WO2022183418A1

WO2022183418A1 - Anti-vegf antibody formulation

Info

Publication number: WO2022183418A1
Application number: PCT/CN2021/078974
Authority: WO
Inventors: 罗晓萍; 吴用; 吴晓云; 李志强; 刘育杰; 王盛武; 刘翠华; 李胜峰
Original assignee: 百奥泰生物制药股份有限公司
Priority date: 2021-03-04
Filing date: 2021-03-04
Publication date: 2022-09-09
Also published as: CN116897052A

Abstract

Provided is an anti-VEGF antibody formulation. The antibody formulation targets and acts on human VEGF, can specifically bind to the human VEGF with high affinity, and then inhibit angiogenesis. The antibody formulation is mainly a liquid formulation. The liquid formulation can enhance the stability of a drug containing a recombinant anti-VEGF humanized monoclonal antibody, and prolong the storage period of the drug in the formulation. For example, the liquid formulation remains stable after storage for 20 days at a high temperature of 40 °C, at least 5 cycles of freezing-thawing, storage for 6 months at 25 °C, or storage for at least 24 months at 2-8 °C. The formulation can be used for intravenous, subcutaneous, intraocular, intramuscular, or intravitreal injection.

Description

Anti-VEGF Antibody Preparations

technical field

The invention belongs to the field of biomedicine, and relates to an anti-VEGF antibody preparation and a preparation method and application thereof.

Background technique

Vascular endothelial growth factor (VEGF) is considered to be the most critical factor in physiological and pathological angiogenesis. In addition to being an angiogenic factor in angiogenesis and vasculogenesis, VEGF is a pleiotropic growth factor that exhibits a variety of functions in endothelial cell survival, vascular permeability and vasodilation, monocyte chemotaxis, and calcium influx. Biological effects, such as VEGF have been reported to promote cell division on retinal pigment endothelial cells and neural membrane cells. Extensive data show that VEGF plays a key role in the development of diseases involving pathological angiogenesis, VEGF mRNA is overexpressed in most human tumors, and the concentration of VEGF in aqueous humor is comparable to that in patients with diabetes or other ischemic retinal diseases. The vasoactive hyperplasia found in patients with age-related macular degeneration (AMD) is highly correlated, especially in the neovascularization membrane of the choroid plexus in patients with age-related macular degeneration (AMD). Overexpression of VEGF is not necessarily a key factor in wet AMD, but high levels of VEGF expression have been found both in experimental models and in pathological angiogenesis of wet AMD.

There is a considerable amount of literature reporting that angiogenesis and vascular leakage can be inhibited by neutralizing VEGF. There are also many reports in the literature that inhibition of VEGF can treat a variety of tumors. Therefore, anti-VEGF antibodies or VEGF inhibitors are effective candidates for the treatment of solid tumors and various intraocular neovascular disorders and other diseases associated with VEGF overexpression. In the prior art, commercialized anti-VEGF antibodies such as Bevacizumab (trade name:

), ranibizumab (Ranibizumab, trade name:

), aflibercept

Compaq

However, as proteins, antibodies are more complex than traditional inorganic and organic drugs. Protein pharmaceutical preparations are prone to chemical instability (such as deamidation, racemization, hydrolysis, oxidation, beta elimination or disulfide bond exchange, etc.) or physical instability (such as denaturation, aggregation, precipitation, etc.) during storage. or adsorption). The instability of antibody preparations will not only bring about a reduction or disappearance of drug efficacy, but also adversely affect the health of patients. Accordingly, antibody formulations need to be long-term stable, containing safe and effective amounts of pharmaceutical compounds.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a formulation suitable for use with specific antibodies.

Another object of the present invention is to provide an antibody formulation that is stable during storage and delivery.

Another object of the present invention is to provide stable antibody formulations that can contain high concentrations of antibody.

Another object of the present invention is to provide antibody formulations suitable for intraocular administration.

Another object of the present invention is to provide antibody formulations suitable for vitreous administration.

The above-mentioned purpose of the present invention is achieved by the following technical means:

In one aspect, the invention provides antibody formulations comprising an anti-VEGF antibody or fragment thereof, and a buffer. The heavy chain variable region of the anti-VEGF antibody or fragment thereof contains the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region of the anti-VEGF antibody or fragment thereof contains the amino acid sequence shown in SEQ ID NO: 2 amino acid sequence.

In some embodiments, the heavy chain of the anti-VEGF antibody contains the amino acid sequence set forth in SEQ ID NO:3 and the light chain of the anti-VEGF antibody contains the amino acid sequence set forth in SEQ ID NO:4.

In some embodiments, the antibody is a humanized full-length anti-VEGF IgGl antibody. In some embodiments, the antibody is BAT5906, for details of which, please refer to Chinese Patent 201810011151.1.

In some embodiments, the antibody is glycosylated and/or aglycosylated.

In some embodiments, the amount of the antibody is 5-100 mg/ml; or 6-90 mg/ml; or 6-80 mg/ml; or 25-80 mg/ml; or 25-50 mg/ml. In some embodiments, the amount of antibody is any value within those ranges recited above, eg, 6 mg/ml, 12 mg/ml, 25 mg/ml, 50 mg/ml, 60 mg/ml, 80 mg/ml, 90 mg/ml, or 100 mg /ml and so on.

In some embodiments, the pH of the formulation is between 4.0 and 7.0, and the antibody remains stable in this pH range. In some embodiments, the pH of the formulation is between 5.0 and 6.5. In some embodiments, the pH of the formulation is 5.5-6.5; in some embodiments, the pH of the formulation is 5.4-6.4. In some embodiments, the pH of the formulation is between 5.8 and 6.2. In some embodiments, the pH is any pH value within those pH ranges listed above, eg, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, and the like.

In some embodiments, the buffer is selected from the group consisting of histidine buffer, succinate buffer, acetate buffer, citrate buffer, phosphate buffer, or combinations thereof. In some embodiments, the buffer is selected from a histidine buffer, a succinate buffer, an acetate buffer, or a combination thereof. In some embodiments, the buffer is selected from histidine buffers. In some embodiments, the histidine buffer is a buffer composed of L-histidine and L-histidine hydrochloride in a buffer system.

In some embodiments, the amount of the buffer is about 5-50 mM; in some embodiments, the amount of the buffer is about 5-30 mM; in some embodiments, the amount of the buffer is about 5 to 20 mM; in some embodiments, the amount of the buffer is about 5 to 15 mM; in some embodiments, the amount of the buffer is about 5 to 10 mM. In some embodiments, the amount of buffer is any value within those ranges recited above, e.g. 18mM, 19mM, 20mM, 22mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM, 30mM and the like. In some embodiments, the amount of buffer is about 10 mM. In some embodiments, the buffer is about 10 mM histidine buffer.

In some embodiments, when the buffer system is selected from a combination of two, the molar ratio of the former to the latter in the system is 1:2 to 2:1; or 1:1.

The antibody preparation of the present invention further contains a stabilizer.

In some embodiments, the stabilizer is selected from trehalose, sucrose, mannitol, sodium chloride, sorbitol, proline, glycine, methionine, or a salt or hydrate thereof, or a combination thereof.

In some embodiments, the stabilizer is selected from sucrose and/or trehalose.

In some embodiments, the stabilizer is trehalose.

In some embodiments, the amount of the stabilizer is 0.5% to 20%. In some embodiments, the amount of the stabilizer is about 0.5%-15%. In some embodiments, the amount of the stabilizer is about 1% to 15%. In some embodiments, the amount of the stabilizer is about 2% to 10%; in some embodiments, the amount of the stabilizer is any value within those ranges listed above, such as 10%, 9%, 8.9%, 8.8%, 8.7%, 8.6%, 8.5%, 8.4%, 8.3%, 8.2%, 8.1%, 8%, 7.9% or 7.8%. In some embodiments, the stabilizer is trehalose in an amount equivalent to about 8.8% trehalose dihydrate.

The antibody preparation of the present invention may further contain a surfactant.

In some embodiments, the surfactant selected for the antibody formulation is a non-ionic surfactant.

In some embodiments, the surfactant is selected from Tween and/or Poloxamer.

In some embodiments, the surfactant is selected from Tween 20, Tween 80 and/or Poloxamer 188.

In some embodiments, the Tween is selected from Tween 20 and/or Tween 80.

In some embodiments, the surfactant is selected from Tween 20.

In some embodiments, the amount of the surfactant is 0.001% to 0.2%; or 0.01% to 0.2%. In some embodiments, the amount of the surfactant is 0.01% to 0.1%. In some embodiments, the amount of the surfactant is any value within those ranges recited above. In some embodiments, the amount of surfactant is 0.04%. In some embodiments, the surfactant is about 0.04% Tween 20.

In some embodiments, the formulation contains the following components:

(1) 5～100mg/ml anti-VEGF antibody or fragment

(2) 5～50mM buffer

(3) 0.5-20% stabilizer

(4) 0.001%～0.2% surfactant

(5) water for injection;

The pH of the formulation is 4.0 to 7.0.

In other embodiments, the formulation contains the following components:

(1) 6～90mg/ml anti-VEGF antibody or fragment

(2) 5～30mM buffer

(3) 0.5%～10% stabilizer

(4) 0.01%～0.2% surfactant

(5) water for injection;

The pH of the formulation is 5.0-6.5.

Wherein, the buffer, stabilizer, and surfactant are as described above.

