WO2020060183A1 - Liquid formation for stabilizing trastuzumab antibody - Google Patents

Abstract

The present invention relates to a liquid formulation comprising: a trastuzumab or an antigen binding fragment thereof; a stabilizer; and a buffering agent, wherein the liquid formulation is free of a surfactant, and a method for preparing same.

Description

Trastuzumab antibody stabilized liquid formulation

The present invention relates to a stable liquid formulation comprising trastuzumab or an antigen-binding fragment thereof, and more specifically, a liquid formulation capable of stabilizing trastuzumab or an antigen-binding fragment thereof that does not contain a surfactant, and its preparation It's about how.

The need to develop high-concentration, e.g., 50 mg / mL or higher antibody drugs to enhance patient convenience is emerging, and Herceptin ^® SC (120 mg / mL), Xolair ^{® (} 125 mg / mL), Simponi ^{® (} 100 mg / mL), and drugs such as Humira ^{® (} 50 mg / mL, 100 mg / mL) are commercially available. Antibody pharmaceuticals may be exposed to various environments during manufacture, transportation, storage and administration or undergo phase changes such as air-liquid, solid-liquid, or liquid-liquid. In particular, when the antibody contacts the hydrophobic interface, problems such as protein aggregation or particle formation may occur. To solve this problem, surfactants such as polysorbate 20 or polysorbate 80 are mainly used. Antibody formulations comprising polysorbate 20 are known to reduce protein uptake or aggregation. However, polysorbates are known to degrade through oxidation and hydrolysis, which in turn produces reactive oxygen species (ROS), resulting in oxidative damage to antibody proteins.

Therefore, there is still a need to develop stable formulations that can solve the problems of polysorbates discussed above.

One aspect of the present invention is to provide a liquid formulation capable of maintaining stability without inducing aggregation of the antibody without including a surfactant capable of decomposing by oxidation and hydrolysis to generate ROS.

Another aspect of the present invention, adding a stabilizer and a buffering agent to the solvent to prepare a mixed solution; And adding trastuzumab to the mixed solution.

One aspect of the invention, (a) Trastuzumab or an antigen-binding fragment thereof; (b) stabilizer; And (c) a buffering agent and no surfactant.

Another aspect of the present invention, adding a stabilizer and a buffering agent to the solvent to prepare a mixed solution; And adding trastuzumab to the mixed solution.

It has been found that the liquid formulation according to one aspect of the present invention can maintain the stability of the antibody without including polysorbate, which may cause ROS under stress conditions.

1 is a change in the percent high molecular weight material (percent high molecular weight,% HMW) of Example 1 and Comparative Example 1 under temperature stress conditions by size exclusion-high performance liquid chromatography (Size-Exclusion HPLC, SE-HPLC) It is a graph showing one result.

Example:

Hereinafter, the present invention will be described in more detail by examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereby.

Preparation Example: Preparation of Examples and Comparative Examples

The formulations of Example 1 and Comparative Example 1 were prepared.

First, in the composition of Table 1, trastuzumab (5 mM histidine buffer (pH 6.0), 40 mg / mL, Samsung Bioepis) and a buffering agent and a stabilizer, except for the surfactant, are made of polycarbonate material of 5L baotainer (biotainer) ) 5L buffer solution was prepared by adding to sterile distilled water in a container. 3 mL of 40 mg / mL trastuzumab solution in 5 mM histidine buffer at pH 6.0 was placed in a dialysis cassette (MWCO 10 kDa) (Slide-A-Lyzer cassette, Thermo Fisher Scientific), and then a cassette containing this trastuzumab solution Was placed in a 2L beaker containing 1,600 mL of the buffer solution to make dialysis, and the 5 mM histidine buffer at pH 6.0 was exchanged with the buffer solution. Thereafter, trastuzumab was concentrated to 120 mg / mL or more. Concentration was achieved by passing the dialysis trastuzumab containing solution through an Amicon Filter with a cutoff molecular weight of 30 kDa. Finally, to the concentrated trastuzumab solution, a surfactant prepared at a concentration of 20x, that is, 0.8 w / v%, in the buffer solution prepared above was added to a concentration of 1x. The composition of Comparative Example 1 is also listed in Table 1. The formulation of Example 1 had a pH of about 5.5.

In addition, the preparation of Comparative Example 1 was prepared in the same manner as in Example 1, except that polysorbate 20 was added as a surfactant. The pH of the formulation obtained after adding the surfactant in Comparative Example 1 was about 5.5. Table 1 shows the composition of the formulations of Example 1 and Comparative Example 1.

		Example 1	Comparative Example 1
Trastuzumab (mg / mL)		120	120
Buffering agent	Histidine (mM)	20	20
Stabilizer	Methionine (mM)	10	10
	Trehalose (w / v%)	8	8
Surfactants	Polysorbate 20 (w / v%)	-	0.04

Test Example 1: Check the stability of the surfactant-free formulation

Although the preparation of Example 1 prepared does not contain a surfactant, it was confirmed by SE-HPLC whether it can maintain stability under temperature stress and freeze-thaw stress.

