.16 9482 0.55 4W C4 1.10 96.24 2.67 40.12 51.77 8.11 435 1.04 9540 0.25

C5 1.08 96.24 2.68 40.25 51.66 8.09 453 1.03 9518 0.29

C6 1.20 96.07 2.73 40.39 51.36 8.25 480 1.05 9490 0.30

C7 4.82 92.48 2.70 49.96 41.63 8.41 593 1.28 93.17 0.90

By examining the antibody binding activity at day 6 of illumination (GZ) and week 3 of high 32

BL-5733 . cpa Co . LU504834 temperature (GW), the results were shown in Table 20, with little variation in activity of each formulation.

Table 20. Detection results of activity for different formulation samples

Formulation Binding activity (%)

Co Formulation name —————_—_—__ abbreviation 0D GZ-6D GW-3W

Cl 01-His-25 102 86 97

C2 02-His-50 104 86 109

C3 03-His-80 106 84 102

C4 04-His+5mM Met 104 87 108

C5 05-His+10mM Met 111 103 101

C6 06-His +EDTA 106 87 98

C7 07-PB 104 83 91

On day 6 under the illumination condition (GZ), there was no significant difference in the polysorbate content decrease among formulations.

Under the high temperature condition, the higher the concentration of the antibody protein in the His formulation, the more the PS80 content in the sample was reduced. The addition of Met had no significant inhibitory effect on the degradation of PS80, and the formulation added with

EDTA (formulation C6) had smallest decrease in the PS80 content, indicating that EDTA had a certain effect on inhibiting the degradation of PS80.

Table 21. Detection results of the polysorbate content of different formulation samples

Formulation Formulation name Polysorbate content (ng/mL) abbreviation 0D GZ-6D GW-1W GW-3W

Cl 01-His-25 377 353 364 358

C2 02-His-50 367 357 344 327

C3 03-His-80 382 355 343 332

C4 04-His+5mM Met 368 345 354 354

C5 05-His+10mM Met 366 349 353 350

C6 06-His +EDTA 360 343 345 358

C7 07-PB 353 344 341 352

The His formulation with the high concentration of 80 mg/mL antibody protein had slightly poorer stability than the His formulation with the concentration of 25 mg/mL, but had better quality than the PB formulation with the concentration of 25 mg/mL under the same investigation condition. The addition of Met or EDTA had a certain effect on improving the 33

BL-5733 . oo or . ... Ce LU504834 illumination stability of the formulation.

The addition of EDTA had a certain inhibitory effect on the degradation of PS80. 34

Claims

BL-5733 CLAIMS LU504834

1. À formulation comprising (1) 20-100 mg/mL anti-VEGF antibody; (2) a histidine buffer; (3) a stabilizer; and (4) a surfactant; wherein the formulation has a pH value of 4.0-6.5, and the anti-VEGF antibody comprises a heavy chain variable region set forth in SEQ ID NO: 1 and a light chain variable region set forth in SEQ ID NO: 2.

2. The formulation according to claim 1, wherein the histidine buffer is at a concentration of 1-30 mM; and preferably, the histidine buffer is at a concentration of 8-15 mM.

3. The formulation according to claim 1 or 2, wherein the stabilizer is a saccharide; and preferably, the saccharide is trehalose or sucrose.

4. The formulation according to any one of claims 1-3, wherein the stabilizer is at a concentration of 10-100 mg/mL or 50-300 mM.

5. The formulation according to any one of claims 1-4, wherein the surfactant is selected from polysorbate and poloxamer; and preferably, the surfactant is polysorbate 20 or polysorbate 80.

6. The formulation according to any one of claims 1-5, wherein the surfactant is at a concentration of 0.001-10 mg/mL; and preferably, the surfactant is at a concentration of 0.01- 2 mg/mL.

7. The formulation according to claim 1, comprising: (1) 20-100 mg/mL anti-VEGF antibody; (2) 8-15 mM histidine buffer; (3) 10-100 mg/mL trehalose; and (4) 0.1-2 mg/mL polysorbate, wherein the formulation has a pH value of 4.0-6.5.