In some embodiments, the formulation contains the following components:

(1) 6～80mg/ml anti-VEGF antibody or fragment

(2) 10～20mM histidine buffer

(3) 2%～10% trehalose

(4) 0.01%～0.1% Tween 20

(5) Water for injection

The pH of the formulation is 5.5-6.5.

In some embodiments, the formulation contains the following components:

(1) 6～80mg/ml anti-VEGF antibody or fragment

(2) 10mM histidine buffer

(3) 2%～10% trehalose

(4) 0.01%～0.1% Tween 20

(5) Water for injection

The pH of the preparation is 5.5-6.5;

In other embodiments, the formulation contains the following components:

(1) About 6, 12, 25, 50 or 80 mg/ml of anti-VEGF antibody or fragment

(2) About 10mM histidine buffer

(3) about 9% trehalose

(4) About 0.04% Tween 20

(5) Water for injection

The pH of the formulation is 6.0 ± 0.4;

In other embodiments, the formulation contains the following components:

(1) About 6 or 12 or 25 or 50 or 80 mg/ml of anti-VEGF antibody or fragment

(2) About 10mM histidine buffer

(3) About 8% trehalose

(4) About 0.04% Tween 20

(5) Water for injection

Wherein, the pH of the formulation is 6.0±0.4.

In some embodiments, the osmotic pressure of the antibody preparation is 150-400 mOsm/Kg. In some embodiments, the osmotic pressure of the antibody preparation is 200-350 mOsm/Kg. In other embodiments, the osmotic pressure of the antibody preparation is 260-340 mOsm/Kg.

The mode of administration of the antibody preparation of the present invention is intravenous, subcutaneous, intraocular or intramuscular, and the like. In some embodiments, the intraocular administration is by injection, or by direct instillation. In some embodiments, the aqueous liquid antibody formulations of the invention are administered by intraocular injection such as intravitreal injection.

The antibody preparation provided by the present invention is a liquid preparation. In some embodiments, the solvent for the antibody formulation is water. In some embodiments, the solvent for the antibody formulation is sterile water for injection.

Another aspect of the present invention provides a delivery device containing the antibody formulation described above. In some embodiments, the delivery device is a prefilled syringe that provides convenience, accuracy, sterility, and safety for site delivery, such as intravitreal, to administer the antibody formulation. In one embodiment, the delivery device is a prefilled syringe with an accompanying or integral needle. In other embodiments, the delivery device is a prefilled syringe without an accompanying needle. In one embodiment, the delivery device is an automatic injection device. Other alternative delivery devices include stents, catheters, microneedles, and implantable controlled release devices, among others.

Another aspect of the present invention provides the use of the antibody preparation, or the delivery device, in the manufacture of a medicament for treating, preventing or ameliorating VEGF-related diseases.

Another aspect of the present invention provides a method of treating a VEGF-related disease, the method comprising administering the antibody formulation to a patient.

In some embodiments, the VEGF-related disease is a VEGF overexpression-related disease.

In some embodiments, the disease that the antibody preparation can treat is selected from the group consisting of: branch retinal vein occlusion, central retinal vein occlusion, diabetic macular edema, diabetic retinopathy, choroidal neovascularization secondary to pathological myopia, age-related Macular degeneration, neovascular glaucoma, diabetic retinopathy, retinopathy of prematurity, retropharyngeal fibrosis, breast cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, liver cancer, ovarian cancer, coma, androgenoma, cervix Carcinoma, endometrial cancer, endometrial hyperplasia, endometriosis, fibrosarcoma, choriocarcinoma, head and neck cancer, nasopharyngeal cancer, laryngeal cancer, hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinoma, hemangioma, cavernous hemangioma, hemangioblastoma, pancreatic cancer, retinoblastoma, astrocytoma, glioblastoma, schwannoma, oligodendroglioma, medulloblastoma, Neuroblastoma, rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, urinary tract cancer, thyroid cancer, Wilms tumor, renal cell carcinoma, prostate cancer, abnormal vascular proliferation associated with macular hamartoma, edema, McGonagall syndrome, rheumatoid arthritis, psoriasis, or atherosclerosis.

In some embodiments, the disease that the antibody formulation can treat is selected from the group consisting of: age-related macular degeneration, choroidal neovascularization secondary to pathological myopia, diabetic retinopathy, branch retinal vein occlusion, central retinal vein occlusion, or diabetic retinopathy Macular edema.

In some embodiments, the patient is a mammal.

In some embodiments, the mammal is a human.

Another aspect of the present invention provides a method for preparing the antibody preparation, which comprises the following steps:

(1) prepare buffer solution;

(2) by UF/DF ultrafiltration, using the buffer solution prepared in step (1) to carry out ultrafiltration and exchange liquid on the antibody, and then concentrating to obtain an antibody solution;

(3) preparing an excipient mother liquor containing a stabilizer and/or a surfactant, and adding the excipient mother liquor to the antibody solution prepared in step (2) to obtain an antibody preparation.

In some embodiments, the preparation method of the antibody preparation, the step further comprises:

Sterilize and filter the antibody preparation prepared in step (3).

A specific amount of surfactant is added to the antibody solution prepared in step (2) to achieve the desired concentration of surfactant.

In some embodiments, the preparation method of the antibody preparation, it comprises the following steps:

(1) prepare buffer solution;

(3) Prepare an excipient mother solution containing 4 times the target concentration of stabilizer and/or 4 times the target concentration of surfactant, and add the excipient mother solution to the antibody solution prepared in step (2), so that the excipient concentration finally reaches the target concentration , and bring its antibody concentration to the final target concentration. If the antibody concentration needs to be diluted after the addition of the excipient stock solution, use a solution containing stabilizers, surfactants and buffers for dilution treatment to keep the concentration of the excipients unchanged.

(4) Sterilize and filter the antibody solution prepared in (3).

(1) prepare histidine buffer;

(2) by UF/DF ultrafiltration, using the buffer prepared in step (1) to carry out ultrafiltration and liquid exchange on the antibody, and then concentrating to obtain an antibody solution;

(3) preparing an excipient mother solution containing trehalose at 4 times the target concentration and Tween 20 at 4 times the target concentration, adding the excipient mother solution to the antibody solution prepared in step (2), so that the excipient concentration finally reaches the target concentration, so that Its antibody concentration reaches the final target concentration. If the antibody concentration needs to be changed after the addition of the excipient mother liquor, use a buffer containing trehalose and Tween 20 for dilution treatment, so that the concentration of the excipient remains unchanged.

(4) Sterilize and filter the antibody solution prepared in (3).

In some embodiments, the method further includes sterile filtration followed by filling into a container.

The adjuvant is added to the antibody solution in the form of liquid, and the effect is better than that in the form of solid.

The formulation of the present invention may be suitable for intravitreal injection use.

The antibody preparation of the present invention can enhance the stability of the recombinant anti-VEGF humanized monoclonal antibody drug containing a large concentration range (low concentration to high concentration), prolong its storage period in the liquid preparation, and meet the requirements for vitreous injection with different doses need for antibodies.

The antibody preparation of the present invention remains stable after being placed at a high temperature of 40°C for 20 days, at least 5 cycles of freezing and thawing, and placed at 25°C for 6 months, and then stored at 2-8°C for at least 12 months, such as 24 months.

Description of drawings

1A and B are the variation trend diagrams of SEC monomer purity and IEC main peak under the condition of different pH and buffer in Example 1 at 50°C; FIG. 1A is the variation trend diagram of SEC monomer purity, and FIG. 1B is the variation trend diagram of IEC main peak.

2A and B are the variation trend diagrams of SEC monomer purity and IEC main peak under the condition of different pH and buffer in Example 2 at 50°C; FIG. 2A is the variation trend diagram of SEC monomer purity, and FIG. 2B is the variation trend diagram of IEC main peak.

3A and B are the variation trend diagrams of SEC monomer purity and IEC main peak under the condition of 40° C. for different stabilizers in Example 4; FIG. 3A is the variation trend diagram of SEC monomer purity, and FIG. 3B is the variation trend diagram of IEC main peak.

4A and B are the variation trend diagrams of the SEC monomer purity and IEC main peak under illumination conditions for different stabilizers in Example 4; FIG. 4A is the SEC monomer purity variation trend diagram, and FIG. 4B is the IEC main peak variation trend diagram.

Fig. 5 is the variation trend diagram of SEC monomer purity and IEC main peak for different amino acid stabilizers in Example 4 at 40°C; Fig. 5A is the variation trend diagram of SEC monomer purity, and Fig. 5B is the variation trend diagram of IEC main peak.

6A and B are the variation trend diagrams of SEC monomer purity and IEC main peak under illumination conditions for different amino acid stabilizers in Example 4; FIG. 6A is the variation trend diagram of SEC monomer purity, and FIG. 6B is the IEC main peak variation trend diagram.

7A and B are the variation trend diagrams of the SEC monomer purity at 50°C and the IEC main peak at 40°C of the two stabilizers in Example 4; FIG. 7A is the SEC monomer purity variation trend diagram, and FIG. 7B is the IEC main peak variation diagram. Trend.

Figure 8 is the variation trend diagram of the SEC monomer purity and IEC main peak of the mixed protein solution of the two stabilizers in Example 4 under light conditions; Figure 8A is the variation trend diagram of the SEC monomer purity, and Figure 8B is the IEC main peak variation trend diagram.