For the temperature stress, 1 mL of each of the formulations of Example 1 and Comparative Example 1 was placed in a 1.5 mL microtube made of polypropylene (Axygen), and the tube was heated to 40 ° C. in a stability thermostat (JEIO TECH). They were exposed to temperature stress conditions for 4 weeks. Specifically, the tube was placed in the stability thermostat and placed for 4 weeks under conditions of temperature 40 ± 2 ° C. and relative humidity 75 ± 5%. % HMW,% monomer and% LMW were measured using SE-HPLC for formulations stored for 4 weeks. When the antibody in the formulation loses stability, aggregation is induced to increase the proportion of high molecular weight (HMW) substances, or decrease the monomer ratio due to decomposition of the antibody or destabilization of disulfide bonds, or low molecular weight ( It will be observed that the proportion of low molecular weight (LMW) material increases.

The results are shown in Table 2 and FIG. 1. The average value and standard deviation (SD) of the data obtained by analyzing each formulation prepared in 3 replicates per formulation were obtained, and Table 2 shows the average per item for each formulation and the standard deviation in the lower row. As shown in Table 2, in Example 1, Δ% HMW was 5% lower than in Comparative Example 1. Thus, formulations without surfactant were more stable than formulations with surfactant.

	Starting point	% HMW 40 ℃ / 4 weeks	Δ% HMW 40 ℃ / 4 weeks
Example 1	1.17	2.32	1.15
	SD: 0.01	SD: 0.04	SD: 0.04
Comparative Example 1	0.83	2.04	1.21
	SD: 0.07	SD: 0.13	SD: 0.06

In addition, for freeze-thaw stress, 1 mL of each of the formulations of Example 1 and Comparative Example 1 was placed in a 1.5 mL microtube (Axygen) made of polypropylene, and the tube was maintained at -70 ± 10 ° C. After placing it in a deep freezer (Thermo scientific) and maintaining it for 18 hours in a frozen and frozen state, the tube was taken out and left at room temperature for 1 hour to keep it in a thawed and thawed state. This process was repeated 5 times. If the antibody in the formulation loses stability, aggregation will be observed to increase the proportion of high molecular weight (HMW) material.

For the finally thawed formulation,% HMW was measured using SE-HPLC. The results are shown in Table 3 below. The average value and standard deviation (SD) of the data obtained by analyzing each formulation prepared in 3 replicates per formulation were obtained, and Table 3 shows the average per item for each formulation and the standard deviation in the lower row. 2 is a view showing the results of measuring the change in% HMW using SE-HPLC after exposing the formulations of Example 1 and Comparative Example 1 to freeze-thaw stress.

As shown in Table 3, the formulation of Example 1 showed better stability than Comparative Example 1 in freeze-thaw stress. Specifically, the formulation of Example 1 reduced Δ% HMW by 33% compared to the formulation of Comparative Example 1 in freeze-thaw stress.

Formulation	% HMW (0 freeze-thaw)	% HMW (5 times freeze-thaw)	△% HMW
Example 1	1.17	1.15	-0.02
	[N = 3, SD: 0.01]	[N = 3, SD: 0.01]	[N = 3, SD: 0.01]
Comparative Example 1	0.83	0.89	0.06
	[N = 3, SD: 0.07]	[N = 3, SD: 0.06]	[N = 3, SD: 0.01]

The surfactant-free formulations identified by this example were more stable than the surfactant-containing formulations, which is a remarkable remarkable effect by those skilled in the art.

All technical terms used in the present invention, unless defined otherwise, are used in the sense as commonly understood by those skilled in the art in the relevant field of the present invention. In addition, although a preferred method or sample is described herein, similar or equivalent ones are included in the scope of the present invention. In addition, the numerical values described in this specification are considered to include the meaning of “about” even if not specified. The contents of all publications described by reference herein are incorporated herein by reference in their entirety.

One aspect of the invention, (a) Trastuzumab or an antigen-binding fragment thereof; (b) stabilizer; And (c) a buffering agent and no surfactant.

The term “antigen binding fragment” is a fragment of a portion capable of binding to an antigen in a Trastuzumab antibody, including, but not limited to, Fab, F (ab ′) 2 and Fv, for example. The antigen can be a HER2 receptor.

Trastuzumab is a monoclonal antibody used to treat cancer, including breast cancer, and is marketed under the name Herceptin ^™ under one of several trade names. Trastuzumab is used for breast cancer that is positive for HER2 receptors. In addition, trastuzumab can be used alone or in combination with other chemotherapeutic agents. Trastuzumab is administered by intravenous injection and subcutaneous injection. Trastuzumab works by binding to the HER2 receptor and slows cell replication. Trastuzumab is a recombinant IgG1 kappa humanized from mice that binds to the extracellular domain of human epidermal growth factor receptor protein (HER2), a whole antibody.