8. A formulation comprising (1) 20-100 mg/mL anti-VEGF antibody; (2) about 10 mM histidine buffer; (3) about 60 mg/mL trehalose dihydrate;

BL-5733 and (4) 0.1-2 mg/mL polysorbate 20 or polysorbate 80, 7006808 wherein the formulation has a pH value of 4.0-6.5; and preferably, the formulation has a pH value of 4.9-5.7, and the anti-VEGF antibody comprises a heavy chain variable region set forth in SEQ ID NO: 1 and a light chain variable region set forth in SEQ ID NO: 2.

9. The formulation according to claim 8, comprising: (1) about 25 mg/mL anti-VEGF antibody; (2) about 10 mM histidine buffer; (3) about 60 mg/mL trehalose dihydrate; and (4) about 0.4 mg/mL polysorbate 20 or polysorbate 80; or (1) about 50 mg/mL anti-VEGF antibody; (2) about 10 mM histidine buffer; (3) about 60 mg/mL trehalose dihydrate; and (4) about 0.4 mg/mL polysorbate 80; or (1) about 80 mg/mL anti-VEGF antibody; (2) about 10 mM histidine buffer; (3) about 60 mg/mL trehalose dihydrate; and (4) about 0.4 mg/mL polysorbate 80, wherein the antibody formulation has a pH value of 4.0-6.5; and preferably, the formulation has a pH value of 4.9-5.7; and the anti-VEGF antibody comprises a heavy chain variable region set forth in SEQ ID NO: 1 and a light chain variable region set forth in SEQ ID NO: 2.

10. The formulation according to any one of claims 1-9, wherein the formulation does not comprise hyaluronidase, EDTA, methionine, or a combination thereof.

11. The formulation according to any one of claims 1-9, further comprising an antioxidant or a chelating agent.

12. The formulation according to claim 11, wherein the antioxidant or the chelating agent is methionine or EDTA. 36

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13. The formulation according to claim 11 or 12, comprising 1-20 mM methionine or 0.01-0.1 17006886 mM EDTA; and preferably, comprising 5-10 mM methionine or about 0.05 mM EDTA.

14. The formulation according to any one of claims 11-13, comprising: (1) 20-100 mg/mL anti-VEGF antibody; (2) about 10 mM histidine buffer; (3) about 60 mg/mL trehalose dihydrate; (4) 0.1-2 mg/mL polysorbate 20 or polysorbate 80; and (5) 1-20 mM methionine or 0.01-0.1 mM EDTA, wherein the antibody formulation has a pH value of 4.0-6.5, and the anti-VEGF antibody comprises a heavy chain variable region set forth in SEQ ID NO: 1 and a light chain variable region set forth in SEQ ID NO: 2.

15. The formulation according to any one of claims 11-14, comprising: (1) about 25 mg/mL anti-VEGF antibody; (2) about 10 mM histidine buffer; (3) about 60 mg/mL trehalose dihydrate; (4) 0.1-2 mg/mL polysorbate 20 or polysorbate 80; and (5) about 5 mM methionine; or (1) about 25 mg/mL anti-VEGF antibody; (2) about 10 mM histidine buffer; (3) about 60 mg/mL trehalose dihydrate; (4) 0.1-2 mg/mL of polysorbate 20 or polysorbate 80; and (5) about 10 mM methionine; or (1) about 25 mg/mL anti-VEGF antibody; (2) about 10 mM histidine buffer; (3) about 60 mg/mL trehalose dihydrate; (4) 0.1-2 mg/mL polysorbate 20 or polysorbate 80; and (5) about 0.05 mM EDTA, wherein the antibody formulation has a pH value of 4.0-6.5, 37

BL-5733 and the anti-VEGF antibody comprises a heavy chain variable region set forth in SEQ ID NO: 7006808 1 and a light chain variable region set forth in SEQ ID NO: 2.

16. The formulation according to any one of claims 1-15, wherein the anti-VEGF antibody comprises a heavy chain set forth in SEQ ID NO: 3 and a light chain set forth in SEQ ID NO: 4; or the anti-VEGF antibody is bevacizumab.

17. The formulation according to any one of claims 1-16, wherein the formulation is a liquid formulation.

18. A solid formulation obtained by lyophilizing the formulation according to any one of claims 1-17. 38