FIG. 9 is a graph showing the variation trend of turbidity over time after the shaking of samples with different contents of Tween in Example 5. FIG.

Figures 10A and B show the number of particles after dilution of samples containing Tween with different contents in Example 5; Figure 10A shows the first detection, and Figure 10B shows the second detection.

Figures 11A and B are the variation trend diagrams of the SEC monomer purity and IEC main peak of the trehaloprotein solution containing different amounts in Example 6 at 40°C; Figure 11A is the variation trend diagram of the SEC monomer purity, and Figure 11B is the IEC main peak Change trend graph.

Figures 12A and B are graphs of changes in protein content with time at different pHs in Example 7 at 40°C; Figure 12A is a graph of changes in SEC monomer purity, and Figure 12B is a graph of changes in IEC main peaks.

Figures 13A and B are graphs of changes in protein content with time at different pHs in Example 7; Figure 13A is a graph of changes in SEC monomer purity, and Figure 13B is a graph of changes in IEC main peaks.

Detailed ways

The technical solutions of the present invention are further described below through specific embodiments, which do not represent limitations on the protection scope of the present invention. Some non-essential modifications and adjustments made by others according to the concept of the present invention still belong to the protection scope of the present invention.

In addition, in this invention, "%" of a component is involved. Specifically, it refers to the weight-to-volume (w/v) percentage, wherein the weight unit can be g, and the volume unit can be ml. For example, 1% stabilizer in the solution means that 100ml of the solution contains 1g of stabilizer, or the stabilizer content is 0.01g/ml.

"About" or "approximately" refers to the conventional error range of the corresponding numerical value readily known to those skilled in the relevant art. In some embodiments, references herein to "about" or "approximately" refer to the recited value and ranges of ±10%, ±5%, ±1%, or ±0.1% thereof.

The term "buffer", also referred to in some literature as a buffer system or buffer system, includes, but is not limited to, organic acid salts such as succinic acid, acetic acid, citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid or benzene Diformic acid and its salts; Tris, thethamine hydrochloride, or phosphate buffer. In addition, amino acids and their salts can also be used as buffers. Such amino acid components include, but are not limited to, glycine, histidine, arginine, lysine, ornithine, isoleucine, leucine, alanine, glutamic acid, or aspartic acid. In some embodiments, the buffer is a histidine buffer.

The amount of buffer in the present invention refers to the total amount of buffer pairs in the buffer system that constitutes the buffer. In some embodiments, molarity is employed as the unit for the amount of buffer, the value of which refers to the molarity of the buffer pair in the buffer system of the buffer. For example, when a histidine buffer consisting of L-histidine and L-histidine hydrochloride is used as a buffer, a given concentration of histidine buffer (eg, 10 mM) is L-histidine and L-histidine hydrochloride in combined concentrations (e.g. 5 mM for L-histidine and 5 mM for L-histidine hydrochloride; or 6 mM for L-histidine and 6 mM for L-histidine hydrochloride 4mM.)

The term "stabilizer" includes, but is not limited to, monosaccharides, such as fructose, maltose, galactose, glucose, sorbose, etc.; disaccharides, such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides, such as raffinose, pine Trisaccharides, maltodextrin, dextran, starch, etc.; and sugar alcohols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), etc.; ionic stabilizers, which include Salts such as NaCl or amino acid components such as arginine-HCl, proline, glycine, methionine.

The stabilizer in the preparation of the present invention has the function of adjusting the osmotic pressure, so the stabilizer can also be called an osmotic pressure regulator.

The term "surfactant" includes, but is not limited to, Tween (eg, Tween 20 and Tween 80); Poloxamers (eg, Poloxamer 188); Triton; Sodium Dodecyl Sulfate (SDS); Sodium sulfate; octylglycoside sodium salt; lauryl sulfobetaine, myristyl sulfobetaine, linolenyl sulfobetaine or stearyl sulfobetaine; lauryl sarcosine, meat Myristyl sarcosine, linolenyl sarcosine or stearyl sarcosine; linolenyl betaine, myristyl betaine or cetyl betaine; lauroamidopropyl betaine, coconut oil amidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmitamidopropyl betaine or isostearamidopropyl betaine (e.g. lauroamidopropyl) ; myristamidopropyl dimethylamine, palmitamidopropyl dimethylamine or isostearamidopropyl dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleyl taurate ; polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (eg Pluronics, PF68, etc.); and the like. In some embodiments, the surfactant is Tween 20.

Tween is also known as polysorbate (eg, Tween 20 is known as polysorbate 20 and Tween 80 is known as polysorbate 80).

The preparation of the present invention can be formulated with the adjuvant or its hydrate. For example, histidine hydrochloride, also known as histidine hydrochloride, can be anhydrous histidine hydrochloride or histidine hydrochloride hydrate, such as histidine hydrochloride monohydrate. As described in "5mM histidine hydrochloride", it is 5mmol histidine hydrochloride or histidine hydrochloride monohydrate dissolved in a solvent to form a 1L solution; 1.06mg histidine hydrochloride monohydrate containing 1.06 mg of histidine hydrochloride monohydrate or a corresponding amount of histidine hydrochloride. Another example is trehalose, which can be anhydrous trehalose or trehalose hydrate, such as trehalose dihydrate. As described in "8% trehalose", 8g trehalose (or corresponding amount of trehalose hydrate (eg 8.8g trehalose dihydrate)) is dissolved in a solvent to form a 100ml solution, unless otherwise specified.

The term "therapeutically effective amount" is an amount that can treat a disease or disorder.

The dosage of the antibody preparation of the present invention to the human body will vary with the age, weight, sex, medication form, health status and severity of the disease of the patient.

In some embodiments, the antibody formulations of the invention can be administered to patients suffering from a disease associated with VEGF overexpression. In some embodiments, the antibody formulations of the invention can be administered to patients diagnosed with wet age-related macular degeneration who still have active disease. Among them, active lesions are any of the following lesions in the macular area of the patient: ① intraretinal fluid; ② intraretinal lipid exudation; ③ subretinal fluid; ④ subretinal hemorrhage; ⑤ retinal pigment epithelium detachment; ⑥ choroidal neovascularization. In some embodiments, the antibody formulations of the invention can be administered to patients with a total area of ≤ 30 ^mm2 (12 optic disc areas) of all types of lesions. In some embodiments, the antibody formulations of the invention can be administered to patients with best corrected visual acuity ≤ 70 letters (ETDRS visual chart) (equivalent to snellen visual acuity ≤ 20/40).

In some embodiments, the antibody is used in an amount of about 0.3 mg/eye to about 4.0 mg/eye for a disease associated with VEGF overexpression. In some embodiments, the antibody is used in an amount of about 0.6 mg/eye to about 2.5 mg/eye for a disease associated with VEGF overexpression. In some embodiments, the antibody is used in an amount of about 1.25 mg/eye for a disease associated with VEGF overexpression. In some embodiments, the unit dose of the antibody is about 0.3 mg/eye, or 0.6 mg/eye, or 1.25 mg/eye, or 2.5 mg/eye, or 4.0 mg/eye. In some embodiments, the unit dose of antibody is about 1.25 mg/eye. In some embodiments, the unit dose of antibody is about 2.5 mg/eye. In some embodiments, the unit dose of antibody is about 4 mg/eye.

In some embodiments, for diseases related to VEGF overexpression, for antibody formulations applied to the eye, the administration volume is about 0.01 ml/eye to 0.5 ml/eye; in some embodiments, for diseases related to VEGF overexpression Disease, for antibody formulations applied to the eye, the dosing volume is approximately 0.05 ml/eye. In some embodiments, for diseases related to VEGF overexpression, for antibody formulations applied to the eye, the administration volume is about 0.1 ml/eye to 0.3 ml/eye; in some embodiments, for diseases related to VEGF overexpression Disease, for antibody formulations applied to the eye, the dosing volume is approximately 0.2 ml/eye. Application to the eye includes intraocular injection, intravitreal injection.

The term "mammal" refers to any animal classified as a mammal, including, but not limited to, humans, dogs, horses, cats, rabbits, pigs, cows, mice, and the like. In some embodiments, the mammal is a human.

"Treatment" of a patient's disease means (1) preventing the occurrence of the disease in a patient who is predisposed or not yet showing symptoms of the disease; (2) inhibits the disease or symptom or prevents its progression; or (3) alleviates the disease or symptom or causes it. its degradation.

As used herein, "stability", "stable" means that in a liquid formulation comprising an antibody, the antibody (including antibody fragments thereof) does not occur, or only minimally, under the given production, preparation, transportation and/or storage conditions Aggregation, degradation or fragmentation occurs. A "stable" formulation retains biological activity under given conditions of manufacture, preparation, transportation and/or storage. The degree of aggregation, degradation or fragmentation of the formulation, etc., which can be measured by techniques such as SEC-HPLC, IEC-HPLC, CE-SDS (NR), light detection and turbidity, insoluble particles, DLS detection of particle size, etc., thereby assess the stability of the antibody). In some embodiments, "stable" refers to a decrease in monomer purity of no more than 10%, 5%, or 2% over a period of time under certain storage conditions.