In one embodiment, the concentration of trastuzumab or an antigen-binding fragment thereof in the liquid formulation may be 2 to 300 mg / mL. The concentration of the antibody can be freely adjusted within a range that does not substantially affect the stability, viscosity and desired pH of the liquid formulation of the present invention. In one embodiment, the concentration of the trastuzumab or antigen binding fragment thereof is, for example, 10 to 300 mg / mL, 20 to 300 mg / mL, 30 to 300 mg / mL, 50 to 300 mg / mL, 80 To 300 mg / mL, 100 to 300 mg / mL, 80 to 250 mg / mL, 80 to 200 mg / mL, 80 to 180 mg / mL, 80 to 150 mg / mL, 100 to 150 mg / mL, 100 to 140 mg / mL, 105-135 mg / mL, 110-130 mg / mL, 115-125 mg / mL, 100-250 mg / mL, 100-200 mg / mL, 100-180 mg / mL, 100-150 mg / mL, 100-140 mg / mL, 150-300 mg / mL, 200-300 mg / mL, 220-300 mg / mL or 250-300 mg / mL. Proper antibody concentrations can contribute to their stability in liquid formulations, the viscosity of liquid formulations suitable for administration, and the pH maintenance of liquid formulations.

The liquid formulation of the present invention does not contain a surfactant. The liquid formulations of the present invention may or may not contain surfactant at all. When substantially free of surfactant, it can mean, for example, that the surfactant comprises less than 0.04 w / v%, less than 0.02 w / v%, or less than 0.01 w / v%. In one embodiment, surfactants not included in the liquid formulation of the present invention include polyoxyethylene sorbitan fatty acid ester (Tween), polyethylene-polypropylene glycol, polyoxyethylene stearate, polyoxyethylene alkyl ether, polyoxyethylene- Polyoxypropylene copolymer (poloxamer), or sodium dodecyl sulfate. The polyoxyethylene alkyl ether may be, for example, polyoxyethylene monolauryl ether, alkylphenylpolyoxyethylene 30 ether (Triton-X). The polysorbate is a polyoxyethylene sorbitan fatty acid ester or a derivative thereof. Polysorbates are known to degrade through oxidation and hydrolysis, which in turn produces ROS, which is known to cause oxidative damage to antibody proteins. The liquid formulation of the present invention can maintain the stability of the antibody and antigen-binding fragment thereof without containing a surfactant such as polysorbate. Further, in one embodiment, the surfactant that is not included in the liquid formulation of the present invention may be polysorbate.

The polysorbate may mean polysorbate 20 or polysorbate 80. Thus, in one embodiment, the liquid formulation of the present invention may be one that does not include polysorbate 20 and polysorbate 80.

In one embodiment, the stabilizer may be one or more selected from the group consisting of sugar, sugar alcohol, sugar acid, polyol, metal salt and amino acid.

The sugar may be selected from monosaccharides, disaccharides, oligosaccharides, polysaccharides, or derivatives thereof. The sugar derivative may mean sugar alcohol or sugar acid. In one embodiment, the sugar, sugar alcohol or sugar acid is glucose, fructose, galactose, sucrose, lactose, maltose, trehalose, fructooligosaccharide, galactoligosaccharide, mango-oligosaccharide, starch, glycogen, cellulose, chitin, pectin , Glycerol, erythritol, thritol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fusitol, iditol, inositol, boletitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol , Polyglycitol, aldonic acid, ulonic acid, uronic acid, aldaric acid, stachyose, sorbose, xylose, ribose, myoinisitos, myoinisitol, and one selected from polyethylene glycol It may be abnormal.

In one embodiment, the concentration of the sugar, sugar alcohol or sugar acid may be 1 to 20w / v%. In another embodiment, the concentration of the sugar, sugar alcohol or sugar acid is 2 to 20w / v%, 4 to 20w / v%, 6 to 20w / v%, 6 to 12w / v%, 4 to 14w / v%, respectively. , 2 to 18w / v%, 7 to 9w / v%, 1 to 18w / v%, 1 to 15w / v%, 1 to 12w / v%, 1 to 10w / v%, 2 to 18w / v%, 2 to 15 w / v%, 2 to 12 w / v%, 2 to 10 w / v%, 4 to 18 w / v%, 4 to 15 w / v%, 4 to 12 w / v% or 4 to 10 w / v% have. The concentration of sugar, sugar alcohol or sugar acid can be freely adjusted within a desired range to maintain the stability of the trastuzumab or antigen-binding fragment thereof in the liquid formulation of the present invention and the viscosity of the liquid formulation, and each specific sugar, sugar alcohol or It can vary individually depending on the sugar acid.

The term "polyol" can mean an organic compound having two or more hydroxyl groups (-OH) in its molecule. In one embodiment, the polyol is any one selected from the group consisting of propanediol, glycerin, butylene glycol, propylene glycol, dipropylene glycol, pentylene glycol, hexylene glycol, polyethylene glycol, and sorbitol You can.