The detection method in the following embodiment of the present invention is as follows:

The analytical method of SEC-HPLC (referred to as SEC):

1. Prepare the liquid chromatography system and TOSOH Biotech TSK-GEL G3000SWXL chromatographic column (column size is 7.8×300mm, 5μm). Set the wavelength of the UV detector to 280nm, set the column temperature to 30°C or room temperature, adjust the flow rate of the mobile phase to 0.5ml/min, and equilibrate for about 30-60min until the baseline is stable.

2. Inject the sample, record the chromatogram, and calculate the percentage of monomer or polymer in the test solution according to the peak area normalization method after integration.

Analysis method of IEC-HPLC (referred to as IEC):

1. Prepare the liquid chromatography system and the ProPacTMWCX weak cation exchange separation column. Equilibrate the column with 50% mobile phase A and 50% mobile phase B, 0.3ml/min initial conditions, set the wavelength of the UV detector to 280nm, and set the column temperature to 30°C. After the pressure is stabilized, slowly adjust the flow rate to 0.8ml/min to The pressure is stable, and then use 84% mobile phase A to balance for about 30 to 60 minutes until the baseline is stable.

2. Inject the sample, manually integrate the detected chromatogram, and calculate the percentages of acidic peaks, main peaks and basic peaks according to the peak area normalization method.

Analytical method of CE-SDS(NR) (CE-SDS non-reducing):

The capillary electrophoresis instrument adopts Agilent's CE 7100. After the sample is injected, the chromatogram is recorded, the data is integrated and processed, and the percentage content of the monomer peak is calculated by the area normalization method.

Light inspection: Using a clarity detector, turn the light source up and down at 1000-1500Lx, and visually observe whether the inspected sample has opalescence or visible foreign matter.

Turbidity (OD340 value): Using an ultraviolet spectrophotometer, the sample is detected at UV340nm, and the detection result is recorded as the sample turbidity OD340.

Insoluble Particles: Use the HIAC Insoluble Particle Detector or Flowcam Insoluble Particle Detector to detect particles of different particle sizes that are invisible to the naked eye.

DLS detection of particle size: The sample was subjected to particle size detection using DLS (Dynamic Light Scattering Detector).

The present invention provides a liquid preparation containing VEGF antibody. Specifically, the preparation provided by the present invention is a liquid aqueous preparation with pH 4.0-7.0. In some embodiments, the preparation contains an antibody at a concentration of 5-100 mg/ml, and has the ability to enhance the recombinant anti-VEGF humanized monoclonal antibody. Stability of VEGF antagonist antibody drugs such as clonal antibodies. In some embodiments, the formulations of the present invention include the following components: an antibody that binds human VEGF with high affinity, low dissociation rate, high neutralizing ability, buffers, including L-histidine and histidine buffers; Osmotic pressure regulators, trehalose, sucrose; surfactants, including Tween 20.

In some embodiments, the antibody in the antibody formulation is an anti-VEGF antibody or fragment thereof. In some embodiments, the heavy chain variable region of the anti-VEGF antibody or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 1, and the light chain variable region of the anti-VEGF antibody or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 1 The amino acid sequence shown in SEQ ID NO:2. In some embodiments, the heavy chain of the anti-VEGF antibody contains the amino acid sequence set forth in SEQ ID NO:3 and the light chain of the anti-VEGF antibody contains the amino acid sequence set forth in SEQ ID NO:4.

In some embodiments, the concentration of the antibody in the liquid aqueous pharmaceutical formulation is about 6-90 mg/ml; in some embodiments, the concentration of the antibody in the liquid aqueous pharmaceutical formulation is about 25-80 mg/ml; In some embodiments, the concentration of the antibody in the liquid aqueous pharmaceutical formulation is about 6 mg/ml, or 12 mg/ml, or 25 mg/ml, or 50 mg/ml, or 80 mg/ml, or a range between any two concentrations , including endpoint values.

In some embodiments, the present invention provides aqueous antibody formulations of various buffers, including succinate buffers, citrate buffers, histidine buffers, acetate buffers, etc., and water for injection, with a pH of About 5.0 to 7.0.

The present invention provides liquid aqueous antibody formulations comprising buffering agents. The buffer includes histidine buffer, acetate buffer or phosphate buffer. In some aspects of the formulation of the present invention, the buffering agent is a histidine buffer, and the concentration of the histidine buffer is in the range of 5 to 15 mM. In some aspects, the concentration of the histidine buffer is about 10mM.

The present invention provides a liquid aqueous antibody formulation comprising a stabilizer to adjust the osmotic pressure of the liquid system to stabilize the antibody. The stabilizer is added to the antibody in an amount that can be varied according to the desired isotonicity of the formulation. In some embodiments, trehalose is used in the formulation as an osmotic pressure regulator, and in some embodiments of the invention, the stabilizer comprises 3.0% to 10% sucrose, and/or 3% to 10% % trehalose, or 5% to 10% trehalose, such as about 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two concentrations, inclusive.

In a solid state, trehalose usually exists as trehalose dihydrate, so in some embodiments, trehalose dihydrate is used when formulating preparations, and other forms of trehalose can also be used for preparation.

The present invention provides liquid aqueous antibody formulations that include a surfactant. In some embodiments, the surfactant is a nonionic surfactant, such as Tween 20. Surfactants can reduce aggregation of the prepared antibody and/or reduce particle formation in the formulation and/or reduce adsorption. In some embodiments, the formulation has Tween 20 as the surfactant. In some embodiments, the formulation comprises about 0.01% to 0.2% Tween 20, or about 0.01% to 0.1%. In some embodiments, about 0.04% Tween 20 is present in the formulations of the invention.

In some embodiments, the administration mode of the liquid formulation provided by the present invention is intravenous, subcutaneous, intraocular or intramuscular administration; in some embodiments, the administration mode of the liquid formulation is intraocular administration; in some embodiments, the liquid formulation The formulation is administered by intravitreal injection. If the formulation is to be used for intravenous, intraocular or intravitreal injection, the osmolarity of the liquid formulation is a critical parameter. In some embodiments, the osmotic pressure of the antibody preparation is about 150-400 mOsm/Kg; in some embodiments, the osmotic pressure of the antibody preparation is about 200-350 mOsm/Kg; in some embodiments, the osmotic pressure of the antibody preparation is about It is 260～340mOsm/Kg.

In some embodiments, the liquid formulation of the present invention can maintain the osmotic pressure at any concentration in the range of about 6 mg/ml to 80 mg/ml of the antibody concentration, and maintain the osmotic pressure at about 260 to 340 mOsm/Kg.

In some embodiments, the antibody formulation contains the following components: 5-100 mg/ml anti-VEGF antibody, 5-50 mM buffer, 0.5%-20% stabilizer, 0.001%-0.2% surfactant, water for injection , the pH of the preparation is 4.0-7.0. In some embodiments, the antibody formulation contains the following components: 6-90 mg/ml anti-VEGF antibody, 5-30 mM buffer, 0.5-10% stabilizer, 0.01-0.2% surfactant, water for injection , the pH of the preparation is 5.0-6.5. In some embodiments, the antibody preparation contains the following components: 6-80 mg/ml anti-VEGF antibody, 10-20 mM histidine buffer, 2%-10% trehalose, water for injection, the preparation of The pH is 5.5 to 6.5. In some embodiments, the antibody preparation contains the following components: 6-80 mg/ml anti-VEGF antibody, 10 mM histidine buffer, 2%-10% trehalose, 0.01%-0.1% Tween 20, Water for injection, the pH of the formulation is 5.5-6.5. In some embodiments, the formulation contains the following components: about 6, 12, 25, 50 or 80 mg/ml of anti-VEGF antibody, about 10 mM histidine buffer, about 9% trehalose (about 10% available Trehalose dihydrate preparation), about 0.04% Tween 20, water for injection, the pH of the preparation is 5.6-6.4. In some embodiments, the formulation contains the following components: about 6, 12, 25, 50 or 80 mg/ml anti-VEGF antibody, about 10 mM histidine buffer, about 8% trehalose (about 8.8% available Trehalose dihydrate preparation), about 0.04% Tween 20, water for injection, wherein, the pH of the preparation is 5.6-6.4.

In some embodiments, the formulation buffer includes a histidine buffer, such as L-histidine buffer, the formulation stabilizer includes trehalose and/or sucrose, and the formulation surfactant Including Tween 20, the pH value of the preparation is in the range of 4.0 to 7.0, and the osmotic pressure of the preparation is in the range of 150 to 400 mOsm/Kg. In one embodiment of the present invention, the buffer system is a histidine buffer with a concentration range of 5-15 mM, or about 10 mM, and a pH value of 4.0-7.0, or a pH of 5.6-6.4.

The present invention provides liquid aqueous pharmaceutical formulations, including antibodies suitable for therapeutic use, which are convenient to administer and may contain a wide range of protein concentrations from low to high, primarily for the treatment of disorders caused by VEGF. In one embodiment, the pharmaceutical formulation has a stability enhancing effect. In another embodiment, the formulations of the present invention are stable over at least 5 freeze-thaw cycles. In another embodiment, the formulations of the present invention are stable over 1 month at room temperature. In another embodiment, the formulation of the present invention is stable at 2-8°C for at least 24 months. In another embodiment, the antibody is directed against human VEGF. In another embodiment, the antibody is recombinant anti-VEGF humanized monoclonal antibody BAT5906.

The present invention provides an aqueous pharmaceutical preparation, comprising an effective amount of an antibody component, the buffer is histidine buffer, the osmotic pressure stabilizer is sucrose or trehalose, the surfactant is Tween 20, and the pH is about 4.0-7.0. In one embodiment of the invention, the formulation is suitable for intravitreal injection. In one embodiment of the invention, the concentration of the antibody in the liquid aqueous pharmaceutical formulation is about 80 mg/ml.

The present invention also provides a method of treating a VEGF-positive associated disease, the method comprising administering the antibody formulation to a patient.

In some embodiments of the invention, the prepared reagent is a 0.2 ml solution containing the ingredients shown in Table 1 in a vial. In some embodiments, the ingredients contained in the formulation may be 25 mg/ml anti-VEGF antibody; 9% trehalose, 0.04% Tween 20, 10 mM histidine buffer (prepared according to formulation A in Table 1). In other embodiments, the ingredients contained in the formulation may be 6, 12, 25, 50, or 80 mg/ml of anti-VEGF antibody; 8% trehalose, 0.04% Tween 20, 10 mM histidine buffer ( Prepared according to the formulation of Formulation B in Table 1). In other embodiments, the ingredients contained in the formulation may be 50 mg/ml or 80 mg/ml of anti-VEGF antibody; 8.5% trehalose, 0.04% Tween 20, 10 mM histidine buffer. In other embodiments, the formulation comprises 50 mg/ml BAT5906, 8.5% trehalose, 0.04% Tween 20, 10 mM histidine buffer. In other embodiments, the formulation comprises 80 mg/ml BAT5906, 8.5% trehalose, 0.04% Tween 20, 10 mM histidine buffer.

Table 1. List of formulations of recombinant anti-VEGF humanized monoclonal antibody BAT5906

Example

Example 1: Buffer and pH screening studies

Three different pH values (pH 5.5, pH 6.0, pH 6.5) samples were prepared with three buffers: 10 mM phosphate buffer, 10 mM citrate buffer, 10 mM histidine buffer, protein (Example Medium protein refers to BAT5906) with a concentration of 8 mg/ml, and the specific composition is shown in Table 2. Observation was carried out for 20 days under the condition of high temperature 50°C, and sampling was carried out on the 0th day (0D), the 10th day (10D), and the 20th day (20D) for the detection of SEC-HPLC and IEC-HPLC. The results are shown in Table 3, Fig. 1A, 1B.

Table 2. Buffer Composition Information

Table 3. Changes of SEC and IEC of different pH samples with time at 50℃

It can be seen from the SEC-HPLC data in Table 3 that the SEC monomer purity of the samples at different pH decreased with the prolongation of time, while the multimer and fragment showed an increasing trend. It can be seen from the change trend diagram of SEC monomer purity in Figure 1 that the SEC value of the sample in PB buffer is not as stable as that in citrate buffer and histidine buffer.

It can be seen from the variation trend graph of IEC main peak in Figure 1 that the main peak of the sample has different descending trends under different pH and buffers. The main peak of the sample is arranged in order of the descending speed of the main peak. The samples were slower than pH 5.5 and pH 6.5; the falling speed of the IEC main peak of the buffer had the following rules: histidine buffer < citrate buffer < phosphate buffer. It can be seen from the IEC acid region change data in Table 3 that the acid region changes in the following order: His6.0<His5.5<NMS6.0<His6.5<PB6.0. At the same time, it can be seen that the IEC main peak content of the protein solution at different pH decreased with the prolongation of time, the acid peak content increased with the prolongation of time, and the alkali peak content changed little. It can be seen from Figure 1 that the content of IEC main peak in pH 6.5 and pH 5.5 samples decreased faster than that in pH 6.0 samples; the content of IEC main peak in the samples in histidine buffer was lower than that in citrate buffer and PB buffer. Slow down. Antibodies can maintain good stability in citrate buffer and histidine buffer at pH 5.5-6.5; the best performance is at pH 6.0. Histidine buffers are slightly better than citrate buffers. According to the above results, it can be seen that the pH 6.0 histidine buffer can be used as a relatively stable buffer for this sample.

It can be seen from the experimental results that the target protein exhibits different stability in buffers with different pH values, but when the pH value fluctuates within a certain range, it does not have a significant effect on the quality of the target protein. And considering the long-term stability inspection requirements and industrialization requirements, the pH value of the formulation must provide an effective range, rather than a fixed value. According to the analysis results of the changes of the two key quality attributes, when the pH is higher than 6.5 and lower than 5.5, the quality of the BAT5906 antibody decreases rapidly and the performance is unstable. To sum up, it is preliminarily determined that pH6.0 is relatively stable in histidine buffer.

Example 2. Buffer Screening Assay

Different buffers have different stabilizing effects on the amount of protein. In this study, acetate buffer (10mM) commonly used in monoclonal antibody preparations was selected, and its pH range was between 5.0 and 6.0, which was better than succinate buffer (10mM) at pH 6.0 and in the pH screening experiment. A histidine buffer (10 mM) at pH 6.0 was used for a comparative test to investigate the changes in the mass of the target protein (taking the protein concentration of 10 mg/ml as an example) in different buffers. The specific formula is shown in Table 4. The test was carried out under the condition of 50°C, on the 0th day (0D), the 5th day (5D), the 10th day (10D), the 20th day (20D) and the 30th day (30D) The samples after the test were detected by SEC-HPLC and IEC-HPLC. The results are shown in Table 5 and Figures 2A and 2B.

Table 4. Buffer Composition Information

Table 5. Changes of protein SEC and IEC in different buffers with time at 50℃

From the data in Table 5 and Figure 2, it can be seen that among the three different buffers selected, the rate of change of the target protein is slightly different. In terms of SEC-HPLC, histidine buffer and succinate buffer have more Aggregate increases slightly slower than with acetate buffer. Under the three different pH values of acetate buffer, pH 5.0, 5.5, and 6.0, the SEC monomer purity decreased faster than that of histidine and succinate buffer, mainly because the polymer increased faster.

In terms of IEC-HPLC, the rate of decline of the main peak was slower in histidine and succinate buffers than in acetate buffers. The protein in the acetate buffer pH 5.0, 5.5 and 6.0 was better in the acetate pH 5.5 buffer, but the decline rate of the IEC main peak was still faster than that of histidine and succinate buffer. It is shown that the acid region and the alkali region increase rapidly. Thus, histidine and succinate buffers were chosen as a relatively good buffer for this formulation.

The experimental results of comprehensive pH and buffer screening experiments can also confirm that histidine buffer can be used as a stable buffer for the target protein.

Example 3 Buffer concentration screening

Prepare 10 mM, 20 mM, 30 mM histidine buffers, pH 6.0, respectively. The BAT5906 was ultrafiltered and exchanged into the three groups of buffers, and the protein concentration was 10 mg/ml. After the samples were sterilized and filtered, they were placed at 40°C for 5, 10, 20, and 30 days for detection and at 25°C for 1, 2, and 3 months. The experimental results are as follows:

Table 6. 40°C-SEC Results

Table 7. 40℃-IEC Results

Table 8. 25℃-SEC test results

Table 9. 25℃-IEC experimental results

From the results in the above table, it can be seen that the buffer concentration is between 10 and 30 mM, and the protein has good stability. In contrast, 10 mM histidine buffer works better, so 10 mM histidine buffer can be considered.

Example 4: Stabilizer Screening Study

Excipients are all components of the formulation except the active ingredient. The excipients mentioned in this section mainly refer to ingredients such as stabilizers, surfactants, and osmotic pressure regulators that provide specific effects.

Screening of stabilizers

Select stabilizers trehalose, sucrose, mannitol, sodium chloride, sorbitol and maltose to prepare a protein solution containing these stabilizers in 10mM histidine buffer (hereinafter referred to as His) (with a protein concentration of 55mg). /ml as an example), the specific formula is shown in Table 10, and the experimental results are shown in Table 11 and Figures 3A, 3B, 4A, and 4B. The samples were subjected to high temperature experiments, sampling and testing on the 0th day (0D), the 5th day (5D), the 10th day (10D), the 20th day (20D), and the light experiment, on the 0th day (0D), the first 5 days (5D), 10 days (10D), and 14 days (14D) were sampled and tested, and the samples with better stability were compared. The trehalose weight percentage in this example is the percentage calculated by the weight of trehalose dihydrate.

Table 10. Different stabilizer solution formulations

Table 11. Samples with different stabilizers at 40°C as a function of time

From the results of the SEC experiment at 40°C, the SEC of sample 6 (containing maltose) changed the fastest, indicating that the reducing sugar maltose was added to the sample, which has poor protein stability. The SEC changes for the remaining samples were comparable.

From the IEC test results at 40°C, the main peak of sample 6 (maltose) decreased the fastest, and the other stabilizers changed at the same speed. Of these, sample 4 (sodium chloride) is relatively good.

Table 12. Changes over time for samples with different stabilizers under light conditions (25°C, 4000lx)

From the results of the light experiment, the main peak of sample 6 (maltose) declined the fastest in SEC, followed by sample 4 (sodium chloride), and the rest of the samples had similar trends.

In terms of IEC, the main peak of sample 6 (maltose) decreased the fastest, and the main peaks of other samples decreased close to each other.

From the results of high temperature and light experiments, trehalose does not have the same effect on the stability of samples as maltose accelerates the degradation of samples, and it can keep the samples stably stored. Therefore, among the above-mentioned stabilizers, trehalose is selected as a suitable stabilizer.