In one embodiment, the metal salt may be NaCl, KCl, NaF, KBr, NaBr, Na ₂ SO ₄ , NaSCN, or K ₂ SO ₄ , but is not limited thereto. In one embodiment, the metal salt may be NaCl. The concentration of the metal salt may be 0.1 to 10w / v%. In another embodiment, the concentration of the metal salt is 0.1 to 8w / v%, 0.1 to 6w / v%, 0.1 to 5w / v%, 0.1 to 3w / v%, 0.1 to 1w / v%, 0.3 to 10w / v %, 0.3 to 8 w / v%, 0.3 to 6 w / v%, 0.3 to 5 w / v%, 0.3 to 3 w / v% or 0.3 to 1 w / v%, 0.5 to 10 w / v%, 0.5 to 8 w / v% , 0.5 to 6w / v%, 0.5 to 5w / v%, 0.5 to 3w / v% or 0.5 to 1w / v%. The concentration of the metal salt can be freely adjusted within a range in which the stability of trastuzumab or an antigen-binding fragment thereof in the liquid formulation of the present invention is not precipitated, and may be individually changed for each specific metal salt.

In one embodiment, the amino acid is glycine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, histidine, glutamine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine It may be one or more selected from the group consisting of. The concentration of amino acids can be freely adjusted within a range that does not affect the desired pH of the liquid formulation of the present invention, and does not affect the stability of trastuzumab or antigen-binding fragments thereof, and can be individually adjusted for each specific amino acid. It may vary.

In one embodiment, the amino acid may be methionine.

In one embodiment, the stabilizer may include two or more stabilizers.

In one embodiment, the stabilizer may be one or more selected from the group consisting of trehalose and methionine.

In one embodiment, the stabilizer may be one or more selected from the group consisting of 1 to 15w / v% trehalose and 1 to 50 mM methionine. The stabilizer may be at least one selected from the group consisting of 2 to 14w / v% trehalose and 1 to 30 mM methionine. The stabilizer may be at least one selected from the group consisting of 3 to 13w / v% trehalose and 1 to 20 mM methionine. The stabilizer may be at least one selected from the group consisting of 4 to 12w / v% trehalose and 1 to 20 mM methionine. The stabilizer may be at least one selected from the group consisting of 5 to 11w / v% trehalose and 2 to 18 mM methionine. The stabilizer may be at least one selected from the group consisting of 6 to 10w / v% trehalose and 2 to 18 mM methionine. The stabilizer may be at least one selected from the group consisting of 6 to 10w / v% trehalose and 4 to 16 mM methionine. The stabilizer may be at least one selected from the group consisting of 6 to 10w / v% trehalose and 6 to 14 mM methionine. The stabilizer may be at least one selected from the group consisting of 7 to 9w / v% trehalose and 8 to 12 mM methionine. The stabilizer may be at least one selected from the group consisting of 2 to 8w / v% trehalose and 1 to 20 mM methionine.

In one embodiment, the buffering agent may be to provide a pH of 4.0 to 7.0. The liquid formulation may have a pH of 4.0 to 7.0. In other embodiments, the pH of the liquid formulation may be 4.5 to 6.5, 5.0 to 6.0 or 5.5 ± 0.2. The pH of the liquid formulation can be freely adjusted within a range suitable for preventing aggregation of the antibody and maintaining activity.

The liquid formulation of the present invention may contain a buffering agent to maintain the desired pH for stabilizing trastuzumab or an antigen-binding fragment thereof. The term "buffer" refers to a neutralizing material that minimizes the change in pH by acid or alkali, and in one embodiment, the buffering agent is histidine, phosphoric acid, citric acid, maleic acid, tartaric acid, succinic acid, citrate, acetate , Carbonate and salts thereof, but are not limited thereto.