Amino Acid Stabilizer Screening Experiment

Select amino acids as stabilizers to prepare protein solutions in 10 mM histidine buffer (hereinafter referred to as His) containing these stabilizers (with a protein concentration of 77 mg/ml as an example). The specific formula is shown in Table 13. The samples were subjected to high temperature experiments, sampling and testing on the 0th day (0D), the 5th day (5D), the 10th day (10D), the 20th day (20D), and the light experiment, on the 0th day (0D), the first The 5th day (5D) and the 10th day (10D) were sampled and tested, and the samples with better stability to the samples were compared. The specific experimental results are shown in Tables 13, 14 and Figures 5A, 5B, 6A, and 6B.

Table 13. Different stabilizer solution formulations

Table 14. Changes over time for samples with different stabilizers at 40°C

From the results of SEC and IEC at 40°C, it can be seen that formulation 3 (glycine) is inferior, and the rest of the differences are not particularly large, and formulation 5 (trehalose) and formulation 6 (sucrose) have a slight advantage. It shows that under high temperature conditions, the use of trehalose will not affect the stability of the sample.

Table 15. Changes over time for samples with different stabilizers under light conditions (25°C, 4000lx)

From the light SEC results, it can be seen that samples No. 4 (methionine), No. 7 (trehalose + methionine), No. 8 (sucrose + methionine), and No. 3 (glycine) under light conditions, the SEC The monomer purity decreased the slowest, and the aggregate increased the slowest, while No. 5 (trehalose) and No. 6 (sucrose) SEC main peaks did not have a dominant decline rate, indicating that methionine and glycine have a significant effect on the samples under light conditions. certain protection.

From the light IEC results, it can be seen that samples No. 4 (methionine), No. 7 (trehalose + methionine), No. 8 (sucrose + methionine), and No. 3 (glycine) under light conditions, IEC The main peak decreased the slowest, while No. 5 (trehalose) and No. 6 (sucrose) SEC main peaks did not have a dominant decline rate, indicating that methionine and glycine have a certain protective effect on the samples under light conditions. From the perspective of lighting, these can be selected as accessories.

Based on the results of light and high temperature experiments, No. 3 (glycine) can protect the samples under light conditions and reduce the decline rate of the main peaks of SEC and IEC of the samples, but the performance is poor under high temperature conditions, and the decline rate of the main peak of the sample is faster. Methionine, on the other hand, has an obvious advantage in protecting protein when exposed to light, and is similar to trehalose at high temperature. Considering that the finished product will be stored in the dark, methionine may not be selected as a stabilizer. Based on the above-mentioned stabilizer screening experiments and accelerated long-term stability data, it is shown that selecting trehalose as the stabilizer can make the finished product stably stored at 2-8°C for 2 years or more.

Mixed Screening Test of Two Stabilizers

To investigate the effect of different stabilizer concentrations on the protein, experiments were performed to determine the appropriate stabilizer concentration. Set different contents of trehalose (the weight percentages of trehalose in the table herein are all percentages calculated by the weight of trehalose dihydrate) and mixed with sodium chloride, and investigated at high temperature of 50°C and 40°C. Considering that the injection is a vitreous injection, it is necessary to pay attention to the osmotic pressure of the sample. After calculation and determination, the protein in 10 mM histidine buffer needs to be added with 10% trehalose. The osmotic pressure can make the protein concentration less than or equal to 25 mg The osmotic pressure range of 240-360 mmol/L of the protein solution per ml, this experiment takes the protein concentration of 5 mg/ml as an example. In the stabilizer screening test, sodium chloride can reduce the increase of the IEC acid area under high temperature conditions, and the osmotic pressure of 0.9% sodium chloride can also reach the plasma osmotic pressure, so trehalose and sodium chloride were selected in different proportions and reached the osmotic pressure. The test was carried out in a mixed manner, placed on the 0th day (0D), 10th day (10D), 20th day (20D) under high temperature conditions, and on the 0th day (0D), 1st week (1W), In the second week (2W) and the third week (3W), samples were taken for SEC-HPLC protein purity and IEC-HPLC analysis of charge isomers. The specific formulation of the stabilizer is shown in Table 16. The experimental results are shown in Table 17, Table 18 and Figures 7A, 7B, and 8A, 8B.

Table 16. Two stabilizer mixed solution formula table

Table 17. Sample changes with time at high temperature (50°C and 40°C)

From the SEC-HPLC data, under the condition of high temperature 50 ℃, the main peak of prescription 1 (without stabilizer) decreased the fastest, and the main peak of prescription 2 (10% trehalose) decreased the slowest, and the formation of multimers was the least. After formulation 3, formulation 4, and formulation 5 trehalose mixed with sodium chloride, the main peak decline speed can be reduced, but the decline speed of the main peak is not as slow as formulation 2 (10% trehalose).

From the IEC-HPLC data, it can be seen that under the condition of high temperature of 40 °C, the IEC main peak of prescription 1 (without stabilizer) has a relatively rapid decline. It shows that the stabilizer has the effect of reducing the descending speed of the main peak. Among them, prescription 4 (5% trehalose + 0.45% sodium chloride) and prescription 5 (3% trehalose + 0.6% sodium chloride) can reduce the descending speed of the main peak and slow down the transition of the sample to the acid region.

Table 18. Changes of samples with time under the condition of light (25℃, 4000lx)

From the SEC-HPLC data, it can be seen that the main peak of SEC changes significantly under light conditions. The main peak of prescription 6 (0.9% sodium chloride) and prescription 1 (without stabilizer) decreased the fastest. The best way to reduce the decline rate of the main peak of the sample is formulation 2 (10% trehalose) and formulation 3 (7% trehalose + 0.3% sodium chloride).

From the IEC-HPLC data, it can be seen that under light conditions, the main peak of prescription 4 (5% trehalose + 0.45% sodium chloride) and prescription 1 (without stabilizer) decreased the fastest, indicating that prescription 4 (5% trehalose + 0.45% sodium chloride) can not effectively slow down the falling speed of IEC main peak when illuminated. However, prescription 2 (10% trehalose) and prescription 3 (7% trehalose + 0.3% sodium chloride) can effectively slow down the transition of the IEC main peak into the acid region and the alkali region under light conditions, and the decline rate of the main peak is slowest.

Based on the above experimental results of light and high temperature, the presence of stabilizer can inhibit the production and increase rate of acidic isomers and increase the content of the main peak. Recipe 2 (10% trehalose) has a certain effect in slowing down the decline rate of the main peak of protein SEC-HPLC under high temperature and light conditions, and slowing down the transition of the main peak of protein IEC-HPLC to acid region and alkali region, and compared with other prescriptions There are certain advantages, so the stabilizer and its content under the prescription can be selected and used as a suitable stabilizer and its content for the protein.

Example 5: Tween content screening test

Tween 20 was selected as the surfactant, and 10mM histidine buffer (hereinafter referred to as His) was used as the buffer to prepare protein solutions containing different amounts of Tween 20 (protein concentration: 1.8 mg/ml). The specific formula is shown in the table. 19. At room temperature, the sample was placed on the shaker at a speed of 200 rpm, and the protein solution was shaken. At 0h, 2h, 7h, 24h, 48h, and 144h, the solution was subjected to lamp inspection and turbidity (OD ₃₄₀ value) detection to investigate the changes in the quality of the protein solution. The results are shown in Figure 9. (Note: The OD value is the value obtained by detecting the protein solution at UV _340nm .)

Table 19. Formulas of Tween Solutions with Different Contents

From the turbidity of the solution, it can be seen that after being placed under shaking conditions for 2 hours, sample 1 begins to appear turbid, and samples 2, 3, and 4 remain clear after being placed for 144 hours, indicating that Tween has certain protection for samples under shaking conditions. It can prevent the turbidity of protein agglomeration and play a role in solubilization.

Too little Tween may affect protein stability, and too much Tween may cause safety problems, while about 0.04% Tween 20 can keep the protein stable and prevent the protein from turbidity under shaking conditions , 0.04% Tween 20 can be selected as a suitable Tween concentration.

In the surfactant screening experiment, 0.04% Tween 20 was finally selected as the surfactant of this protein. In order to prove that this content has good stability to the protein, 10mM histidine buffer (hereinafter referred to as His) was used as the buffer to prepare protein solutions containing different surfactants (taking the protein concentration of 77mg/ml as an example), the specific See Table 20 for the recipe. At room temperature, the sample was diluted with purified water so that the final sample concentration was 0.5 mg/ml. The change in the quality of the protein solution was investigated by detecting the number of particles contained in the diluted sample, and it was proved that 0.04% Tween 20 can be used as a suitable surfactant. The data are the results of repeated detection of the same sample. The detailed results are shown in Figures 10A and 10B.

Table 20. Formulations of Tween Solutions with Different Contents

From the results of the microparticles detected after dilution of protein solutions containing different contents of Tween 20, the protein solution without Tween 20 has the highest microparticles, which also shows that Tween can effectively prevent protein aggregation to form microparticles and play a role in antiflocculation. In the protein solution with different contents of Tween, in which the protein solution of 0.04% Tween 20 was added, the number of particles of 2 μm, 5 μm, 10 μm and 25 μm was the least, indicating that adding 0.04% Tween 20 can indeed make the protein The solution remained stable.

Embodiment 6: Trehalose content screening test

In the samples containing 0.04% Tween 20, different amounts of trehalose were added, and the protein concentration was 10 mg/ml, and high temperature experiments were carried out. Sampling and testing to observe whether different contents of trehalose have an effect on protein stability. Table 21 for the specific ingredients and formula, and Table 22 for the experimental results, Figures 11A and 11B. The trehalose weight percentage in this example is the percentage calculated by the weight of trehalose dihydrate.

Table 21. Recipe table of trehalose content screening solution

Table 22. Sample changes over time at high temperature (40°C)

It can be seen from recipes 1 to 5 that there is basically no difference between SEC and IEC in recipes containing different amounts of trehalose when placed at a high temperature of 40°C for a certain period of time. Therefore, it can be seen that the content of trehalose has little effect on SEC and IEC. In the following experiments, the amount of trehalose is only used to adjust the osmotic pressure.

Example 7: Further screening of pH ranges

The final selected samples of different pH around pH6.0, i.e. pH5.6, pH5.8, pH6.0, pH6.2, pH6.4 samples were prepared. The sample concentration is 25mg/ml, and the samples all contain the final selected formulation formulation ingredients, namely, the histidine buffer contains trehalose (the weight percentage is calculated as 10% with trehalose dihydrate) and 0.04% Tween 20, It can be seen from the above experiments that the trehalose content has little effect on the changes of the SEC and IEC of the samples, so the following experimental results are also applicable to the formulation of trehalose (8.8% by weight calculated as trehalose dihydrate). The samples were subjected to accelerated experiments at high temperature, placed at 40°C on day 0 (0D), day 5 (5D), day 10 (10D), day 20 (20D), day 30 (30D), and After being placed at 25°C on day 0 (OD), month 1 (1M), and month 2 (2M), samples were taken out for SEC-HPLC and IEC-HPLC detection. The experimental results at 40° C. are shown in Table 23 and FIGS. 12A and 12B , and the experimental results at 25° C. are shown in Table 24 and FIGS. 13A and 13B .

Table 23. Changes of protein content with time at different pH at 40°C

From the SEC-HPLC data, it can be seen that under the condition of 40 °C, the SEC changes of the samples at different pHs are basically the same, the decrease rate of monomer purity is similar, and the generation rate of fragments and aggregates is also similar. It shows that the protein has no obvious difference in SEC in the range of pH 5.6-6.4, and can basically keep at a stable level.

From the IEC-HPLC data, it can be seen that under the condition of 40 ℃, the main peak decline speed of the sample is consistent between pH 5.6 and 6.4. The sample with pH 6.4 was placed at 40 °C until the 10th day, and the main peak of IEC began to decline faster than that of other pHs, but it was not significantly different from the main peaks of samples at other pHs. It can be considered that the antibody protein is basically stable between pH 5.6 and 6.4.

Table 24. Changes in the amount of protein at different pH with time at 25°C (D represents day, M represents month)

From the SEC-HPLC data, it can be seen that the SEC changes of samples with different pH are basically the same after being placed at 25 °C for 2 months. The monomer purity was still high, indicating that the protein could remain stable between pH 5.6 and 6.4.

From the IEC-HPLC data, it can be seen that when the samples are placed at 25°C for 2 months, the pH of the samples is between 5.6 and 6.4, and the decline rate of the IEC main peak is basically the same. The samples with pH 6.4 were placed at 25℃, and the main peak of IEC decreased faster than the samples at other pHs, mainly because the acid region increased faster than the samples at other pHs. However, the difference between the drop of the main peak of the sample at pH 6.4 and the drop of the main peak of the samples at other pHs is within the measurement error range, so it can be considered that the drop rates of the samples at pH 6.4 and other pHs are consistent. Therefore, it can be seen that the samples can remain stable between pH 5.6 and 6.4.

Example 8: Sample stability data at different concentrations

The stability of the samples at different concentrations under high temperature 50 °C, 40 °C and light conditions was compared. The set concentrations are 6mg/ml, 12mg/ml, 25mg/ml, 50mg/ml and 80mg/ml. Except for the different protein concentrations, other compositions are the same, that is, it also includes trehalose (weight percentage calculated as trehalose dihydrate is 8.8%), 0.77mg/ml L-histidine, L-histidine hydrochloride (content Calculated as L-histidine hydrochloride monohydrate (1.06 mg/ml), 0.04% Tween 20.

Table 25. Changes of SEC and IEC of different concentrations of protein with time at 50℃

It can be seen from the change of SEC monomer purity at 50 °C that with the extension of storage time, the SEC monomer purity of samples with different concentrations has a different downward trend. The higher the sample concentration, the faster the SEC monomer purity decreases, and the SEC more The faster the aggregates are formed. Among the samples with concentrations of 6, 12, 25, 50 and 80 mg/ml, the 6 mg/ml sample had the slowest decrease in SEC monomer purity and the slowest SEC multimer formation.

From the change of IEC at 50°C, with the extension of storage time, the samples with different concentrations all showed a similar downward trend, and the main peak of IEC decreased and the speed of the generation of IEC acid peak was not very different between samples of different concentrations.

Table 26. Changes of SEC and IEC of different concentrations of protein with time at 40℃

From the change trend of SEC monomer purity at 40℃ to 28 days, with the extension of storage time, the SEC monomer purity of samples with high concentration decreased rapidly, and the decreasing trend was similar, but there was little difference between samples with different concentrations. In SEC multimers, samples with high concentrations formed multimers faster than samples with low concentrations.

From the change of IEC at 40°C, with the extension of storage time, the main peak of IEC decreases, the decreasing trend of different concentrations is close, and the increasing trend of IEC acid region is close.

Table 27. Changes of SEC and IEC of different concentrations of protein with time under light conditions (25℃, 4000lx)

From the change of light SEC, the purity of SEC monomer decreased to varying degrees with the prolongation of storage time, and the degree of decrease in the purity of SEC monomer was related to the concentration of the sample. The results showed that the sample SEC monomer of 80 mg/ml The steepest drop in purity, ie the higher the concentration of the sample, the faster the drop in SEC monomer purity and the faster the formation of SEC multimers.

From the change of IEC under light conditions, the decrease degree of IEC main peak was correlated with the concentration. The higher the concentration of the sample, the faster the IEC main peak decreases and the faster the IEC acid peak increases.

The insoluble particles of 5 samples of different concentrations were detected, and the detection results of the insoluble particles are as follows in Table 28 (units are "pieces/ml").

Table 28. Detection of protein insoluble particles at different concentrations

It can be seen from the test results of insoluble particles that the number of insoluble particles ≥ 10um is relatively small, and all meet the standard.

The particle size of the sample was tested by DLS, and the test results at 25°C are shown in Table 29 below:

Table 29. Detection of protein particle size at different concentrations

蛋白浓度(mg/ml)Protein concentration (mg/ml)	radius(nm)radius(nm)
66	6.16.1
1212	6.16.1
2525	5.25.2
5050	4.54.5
8080	4.44.4

From the perspective of sample radius, the sample radius is smaller when the sample concentration is 50, 80 mg/ml than that of 6, 12, and 25 mg/ml.

When the concentration of protein increases, the osmotic pressure of the solution will increase, and the present invention reduces the trehalose content so that the osmotic pressure of the solution is within the osmotic pressure range. Different concentrations of protein were added with the same content of trehalose, and the osmotic pressure was measured, so that the osmotic pressure of all samples was within the osmotic pressure range, and then the most appropriate trehalose concentration was selected as the appropriate protein concentration for the prescription. As the above experiments (trehalose content screening experiment) have proved that the trehalose content has little effect on protein stability, but has a greater effect on osmotic pressure. Therefore, the selection of trehalose content is based on the osmotic pressure as the selection criterion.

Table 30. Sample osmotic pressure of different concentrations of protein with different trehalose content (weight percent calculated as trehalose dihydrate)

According to the results of the above-mentioned measured osmotic pressure, the trehalose content in the above-mentioned contents can basically maintain the ideal osmotic pressure of 300±40mOsmol/Kg of the preparations with these 5 antibody concentrations. Among them, trehalose dihydrate is preferred. The amount was calculated to be around 8.8% (8.7%-8.9%).

Example 9: Sample Stability Data

According to the prescription: 10mM histidine buffer + 0.04% Tween 20 + trehalose (the dosage is 8.8% trehalose dihydrate), the protein concentration is 6, 12, 25, 50, 80 mg/ml, and the ultrafiltration is changed. The liquid was finally filled into samples containing the above formulations. The 25 mg/ml stability data for filling are shown in Table 31 below:

Table 31. Sample Stability

According to the stability data of the above samples, it can be known that the samples placed at 25°C for 6 months still meet the standard, and the samples placed at 4°C for 9 months are also within the standard.

Example 10: Study on influence factors of samples

The stability of the samples in Example 9 was investigated under the conditions of light and 25°C.

Table 32. Light and high temperature test results

From the data in Table 32, it can be seen that after the sample was placed at 25°C for 1 month or placed under light for 14 days, compared with the 0th day, the sample was still a clear and transparent liquid, no visible foreign matter appeared, and the protein concentration did not change significantly. After being placed at 25 °C for 1 month, the CE-SDS, SEC-HPLC purity and IEC-HPLC main peak content of the protein did not change significantly, indicating that the sample can be stored at 25 °C for a short time without affecting the protein. quality. Under light conditions, the content of the main peak of CE-SDS decreased with the prolongation of time, and the purity of SEC-HPLC monomers decreased slightly with the prolongation of time, and the multimers increased slowly, indicating that the protein in the light conditions There will be a tendency to aggregate; the content of the main peak of IEC-HPLC decreases rapidly with the extension of time, indicating that the quality of the target protein is easily affected by light and should be stored in the dark. In addition, after 14 days of testing, the activity detection data of the protein were all within the qualified standard.