In one embodiment, the buffering agent is histidine. In one embodiment, the buffering agent is 1 to 100 mM histidine. The concentration of histidine is, for example, 1 to 80 mM, 1 to 70 mM, 1 to 60 mM, 1 to 50 mM, 1 to 40 mM, 1 to 30 mM, 2 to 80 mM, 2 to 70 mM, 2 To 60 mM, 2 to 50 mM, 2 to 40 mM, 2 to 30 mM, 2 to 25 mM, 3 to 80 mM, 3 to 70 mM, 3 to 60 mM, 3 to 50 mM, 3 to 40 mM, 3 To 30 mM, 3 to 25 mM, 4 to 80 mM, 4 to 70 mM, 4 to 60 mM, 4 to 50 mM, 4 to 40 mM, 4 to 30 mM, 4 to 25 mM, 5 to 80 mM, 5 To 70 mM, 5 to 60 mM, 5 to 50 mM, 5 to 40 mM, 5 to 30 mM, 5 to 25 mM, 6 to 80 mM, 6 to 70 mM, 6 to 60 mM, 6 to 50 mM, 6 To 40 mM, 6 to 30 mM, 6 to 25 mM, 8 to 80 mM, 8 to 70 mM, 8 to 60 mM, 8 to 50 mM, 8 to 40 mM, 8 to 30 mM, 8 to 25 mM, 10 To 80 mM, 10 to 70 mM, 10 to 60 mM, 10 to 50 mM, 10 to 40 mM, 10 to 30 mM, 10 to 25 mM, 12 to 80 mM, 12 to 70 mM, 12 to 60 mM, 12 To 50 m M, 12 to 40 mM, 12 to 30 mM, 12 to 25 mM, 14 to 80 mM, 14 to 70 mM, 14 to 60 mM, 14 to 50 mM, 14 to 40 mM, 14 to 30 mM, 14 to 25 mM, 16 to 80 mM, 16 to 70 mM, 16 to 60 mM, 16 to 50 mM, 16 to 40 mM, 16 to 30 mM, 16 to 25 mM, 16 to 24 mM, 18 to 80 mM, 18 to 70 mM, 18-60 mM, 18-50 mM, 18-40 mM, 18-30 mM, 18-25 mM, 18-24 mM, 18-22 mM, 19-80 mM, 19-70 mM, 19-60 mM, 19-50 mM, 19-40 mM, 19-30 mM, 19-25 mM, 19-23 mM, 19-21 mM, 10-100 mM, 15-100 mM, 20-100 mM, 15-80 mM, 20 to 80 mM, 15 to 50 mM, 20 to 50 mM, 15 to 30 mM, 15 to 25 mM, 17 to 23 mM, or 20 to 30 mM. The histidine may be in the form of histidine and its conjugate acid. The conjugate acid may be histidine HCl. The ratio of histidine and its conjugate acid may vary depending on the pH of the selected agent. For example, when the pH of the formulation is 5.5 ± 0.2, the concentration ratio of histidine and histidine HCl monohydrate may be about 1: 6.

In one embodiment, the stabilizer, buffering agent, and surfactant may be different.

In the liquid formulation of the present invention, the trastuzumab or antigen-binding fragment thereof may be stabilized. The liquid formulation may be a stable liquid pharmaceutical formulation. The term “stabilization” means that trastuzumab or an antigen-binding fragment thereof substantially retains its physical stability, chemical stability and / or biological activity before and after administration, during further manufacturing processes, storage or storage. Physical stability, chemical stability and / or biological activity can be assessed by commonly known methods.

Stability can be measured for a selected period of time at a selected temperature. For example, in one embodiment, a stable formulation is one in which no significant change is observed at 2-8 ° C. for 12 months or longer. In another embodiment, a stable formulation is one that does not exhibit significant changes at 2-8 ° C. for 18 months or longer. In another embodiment, a stable formulation is one where no significant change is observed for 3 months or longer at 23-27 ° C. In another embodiment, a stable formulation is one where no significant changes are observed for 6 months or longer at 23-27 ° C. In another embodiment, a stable formulation is one where no significant change is observed at 23-27 ° C. for 12 months or longer. In another embodiment, a stable formulation is one where no significant change is observed at 23-27 ° C. for 18 months or longer. Stability criteria for antibody formulations are as follows. When measured by SE-HPLC, the antibody monomer is 10% or less, for example, 5% or less, or 2.5% or less relative to the initial monomer amount. The antibody potency is within 60 to 140%, or 80 to 120%, of the control or standard antibody. For example, when measuring low molecular weight (LMW) species by SE-HPLC, the change in the amount of the low molecular weight species is less than 10%, less than 5%, or less than 2.5% relative to its initial amount. It may have. For example, when high molecular weight (HMW) species are measured by SE-HPLC, the change in the amount of the high molecular weight species is 10% or less, 5% or less, or 2.5% or less relative to its initial amount. It may have. In addition, the concentration of the formulation, and the pH may have a change of 0% or less, ± 5% or less, or ± 2.5% or less relative to the initial value.

In one embodiment, when the formulation is placed in a stable thermostat and placed for 4 weeks at a temperature of 40 ± 2 ° C. and a relative humidity of 75 ± 5%, the initial pH is 10% or less, 5% or less, or 2.5% or less The pH changes within the range.

In one embodiment, the formulation is placed in a freezer maintained at -70 ± 10 ° C and maintained for 18 hours to maintain a frozen state, and then the process of removing the tube and leaving it at room temperature for 1 hour to maintain it in a thawed state is repeated 5 times. When done, the pH changes within the range of 10% or less, 5% or less, or 2.5% or less of the initial pH.

In one embodiment, the preparation is when 1 mL is placed in a 1.5 mL microtube (Axygen) made of polypropylene, and the tube is mounted on a stirrer (Heidolph) and stirred at 400 rpm for 72 hours at room temperature. , PH is changed within the range of 10% or less, 5% or less, or 2.5% or less with respect to the initial pH.

The trastuzumab or antigen-binding fragment thereof retains chemical stability within the pharmaceutical agent when it is chemically stable at a predetermined time that appears to still retain biological activity. Chemical stability can be assessed by detecting and quantifying a chemically modified form of the antibody. Chemical modification includes size modification or charge change. The charge change can be, for example, a charge change that occurs as a result of deamidation.