From the data of insoluble particles at 25°C, it can be seen that the test results of different days fluctuated to a certain extent, but the data did not show a gradual increase trend, nor did there appear a sudden increase in particles, and the number of particles in the solution remained very low. s level. The pH variation of the samples also varied within the margin of error. Under light conditions, from the change of sample pH, the pH of the sample basically changed little, and the particles also changed little, within the error range, it can be considered that the pH and the particles are unchanged.

Example 11: Samples subjected to repeated freeze-thaw studies

Repeated freezing and thawing of samples of the formulated formulation: 25 mg/ml recombinant anti-VEGF humanized monoclonal antibody (BAT5906), 10 mM histidine buffer, 0.04% Tween 20, 10% trehalose dihydrate Experiment, the experimental results are shown in Table 33:

Table 33. Repeated freeze-thaw test results

It can be seen from the data in Table 33 that after repeated freezing and thawing of the sample for 5 times, compared with the 0th time, the sample is still a clear and transparent liquid, no visible foreign matter appears, and the pH value and protein concentration of the sample have no significant changes, indicating that the repeated freezing and thawing are performed. In the melting test, the sample was stable and had no precipitates. In addition, the SEC-HPLC purity, IEC-HPLC main peak content, CE-SDS purity and activity of the samples did not change significantly.

From the data of insoluble particles, repeated freeze-thaw experiments did not lead to an increase in the number of particles in the sample. After repeated freezing and thawing of the sample for 5 times, the sample particles are still within the qualified range, indicating that the sample can be stored stably. Judging from the changes in pH after repeated freezing and thawing 5 times, the pH did not change much, and all fluctuated within the error range.

Judging from the influencing factors and the results of repeated freezing and thawing experiments, the selection of histidine buffer as the buffer system of the protein, trehalose as the stabilizer or osmotic pressure regulator, and 0.04% Tween 20 as the surfactant can make protein remains stable.

For the formulation: 50mg/ml recombinant anti-VEGF humanized monoclonal antibody (BAT5906), 10mM histidine buffer, 0.04% Tween 20, 8.8% trehalose dihydrate, the prepared preparation was subjected to repeated freeze-thaw experiments . The experimental results are shown in Table 34:

Table 34. Repeated freeze-thaw test results

As can be seen from Table 34, after repeated freezing and thawing of the preparation for 5 times, compared with the 0th time, the preparation is still a clear and transparent liquid, no visible foreign matter appears, the pH value of the preparation has no significant change, and the sample concentration fluctuates within the normal error range , indicating that the sample is stable in repeated freeze-thaw tests.

From the data of insoluble particles, the number of insoluble particles in 5 repeated freeze-thaw tests is higher than that in 4 repeated freeze-thaw tests, but after 5 repeated freeze-thaw cycles, the sample particles are still within the qualified range. From the change of pH after repeated freezing and thawing 5 times, the pH did not change much and remained stable.

Example 12: In vivo pharmacodynamic studies

The formulated BAT5906 formulation of Example 9 was used: 25 mg/ml antibody, 10 mM histidine buffer, 0.04% Tween 20, trehalose (8.8% trehalose dihydrate).

In this experiment, laser photocoagulation around the fovea of the rhesus macaque was used to induce choroidal angiogenesis, and an animal model similar to human choroidal neovascularization was established. 20 days after photocoagulation, fluorescein fundus angiography was performed to determine the condition of modeling, and 20 rhesus monkeys (9 females and 11 males) with successful modeling were selected and divided into 5 groups, which were the model control group (normal saline) and the positive control group ( Lucentis (ranibizumab 0.5 mg/eye group) and recombinant anti-VEGF humanized monoclonal antibody BAT5906 injection 0.25, 0.5, 1.25 mg/eye group, 4 monkeys in each group, both male and female.

21 days after photocoagulation, monkeys in the model control group, Lucentis group and BAT5906 injection 0.25, 0.5, 1.25 mg/eye group were given 0.9% sodium chloride injection, 10 mg/ml Lucentis by a single injection of 50 μl/eye through the vitreous body of both eyes. 25mg/ml BAT5906 injection was given by single injection of 10μl/eye, 20μl/eye, 50μl/eye through the vitreous body of both eyes, and fundus color photography, fluorescein fundus angiography, optical fundus were performed 14 and 28 days after administration, respectively Coherence tomography (OCT) examination was performed to observe the inhibition of choroidal neovascularization by the test product; 29 days after administration, aqueous humor was extracted from both eyes of all monkeys for VEGF detection, and after euthanasia, both eyes were taken for HE staining, and the left eye was stained with HE. Masson's trichrome staining was performed, and CD31 immunohistochemical staining was performed on the right eye for histopathological examination. The main results are as follows (see Tables 35-36 for data, n=8):

Under the experimental conditions, rhesus monkeys in the laser-induced choroidal neovascularization (CNV) model were given 0.25, 0.5, 1.25 mg/eye doses of recombinant anti-VEGF humanized monoclonal antibody BAT5906 injection and 0.5 mg through the vitreous of both eyes. Lucentis per eye, retinal angiography, OCT examination and ocular histopathological examination showed that recombinant anti-VEGF humanized monoclonal antibody injection at doses of 0.25, 0.5 and 1.25 mg/eye had significant inhibition on monkey CNV effect. The efficacy of recombinant anti-VEGF humanized monoclonal antibody injection at doses of 0.5 and 1.25 mg/eye was slightly better than that of Lucentis.

Rhesus monkeys were given 0.25, 0.5, 1.25 mg/eye dose of recombinant anti-VEGF humanized monoclonal antibody injection by single injection into the vitreous of both eyes, showing linear kinetic characteristics.

Table 35. Effect of vitreous injection of BAT5906 on the reduction and improvement rate of rhesus monkey fluorescein penetration area

*Compared with the model control group, the difference in mean is statistically significant (P≤0.05)

Table 36. Effect of vitreous injection of BAT5906 on the reduction and improvement rate of retinal thickness in rhesus monkeys

Claims

An antibody preparation, characterized in that the antibody preparation contains an anti-VEGF antibody or a fragment thereof; the heavy chain variable region of the anti-VEGF antibody or a fragment thereof contains the amino acid sequence shown in SEQ ID NO: 1, and the The light chain variable region of the anti-VEGF antibody or fragment thereof contains the amino acid sequence set forth in SEQ ID NO: 2; and, the antibody formulation further comprises one or more of a buffer, a stabilizer, and a surfactant.
The antibody preparation according to claim 1, wherein the amount of the antibody is 5-100 mg/ml.
The antibody preparation according to claim 1, wherein the pH of the antibody preparation is 4.0 to 7.0.
The antibody preparation according to claim 1, wherein the buffer is selected from the group consisting of histidine buffer, succinate buffer, acetate buffer, citrate buffer and phosphate buffer one or more; preferably, the amount of the buffer is 5-50 mM.
The antibody preparation according to claim 1, wherein the stabilizer is selected from one or more of trehalose, sucrose, mannitol, sodium chloride, sorbitol, proline, glycine and methionine species; preferably, the amount of the stabilizer is 0.5% to 20%.
The antibody preparation according to claim 1, wherein the surfactant is selected from Tween and/or Poloxamer; preferably, the amount of the surfactant is 0.001% to 0.2%.
The antibody preparation according to claim 1, wherein the antibody preparation contains the following components:

(1) 5～100mg/ml anti-VEGF antibody or fragment

(2) 5～50mM buffer

(3) 0.5%～20% stabilizer

(4) 0.001%～0.2% surfactant

(5) water for injection;

The pH of the formulation is 4.0 to 7.0.
The antibody preparation according to claim 7, wherein the preparation contains the following components:

(1) 6～90mg/ml anti-VEGF antibody or fragment

(2) 10mM histidine buffer

(3) 2%～10% trehalose

(4) 0.01%～0.1% Tween 20

(5) water for injection;

The pH of the formulation is 5.5-6.5.
The antibody preparation according to any one of claims 1 to 8, wherein the osmotic pressure of the antibody preparation is 150-400 mOsm/Kg.
The antibody preparation according to claim 1, wherein the administration mode of the antibody preparation is intravenous, subcutaneous, intraocular or intramuscular administration.
A delivery device comprising the antibody preparation of any one of claims 1-10.
Use of the antibody preparation according to any one of claims 1 to 10, or the delivery device according to claim 11, in the preparation of a medicament for the treatment of VEGF-related diseases.
The preparation method of the antibody preparation described in any one of claims 1 to 10, characterized in that it comprises the following steps:

(1) prepare buffer solution;

(2) by UF/DF ultrafiltration, using the buffer solution prepared in step (1) to carry out ultrafiltration and exchange liquid on the antibody, and then concentrating to obtain an antibody solution;

(3) preparing an excipient mother liquor containing a stabilizer and/or a surfactant, and adding the excipient mother liquor to the antibody solution prepared in step (2) to obtain an antibody preparation.