The size modification is size exclusion chromatography (SE-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis-sodium dodecyl sulfate, CE-SDS ) Analysis, and substrate-assisted laser desorption / ionization / time-of-flight mass spectrometry (MALDI / TOF MS). In addition, charge changes can be evaluated by ion exchange chromatography (IEC), and imaged capillary isoelectric focusing (icIEF). Activity can be determined through HER2 receptor binding assay. The HER2 receptor binding assay can be performed by Enzyme-linked immuonsorbent assay (ELISA). ELISA analysis measures the ratio (%) of trastuzumab binding to the HER2 receptor. ELISA can be performed using the Her2 ELISA kit (R & D system). After reacting with the trastuzumab sample and the HER2 receptor on the ELISA substrate, the binding degree can be determined by measuring the absorbance at 450 nm to obtain a relative binding rate (%).

The trastuzumab or antigen-binding fragment thereof retains biological activity in the pharmaceutical agent when the trastuzumab or antigen-binding fragment thereof is biologically active for its intended purpose. The biological activity is, for example, about 30%, about 20%, within about 10%, about 5% of the biological activity at which the biological activity of the trastuzumab or antigen-binding fragment thereof in the pharmaceutical agent is indicated at the time of manufacture of the pharmaceutical agent. Biological activity is retained within or within analytical error. The biological activity can be determined, for example, in antigen binding assays.

Stabilization is temperature stress, e.g., 1 to 4 weeks at 40 ° C, freeze-thaw stress, e.g., 5 repetitions of cycles at -70 ° C freeze and thaw at room temperature, or stirring stress, e.g. , Can be evaluated by measuring% HMW,% monomer and / or% LMW using SE-HPLC, by applying 400 rpm rotational force for 72 hours in a stirrer. In one embodiment, the liquid formulations of the present invention may have less Δ% HMW, Δ% monomer and / or Δ% LMW values compared to Herceptin®.

The liquid formulation provided herein has a change in% HMW measured when stored at 40 ° C for 4 weeks at 40 ° C when the antibody content is 120 mg / ml (pH 5.5), that is,% HMW at week 4 of storage- Week 0% HMW value is about 5% or less, about 3% or less, about 2.5% or less, about 2.3% or less, about 2.0% or less, about 0.5 to about 5%, about 0.8 to about 3%, about 0.1 to About 2.25%, about 1.00 to about 2.23%, about 1.15% ± 0.08%, and also about 1.15% ± 0.04%. The "week 0" indicates the initial time of storage.

In another example, the liquid formulation provided herein is thawed by maintaining the liquid composition at -70 ± 10 ° C for 18 hours, and then standing at room temperature for 1 hour when the antibody content is 120 mg / ml (pH 5.5). After repeating the process 5 times, the change amount of% HMW measured by conventional SEC is about 2% or less, about 1.5% or less, about 1.0% or less, about 0.5% or less, about 0.1% or less, about 0.05% or less, or about 0.01% or less, about 0.001% or less, about 0.1 to about 1%, about -0.05 to about 0.05%, about -0.03 to about 0.03%, about -0.02% ± 0.02%, or about -0.02% ± 0.01% have.

In one embodiment, the liquid formulation may be for subcutaneous or intravenous injection. The liquid formulation may further include an aqueous carrier suitable for injection. The aqueous carrier may be a safe and non-toxic pharmaceutically acceptable drug when administered to humans. The aqueous carrier includes, but is not limited to, sterile alcohol, for example, sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), sterile saline solution, Ringer's solution, and dextrose.

In one embodiment, the liquid formulation of the present invention may have an appropriate range of osmolality upon subcutaneous or intravenous injection. Osmolarity may be, for example, 200 to 400 mOsm / kg, 250 to 380 mOsm / kg, or 270 to 360 mOsm / kg. The osmolarity concentration can be appropriately adjusted to minimize pain that may occur during administration.

In one embodiment, the liquid formulation of the present invention may have an appropriate range of viscosity when subcutaneously or intravenously injected. The viscosity, for example, 0.5 to 50 cp, can be appropriately adjusted to minimize pain that may occur upon administration.

In one embodiment, the liquid formulation of the present invention may include a hyaluronidase enzyme. The amount of the hyaluronidase enzyme may vary depending on the hyaluronidase enzyme used. The amount of hyaluronidase enzyme can be an amount sufficient to increase the dispersion and absorption of an anti-HER2 antibody, e.g., trastuzumab or an antigen binding fragment thereof, administered together. The amount of hyaluronidase enzyme can be 150 U / ml or more. An effective amount of hyaluronidase enzyme is, for example, about 1,000 to 16,000 U / ml, where the amount corresponds to about 0.01 mg to 0.16 mg based on 100,000 U / mg of estimated specific activity. Alternatively, the concentration of the hyaluronidase enzyme may be about 1,500 to 12,000 U / ml, or about 2,000 to 12,000 U / ml. This amount corresponds to the amount of hyaluronidase enzyme added to the formulation. The amount of hyaluronidase enzyme measured in the final formulation can vary within a specific range. The ratio (w / w) of hyaluronidase enzyme to anti-HER2 antibody is 1: 5,00 to 1: 100,000, 1: 1,000 to 1: 50,000, 1: 1,000 to 1: 30,000, 1: 1,000 to 1: 20,000, 1: 1,000 to 1: 15,000, 1: 1,000 to 1: 13,000, 1: 1,000 to 1: 12,000, 1: 1,000 to 1: 10,000, 1: 1,000 to 1: 8,000, 1: 4,000 to 1: 5,000, Or about 1: 6,000.

The hyaluronidase enzyme may be derived from an animal, bacterial, or human sample or prepared by recombinant DNA technology.

The hyaluronidase enzyme may be soluble hyaluronidase glycoproteins (sHASEGPs). These soluble hyaluronidase glycoproteins can be administered together with the agent or separately. The addition of the soluble hyaluronidase glycoprotein can facilitate the subcutaneous administration of the therapeutic drug. By rapidly depolymerizing hyaluronan (HA) in the extracellular space, the soluble hyaluronidase glycoprotein lowers the viscosity of the interstitium, increasing hydraulic conductance, making it easier for large volumes to subcutaneous tissue. Make it administered comfortably. The increased hydraulic conductivity induced by soluble hyaluronidase glycoproteins through reduced interstitial viscosity can result in better dispersion, thereby increasing the systemic bioavailability of the subcutaneously administered therapeutic drug.

The hyaluronidase enzyme may be licensed for use in humans in any one or more countries. The hyaluronidase enzyme may be, for example, Hylase ^TM , Dessau, Hyalase ^TM , EU approved, Vitrase ^TM , Hydase ^TM , Amphadase ^TM , or Hylenex ^TM , licensed in the United States. The hyaluronidase enzyme may be a human hyaluronidase enzyme such as PH20 or rHuPH20. Recombinant human PH20 (rHuPH20) is a member of the neutral and acid-active β-1,4 glycosyl hydrolase family. rHuPH20 depolymerizes hyaluronan by hydrolysis of a β-1,4 bond between the C1 position of N-acetyl glucosamine and the C4 position of glucuronic acid. rHuPH20 may be a truncated deletion variant without amino acid at the carboxy terminal required for lipid attachment developed by Halozyme.

Another aspect of the present invention provides a liquid formulation, wherein the trastuzumab or antigen-binding fragment thereof is 2 to 300 mg / mL, the stabilizer is a combination of trehalose and methionine, and the buffering agent is histidine. .

In one embodiment, the stabilizer is a combination of 1 to 20 w / v% trehalose and 1 to 20 mM methionine, and the buffering agent is 10 to 50 mM histidine. The concentration of the trehalose is 2 to 20w / v%, 4 to 20w / v%, 6 to 20w / v%, 1 to 18w / v%, 1 to 15w / v%, 1 to 12w / v%, 1 to 10w / v%, 2 to 18w / v%, 2 to 15w / v%, 2 to 12w / v%, 2 to 10w / v%, 4 to 18w / v%, 4 to 15w / v%, 4 to 12w / v% or 4 to 10 w / v%. The concentration of the methionine is 1 to 20 mM, 2 to 20 mM, 5 to 20 mM, 8 to 20 mM, 1 to 18 mM, 2 to 18 mM, 5 to 18 mM, 1 to 15 mM, 2 to 15 mM, 5 to 15 mM, 8 to 15 mM, 1 to 12 mM, 2 to 12 mM, 5 to 12 mM, or 8 to 12 mM. The histidine may be 10 to 45 mM, 10 to 40 mM, 10 to 35 mM, 10 to 30 mM, 15 to 25 mM, or 18 to 22 mM. The formulation may be pH 5.5 ± 0.2.

One embodiment of the present invention (a) Trastuzumab or an antigen-binding fragment 80 to 160 mg / ml thereof; (b) 1 to 20 mM methionine and 1 to 15 (w / v)% of trehalose as stabilizers; And (c) 1-50 mM histidine as a buffering agent. The formulation may be pH 5.5 ± 0.2.

One embodiment of the present invention is (a) Trastuzumab or an antigen-binding fragment 80 to 160 mg / ml thereof; (b) 1 to 20 mM methionine and 1 to 15 (w / v)% trehalose as stabilizers; And (c) 1 to 40 mM histidine as a buffering agent. The formulation may be pH 5.5 ± 0.2.

One embodiment of the present invention is (a) Trastuzumab or an antigen-binding fragment 100 to 140 mg / ml thereof; (b) 3 to 18 mM of methionine and 2 to 13 (w / v)% of trehalose as stabilizers; And (c) 10-30 mM histidine as a buffering agent. The formulation may be pH 5.5 ± 0.2.

One embodiment of the present invention (a) Trastuzumab or an antigen-binding fragment 110 to 130 mg / ml thereof; (b) 5 to 15 mM methionine as stabilizer and 4 to 12 (w / v)% trehalose; And (c) 15 to 25 mM histidine as a buffering agent. The formulation may be pH 5.5 ± 0.2.

One embodiment of the present invention (a) Trastuzumab or an antigen-binding fragment 110 to 130 mg / ml thereof; (b) 8 to 12 mM methionine and 6 to 10 (w / v)% threonyl as stabilizers; And (c) 16 to 24 mM histidine as a buffering agent. The formulation may be pH 5.5 ± 0.2.

One embodiment of the present invention (a) Trastuzumab or an antigen-binding fragment thereof 120 mg / ml ± 5%; (b) 10 mM ± 5% methionine as stabilizer and 8% (w / v)% ± 5% trehalose; And (c) 20 mM ± 5% histidine as a buffering agent. Here, “± 5%” indicates that there is a variation of “± 5%” with respect to the basic amount. The liquid formulation may be pH 5.5 ± 0.2. The histidine may include histidine and its conjugate acid, for example, histidine HCl. The buffering agent may include only histidine and its conjugate acid. The formulation may be one that does not contain a surfactant, for example, a nonionic surfactant. The formulation may not contain polysorbate 20 or 80.

Another aspect of the present invention, adding a stabilizer and a buffering agent to the solvent to prepare a mixed solution; And adding trastuzumab to the mixed solution.

It is understood that any of the terms or elements mentioned in the liquid formulations, such as those mentioned in the description of the method for preparing the claimed liquid formulation, is as mentioned in the description of the liquid formulation claimed above.

Claims (19)

1. (a) Trastuzumab or an antigen-binding fragment thereof;

   (b) stabilizers; And

   (c) a buffering agent,

   Liquid formulations containing no surfactant.
2. The liquid formulation of claim 1, wherein the surfactant is polysorbate.
3. The liquid formulation of claim 1, wherein the surfactant is polysorbate 20 and polysorbate 80.
4. The method according to claim 1, wherein the concentration of the trastuzumab or antigen-binding fragment thereof is 80 to 300 mg / mL liquid formulation.
5. The method according to claim 1, wherein the stabilizer is a liquid formulation of one or more selected from the group consisting of sugar, sugar alcohol, sugar acid, polyol, metal salt and amino acids.
6. The liquid formulation of claim 5, wherein the amino acid is methionine.
7. The method according to claim 5, wherein the sugar, sugar alcohol or sugar acid is glucose, fructose, galactose, sucrose, lactose, maltose, trehalose, fructooligosaccharide, galactoligosaccharide, met oligosaccharide, starch, glycogen, cellulose, chitin, pectin , Glycerol, erythritol, thritol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fusitol, iditol, inositol, boletitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol , Polyglycitol, aldonic acid, ulonic acid, uronic acid, aldaric acid, stachyose, sorbose, xylose, ribose, myoinisitos, myoinisitol, and one selected from polyethylene glycol Liquid formulations that are ideal.
8. The method according to claim 5, The concentration of the sugar, sugar alcohol or sugar acid is 1 to 20w / v% liquid formulation.
9. The method according to claim 1, wherein the stabilizer is a liquid formulation that is at least one selected from the group consisting of trehalose and methionine.
10. The method according to claim 1, wherein the stabilizer is a liquid formulation of at least one selected from the group consisting of 1 to 20w / v% trehalose and 1 to 50 mM methionine.
11. The liquid formulation of claim 1, having a pH of 4.0 to 7.0.
12. The liquid formulation of claim 1, wherein the buffering agent is histidine.
13. The method according to claim 1, wherein the buffering agent is a liquid formulation of 1 to 50 mM histidine.
14. The method according to claim 1, wherein the trastuzumab or antigen-binding fragment thereof is a liquid formulation that is stabilized in the liquid formulation.
15. The liquid formulation of claim 1, wherein the liquid formulation is for subcutaneous or intravenous injection.
16. The method according to claim 1, The trastuzumab or antigen-binding fragment thereof is 80 to 300 mg / mL, the stabilizer is a combination of 1 to 20w / v% trehalose and 1 to 50 mM methionine, and the buffering agent is 1 to A liquid formulation that is 50 mM histidine.
17. The method according to claim 1, The trastuzumab or antigen-binding fragment thereof is 80 to 160 mg / mL, the stabilizer is a combination of 1 to 15w / v% trehalose and 1 to 20 mM methionine, and the buffering agent is 1 to A liquid formulation that is 40 mM histidine.
18. The method according to claim 1, wherein the trastuzumab or antigen-binding fragment thereof is 100 to 140 mg / mL, the stabilizer is a combination of 2 to 13% trehalose and 3 to 18 mM methionine, and the buffering agent is 10 to A liquid formulation that is 30 mM histidine.
19. Preparing a mixed solution by adding a stabilizer and a buffering agent to the solvent; And

    A method of preparing a liquid formulation comprising adding trastuzumab to the mixed solution.

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